ozbio2010 poster abstracts

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 99

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010


POSTERS MONDAY & TUESDAY

POS-MON-01 POS-TUE-02


DIGESTIVE VACUOLE GENESIS AND ENDOCYTIC PROCESSES IN THE EARLY INTRAERYTHROCYTIC STAGES OF PLASMODIUM FALCIPARUM

Abu Bakar N.1, Klonis N.1, 2, Hanssen E.1, 2, Chan C.1 and Tilley L.1, 2 1Department of Biochemistry, La trobe University. 2Centre of Excellence for Coherent X-ray Science, La Trobe University.

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of hemoglobin digestion and heme detoxification and is the target of chloroquine and other antimalarials. The mechanisms for genesis of the digestive vacuole and transfer of hemoglobin from the host cytoplasm are still debated. In the present study, we use live-cell imaging and photobleaching to monitor the uptake of the pH-sensitive fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in ring-stage and trophozoite-stage parasites. We compare these results with electron tomography of serial sections of parasites at different stages of growth. We show that uptake of erythrocyte cytoplasm is initiated in mid-ring-stage parasites. The host cytoplasm is internalised via cytostome-derived invaginations and concentrated into several acidified peripheral structures. Hemoglobin digestion and hemozoin formation take place in these vesicles. The ring-stage parasites can adopt a deeply invaginated cup shape but do not take up hemoglobin via macropinocytosis. As the parasite matures, the hemozoin-containing compartments coalesce to form a single acidic digestive vacuole that is fed by hemoglobin-containing vesicles. There is also evidence for hemoglobin degradation in compartments outside the digestive vacuole. The work has implications for the stage specificity of quinoline and endoperoxide antimalarials.

ROLES OF WNT SIGNALLING IN SPERMATOGENESIS

Abud H.E.1, Kerr G.1, Horvay K.1 and Loveland K.L.1, 2, 3 1Monash University, Department of Anatomy and Developmental Biology, Clayton Vic 3800. 2Monash University, Department of Biochemistry, Clayton Vic 3800. 3ARC Centre of Excellence in Biotechnology and Development.

Male infertility is a worldwide problem with increasing incidence and may be caused in part by disruptions in the development of male germ cells and their supporting somatic cells. We have discovered that testicular cell communication via the Wnt signalling pathway is required for sperm development, because genetically altered mice with perturbed Wnt signalling exhibit interrupted spermatogenesis and appear to have reduced fertility. We are currently using two unique mouse models to understand the precise functional role of Wnt signalling during spermatogenesis. We are studying the effect of blocking Wnt signalling by conditional mutation of beta-catenin, a key mediator of canonical Wnt signalling. In contrast, the effects of constitutively active Wnt signalling will be studied by conditional mutation of the negative regulator, Adenomatous Polyposis Coli (APC). In addition, the endogenous Wnt signalling components and downstream target genes present in the mouse testis are being examined using a combination of immunohistochemical and qRT-PCR techniques. Our results to date implicate canonical Wnt signalling in post-mitotic germ cell development, and ongoing work with human clinical specimens will reveal the potential contribution of disturbances in this pathway to male infertility.

L1CAM ACTS AS A SOX10 MODIFIER GENE DURING ENTERIC NERVOUS SYSTEM DEVELOPMENT

Anderson R.B.1, Wegner M.2 and Wallace A.S.1 1University of Melbourne, Australia. 2University of Erlangen-Nurnberg, Germany.

During development, the enteric nervous system (ENS) is derived from neural crest cells that emigrate from the hindbrain, enter the foregut and migrate caudally to colonise the entire gut. Failure of neural crest cells to fully colonise the gastrointestinal tract results in an ‘aganglionic zone’ that lacks an enteric nervous system over a variable length of the distal bowel, a condition in humans known as Hirschsprung’s disease. The variability observed in the penetrance and severity of Hirschsprung’s disease strongly suggests a role for modifier genes. Human clinical and animal model studies have suggested that the X-linked gene, L1CAM, may act as a modifier gene for the development of Hirschsprung’s disease. To examine whether L1cam interacts with the Hirschsprung’s associated gene, Sox10, we used a two-locus complementation approach. To assay the effects of an interaction, we examined whether the migration of enteric neural crest cells was altered in L1cam null mutant mice when combined with a heterozygous mutation in Sox10. We show that interactions between L1cam and Sox10 significantly delay neural crest migration within the developing gut, and that neural crest cells undergo excessive cell death prior to gut entry. Using a doxycycline inducible system, we show that Sox10 can regulate the expression of L1cam. Thus, L1cam can act as a modifier gene for the Hirschsprung’s associated gene, Sox10, and is likely to play a role in the aetiology of Hirschsprung’s disease.

SEXUALLY-DIMORPHIC EXPRESSION OF MIR202* IS ASSOCIATED WITH TESTIS DIFFERENTIATION IN THE MALE CHICKEN EMBRYOBannister S.C.1, 3, Smith C.A.2, Buermans H.4, Doran T.J.1, Sinclair A.H.2, 3 and Tizard M.L.V.1 1CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Australia, 3220. 2Early Development & Disease, Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia, 3052. 3Department of Paediatrics, The University of Melbourne, Parkville, Victoria, Australia, 3052. 4Center for Human and Clinical Genetics, Leiden University Medical Centre, Leiden, Netherlands.

In the chicken, sex is determined genetically by the inheritance of sex chromosomes at fertilization. In birds the sex chromosomes are Z and W and assort as ZW in females and ZZ in males. Establishment of the sexual phenotype of the embryonic gonads is subsequently driven by the expression of sex-determining genes. In the chick, the gonads begin to differentiate into testes and ovaries at embryonic day 6.5 (E6.5). After this point, testes develop bilaterally in the male, whilst asymmetric development proceeds in the female, with only the left gonad forming a functional ovary. We are studying the chicken embryo as a model for vertebrate gonadal development and aim to identify how microRNAs (miRNAs) may be involved in defining, or maintaining the differentiation of embryonic testes and ovaries. MiRNAs are 21-24nt non-coding RNAs which potentiate sequence-specific, post-transcriptional gene repression during development. We have used microarray and next-generation sequencing technologies to compare male and female miRNA expression profiles across three stages of gonadal sex differentiation in the chick. We have since focused our studies on chicken MIR202*, which was found to be up-regulated in testes cords of male gonads, from the onset of sex differentiation. Our recent work shows MIR202* expression is suppressed during estrogen-induced feminization of male embryonic gonads and promoted in female gonads when estrogen synthesis is blocked by an Aromatase inhibitor. These findings suggest MIR202* expression is associated with male embryonic testes development. Our continuing studies are focused on elucidating the cellular and molecular function of MIR202* during sexual differentiation. We are currently using in ovo retroviral-mediated delivery to optimise over-expression of MIR202* during early gonadogenesis. This will allow us to identify and validate MIR202* gene targets and understand the sex-specific regulation of MIR202* expression and processing.





TWIST1 IS REQUIRED BOTH IN THE CRANIAL NEURAL CREST AND THE CRANIAL MESODERM FOR PROPER HEAD DEVELOPMENT

Bildsoe H.1, 2, Loebel D.A.F.1, 2, Jones V.1, Hor A.1, Steiner K.1, Chen Y.T.3, Beringer R.R.3 and Tam P.P.1, 2 1Embryology Unit, Children’s Medical Research Institute, Locked Bag 23 Wentworthville, NSW 2145, Australia. 2Sydney Medical School, University of Sydney, NSW, Australia. 3Department of Molecular Genetics, University of Texas, M.D.Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA.

The basic helix-loop helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development. The mutant embryos die at E11.0 preventing studies beyond midgestation. We have used Cre-loxP conditional deletions to dissect the function of Twist1 specifically in the cranial neural crest (CNC) and cranial mesoderm (CM). Deletion of Twist1 in CNC cells resulted in loss or malformations of the CNC-derived skeleton, including the frontal bone, upper jaw and snout, due to impaired CNC cell survival and differentiation. Parietal and interparietal bones are also affected although these structures are primarily of mesodermal origin. Loss of Twist1 in the CM leads to incomplete closure of the cephalic neural tube but normal development of 1st branchial arch and frontonasal tissues. The majority of these embryos died at mid-gestation. Embryos developing past midgestation show loss or reduction of parts of the chondrocranium and poor development of the posterior skull base, while the viscerocranium was not affected. Taken together, the phenotypes of these two tissue-specific conditional deletions recapitulate that of the null mutation. Our results further show that tissue-specific loss of Twist1 function in one of the two cell populations that make up the craniofacial skeleton can impact on both tissues, suggesting that interaction between primordial tissues is critical for proper craniofacial development.

FGF9 ANTAGONISES FEMALE AND PROMOTES MALE GERM CELL FATE IN MICE

Bowles J., Feng C.-W., Davidson T.-L., Spiller C. and Koopman P. Institute for Molecular Bioscience, University of Queensland, Australia.

In the fetal gonad, germ cells commit to a female or male sexual fate on the basis of environmental cues, rather than XX or XY chromosome constitution. Germ cells in a developing ovary enter meiosis, hence committing to the female fate or oogenesis. In a developing testis, germ cells do not enter meiosis during fetal life but they do stop proliferating and arrest in G0/G1 (mitotic quiescence), hence committing to the male fate or spermatogenesis. In recent years we showed germ cells in a female mouse embryonic gonad are triggered to enter meiosis by the potent signaling molecule retinoic acid (RA). RA induces germ cells to express a key gene, Stra8, which encodes a protein essential for initiation of meiosis. In the developing testis, germ cells avoid entering meiosis because RA is actively degraded by a cytochrome P450 enzyme, CYP26B1. Hence, CYP26B1 is a ‘meiosis-inhibiting’ substance by virtue of its ability to degrade the meiosis inducer, RA. Here, we report that a second ‘meiosis-inhibiting’ substance, FGF9, acts directly on germ cells to prevent up-regulation of Stra8 and hence their entry into meiosis. In addition, FGF9 maintains germ cell pluripotency and promotes expression of male fate markers. Since RA is more abundant in the developing ovary and FGF9 is more abundant in the developing testis, the model we propose allows for ‘back-up’ and hence robustness in the oogenic/spermatogenic fate decision. Antagonistic interplay between FGFs and RA is proving to be a recurring theme in development, in each instance being associated with key cell lineage decisions. Our findings provide a new example of this phenomenon.

SIDEROPHORE DISCOVERY BY APPLICATION OF IMMOBILIZED METAL ION AFFINITY CHROMATOGRAPHY

Braich N.E. and Codd R. School of Medical Science (Pharmacology) and Bosch Institute, The University of Sydney, NSW 2006, Australia.

Siderophores are low-molecular-weight Fe(III) binding secondary metabolites produced by bacteria and plants. Siderophores are pharmacologically important and have numerous applications. Currently they are clinically used for the treatment of iron overload diseases. However, they are difficult to synthesize and/or purify. Thus, studies were conducted on the use of Ni2+-charged immobilized metal ion affinity chromatography (IMAC) for the capture of hydroxamate-based siderophores as a fast and effective means of biodiscovery. Results showed the successful capture of monohydroxamate, dihydroxamate, and trihydroxamate based siderophore standards and optimal capture conditions were also established. IMAC was successfully used for the purification of desferrioxamine B (DFO B), a trihydromate, from crude samples of culture supernatant of the DFOB-producing bacterium Streptomyces pilosus. RP-HPLC traces indicate a significant purification of >50 species reduced to 3 species, of which 2 are media derived. Salinispora triopica is an actinomycete bacteria which is predicted by bioinformatics to produce multiple siderophores including DFO and yersiniabactin like siderophores. Preliminary results show that S. tropica produces DFOB, but also produces several unidentified siderophore-like molecules. Currently, investigations are underway on identifying the remaining siderophores. This work opens the door for the use of IMAC in discovering a range of secondary metabolites with metal ion binding affinity.

TWIST FUNCTION IN CRANIOFACIAL MORPHOGENESIS

Brinas I.B., Wade C.W. and Farlie P.F. Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Rd, Parkville, Victoria, 3052.

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of sites. We have used RCAS-mediated overexpression to investigate the function of Twist genes in craniofacial development. Sustained expression of Twist1 or 2 results in failure of fusion between the maxillary, lateral nasal and frontonasal processes. The Twist1 phenotype tends to be more severe than the Twist 2 phenotype. Analysis of the craniofacial skeleton reveals a failure of fusion between the premaxilla and the maxillary/palatine bones. In addition, the premaxilla, maxilla and palatine bones were hypoplastic. These facial clefts were either uni- or bilateral in nature and exhibited substantial variation in the severity in terms of the width of the cleft and the extent of hypoplasia. Apart from some skeletal hypoplasia, the mandible appears to be predominantly unaffected by Twist1 or 2 overexpression. There are no consistent changes in either Fgf or Shh or Bmp signalling around the time of facial fusion. The molecular basis of this phenotype remains unclear. Apoptosis and proliferation was quantified to determine the cause of facial clefts in Twist1 overexpressed embryos. There was no change in apoptosis of Twist1 overexpressed and control embryos. There was, however, an unexpected 3-fold increase in the number of proliferating cells within the lateral nasal and frontonasal masses of Twist1 overexpressed embryos. It is likely that Twist2 overexpression is also upregulating proliferation but we will test this. Overall, Twist1 and 2 appear to have important roles in craniofacial morphogenesis and particularly in the fusion of the facial processes.





DISCOVERY OF A NOVEL A-TYPE LAMIN INTERACTING TRANSCRIPTION FACTOR, LITF, REQUIRED FOR MYOGENESIS

Ahmady E.1, Deeke S.A.1, Rabaa S.1, Kouri L.1, Krotneva S.1, Kenney L.1, Blais A.2, Stewart A.F.R.1 and Burgon P.G.1 1University of Ottawa Heart Institute, Ottawa ON Canada. 2University of Ottawa, Ottawa ON Canada.

Lamins are intermediate filament proteins of the inner nuclear membrane fundamentally important for nuclear architecture, chromatin organization and transcriptional regulation of gene expression. However, the molecular mechanisms that couple lamins to transcription are poorly understood. Here, we report the identification of an A-type Lamin-interacting Transcription Factor, LITF, a unique single copy amniota gene. Chromatin immunoprecipitation and gel mobility shift assays demonstrated that LITF binds to DNA within close proximity of genes encoding many transcription factors that control tissue-specific differentiation. Transient transfections of GAL4-LITF fusion constructs with a GAL4 responsive reporter showed that LITF contains a strong transactivation function within its conserved C-terminal domain. LITF protein is primarily expressed in cardiac and skeletal muscle and is expressed as early as E8.5 in the mouse embryo. Using the well-characterized C2C12 model of in vitro muscle differentiation, we found that blocking LITF expression in myoblasts also prevented the expression of myogenic transcription factors (Mef2C, MyoD, Myf6 & MyoG) and subsequently inhibited myogenic differentiation. In addition, LITF mediated chromatin immunoprecipitation studies revealed that LITF interacts with many DNA regions in close proximity of transcriptions factors (GATA4, Pitx2, Mef2c, RXRα and Sox4) that are important for normal muscle formation and differentiation. Data will be presented from the phenotypic analysis of a muscle specific LITF null. These observations coupled with the molecular uniqueness of LITF suggest that LITF lies in a prominent position within the regulatory process of muscle development. Our discovery of LITF provides the first direct molecular link between the Lamin A/C gene and gene expression.

PROLIFERATION OF NEURONS AND NON-NEURONAL CELLS IN THE DEVELOPING MOUSE STELLATE GANGLION

Gonsalvez D.G., Cane K.N. and Anderson C.R. University of Melbourne. Parkville VIC, 3010.

Mouse sympathetic ganglia appear around embryonic day (E)9.5, with differentiated neurons present at E10.5 and the first glial cells at E11.5. From E10.5 onwards, the number of cells in the ganglion increases dramatically. We have used immunoreactivity to Sox10, to mark uncommitted neural crest cells and glia, and tyrosine hydroxylase (TH), to mark sympathetic neurons in E10.5 to E18.5 stellate ganglia (n=4 in each case). We have also estimated cell division rates with bromodeoxyuridine (BrdU) and S-phase length with BrdU and ethyldeoxyuridine(EdU). At E10.5, 99% of all cells are positive for Sox10 expression, with half of this population also expressing TH; only 1% of cells express TH alone. By E11.5, 19% of cells express Sox10 alone, 6% express both Sox10 and TH and 76% of cells express TH only. The proliferation rate of both TH-IR and Sox10-IR cells was highest around E12.5, when around 50% of both neuronal and non-neuronal cells in the ganglion contained BrdU after a two hour pulse. We have determined that S-phase lengths for neuronal and non-neuronal cells differ during this period (TH+-6hrs, Sox10+-3hrs). Total cell cycle lengths for the two cell populations do not differ greatly at either of the two time points considered. Proliferation of non-neuronal cells overtook that of neurons on E16.5, when neuronal division had dropped to a low rate. Between E11.5 and E14.5, the growth of the ganglion is largely due to the division of existing neurons. The increasing disparity between the number of neurons and non-neuronal cells during this period does not depend on differences in proliferation rate or cell cycle length, but solely on the relative starting numbers of neurons versus non-neuronal cells, which is established on E10.5 when most, but not all, of the neural crest precursor cells differentiate into neurons.

POSITIONAL CLONING OF LYMPHATIC MUTANTS IN ZEBRAFISH

Coxam B.1, Neyt C.1, Shulte-Merker S.2 and Hogan B.1 1Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St Lucia, 4072, Brisbane, QLD, Australia. 2Hubrecht Institute Uppsalalaan 83584 CT UTRECHT,The Netherlands.

Lymphatic vessels play an essential role in fluid homeostasis, immune responses, fat absorption, and in pathological processes such as lymphedema and cancer metastasis. Despite recent breakthroughs in unravelling the complexity of lymphatic vessel development (called lymphangiogenesis), a lot is left to discover about the early steps of this process and its underlying cellular dynamics and molecular regulation. Using a genetic screen in zebrafish, we have identified two mutants presenting novel lymphatic phenotypes. One mutant presented a block in lymphangiogenesis associated with larval stage cardiac dysfunction and the other exhibited pleiotropic developmental defects encompassing a block in lymphangiogenesis, blood vasculature hyper-branching and a dysmorphic head. Initial molecular genetic data and a detailed phenotypic analysis at the cellular level using vascular transgenic fish lines will be presented. Keywords: Lymphatic system, Zebrafish, Positional Cloning.

CHARACTERISATION OF THE DEVELOPMENTAL ROLES OF THE DROSOPHILA MACPF PROTEIN TORSO-LIKE

Crossman T.1, 2, Johnson T.K.1, 2, Herr A.1, 2, Whisstock J.C.2 and Warr C.G.1 1School of Biological Sciences, Monash University, Clayton, Victoria, Australia 3800. 2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3800.

Torso-like (tsl) is the sole Drosophila member of the Membrane Attack Complex and Perforin (MACPF) protein superfamily. While members of the MACPF family are typically involved in immunity and defence, tsl has long been established as a maternally expressed gene and key component of the pathway responsible for patterning the anterior and posterior poles of the developing oocyte. However, our recent research has indicated additional roles for tsl in development. Expression studies using in situ hybridization experiments and a reporter gene line have shown that tsl is expressed in a number of specific tissue and cell types. In the developing embryo we see tsl expression in the midline glia of the central nervous system, a specialized set of glia that are analogous to the vertebrate floor plate. During larval stages we see tsl expression in the prothoracic gland, a part of the ring gland, which is the hormonal control centre of the developing fly. In adults we see expression of tsl in a subset of cells in the retina. Functional studies on tsl mutant strains have shown serious central nervous system defects, indicating a role for tsl in midline glia survival or function. To study the role of tsl in the prothoracic gland we are performing tissue-specific RNA interference experiments. The role of tsl in the adult retina is being characterised using available mutant strains and the many markers available for different cell types in the retina. Overall these approaches aim to enable the complete characterisation of the roles of tsl from embryogenesis to adulthood, and to provide insight into the roles of MACPF proteins in development.





INTEGRIN LINKED KINASE (ILK) IS REQUIRED FOR LENS EPITHELIAL CELL SURVIVAL AND PROLIFERATION

Teo J.1, McQueen-Miscamble L.1, Turner K.1, Martinez G.1, Reneker L.2, Dedhar S.3, Robinson M.L.4 and De Iongh R.U.1 1Anatomy & Cell Biology, University of Melbourne, Parkville, Victoria, Australia. 2Ophthalmology, University of Missouri. 3British Columbia Cancer Research Center, Vancouver, Canada. 4Zoology, Miami University, Oxford, Ohio.

The role of growth factors in lens development has been investigated extensively; the role of ECM signaling is less well understood. The laminin-binding integrins (α3β1, α6β1) are cooperatively required for epithelial cell survival during embryonic development. In this study we investigated the role of integrin linked kinase (ILK), a downstream mediator of ECM-integrin signaling, in lens development. We generated mice that were conditionally null for Ilk in the lens or expressed a hyperactive ILK in the lens. Consistent with its epithelial expression, Ilk deletion only resulted in a phenotype if deleted in lens epithelium, not when deleted in fibres only. The phenotype was characterised by thinning of the epithelium by E17.5, loss of central epithelial cells by P2 and extensive fibre cell degeneration and apoptosis by P10. At E17.5 there was significant inhibition (~50%) of epithelial cell cycle progression (reduced BrdU, cyclin D1, cyclin D2 and phospho-histone3 staining), but no significant changes in epithelial markers (E-cadherin, Pax6). Loss of Ilk also affected deposition of ECM (laminin and collagen IV), with retention of collagen in exocytic pathway. Erk and Akt phosphorylation were markedly decreased in Ilk null lenses. Postnatally, there was reduced and delayed expression of fibre cell markers, β-crystallin, c-Maf, p57kip2 and p27kip1, but not Prox1, and abnormal accumulation of fibronectin and α-smooth muscle actin. Ectopic expression of hyperactive kinase ILK (S343D) did not affect lens development with only subtle changes in epithelial cell morphology detected in vitro (increased formation of lamellipodia, enriched ILK at focal contacts). The hyperactive ILK transgene completely rescued lens morphology in Ilk null mice cell, but proliferation was incompletely rescued. These data indicate IK is required for epithelial proliferation, survival and subsequent fibre cell differentiation. ILK is required for epithelial proliferation and survival, but is insufficient to initiate cell proliferation.

NARINGIN PROMOTES ANTICARCINOGENIC EFFECTS ON RATS BEARING WALKER 256 TUMOR

Camargo C.A.1, Wutzki N.C.1, Bueno L.G.M.1, Gomes-Marcondes M.C.C.2, Da Silva M.E.F.3 and Aoyama H.1 1Departamento de Bioquímica, Instituto de Biologia, UNICAMP – Campinas, São Paulo, Brazil. 2Departamento de Anatomia, Biologia Celular e Fisiologia, Instituto de Biologia, UNICAMP – Campinas, São Paulo, Brazil. 3Instituto de Ciências da Saúde, UNIP - Campinas, São Paulo, Brazil.

Naringin (NAR), a plant polyphenolic compound, is a naturally occurring citrus flavonone (oranges and grapefruits). Flavonoids have been reported to exhibit numerous biological and pharmacological effects, such as antioxidant, anticarcinogenic, antiinflammatory and cardioprotective. Cachexia is a poorly understood syndrome characterized by generalized host wasting, anorexia and a variety of metabolic alterations that result in death. One characteristic of Walker 256 tumor (W256) is to provoke cachexia in infected animals. More than 80% of the patients with malignant disease present features of malnutrition and cachexia as the cause of death. This study was designed to describe an in vivo naringin therapeutic treatment of rats bearing W256. Rats bearing W256 were treated with different daily doses (10, 25 and 35 mg/kg) of naringin i.p. for 50 days or until the obit. Survival, dose-response (ED50) and tumor regression curves were obtained and 25 mg/kg was taken as the effective dose. Our studies showed that daily administrations of naringin, in low doses (10-25 mg/kg), had inhibited tumors growth (about 50%) in comparison with the control animals. Moreover, two animals of this group had presented complete tumor regression and when inoculated again with the carcinosarcoma, no tumor development was observed. However, at a higher concentration (35 mg/kg) this flavonoid provoked corporal weakening and cachexia process in the animals. Our studies suggest that naringin can be used as an effective drug to treat cancer and to prevent against the effects of cachexia. Financial Support: CAPES, CNPq and FAPESP.

REGULATION OF INTERNEURON DEVELOPMENT BY SUPPRESSOR OF CYTOKINE SIGNALLING-2 (SOCS2)

Faux C.H. and Turnley A.M. Centre for Neuroscience, The University of Melbourne.

Gamma-aminobutyric acid (GABA)ergic interneurons play a vital role in modulating the activity of the cerebral cortex. Comprising approximately 20% of the total cortical neuron population, they are an extremely diverse population of cells, differing in their morphology, physiology and molecular characteristics. Recent studies have shown that a disruption to the function of GABAergic interneurons can directly contribute to neurological and mental health issues such as schizophrenia, epilepsy and depression. Often such disruptions arise during development, with alterations to interneuron number, distribution or connectivity leading to functional neural impairment. Currently, however, little is known about the signalling mechanisms that regulate interneuron specification, migration or maturation. We have previously shown that the regulatory protein suppressor of cytokine signalling-2 (SOCS2) is a key player in interneuron development. SOCS2-overexpressing mice have large increases in numbers of calbindin and calretinin-expressing interneurons in the adult cortex. In contrast, SOCS2 knockout mice show a reduction in the number of parvalbumin positive cells. We have found that during development SOCS2 is highly expressed in the ganglionic eminence (GE) of the ventral telencephalon, which is the primary source of cortical interneurons. In addition, SOCS2 is expressed in the developing cortical plate and in the subventricular zone, one of the main regions through which interneurons tangentially migrate to enter the cortex. These data suggest that SOCS2 is involved in the early specification and/or in the migration of particular interneuron subtypes. Further elucidation of the precise role that SOCS2 plays in interneuron development will greatly enhance our understanding of the complex mechanisms underlying various neurological disorders.

REPAIRING HYPEROXIA-MEDIATED RESPIRATORY DEFICIT WITH ENDOTHELIAL PROGENITOR CELLS

Firsova A.B., Cole T.J. and Mollard R.A. Monash University, Victoria, Australia.

High tension oxygen treatment is performed to assist breathing of very prematurely born babies. However, such treatment can result in disruptions to lung vascularization and alveolarization and eventually lead to chronic lung disease. Steps are taken to reinforce cell differentiation and repair lung tissue injured following oxygen treatment, but these steps remain sub-optimal. Previous results suggest that administration of endothelial progenitor cells (EPCs) from bone marrow to the diabetic foot can stimulate neovascularisation and thus lead to beneficial functional outcomes. We have hypothesized therefore that EPCs may engraft the site of hyperoxia-induced lung damage to similarly offer benefit. In this study, a model of hyperoxia-mediated deficit of the neonatal mouse lung has been established to study such EPC engraftment and investigate associated functional outcomes. Newborn mice were treated with 90% oxygen for four days at birth and parameters of lung development were monitored for an eight week period. Relative to untreated controls, we observed (i) temporary changes in lung vascularization (blood vessel number and Pecam1 protein levels decreased) and (ii) persistent changes in lung septation (alveolar number and tissue area decreased and alveoli diameter increased). Treated animals also displayed an accelerated increase in the number of secondary septa between one day and eight weeks after hyperoxia exposure, such that an early secondary septal number deficit was later returned to control levels. Data from bone marrow sorting and subsequent culture have identified Kdr and Tek as key cell surface markers for vessel-like structure formation in vitro. This model recapitulates many aspects of mechanical ventilation-associated alterations to the very preterm birth human lung and permits study of the effects of attenuations to neovascularisation upon high oxygen damaged alveoli.





GENETIC INTERACTION BETWEEN TRANSCRIPTION FACTOR (Lhx1) AND WNT INHIBITOR (Dkk1) IN HEAD FORMATION IN MOUSE EMBRYOS

Fossat N., Khoo P.-L., Jones V., Lewis S.L. and Tam P.P.L.Embryology Unit, Children’s Medical Research Institute, University of Sydney, Westmead, NSW 2145, Australia

In mouse embryos, Lhx1 gene encoding the LIM homeobox 1 transcription factor is expressed in the anterior visceral endoderm (AVE) and the anterior axial mesendoderm (AME). The expression pattern of Lhx1 partly overlaps with that of Dkk1 (Dickkopf-1) that encodes an inhibitor of the WNT signalling pathway. Loss of Lhx1 function in the whole embryo leads to truncation of the head at the mid- to hindbrain level. However, chimeric embryos containing wild type visceral endoderm (including AVE) and Lhx1-deficient embryonic tissues also display head defects, suggesting that Lhx1 function is required more than in the AVE, which is likely to involve the AME. To better characterise the non-AVE related function of Lhx1, the transcriptome of Lhx1-/flox;Meox2+/Cre (Lhx1 CKO) embryos in which Lhx1 is inactivated in the embryo proper but not in the visceral endoderm was analyzed. In the Lhx1-deficient tissues, the gene expression profile was reminiscent of an increase of the WNT signalling activity. Testing the expression of the BATGal transgene where LacZ expression reports WNT signalling, ectopic activation of WNT activity was found in the anterior region of the Lhx1 CKO mutant embryos. A functional connection between the transcriptional function of Lhx1 and WNT signalling was further demonstrated by the manifestation of the head truncation phenotype in compound heterozygous Dkk1+/-;Lhx1+/CKO mutant embryos which was not found in either Dkk1+/- or Lhx1+/CKO simple heterozygous mutants. The synergistic interaction between Lhx1 and Dkk1, principally in the AME, is therefore essential for the formation of the embryonic head through the negative modulation of WNT signalling activity.

DOES GENETIC VARIATION IN METABOLITE PROFILES CORRELATE WITH SUCROSE CONTENT?

Glassop D.1, 2, Zwart A.3 and Bonnett G.D.1, 2 1CSIRO Plant Industry, St Lucia, Queensland, 4067. 2CRC Sugar Industry Innovation through Biotechnology, St Lucia, Queensland, 4067. 3CSIRO Mathematics Informatics and Statistics, Acton, Australian Captial Territory, 2601.

Sugarcane provides most of the world’s supply of sugar. Sugarcane is able to accumulate sucrose in mature stem tissue, reaching concentrations of ~50% of the dry weight. In recent years the yield of sucrose from sugarcane has only increased due to increases in the yield of biomass rather than increased sucrose concentration of the biomass. This has lead the search for new approaches to break this upper limit for the concentration of sucrose stored. The evolution of the ‘omics’ sciences provides an opportunity to extensively examine sugarcane via genomics, transcriptomics, proteomics and metabolomics in the search for alternative strategies for increasing sucrose content. Previous research revealed metabolic profiles specific to stem tissues of different developmental age. Those results encouraged research to determine the variation in metabolite profiles between sugarcane progeny segregating for sucrose content. Using gas chromatography mass spectrometry we have detected and quantified 105 metabolites in sugarcane stem tissue from internodes four (immature) and nine (mature) from five high and five low sucrose genotypes. Of the 105 metabolites detected and measured, 46 could be assigned an identity on the basis of mass spectra and retention time; 59 remained unidentified. Some of the identified metabolites have not been previously identified in sugarcane metabolite profiles. This poster will report on the relationship between metabolites and sucrose content and identify pathways which could potentially be manipulated for increased sucrose production.

DNA DAMAGE-INDEPENDENT FUNCTIONS OF ASCIZ AS AN ESSENTIAL REGULATOR OF PULMONARY ORGANOGENESIS

Jurado S.1, Smyth I.2, Van Denderen B.1, Tenis N.1, Hewitt K.1, Cole T.J.2 and Heierhorst J.1, 3 1St. Vincent’s Institute of Medical Research. 2Monash University. 3Dept of Medicine, The University of Melbourne.

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The Zn2+-finger protein ASCIZ (ATMIN/ZNF822) was originally discovered as a Chk2-interacting ATM substrate with functions in the DNA base damage response and also proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late embryonic lethality in mice. ASCIZ-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but ASCIZ deletion or knock-down does not affect ATM levels and activation in mouse, chicken or human cells. Unexpectedly, ASCIZ-deficient embryos also exhibit severe respiratory tract defects, where lung buds never emerge from the respiratory endoderm and where ascending separation of the tracheal bud from the ventral foregut stalls early. Genetically, the complete pulmonary agenesis and severe tracheal atresia place ASCIZ between Wnt/ß-catenin and FGF10 signaling pathways. The data indicate that, in addition to its role in the DNA damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

FUNCTIONAL IDENTITY OF THE GAMMA TROPOMYOSIN (γ-TM) GENE: SPECIFIC ROLES IN EMBRYONIC DEVELOPMENT, REPRODUCTION AND CELL VIABILITY

Hook J.1, Schevzov G.1, Hardeman E.C.2 and Gunning P.W.1 1Oncology Research Unit, School of Medical Sciences, University of New South Wales. 2Neuromuscular & Regenerative Medicine Unit, School of Medical Sciences, University of New South Wales.

Tropomyosins (Tm) are highly conserved components of actin filaments which differentially regulate filament stability and function. The mammalian Tm family consists of four genes; α-Tm, β-Tm, γ-Tm and δ-Tm. Multiple Tm isoforms (>40) are generated by alternative splicing, and expression of these isoforms is highly regulated during development. We tested the specificity of function of products from the γ-Tm gene in mice using a series of gene knockouts. Mice homozygous for the knockout of all cytoskeletal products from the γ-Tm gene (Tm5NM1-11) were not viable and could not be detected as early as blastocysts. Elimination of just two cytoskeletal products from the γ-Tm gene (NM1,2) resulted in a 50% reduction in embryo viability. We re-targeted the γ-Tm gene in embryonic stem (ES) cells hemizygous for the knockout of all or subsets of isoforms from this gene. It was not possible to create knockout ES cells for the targets which eliminated or reduced embryo viability in mice. In contrast, homozygous ES cells were created for a different set of isoforms (NM3,5,6,8,9,11) which were not required for embryogenesis. We also observed that males hemizygous for the knockout of all cytoskeletal products from the γ-Tm gene preferentially transmitted the minus allele with 80-100% transmission. Since all four Tm genes are expressed in early embryos, ES cells and sperm, we conclude that isoforms of the γ-Tm gene perform specific functions in embryogenesis, ES cell viability and sperm function that cannot be compensated by the other Tm genes.





AMOUNT OF DNA METHYLTRANSFERASE 1 TRANSCRIPTS IN VARIOUS DEVELOPMENTAL STAGES OF MOUSE TISSUES AND THEIR EXPRESSION SITES

Isokane T.1, Ghanem.E M.2, Nishibori M.1, Hiraiwa N.3 and Yasue H.4 1Grad. Sch. of Bio Sci., Hiroshima Univ., Hiroshima, Japan. 2Suez Canal Univ, Egypt. 3RIKEN, Japan. 4Nat. Inst. Agrobiol. Sci, Japan.

DNA cytosine methylation in mammals influences many cellular events, including gene transcription, genomic imprinting, X chromosome inactivation, carcinogenesis and genome stability. A member of DNA methyltransferase (Dnmts) has been identified that catalyzes the reaction of cytosine methylation in DNA. When the maintenance-type DNA methyltransferase, designated as Dnmt1 is absent in mouse, homozygous mutant embryos cannot survive after mid-gestation as their genomic imprinting being canceled. Epigenetic modifications of chromatin consist of DNA methylation and post- translational modifications to histones like acetylation, phosphorylation, glycosylation, ribosylation. These modifications are key regulators of gene expression during growth and differentiation in all tissues including brain. In the present study, to elucidate the functions of Dnmt1, the amounts Dnmt1 sense/antisense transcripts in the mouse’s tissue of different developmental stages were measured by real-time RT-PCR. The localization of Dnmt1 sense transcripts as well as the existence of antisense transcripts was also investigated. In results, mouse brain cells showed a remarkable Dnmt1 gene expression values during different stages of embryonic development. Antisense transcript was largely unaltered. The localization of Dnmt1 transcripts in adult medulla oblongata, sense transcript was observed remarkably in neuron, however antisense transcript observed faintly around glia cell. In Cerebral cortex, the expression of these transcripts was observed. These findings indicated that Dnmt1 has a great role in brain development of adulthood. However, its role during the embryonic stages still needs more investigation.

REGULATION OF WNT SIGNALLING IS CRITICAL IN EARLY EYE DEVELOPMENT

Chen Y.1, Storen R.1, 2, 3, Greenlees R.1, 3, Grigg J.2, 3, Tam P.1, 3 and Jamieson R.1, 2, 3 1Eye Genetics Group, Embryology, Children’s Medical Research Institute. 2The Children’s Hospital at Westmead & Save Sight Institute. 3Sydney Medical School, University of Sydney.

Aberrations in optic vesicle and cup formation lead to disorders affecting vision including microphthalmia (small eyes) and abnormal iris and retinal development. Distinct domains are specified in the distal part of the optic vesicle for retinal progenitors and the dorsal part for retinal pigment epithelium (RPE). In optic vesicles of E9.5 mouse embryos, WNT activity is detected in prospective RPE via assaying the expression of TopGal and BatGal reporters. In contrast, WNT reporter activity is absent in prospective retina, where Dkk1, an inhibitor of canonical/beta-catenin WNT signalling, is expressed. Hence, regionalization of WNT signalling activity may be critical for tissue specification in early eye development. While loss of Dkk1 function and increased canonical WNT signalling leads to head truncation precluding investigation of WNT signalling in eye development, reduction of Wnt3 in compound Dkk1-/-;Wnt3+/- mutant mice rescues the head phenotype and reinstates eye development. Optic vesicle examination of E9.5 compound mutant embryos (Dkk1-/-;Wnt3+/-) harbouring a WNT reporter revealed ectopic response of cells in prospective retina to WNT signalling. Concurrently, there was ectopic expression of RPE marker (Mitf) and reduced expression of neuroretinal marker (Chx10) in WNT-responding cells. Subsequently, iris and retinal development was disrupted and small eyes formed. These results indicate that active canonical WNT pathway is required in specification of RPE fate of optic vesicle cells, and suppression of this signalling pathway in prospective retina is essential for retinal cell fate. Our results demonstrate the role of a key inhibitor, Dkk1, in precise spatial and temporal regulation of canonical WNT signalling in early eye development.

THE ROLE OF THE DROSOPHILA MEMBRANE ATTACK PROTEIN TORSO-LIKE IN EARLY EMBRYONIC PATTERNING

Johnson T.K.1, 2, Bennett M.2, Herr A.1,2, Warr C.G.2 and Whisstock J.C.1 1Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia. 2School of Biological Sciences, Monash University, Clayton VIC 3800, Australia.

Membrane Attack Complex/Perforin-like (MACPF) proteins have important roles in vertebrate immunity and often function by forming oligomeric pores in cell membranes causing cell lysis. However, several MACPF proteins play crucial yet poorly characterised roles in embryonic patterning and neural development. In Drosophila, correct formation of the terminal anterior and posterior structures of the embryo is governed by Torso-like (Tsl), the only known Drosophila MACPF. Tsl is expressed maternally in specific ovarian follicle cells at the poles of the developing oocyte, where it is secreted and deposited in the perivitelline space between the oocyte and future eggshell. After fertilisation Tsl mediates localised activation of Trunk (Trk), a noggin-like growth factor, and putative ligand of the Torso receptor tyrosine kinase. The mechanism of Trk activation, and the role of Tsl, remains elusive, however one hypothesis is that Trk is proteolytically cleaved by an unidentified protease. We have used in vivo functional studies such as RNAi and overexpression approaches to screen Drosophila serine proteases and serine protease inhibitors for roles in terminal patterning. In so doing we have found that over-expression of Serpin 4 from follicle cells causes eggshell and possibly terminal patterning defects. We are also expressing predicted cleavage site mutants of Trk in vivo to test whether Trk is proteolytically cleaved and whether this is Tsl-dependent. Finally, using a known and related MACPF structure, we have designed and are testing mutations in Tsl that target the putative membrane inserting region to determine if membrane insertion is crucial for Tsl-mediated Trk activation and embryonic patterning.

SOX18-REGULATED GENES IMPLICATED IN LYMPHATIC DEVELOPMENT

Kartopawiro J., Neyt C., Francois M. and Hogan B. Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.

Recent discoveries have demonstrated the pivotal role of lymphatic vessels in numerous pathological states, including inflammatory diseases, tumor metastasis and lymphedema. Identification of new molecular markers implicated in lymphatic vascular development (lymphangiogenesis) is therefore essential in order to increase our understanding of the mechanisms that underlie these disease conditions. Recently, the transcription factor Sox18 has been shown to initiate lymphatic endothelial cell specification during mouse embryogenesis. The loss of Sox18 function leads to a complete loss of lymphangiogenesis and embryos die in utero completely devoid of lymphatic vasculature. However, the cascade of Sox18-dependent genes that modulates early lymphatic vascular development is yet to be identified. In order to uncover molecular targets of SOX18, a micro-array analysis has been performed from purified lymphatic endothelial cells, comparing wild type and Sox18 mutant mice. This study has revealed a subset of Sox18-regulated genes with no known function in lymphangiogenesis. Using a zebrafish model system, our goal is to investigate on a large scale the in vivo expression patterns and function of conserved lymphatic modulators regulated by Sox18. In order to perform this analysis we utlize in situ hybridization analysis, heat-shock-inducible transgenic zebrafish and transient gene knock-down (loss-of-function). We expect to identify novel genes with critical functions in lymphatic vascular development. These discoveries may subsequently form the basis for novel therapeutic avenues for lymphatic disorders.





CHIROPTERA TRANSTHYRETIN

Khwanmunee J. and Prapunpoj P. Department of Biochemistry, Faculty of Science, Prince of Songkla University,Hat Yai, Songkhla 90112, Thailand.

Thyroid hormone (TH) is known playing an important role in development, reproduction and basal metabolism of vertebrates. In the plasma of larger mammals, thyroxine-binding globulin, transthyretin (TTR) and albumin take responses to transport/distribute the hormones throughout body of the animals. Among of these plasma proteins, TTR is becoming the most of interest because of its multifunction and association to a genetic metabolic disorder called amyloidosis. In Chiroptera, TH is crucial not only on basal metabolism but also the reproductive cycle. However, the TH transporter/distributor and gene expression have not been studied in this eutherian. In this study, the nucleotide sequence of Chiroptera liver TTR cDNA was first reported. The deduced amino acid sequence showed highly conservation in comparison to those of TTRs from human and other eutherians, except two amino acid residues in the N-terminal region was absent. Parsimony analysis revealed the amino acid sequence of the flying mammal TTR is most similar to that of pig TTR. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized and sequenced, and the location and mRNA splicing in Chiroptera TTR gene was identified. Moreover, the recombinant Chiroptera TTR was produced by using the heterologous expression system of Pichia pastoris. The recombinant protein had general physicochemical properties similar to those of TTRs from human and other eutherians. Its functions including binding to retinol binding protein (RBP) and proteolytic cleavage were also demonstrated.

PLATELET DERIVED GROWTH FACTOR RECEPTOR ALPHA (PDGFRα) AND CORONARY VASCULATURE FORMATION

Biben C.1, Ooi F.1, Hartley L.1 and Prall O.W.J.1, 2 1Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052 VIC, Australia. 2Royal Melbourne Hospital, Grattan Street, Parkville 3052 VIC, Australia.

Coronary artery disease is one of the leading causes of death in the western world. Formation of coronary arteries and veins is still poorly understood. Classical embryology experiments have suggested that they derive from an extra cardiac tissue, the proepicardium (PE), which is located posterior to the heart. PE cells cover the myocardium, forming the epicardium, from which cells invade the subepicardial space and then the myocardium. Whether they contribute most or only some of the lineages of the coronary vasculature is unclear, as well as whether they give rise to additional cardiac lineages, including muscle. We have found that the gene encoding the receptor tyrosine kinase Platelet Derived Growth Factor Receptor Alpha (PDGFRα) is expressed throughout the mouse PE. Pdgfrα is required at multiple steps of embryonic development but its role in the heart is uncharacterized. Pdgfrα remains expressed in epicardial and subepicardial cells but becomes downregulated as those cells migrate out of the subepicardium. To understand the role of this gene in formation and maintenance of the coronary vasculature, we have engineered mouse models with either low Pdgfrα expression or specifically lacking this gene in various tissues, such as the PE, endothelium or cardiac muscle. Our preliminary observations suggest that Pdgfrα is specifically required in subepicardial cells and involved in coronary smooth muscle development.

SOX3 OVEREXPRESSION LEADS TO DEFECTIVE SUBCOMMISSURAL ORGAN DEVELOPMENT WITH CONGENITAL HYDROCEPHALUS

Lee K.P.Y., Cheah P.S., Piltz S.G., Rogers N.A. and Thomas P.Q. The University of Adelaide, Adelaide, SA, Australia.

Abnormalities in central nervous system (CNS) development that impede the rostral to caudal flow of cerebrospinal fluid (CSF) cause congenital hydrocephalus (CH) in mammals. Blockage of the sylvian aqueduct (SA), a narrow constriction connecting the third and fourth ventricles, is a common cause of CH. Genetic studies in mice have indicated that the subcommissural organ (SCO), a brain gland located at the dorsal midline immediately anterior of SA, is critical for clear passage of the SA. However, the development of the SCO is poorly understood. To investigate the function of the CNS transcription factor Sry-related HMG box transcription factor 3 (Sox3), we have generated two independent BAC transgenic mouse lines to overexpress Sox3 in developing CNS using native control elements. These transgenic lines develop overt CH (dome-shaped cranium) with 20% and 99% penetrance in single and double transgenic, respectively. We found endogenous Sox3 expression in the SCO from inception (approximately 11.5 dpc) through to adulthood and its pattern is recapitulated in Sox3 transgenic mice spatially and temporally. Double transgenic embryos invariably exhibit profound SCO dysmorphology at 14.5 dpc and agenesis at 18.5 dpc. BrdU analysis indicated increased SCO primordium proliferation at 12.5 dpc in double transgenic embryos, suggesting Sox3 overexpression may inhibit progenitor differentiation. Comparison of expression profiles of wild type and double transgenic SCO at 12.5 dpc through microarray analysis showed increased dosage of Sox3 alters expression of genes implicated in SCO and/or dorsal midline development, including members of the Wnt signalling pathway. We are extending these studies using qRT-PCR and in situ hybridisation analyses. Ultimately, our work will provide a better understanding of the genetic and molecular basis for CH pathogenesis.

THE ROLE OF FYN KINASE IN THE DEVELOPMENT OF PRE-IMPLANTATION EMBRYOS

Levi M.1, Maro B.1, 2 and Shalgi R.1 1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel. 2CNRS, Paris, France.

Fertilization in mammals triggers the arrested oocytes to exit from meiotic metaphase. The extrusion of the second polar body and the formation of the pronuclei mark the completion of meiosis and the beginning of pre-implantation embryo development. Several lines of evidence imply the involvement of Fyn, a Src family kinase, in somatic cell cycle control. In the current study we demonstrated, using live cell confocal imaging and microinjection of Fyn cRNA, the co-localization of Fyn with tubulin at the spindle poles of mouse oocytes. During the exit from meiotic metaphase the amount of phosphorylated Fyn was reduced, Fyn disappeared from the spindle poles and concentrated around the spindle midzone and co-localized with filamentous actin at the cleavage furrow and contractile ring area during meiosis and mitosis. Inhibition of Fyn by exposure to SFKs inhibitor, SU6656, or by microinjection of DN-Fyn cRNA inhibited the exit from metaphase, nuclear envelope breakdown and preimplantation cell mitosis. Moreover, although microinjection of cortical DN-Fyn did not affect the initiation of the ingression of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of polar body extrusion and the first cleavage and enlarged the average volume of the polar body and the length of the meiotic spindle. We propose that Fyn regulate several key pathways leading to the exit from metaphase, nuclear envelope breakdown, and in the ingression of the cleavage furrow during meiosis and mitosis.

8 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010




IDENTIFYING NOVEL GENES IN SEX DEVELOPMENT

Alankarage D.1, 2, Ludbrook L.1, Svingen T.3, Bagheri-Fam S.1, Koopman P.3 and Harley V.1 1Prince Henry’s Institute, Clayton, VIC, Australia. 2Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia. 3Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia.

Disorders of sexual development (DSDs) are surprisingly common (e.g. XX males, XY females), and often result in infertility, genital abnormalities, gender mis-assignment and long-term psychological trauma. The main pathway of sex development in males centres on the regulation of SOX9 in the developing XY gonad, which is regulated by SF1, SRY and SOX9, as well as PGD and FGF9 signalling pathways. However, most DSD conditions remain unexplained genetically suggesting the presence of additional genes involved in gonadal development that have not yet been identified. A microarray expression study where SF1, SOX9, SRY and DAX1 were over expressed separately in NT2 cells, a human pluripotent testicular EC line, was recently performed in the lab. Microarray analysis of FACS-sorted, transiently transfected SOX9 cells identified candidate genes responsive to SOX9 overexpression. A bioinformatic filtering process reduced the list from 2626 genes to 10. Promising candidate genes such as CLCN7, COL18A1 and ETV5 are being evaluated further in terms of their gene expression in XY Sox9 knockout embryonic mouse gonads, and by CHIP-seq and promoter analysis. This study can potentially lead to the discovery of novel causative DSD genes and regulatory mechanisms during sex development.

THE CREB1 TRANSCRIPTION FACTOR MODULATES SCD1 EXPRESSION DURING MOUSE LUNG DEVELOPMENT

Antony N., Bird A.D., Tan K.H. and Cole T.J. Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.

Transcriptional factors play a crucial role during lung development in regulating gene expression programs which influence branching morphogenesis and cellular proliferation and differentiation. Cyclic AMP Response Element-Binding Protein1 (Creb1) is a transcription factor that mediates cyclic adenosine 3’,5’-monophosphate (cAMP) signaling in target tissues. Creb1-null mice die at birth due to respiratory failure and are phenotypically smaller than their wild type litter mates. The aberrant lung morphology in Creb1-null mice is established by E16.5 and is clearly detected at E17.5 as unexpanded distal and proximal airways. We investigated whole genome expression profiles on embryonic day E17.5 in Creb1-null fetal mice lungs to identify novel gene targets under the control of Creb1 and to elucidate the molecular mechanisms underlying the role of cAMP signaling during airway development. Microarray analysis has identified several gene targets that are down-regulated compared to wild type mice lungs; these included, genes involved in lipid metabolism such as, enzyme fatty acid synthase, paraoxonase-1 (Pon-1) and a lipogenic enzyme, stearoyl-CoA desaturase 1 (Scd-1). In Creb1-null mice lungs the gene expression was significantly reduced, Pon-1 (1.6 fold, p=0.0005, n=4) and Scd-1 (8.6 fold, p=0.0001, n=4); this was also confirmed by quantitative RT-PCR, Pon1 (p=0.0005, n=4) and Scd-1 (p=0.0317, n=4). The gene expression profiles of Pon-1 and Scd-1 are developmentally regulated and the expression were increased dramatically from E16.5 to E18.5, following which Pon-1 levels remained elevated, while Scd-1 levels decreased after birth. The expression of Scd-1 protein was restricted to type II pulmonary epithelial cells and the levels were significantly lower in Creb1-null when compare to wild type mice lungs. Expression levels of Pon-1 and Scd-1 are also affected in other transcription factor knockout mice models suggesting overlapping roles in transcriptional regulatory pathways during lung development.

RAPAMYCIN AND ISCHEMIA REPERFUSION INDUCE HEME OXYGENASE-1 AND PEROXIREDOXIN-1 IN LIVER

Kist A.1, Wakkie J.1, Nikolic A.1, Zeile S.1, Versteeg R.1, Ten Berge J.1, Wilson C.H.1, Nieuwenhuijs V.B.2, Padbury R.T.A.3 and Barritt G.J.1 1Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, South Australia. 2Department of Surgery, University Medical Centre, Groningen, The Netherlands. 3The HPB and Liver Transplant Unit, Flinders Medical Centre and School of Medicine, Flinders University, Adelaide, South Australia.

Liver surgery is associated with ischemia and reperfusion (IR) injury resulting in loss of liver function and hepatocyte death. The onset of hepatocyte damage is chiefly due to the deleterious actions of reactive oxygen species (ROS). Experimental induction of two anti-oxidant enzymes, heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1), reduces ROS-induced damage to the liver. Rapamycin, employed clinically in liver transplantation as an immunosuppressant has also been shown to induce the synthesis of HO-1 in non-liver cell types. The aim of this study was to test whether rapamycin will induce HO-1 and Prx-1 in hepatocytes. Incubation of rat hepatocytes with rapamycin or cobalt protoporphyrin (CoPP) increased HO-1 (assessed by quantitative PCR). Maximal effects were observed at 36 h, and half-maximal at 10-100 ng/ml. Rapamycin and CoPP increased HO-1 expression in livers removed immediately after laparotomy and in livers subject to laparotomy and a sham operation. Livers subject to a sham operation exhibited higher relative expression of HO-1 mRNA than livers rapidly removed. 2D-DIGE revealed two abundant cytosolic proteins which showed a 3-fold increase in IR (P≤0.05). These were identified as reduced and hyperoxidised Prx-1. A 1.5-fold increase in expression of Prx-1 mRNA was observed in IR. It is concluded that rapamycin and IR induce expression of HO-1 and Prx-1 in hepatocytes. Clinical application of rapamycin pre-treatment may offer protection against IR damage.

EXOSOMAL MICRORNA PROFILING IN NEURODEGENERATIVE DISEASEBellingham S.A.1, 2, 3, Kuehlich J.1, 2, Coleman B.M.1, 2, Sharples R.1, 2 and Hill A.F.1, 2, 3 1Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia. 3Mental Health Research Institute of Victoria, Melbourne, Victoria, Australia.

Exosomes are small membrane vesicles of endosomal origin that are released from a variety of cell types into the microenvironment. The secretion of exosomes was initially thought to be a mechanism for removing unnecessary proteins. However, our research implicates exosomes in the potential pathogenesis of neurodegenerative disorders such as prion and Alzheimer’s diseases. We have demonstrated that neuronal cells infected with prions release normal and infectious forms of the prion protein in associated in exosomes. We have also shown that exosomes contain the Alzheimer’s disease precursor protein (APP), the amyloid-β peptide (Aβ), and secretase components required for Aβ generation from APP. These studies identified previously unknown pathways for the conversion and propagation of prion infection and the processing of APP that may contribute to AD pathogenesis. More recently, exosomes have been shown to contain both mRNA and miRNA, termed exosomal RNA (esRNA) that can be transferred between cells in a novel mechanism of cell-cell communication of genetic signals. The transferred esRNA is also functional, as the mRNA can be translated into new proteins in recipient cells, while the miRNA, a class of small RNA ~ 22nt long, play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. We therefore hypothesised that esRNA can contribute to the pathogenesis of prion and Alzheimer’s diseases. Utilising our cell culture models, we aim to identify disease specific signatures from isolated exosomes by miRNA profiling with “next-generation deep sequencing”, miRNA Array’s and quantitative PCR. This research has significant diagnostic potential for prion, Alzheimer’s and other human diseases since circulating exosomes can be isolated from a variety of biological fluids including blood, urine, saliva and CSF.





CYCLIN-DEPENDENT KINASE-LIKE 3 REGULATES ACTIN REMODELLING AND CELL MIGRATION

Brown M.K.1, Forrest A.2, Kerr M.3, Wani S.1, Cloonan N.1, Gabrielli B.4 and Grimmond S.1 1Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, Australia. 2Omics Science Centre, RIKEN, Yokohama Institute, Japan. 3Institute for Molecular Bioscience, University of Queensland, Australia. 4Cell Cycle Laboratory, University of Queensland Diamantina Institute, Princess Alexandra Hospital, Australia.

Organisation of the actin cytoskeleton is central to cell migration, and is largely regulated by members of the mammalian Rho GTPase family. Members of this family have recently been implicated in dendrite formation and branching, with alterations in Rho GTPase-signalling reportedly contributing to mental retardation (MR) disorders. Cyclin-dependent kinase-like 3 (Cdkl3) is a poorly characterised member of the serine/threonine family, with homology to the cell cycle regulator, Cdk1. Recently, a role for Cdkl3 in neuronal morphogenesis has been demonstrated, whereby Cdkl3 knock-down has been shown to decrease dendrite formation and branching. We report the characterisation of two alternative transcripts derived from the human Cdkl3 locus. While the canonical Cdkl3 product contains two putative ERK binding motifs and a Nuclear Localisation Sequence (NLS), the second isoform has a single putative ERK binding motif and no NLS, suggesting an alternative function for this variant. We report a significant decrease in cell migration with Cdkl3 knock-down, as well as hyperphosphorylation of ERK in HeLa cells. Additionally, Cdkl3 knock-down leads to actin remodelling and altered cell morphology. We propose a role for Cdkl3 in the MAPK signalling pathway in a manner that regulates actin cytoskeleton organisation, which affects the ability of cells to migrate correctly. We suggest that alternative splice variants from the Cdkl3 locus encode proteins that differ in their biological activities, and that these alternative components should be considered when investigating Cdkl3 functions.

TAXOL AND BORTEZOMIB INDUCE APOPTOSIS SYNERGISTICALLY IN CHRONIC MYELOGENOUS LEUKEMIA CELLS

Bucur O.1, 2, Stancu A.L.1, Petrescu S.M.2 and Khosravi-Far R.1 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. 2Institute of Biochemistry of the Romanian Academy, Bucharest, Romania.

Bcr-Abl fusion protein plays a critical role in the pathogenesis and progression of Chronic Myeloid Leukemia (CML) and in some Acute Lymphocytic Leukemia cases. We and others have previously established Bortezomib (FDA approved for the treatment of Multiple Myeloma) as a potential important treatment in Bcr-Abl positive leukemias. Taxol (Paclitaxel), a mitotic inhibitor drug (FDA approved for the treatment of a number of cancers) is also now in clinical trials for the treatment of CML. However, to our knowledge, there are no studies regarding the combined treatment of Bortezomib and Paclitaxel in Bcr-Abl positive CML. RESULTS: Combined treatment of Bortezomib and Paclitaxel synergistically induces cell death in human Bcr-Abl positive K562 cell line, by activating the initiator caspase 9 and effector caspase 3, which leads to the cleavage of a number of substrates, such as Poly ADP Ribose Polymerase (PARP), and results in apoptosis. Additionally, the exposure of the Bcr-Abl positive leukemia cells to the Bortezomib/Paclitaxel regimen results in an increase in the activation of the stress-related MAP kinases (such as p38 MAPK and JNK) versus the cytoprotective kinases (ERK1, ERK2), suggesting a possible implication of JNK and p38 MAPK in mediating the effect of the combined treatment. Moreover, we also show that the combined treatment is effective in inducing apoptosis in the murine Baf3 Bcr-Abl and Imatinib-resistant Baf3 Bcr-Abl T315I cell lines. CONCLUSION: Taken together, these findings underline that the combined treatment with Bortezomib and Paclitaxel represents a potentially promising strategy for the treatment of Chronic Myelogenous Leukemia in general, and of Imatinib-resistant CML in particular.

INVESTIGATING THE RNA BINDING PROPERTIES OF KRÜPPEL-LIKE FACTORS

Burdach J.G.1, 2, Mackay J.1 and Crossley M.2 1School of Molecular Bioscience, University of Sydney, Australia. 2School of Biotechnology and Biomolecular Sciences, University of NSW, Australia.

The Krüppel-like factors (KLFs) are a family of mammalian transcription factors involved in the regulation of a diverse range of biological processes including proliferation, apoptosis, differentiation and development (Pearson et al., 2008). These proteins bind to a common CACCC box element within GC-rich regions of DNA to activate or repress transcription (Suske et al., 2005). DNA interaction is mediated by three C-terminal Cys2His2 zinc finger domains which are highly conserved across the family. The N-termini are highly variable and can contain either transcriptional activation or repression domains. Cys2His2 zinc finger domains are known to have a variety of functions including DNA, RNA and protein binding (Iuchi, 2001). As transcription factors, the DNA binding characteristics of the KLFs are well established. Preliminary in vitro data from our laboratory suggests that these proteins may also be capable of binding RNA, though the specificity and affinity of these interactions is still unclear. We are using RNA immunoprecipitation coupled with next generation sequencing technology (RIP-seq) to determine whether these proteins are capable of specific RNA interactions in a cellular context. This powerful technique may shed light on the biological role of such an interaction, leading to a better understanding of the role of KLFs in gene regulation. Iuchi, S. (2001). Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58, 625-635. Pearson, R., Fleetwood, J., Eaton, S., Crossley, M., and Bao, S. (2008). Kruppel-like transcription factors: a functional family. Int J Biochem Cell Biol 40, 1996-2001. Suske, G., Bruford, E., and Philipsen, S. (2005). Mammalian SP/KLF transcription factors: bring in the family. Genomics 85, 551-556.

IDENTIFICATION OF NFIL3 AS A DIRECT GLUCOCORTICOID-REGULATED GENE TARGET IN T-LYMPHOCYTES

Carey K.T.1, Tan K.1, Ng J.1, Liddicoat D.R.1, 2, Godfrey D.I.2 and Cole T.J.1 1Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800. 2Department of Immunology and Microbiology, University of Melbourne, Parkville, Victoria, 3010.

Glucocorticoids (GCs) are homeostatic steroid hormones with essential roles in the regulation of development, integrated metabolism, immune and neurological responses. In the immune system the strong lymphocytolytic actions of GCs are central in treatment of lymphocytic leukemias and lymphomas such childhood acute lymphoblastic leukemia (ALL). GCs act via the glucocorticoid receptor (GR) which is expressed from multiple untranslated exon 1s to yield at least 11 alternatively spliced transcripts in humans and at least five in mice (1A-1H). GR transcripts initiating from the GR1A promoter have previously only been localised to T-lymphocytes and brain cortex. In T-lymphocytes the GR1A promoter is implicated in increasing sensitivity to Glucocorticoid Induced Cell Death (GICD). CD4+CD8+ Double Positive (DP) cells as well as NK cells in particular have been shown to be hypersensitive to GICD. To explore the molecular pathway driving GCID in T-cells we have performed whole genome microarray analysis in mouse GR null T-cells. Interesting direct GR targets included P21 and Bim, in addition to many not previously well characterised, such as Nfil3. Regulation of these targets by GCs has been validated using qRT-PCR in WT T-cells. Nfil3 in particular has been studied further. Previous studies suggest that the development and functional maturation of NK cells requires Nfil3 expression, in addition it has been demonstrated that GC-mediated upregulation of Nfil3 is dependent on intracellular calcium levels, and correlates with GCID of GC-sensitive leukemic cells. In silico promoter analysis revealed a putative Glucocorticoid Response Element in the Nfil3 promoter region which was confirmed by ChIP. Immunohistochemical staining of Nfil3 in whole thymus has localised Nfil3 protein primarily to the medullary region, which contains developing NK cells. It will be of interest to investigate the involvement of GICD in negative selection of sensitive DP cells and correlate this to expression of Nfil3.





A NOVEL FUNCTION OF TRISTETRAPROLIN : INHIBITING THE ACTIVITY OF POLY(A) POLYMERASE (PAP) BY DIRECTLY INTERACTING WITH PABPN1 AND PAP

Wang S.-C.1, Su Y.-L.2, Chiang P.-Y.2, Lin N.-Y.1, Shen Y.-F.2 and Chang C.-J.1, 2 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 2Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.

Tristetraprolin (TTP) is one of AU-rich element (ARE)-binding proteins, which facilitates mRNA destabilization. To realize the detailed mechanism of TTP upon mRNA degradation control, it is necessary to find out its direct interaction proteins. In this report, we performed phage-display biopanning to search the TTP-interacting proteins. One of them, Pabpn1 (poly (A) binding protein nuclear 1), was identified and further characterized. Moreover, their interacting domains were verified and determined with their deletion constructs by pull-down assays. Pabpn1 plays a major role in the polyadenylation of mRNAs, which binds to poly(A) RNA and increases the affinity of the poly(A) polymerase (PAP) to promote the formation of the polyadenylate tail of the 3’ end of mRNA. Interestingly, we found that TTP could bind to both Pabpn1 and PAP and inhibit polyadenylation of ARE-containing RNA in vitro. The poly(A) tail is important for the mRNA export, mRNA stabilization and translation efficiency. Consequently, except the mRNA-degrading function, inhibition of polyadenylation activity of PAP is a novel function of TTP to regulate ARE-containing mRNA expression.

INTESTINAL STEM CELL GENE LGR5 IS REGULATED BY SYNERGISTIC C-MYB AND β-CATENIN COOPERATION

Cheasley D.A.1, 2, Malaterre J.1, Vincan E.3, Lightowler S.1, Pereira L.1 and Ramsay R.G.1 1Differentiation and Transcription Laboratory, Peter MacCallum Cancer Institute, East Melbourne and the Pathology Department, The University of Melbourne. 2The Department of Genetics, La Trobe University, Bundoora. 3The Department of Anatomy and Cell Biology, The University of Melbourne, Parkville.

The regulation of tissue development and the establishment of homeostasis is achieved through the orchestration of transcription factor action and co-ordination of stem and progenitor cell populations. The transcription factor c-Myb has emerged as a key regulator of stem/progenitor cells within the gastrointestinal tract crypt. These structures line the epithelium of the colon and small intestine. This conclusion is based upon our exploitation of c-myb knock-out and hypomorphic mutant mouse models. Recent studies have found that the Wnt target gene, Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), along with being an important intestinal and colon crypt stem cell marker, is also expressed in colon cancer cells. Based on co-incidental expression of lgr5 and c-myb in the GI, we investigated whether lgr5 is a c-Myb target gene. Using both in vitro and in vivo studies, we show that the proto-oncogene c-Myb in combination with β-catenin, is bound to (by chromatin immunoprecipitation studies), and is a more potent regulator of, the lgr5 promoter (by reporter experiments) in the presence of activated β-catenin. These observations parallel our previous studies of the regulation of the c-myc gene by both of these transcription factors (Ciznadija et al 2009) suggesting this is a more general mode of transcriptional co-operation. How c-Myb and β-catenin co-operate at the genetic and physical level will be presented. Collectively our data indicate that the Wnt pathway through β-catenin converge with c-Myb in regulating lgr5 expression in the GI stem/progenitor cells.

ANALYSIS OF THE RELATIONSHIP BETWEEN BIOENERGETIC SIGNATURES AND DIFFERENTIATION POTENTIALS OF HUMAN MESENCHYMAL STEM CELLS FROM DIFFERENT ORIGINS

Chen C.T.1, Lan Y.W. 1, Hsu S.H.1, Hwang S.M.2 and Wei Y.H.1, 3 1Department of Biochemistry and Molecular Biology, National Yang Ming University, Taipei, Taiwan. 2Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. 3Department of Medicine, Mackay Medical College, Taipei, Taiwan.

Our previous studies indicate that the metabolic shift from anaerobic glycolysis to mitochondrial respiration plays critical roles during osteogenic differentiation of human mesenchymal stem cells (hMSCs). Hypoxic signals suppressed the activation of mitochondria and attenuated differentiation of hMSCs. Recent studies also showed the necessity of mitochondrial function for stem cells to differentiate into cardiomyocytes and other types of somatic cells. Therefore, we have hypothesized that the metabolic signatures, namely, the relative contribution of aerobic and anaerobic metabolism to energy production, of stem cells may affect their differentiation potentials. We isolated hMSCs from four different origins including cord blood, bone marrow, amniotic membrane and amniotic fluids. Their metabolic features were analyzed by an automatic bioenergetic analyzer, Seahorse XF24 Extracellular Flux Analyzer. We found that hMSCs form different origins displayed distinct metabolic signatures as revealed by their oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) despite their expression of similar panels of surface markers. These hMSCs also showed distinct preference in differentiating into multiple lineages of progenies such as osteoblasts and adipocytes. Manipulation of their aerobic and anaerobic metabolism by inhibitors against either mitochondrial or glycolytic activities affected their differentiation abilities. Taken together, the distinct metabolic signatures of hMSCs from different origins may serve as an additional biomarker for the evaluation of stem cell properties in terms of their differentiation potentials. Characterization of metabolic signatures could be of great value in the selection of stem cells with superior quality to facilitate future clinical application of stem cells in tissue regeneration.

REGULATION OF SPLICEOSOMAL GENES IN SACCHAROMYCES CEREVISIAE

Chen S.-C.E., Kornfeld G.D. and Dawes I.W. School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW Australia.

Sm-like (Lsm) proteins function in a variety of RNA-processing events, including splicing, post-transcriptional modification and RNA degradation. In yeast, the proteins Lsm2-Lsm8 form a heteroheptameric ring complex and interact with the spliceosomal U6 snRNA to facilitate U4/U6 snRNP formation during the mRNA splicing process; whilst another complex comprised of Lsm1-Lsm7 is involved in mRNA degradation via decapping in the cytoplasm. An increasing number of studies have suggested that there are important functions of introns acting in parallel with the protein-based regulatory systems, possibly related to the complexity of organism. Studies from our laboratory showed that the intron of the LSM7 gene along with the coding sequence is required for the regulation of LSM1-LSM8 expression on different carbon sources. The aim of our study is to identify the mechanism whereby the LSM7 intron regulates the expression of LSM genes in Saccharomyces cerevisiae. The approach involved targeted mutagenesis of the LSM7 intron sequence, in particularly, the splicing elements in the intron, and qRT-PCR analysis for the expression profiles of the LSM genes in these mutants. In addition, microarray analysis has also conducted to examine the genome-wide expression profiles in response to deletion of LSM7 intron.





DISTINCT ROLES OF TTP, HNRNP K AND HUR IN COX-2 MRNA STABILITY INDUCED BY LPS

Chiang P.Y.1, Shen Y.F.1 and Chang C.J.1, 2 1Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan. 2Institute of Biological Chemistry, Acdemic Sinica, Taipei, Taiwan.

Cyclooxygenases (COXs) are key regulatory enzymes catalyzing the rate-limiting step of prostaglandin formation in various biological processes, such as inflammation, and overexpression of the inducible isoform COX-2 is associated with carcinogenesis. In macrophage Raw 264.7 cells, lipopolysaccharide (LPS) induces COX-2 expression at both transcription and posttranscriptional level. To understand the mechanism of posttranscriptional regulation, the RNA binding proteins (RNA-BPs) bound to the 3’ untranslated region (3’UTR) of COX-2 mRNA were studied firstly. By using RNA pull-down and RNA immunoprecipitation assay, tristetraprolin (TTP), heterogeneous nuclear ribonuclear protein K (hnRNP K) and HuR were proven to bind to the distinct regions of COX-2 mRNA 3’UTR respectively. Further reporter assay indicated that LPS-induced TTP destabilizes COX-2 mRNA by binding to the AU-rich element (AREs) located within the first sixty nucleotides of 3’UTR in COX-2 mRNA. On the other hand, HuR binds to both ARE and distal region of COX-2 mRNA 3’UTR and increases mRNA stability. Moreover the RNA stability and translation modulated by hnRNP K are mediated by binding to the distal region of COX-2 mRNA 3’UTR. Although these RNA-BPs function in distinct ways, the combination effect mediated by TTP, hnRNP K and HuR causes the tight control of COX-2 expression during inflammation reaction.

PHOSPHORYLATION ADJACENT TO THE BZIP DOMAIN OF FOS RELATED ANTIGEN-1 REGULATES DNA BINDING

Diesch J.1, Low D.1, Morrice N.2, Tulchinsky E.3, Hannan R.D.4 and Dhillon A.S.1 1Department of Biochemistry and Molecular Biology, University of Melbourne, Vic, Australia. 2MRC Protein Phosphorylation Unit, University of Dundee, UK. 3Department of Cancer Studies and Molecular Medicine, University of Leicester, UK. 4MacCallum Cancer Centre, Melbourne, Vic, Australia.

The Activator Protein-1 (AP-1) transcription factor is a sequence-specific DNA binding protein complex that orchestrates gene expression programs directing cell fate decisions (e.g. proliferation, survival, differentiation, migration). It has a dimeric core, consisting mainly of members of the Fos, Jun and ATF protein families, and operates downstream of many cancer-associated signal transduction pathways. The Fos related antigen-1 (Fra-1) is a Fos family protein frequently over-expressed in cancers of epithelial origin, and is linked to increased tumour cell migration and invasion. There is thus much interest in understanding how the functions of Fra-1 are regulated. Using mass spectrometry, we have identified a novel phosphorylation site in Fra-1 (Ser101) that lies adjacent to the region of the protein involved in DNA binding. We substituted Ser101 with alanine (S101A) or aspartic acid (S101D) residues, and examined the effects of these mutations on the subcellular localisation of Fra-1, its capacity to form dimers with c-Jun in cells, and its ability to bind to oligonucleotides that contain an AP-1 consensus sequence (TRE). While the localization and dimerisation of Fra-1 with c-Jun were not affected by the substitutions, we noted a significant increase in the binding of Fra-1S101A, and decrease in the binding of Fra-1S101D, to the TRE. As these Fra-1 proteins were part of dimer containing endogenous c-Jun, our results suggest that phosphorylation of Ser101 can disrupt the recognition or binding of Fra-1/c-Jun complexes to consensus sites in AP-1 target genes.

BIOPHYSICAL AND STRUCTURAL EXAMINATION OF THE LER TRANSCRIPTION FACTOR

Stone R.D.1, 2, Dogovski C.1, 2, Bailey M.F.1, 2, Ji Y.3, Robins-Browne R.M.3 and Perugini M.A.1, 2 1Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville VIC 3010, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC 3010, Australia. 3Department of Microbiology and Immunology, The University of Melbourne, Parkville, VIC 3010, Australia.

Pathogenic strains of Escherichia coli cause acute and persistent diarrhoea in humans. Several pathotypes express specific virulence factors that mediate adhesion to the intestinal epithelium and production of distinctive pathological changes in the intestine mucosa (A/E phenotype). Virulence genes in pathogenic E. coli are typically organised in operons that are tightly controlled by a network of regulatory proteins. The genes involved in the A/E phenotype are encoded by a 36 kb chromosomal pathogenicity island, named the locus of enterocyte effacement (LEE). LEE carries more than 40 genes which form five transcriptional units, termed LEE1 to LEE5. A number of global and specific regulatory proteins are known to play an essential role in gene expression and pathogenesis. The transcription factor Ler (LEE encoded regulator) a homologue of H-NS, is essential for anti-silencing of LEE operons LEE2-LEE5. Ler is considered to be a key regulatory element and novel antibiotic target. Although much work has been done to establish the role of Ler in E. coli pathogenicity, little is known about the solution properties of the protein and how it prevents silencing of the LEE pathogenicity island. The purpose of this study is to characterize the solution properties and structure of Ler via analytical ultracentrifugation, circular-dichroism spectroscopy and X-ray crystallography.

PROTEOLYSIS OF AN ANTI-SIGMA FACTOR CONTROLS SIDEROPHORE SYNTHESIS AND UPTAKE IN PSEUDOMONAS AERUGINOSA

Draper R.C. and Lamont I.L. University of Otago, Dunedin, New Zealand.

Pseudomonas aeruginosa is an important human pathogen that requires iron for growth and pathogenesis. To acquire iron from the environment or host tissues, this bacterium secretes the iron-binding molecule pyoverdine. Pyoverdine is also a signalling molecule, inducing the expression of genes for pyoverdine synthesis and uptake. In the absence of pyoverdine, sigma factor proteins required for gene expression are inhibited by an anti-sigma factor, FpvR, which spans the inner membrane. When pyoverdine binds to a cell surface receptor, this inhibition is relieved, allowing sigma activity and the expression of target genes. This project investigated the role of FpvR in this signalling pathway, and sought to determine the molecular mechanism underlying this system. We have shown that the FpvR protein is only detectable in strains where sigma factor activity is inhibited. When pyoverdine was added we observed a rapid disappearance of FpvR, suggesting that proteolysis regulates FpvR activity. Candidate proteases were screened for involvement in the degradation of FpvR. The intra-membrane protease RseP was implicated in this process by the accumulation of intermediate FpvR fragments in strains lacking RseP. These strains also exhibited reduced sigma activity as measured by qRT-PCR. Together these data suggest a model whereby the binding of pyoverdine at the cell surface causes the degradation of FpvR, resulting in changes in gene expression.





MECHANISM OF SYNERGISTIC INDUCTION OF GLUT1 TRANSCRIPTION BY ENDOTHELIN-1 AND CYCLIC AMP IN 3T3-L1 ADIPOCYTES

Fong J.C. and Kao Y.S. Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC.

We have shown previously that chronic exposure to both endothelin-1 (ET-1) and cAMP results in a synergistic increase in Glut1 transcription in 3T3-L1 adipocytes. In the present study, we have further examined the molecular mechanism involved. Experimental results indicate that ET-1 through ETAR/Gi/PKCε interacts with cAMP to enhance Glut1 transcription. Although p42/p44 MAPK is required for the stimulatory effect of ET-1 alone, it is not involved in the synergistic effect of ET-1 and cAMP on Glut1 transcription. Further investigation demonstrated that a ternary complex containing transcription factors Sp1, pCREB and AP-1 was mediating the synergistic effect of ET-1 and cAMP on Glut1 transcription. While an increase in nuclear AP-1 can be achieved by either ET-1 or cAMP, the accumulation of nuclear pCREB is only sustainable by stimulation with cAMP. An increase in nuclear Sp1, on the other hand, is dependent on the activation of both PKCε and CREB. Thus it seems that chronic exposure to both ET-1 and cAMP provides a circumstance favoring increases in nuclear Sp1, pCREB and AP-1, and their interaction to form a ternary complex which in turn can greatly facilitate the transcription of glut1 gene.

FUNCTIONAL ANALYSIS OF THE PROTEIN L-ISOASPARTYL/D-ASPARTYL O-METHYLTRANSFERASE (PIMT) GENE PROMOTER

Furuchi T.1, Harada S.1, Shimizu Y.1, Kosugi S.2, Katane M.1, Sekine M.1 and Homma H.1 1Sch. of Pharm. Sci., Kitasato Univ., Tokyo, Japan. 2Sch. of Med., Kanazawa Univ., Kanazawa, Japan.

L-Aspartyl (L-Asp) and L-asparaginyl residues in proteins spontaneously isomerize or racemize to D, L-isoaspartyl (D, L-isoAsp) or D-aspartyl (D-Asp) residues under physiological conditions. These atypical Asp residues can interfere with the protein function and lead to malfunction of cells. Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is a cytosolic protein repair enzyme that initiates the conversion of L-isoAsp and D-Asp to normal L-Asp residues. Mice lacking this enzyme suffer from epileptic seizures and die at early age; thus, formation of the abnormal residues and their subsequent correction by PIMT is widely believed to constitute an important pathway of protein damage and repair. However, regulatory mechanisms of PIMT gene expression have remained unclear. To analyze the regulatory mechanisms controlling the expression of human PIMT, we characterized the 5’-flanking region of the gene. About a 1 kbp fragment of the putative promoter region was PCR-amplified and ligated into a luciferase-expression vector, pGL3-basic. Transfection in HEK293 cells indicate that the 5’-flanking region contains regulatory elements for constitutive expression of PIMT. The minimal region required for the basal activity of the PIMT promoter were determined by generating a series of deletion and point mutation constructs, and were found to be encoded by a sequence around –190 relative to the translation initiation codon. Electrophoretic mobility shift assay suggested the presence of factors that bind to the minimal region in a sequence-specific manner in the nuclear extract of HEK293 cells. Further study is currently in progress to identify the binding factors using liquid chromatography/tandem mass spectrometry (LC-MS/MS).

ARABIDOPSIS GLYCINE-RICH RNA BINDING PROTEIN 8 (ATGRP8) EXPRESSION IN RESPONSE TO PHOSPHATE STARVATION

Gaza H.L.1, Jost R.1, Ludwig M.2 and Finnegan P.M.1 1School of Plant Biology, UWA. 2School of Biomedical, Biomolecular and Chemical Sciences,UWA.

Plant growth and development is restricted by phosphate (Pi) deficiency. This leads to an adaptive response by altering gene expression and metabolism as a result of cell signaling (Schmidt et al, 2010). The expression of the Arabidopsis glycine-rich RNA-binding protein 8 (AtGRP8) gene is regulated by a number of external stimuli including cold, circadian rhythm, ABA, drought and salinity (Carpenter et al., 1994; Kwak et al, 2005). The altered gene expression of AtGRP8 under different stress conditions has led to the hypothesis that it may be involved in the response of plants to phosphate starvation. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) the expression of AtGRP8 gene during phosphate deficiency was determined. Roots from A. thaliana plants grown under phosphate-deficient conditions for seven days showed a decrease in expression compared to the plants grown continuously in phosphate-sufficient media. The transcript level of GRP8 in the shoots however, remained unchanged. GRP8 may function similarly to WRKY6 and WRKY75 transcription factors as a negative regulator of genes whose expression is up-regulated during phosphate starvation. Experiments using knock-out lines to determine the role of GRP8 in the regulation of the phosphate starvation response are underway.

FUNCTIONAL REGULATION OF THE FRA-1/AP-1 TRANSCRIPTION FACTOR VIA INTERACTIONS WITH PROTEIN PHOSPHATASE 2A

Gilan O.1, Jastrezebski K.2, Diesch J.1, Verrills N.3, Hannan R.D.2 and Dhillon A.S.1 1Bio21 Institue Department of Biochemistry and Molecular BIology. 2Research, Peter MacCallum Cancer Centre. 3Faculty of Health, University of Newcastle.

The Activator Protein-1 (AP-1) transcription factor complex regulates gene expression downstream of signal transduction pathways activated by a variety of growth factors, cytokines, hormones and cellular stresses. The products of AP-1-regulated genes are required for the execution of fundamental cellular processes, including proliferation, survival and differentiation. AP-1 complexes consist of a dimeric core, formed mainly by members of the Fos, Jun and ATF protein families. Fos related antigen-1 (Fra-1) is a Fos family protein that is frequently over-expressed in cancers, and is strongly linked to cancer progression. The major post-translational modification regulating Fra-1 is phosphorylation. Using a proteomics-based approach, we have found that Fra-1 associates with components of the hetero-trimeric protein phosphatase 2A (PP2A) complex. This complex consists of a holoenzyme formed by the binding of a 36 kDa catalytic subunit (PP2Acat) to a 65 kDa scaffold subunit, whose activity and targeting to substrates is specified by one of at least 25 regulatory subunits. We specifically identified the PR65a scaffold and PR55alpha regulatory subunits in Fra-1 complexes and have confirmed that both proteins, as well as the catalytic subunit, interact with Fra-1 in HEK293 cells and in vitro. We show that the PR55alpha regulates the recruitment of the PP2A catalytic subunit to Fra-1, and that inhibition of PP2A catalytic activity, or silencing of B55alpha expression perturbs proteasome-mediated degradation of Fra-1. Our results provide new insights into how the functions of Fra-1 are regulated in cells, and reveal a novel mechanism regulating Fra-1 turnover.





A NOVEL ROLE OF DIPEPTIDYL PEPTIDASE 9 IN PI3K/AKT PATHWAY REGULATION

Yao T.W.1, 2, Xia P.1, 2, Kim W.S.3, Yu D.M.T.1, 2, Choi K.Y.3 and Gorrell M.D.1, 2 1Centenary Institute. 2Sydney Medical School, University of Sydney. 3Yonsei University, Seoul, Korea.

Dipeptidyl peptidase (DP) IV, DP8, DP9 and FAP (fibroblast activation protein) are DPIV gene family members of particular interest due to their peptidase and extra-enzymatic activities that have been implicated in various diseases. We report here a novel role of DP9 in cell signal transduction. We found that DP8 and DP9, but not DPIV or FAP, interact with H-Ras, a key signalling molecule that mediates multiple signalling pathways, especially of growth factors (GFX), in both liver tissue and human cervical carcinoma cell line, HeLa. The ERK1/2 and Akt pathways are two major downstream effectors of Ras. Interestingly, Akt activation (determined by Akt phosphorylation status) was significantly inhibited by DP9 overexpression in human hepatoma cells (HepG2), whereas ERK1/2 activity was unaffected, revealing a pathway-specific effect. DP9 induced a significant reduction in GFX - induced Akt activation in HepG2 cells. This is consistent with the observation that DP9 markedly attenuated GFX-dependent mitogenic effects, including apoptosis and proliferation. These findings, along with our previous observation that DP9 overexpression is pro-apoptotic, suggest an important signalling role of DP9 in the regulation of a GFX - dependent survival and/or proliferation pathway.

EVIDENCE THAT 4E-BP, KNOWN AS A COMPLETELY DISORDERED PROTEIN, PARTIALLY FOLDS UPON COMPLEX FORMATION : NEW PERSPECTIVES FOR THE REGULATION OF TRANSLATION

Gosselin P.1, Oulhen N.1,3, Czjzek M.2, Cormier P.1and Cosson B.1 1UPMC Univ Paris 06, CNRS, UMR 7150, Traduction Cycle Cellulaire et Développement, Station Biologique de Roscoff, 29682, Roscoff, France. 2UPMC Univ Paris 06, CNRS, UMR 7139, Végétaux marins et biomolécules, Station Biologique de Roscoff, 29682, Roscoff, France.3Current address: Department of Molecular and Cell Biology and Biochemistry Brown University, Providence RI 02912, USA.

Control of translation is a critical step in the regulation of gene expression involved in embryonic development, and mechanisms responsible for human pathologies. The eukaryotic Initiation Factor 4E (eIF4E) interacts with the 5’ cap structure (m7GTP) and controls the cap-dependant translation. The three eIF4E family members (eIF4E1, 2, 3) have been identified in deuterostomes and interact specifically with different partners, extending the biological impact of this translation factor. Only one isoform of each actor has been found in sea urchin, making this organism a strong model to study the regulation of translation. Release of eIF4E from his repressor 4E-Binding protein (4E-BP) is a key process in translation initiation and is triggered by the phosphorylation of 4E-BP. The understanding of the interactions between these different actors, with functional and structural approaches, is a very important step for cellular biology. Classical structural approaches suggest that 4E-BP is mostly or completely unstructured in both free and bound states, and only the central domain of 4E-BP is known to interact with eIF4E during the complex formation. These data are not sufficient to explain the sharp phosphorylation mechanisms that control the association of 4EBP and eIF4E. Using an original structural technic that consists in measurement of small angles X-ray scattering (SAXS), we show for the first time that 4E-BP adopts a folded structure upon the binding to eIF4E but also that this inhibitor has a transitory structure when it’s free in solution. SAXS allows us to see the rest of 4E-BP chain that is missing in the crystallography complex structure, and reveals a « fuzzy complex », involving a larger surface of interaction between these two actors. The results that we obtained with sea urchins proteins, adopting this new dynamic view of 4E-BP structure, open new perspectives for the understanding of the sharp mechanisms of gene regulation by translation.

ARABIDOPSIS POLLEN TRANSCRIPTION FACTORS

Gibalova A.1, 2, Renak D.1, 2, 3, Duplakova N.1, 2, Solcova K.1, 2 and Honys D.1, 2 1Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic. 2Department of Plant Experimental Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Praha 2, Czech Republic. 3Department of Plant Physiology and Anatomy, Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic.

Haploid male gametophyte plays a key role in plant fertility and crop production. Despite of signifiant progress in recent years, we still have very limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. Therefore, it is necessary to identify transcription factors that are part of such haploid regulatory networks. In our studies, we have focused on basic-leucine zipper and heat shock transcription factors knowing to be involved in stress response and developmental processes. We report the functional characterization of members of these gene families that are expressed in both gametophytic and surrounding sporophytic tissues during flower development. The respective T-DNA insertion mutants showed reduced transmission through male and female gametophytes, pollen morphological defects, lower pollen germination efficiency and slower pollen tube growth both in vitro and in vivo conditions. Transient expression revealed the subcellular localization analysed proteins and in addition to other data, the nucleolar localisation of the heat shock transcription factor suggested its possible involvement in regulation of mRNA/rRNA transcription. Acknowledgment: Authors gratefully acknowledge the financial support from the Czech Science Foundation (grant 522/09/0858) and Ministry of Education, Youth and Sports of the Czech Republic (grants LC06004 and OC10054).

HEPARANASE AS A SIGNALING MOLECULE IN CANCER AND INFLAMMATION

Goodall K.J., Poon I.K.H. and Hulett M.D. Department of Biochemistry, LaTrobe University, Bundoora, VIC, Australia

Heparanase is a β-D-endoglucuronidase which degrades heparan sulfate, a key component of the extracellular matrix and basement membrane. The degradation of the ECM is essential for both physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Heparanase is also known to have non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM and basement membrane, its role in generating an inflammatory response by mediating the release of chemokines, cytokines and growth factors have not been well established. In this study, peripheral blood monocyte cells were stimulated with Heparanase, and cytokine release was examined. Heparanase treatment of cells resulted in the release of a range of pro-inflammatory cytokines including IL-8, IL-10, TNF, IL-6 and IL-1β. Similar results were obtained following the treatment of MyD88-/- mouse spleen cells with Heparanase, suggesting that the cytokine release was not simply due to endotoxin contamination. These data suggests that Heparanase can promote inflammation via the release of proinflammatory cytokines. Whether cytokine release is due to the enzymatic or signalling role of Heparanase is currently being investigated.





IN SILICO SEARCH OF POTENTIAL FUNCTIONAL SITES WITHIN PROMOTERS OF MOUSE O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE

Iatsyshyna A.P., Pidpala O.V. and Lukash L.L. Institute of Molecular Biology and Genetics, 150 Zabolotnogo Str., 03680, Kyiv, Ukraine.

The modulation of expression and activity of O6-methylguanine-DNA methyltransferase, MGMT, in tumors and normal tissues is currently being investigated as possible strategies for improving cancer therapy. At present little is known about organization and expression of mouse Mgmt gene, so the aim of our work was to search potential regulatory sequences within the promoters of this gene. Sequences of the mouse Mgmt gene promoters were taken from the database TRED. Identification of functional sites was performed by using program TFSEARCH. There are 3 promoters of mouse Mgmt gene in TRED database: 77428 (known), 77429 (refseq, predicted), 77430 (refseq). In sequences of given promoters we found gomology with functional sites for 15, 9 and 19 transcription factors (TF) respectively. Common for all promoters were such TFs: Nkx-2.5, Gata-1, SRY, USF. In the known promoter, except characterized by others AP-1 and E2F TFs, we detected binding sites (BS) for such TFs: CRE-BP, CREB, C/EBP, SRF, Oct-1, Tst-1, NF-E2, Lyf-1, c-Myc. Such exclusivity of revealed sites can be an evidence of tissue specificity of the Mgmt expression regulation. The predicted promoter contains BSs for such TFs: LYF-1, HSF2, c-Ets-1, C/EBPβ, Ik-2, except common TFs for all studied promoters. In the refseq promoter sequence we revealed unique BSs for TFs: CRE-BP, CREB, C/EBP, AP-1, c-Ets-1, Ik-2, Oct-1, YY1, Sox-5, TATA, STATx, c-Rel, Ik-1, NF-κB, N-Myc. Thereby, by using in silico analysis of three promoters of the mouse Mgmt gene we revealed individual BSs that can be associated with the tissue-specific regulation of gene expression, and common sites which could play a key role in regulation of this gene.

DEMONSTRATION OF AUTOPHOSPHORYLATION OF THE LYN PROTEIN TYROSINE KINASE A NEW SITE IN THE UNIQUE DOMAIN – THE ROLE OF THE NEW AUTOPHOSPHORYLATION IN LYN KINASE FUNCTION

Jarasrassamee B., Chong Y.P., Williamson N.A., Purcell A.W. and Heung-Chin C. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biology, University of Melbourne.

Lyn is a member of the Src family of protein kinases (SFKs). Since SFKs are important signaling enzymes participating in a variety of cellular processes, activation of SFKs is transient and tightly regulated. It is well known that autophosphorylation of Lyn at the conserved tyrosine (YA) in activation loop of the kinase domain results in activation. In addition to autophosphorylation at this site, we discovered a novel autophosphorylation site (Y32) in the unique domain of Lyn. In this study, mutants of Lyn carrying mutations in one or both autophosphorylation sites including Y32F Lyn, Y32, 397F Lyn, and Y397F Lyn were generated. Biochemical analysis revealed that unlike autophosphorylation in most protein kinases, autophosphorylation of Lyn at this site occurs intramolecularly. Using a phosphospecific antibody against Tyr-32 of Lyn, we were able to demonstrate that Lyn undergoes autophosphorylation at Tyr-32 in rat tissues, confirming the physiological relevance of our findings. To investigate the role of Tyr-32 autophosphorylation in the oncogenic action of Lyn, we set out to study the regulation and function of Lyn in Chronic myelogenous leukaemia (CML). Aberrant activation and expression of Lyn has been reported to contribute to the development of drug resistance in CML – some CML patients fail to sustain hematologic remission with the drug Gleevec. We have initiated studies of the role of the Tyr-32 autophosphorylation in the oncogenic action and regulation of the kinase activity of Lyn in both the Gleevec-sensitive and Gleevec-resistant CML cells. Results of our studies will shed light on how Lyn contribute to the development of drug-resistant CML.

COPPER CHLORIDE INDUCED EXPRESSION OF CYTOGLOBIN AND HIF-1α IN VIVO

Jusman S.W.A.1, 2, Prijanti A.R.1, 2, Iswanti F.C.1, Ferdinal F.3, Suyatna F.D.2, 4, Wanandi S.I.1, 2 and Sadikin M.1, 2 1Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Indonesia. 2Biomedical Science Program, Faculty of Medicine, University of Indonesia. 3School of Medicine, Tarumanegara University. 4Department of Pharmacology, Faculty of Medicine, University of Indonesia.

Copper is known to stabilize HIF-1α under normoxic condition, resulting in induction of HIF-1α target genes. Cytoglobin, the novel globin from vertebrate is suggested as one of HIF-1α regulated genes. This study observed the liver tissue response to copper chloride induction in vivo. Male Sprague-Dawley rats were given 1.25 μmol of copper chloride solution intraperitoneally. The observation are made at 2, 6, 24, 48 and 72 hours after treatment and compared to control group. Liver tissue were analyzed for HIF-1α protein using ELISA technique, cytoglobin mRNA with real time RT-PCR and cytoglobin protein with ELISA. It is showed that cytoglobin protein was up-regulated 48 hours after treatment compared to control group, while the HIF-1α protein and cytoglobin mRNA showed tendencies to be stimulated 24 and 48 hours respectively after treatment. It is concluded that copper chloride stimulated expression of cytoglobin protein, which might be mediated by stabilization of HIF-1α protein due to inhibition of its degradation by prolil hydroxylase.

INVESTIGATING THE CONTRIBUTION OF GENOMIC INSTABILITY TO ALTERED mircoRNA EXPRESSION IN OVARIAN CANCER CELL LINES

Kan C.W.S.1, Hahn M.A.1, Huh J.Y.1, Dykema K.2, Howell V.M.1 and Marsh D.J.1 1Functional Genomics Laboratory, Hormones and Cancer Group, Kolling Institute of Medical Research. 2Van Andel Research Institute, Grand Rapids, MI, USA.

Ovarian cancer is the most lethal gynaecological malignancy and the sixth most common cause of cancer death in Australian women. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are often aberrantly expressed in cancer. This project investigated whether chromosomal losses or gains may contribute to changes in expression of miRNA in ovarian cancer cell lines. Gene and miRNA expression microarrays were performed on four ovarian cancer cell lines and a cell line model of normal ovarian surface epithelial cells (OSEs). Predicted regions of chromosomal loss or amplification were identified by comparative genomic microarray analysis (CGMA) of gene expression data. CGMA predicted chromosomal loss at 5q in all four cancer cell lines but not in OSEs. Loss of heterozygosity in chromosome 5q has been reported to be associated with early development of ovarian cancer. Investigation of miRNA located on chromosome 5q revealed miR-146a to be significantly decreased (ANOVA, P<0.01) in expression across all four cell lines compared to OSEs. Predicted targets of miR-146a include CCBP2, chemokine-binding protein 2. CCBP2 is reported to be over-expressed in vascular tumours and may drive tumour development and growth. In summary, we have shown that ovarian cancer cell lines have gene and miRNA expression profiles that are distinct to those of OSEs. These results suggest that genomic instability may contribute to the altered expression of a subset of miRNA and their target genes in ovarian cancer. Supported by an Australian Postgraduate Award, University of Sydney Cancer Research Fund and the Cancer Institute NSW.





EXTENSION OF DROSOPHILA LIFESPAN BY OVEREXPRESSION OF HUMAN CLUSTERIN

Kang B.H.1, Lee Y.N.2, Park J.J.2 and Min B.H.1 1Department of Pharmacology and BK21 program in Biomedical Sciences, College of Medicine, Korea University, 136-705, Seoul, Korea. 2Department of physiology and BK21 program in Biomedical Sciences, College of Medicine, Korea University, 136-705, Seoul, Korea.

Clusterin (CLU) is a disulfide-linked heterodimeric glycoprotein implicated in diverse biological processes. Expression levels of CLU increase during cellular senescence or normal aging, but it is uncertain whether this is protective against aging, or is a consequence of aging. To better understand the role of clusterin in organismal aging, we generated transgenic Drosophila alleles to induce expression of the secretory form of human clusterin (hCluS) by using the Gal4/UAS system. The hCluS protein sized 50-60 kDa was detected in both adult homogenate and larval hemolymph of the flies over-expressing hCluS ubiquitously (da-Gal4>UAS-hCluS) or in motoneurons (D42-Gal4>UAS-hCluS). Life spans of these hCluS-over-expressing flies were significantly extended (39 days) than control flies that presented no hCluS induction. The mean life-span of CS10 (+/+), D42-Gal4/+, and UAS-hCluS/+ was 31, 33, and 35 days, respectively. In addition, the hCluS-over-expressing flies showed enhanced tolerance to heat shock, wet starvation, and oxidative stress. Furthermore, the amount of reactive oxygen species (ROS) in the whole body was significantly reduced in hCluS-over-expressing flies, compared to control flies. Over-expression of hCluS in a group of neurons was sufficient to recapitulate its effects of whole body expression, implying that hCluS works cell nonautonomously. However, the patterns of their feeding behavior were not affected by hCluS expression. Taken together, these results suggest that hCluS may function as an antioxidant to reduce ROS levels and delay the organismal aging in fruit flies. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009-009-1418) and BAERI (Basic Atomic Energy Research Institute) grant from the National Nuclear R&D Program (20090078713) funded all by the Korean Ministry of Education, Science and Technology (MEST)].

THE CELLULAR SIGNALING PATHWAY MEDIATING INDUCTION OF HBV REPLICATION AND GENE EXPRESSION BY ETHANOL

Kim Y.H.1, 2, Kim B.K.1, 2 and Park Y.G.1, 2 1Dept. of Biochemistry. 2Division of Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Anam-dong, Seongbuk-Gu, Seoul, 136-170, Korea.

Even though alcohol intake has been implicated in the etiology of HBV-related diseases, neither ethanol effect on HBV replication nor the cellular signaling pathway mediating the effect are known. This study is designed to elucidate the effect of alcohol on HBV replication and gene expression. Ethanol treatment increased the HBV promoter/enhancer activity and the levels of HBV transcripts, DNA and viral antigens. In contrast, acetaldehyde, a product of hepatic ethanol metabolism, had no effect on the synthesis of HBV mRNA. Moreover, the ethanol-induced increase in HBV mRNA synthesis was not affected by pretreatment with inhibitors of alcohol dehydrogenase or acetaldehyde dehydrogenase. However, CYP2E1 did not make an effect on ethanol-induced synthesis of HBV mRNA even though ethanol-induced ROS generation is well known to be mainly dependent upon CYP2E1. Ethanol-induced synthesis of HBV mRNA was abolished by pretreatment with DPI and Trolox. Ethanol increased the synthesis of IL-6 mRNA and the release of IL-6 protein via ROS generation, and these events are necessary for ethanol-enhanced synthesis of HBV mRNA and DNA as well as increase of HBV promoter/enhancer activity. Ethanol-induced HBV replication and gene expression was dependent upon IL-6/JAK2 signaling but not upon STAT1 and 3. This study indicates that the enhancement of HBV replication by ethanol treatment requires IL-6 production and JAK2 activation through ROS generation.

SUPPRESSOR OF CYTOKINE SIGNALING (SOCS)5 IS A POTENTIAL REGULATOR OF THE IL-4-JAK/STAT PATHWAY

Kolesnik T.B., Colombus R.E., Chakravorty A., Wilson T.A., Sprigg N.S., Carter W., Zhang J.-G., Babon J.J., Nicola N.A. and Nicholson S.E. The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

SOCS5 has been implicated in regulation of the Th1/Th2 balance by inhibition of IL-4 signaling in Th1 cells through interaction with the IL-4 receptor alpha chain (IL-4Rα)1. However, in SOCS5-deficient mice, Th1/Th2 cells differentiate normally and the mice mount normal B and T cell responses to mitogenic stimuli2. Analogous to the Th1/Th2 paradigm, macrophages differentiate into classically activated or alternate in response to IFNγ or IL-4, respectively. To dissect the role of SOCS5 in IL-4 signaling in macrophages, we initially examined regulation of the SOCS proteins. Socs5 mRNA as well as Socs1, Socs2 and Cis, was rapidly induced in response to IL-4, however, only SOCS1, SOCS5 and CIS could inhibit IL-4 signaling in a STAT6-reporter assay. We further demonstrated that, like SOCS1, SOCS5 was able to directly inhibit JAK1 enzymatic activity. Extensive mutagenesis analysis showed that the SOCS5 N-terminus, SH2 domain and SOCS box were required for inhibition of JAK1 activation, whereas the N-terminus was essential for interaction with the IL-4Rα. Western-blot analysis revealed no differences in the level of IL-4-stimulated JAK1 and STAT6 phosphorylation in SOCS5-deficient macrophages compared to wild-type cells. To address possible redundancy between SOCS1 and SOCS5, we analyzed IL-4 signaling in macrophages lacking both SOCS proteins. JAK1 and STAT6 phosphorylation were up-regulated in SOCS1-deficient cells to the same extend as in double knock-out cells indicating that, at least in primary macrophages, SOCS1 and SOCS5 are not functionally redundant. The biological role of SOCS5 in IL-4 signaling remains to be elucidated. 1Seki et al., PNAS 2002, 99(20): 13003-8; 2Brender et al., 2004 MCB, 24(13): 6094-6103.

FUNCTIONAL ANALYSIS OF TRANSCRIPTION FACTORS REGULATING FRUCTOSYLTRANSFERASES INVOLVED IN THE FRUCTAN SYNTHETIC PATHWAY IN WHEAT

Kooiker M., Xue G.P. and McIntyre C.L. CSIRO, 306 Carmody road, St. Lucia, Australia.

Water soluble carbohydrates (WSCs) are accumulated in the stems and leaf sheath of several cereals like wheat, barley and oats. The main WSCs found in wheat stems at the grain filling stage are fructans, which are linear or branched oligosachharides, synthesised from sucrose in the vacuole. For the synthesis the enzymes sucrose:fructan 6-fructosyltranferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST) are essential. Fructans are an important temporary carbon reserve and are hydrolysed by fructan exohydrolases when the grain needs sugars for grain filling. Under normal conditions stem WSCs can contribute to up to 20% of the grain yield, but under stress conditions (like drought stress) this percentage can increase to greater than 50%. Under water limited conditions a positive correlation between grain yield and WSC content in the stem at anthesis is often observed. Some enzymes that play an important role in stem fructan accumulation are sucrose:fructan 6-fructosyltranferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST), which are positively associated with genotypic variation in stem WSCs in recombinant inbred lines Seri/Babax (SB). We have recently identified a number of candidate transcription factors that are potentially involved in controlling the expression of these fructosyltransferases in wheat and have been evaluating the regulatory role of these candidate genes in transgenic wheat.





TLR REGULATION OF SPSB1 REGULATES INOS INDUCTION

Lewis R.S., Kuang Z., Kolesnik T.B., D’Cruz A., Low A., Norton R.S. and Nicholson S.E. Walter and Eliza Hall Institute.

The mammalian innate immune system has evolved to recognise foreign molecules derived from pathogens via the Toll-like receptors (TLRs). TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing interferon (IFN) β (TRIF), which results in the transcription of a small array of genes, including IFNβ. iNOS is an enzyme that is rapidly induced by a range of stimuli, including cytokines and microbes, and catalyses the production of nitric oxide (NO). NO is a potent source of reactive nitrogen species that play an important role in the killing of intracellular pathogens and forms a crucial component of the host defence. We have recently identified iNOS as a target of the mammalian SPRY domain-containing SOCS box (SPSB) -2 protein. The SOCS box is a peptide motif, which in conjunction with elongins B and C recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin complex. Here we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterise the interaction between SPSB1 and iNOS. Through the use of SPSB-1 transgenic macrophages and shRNA knockdown of SPSB-1 we have shown that SPSB-1 controls the induction of iNOS and the subsequent production of NO downstream of TLR3 and 4. Further, we demonstrate that regulation of iNOS by SPSB-1 is dependent on the proteasome via the SPSB-1 C-terminal SOCS box. These data suggest that SPSB-1 acts through a negative-feedback loop that together with SPSB2 controls the extent of iNOS induction and NO production.

GENERATION OF A GRANZYME B/EGFP REPORTER FOR LIVE-CELL IMAGING

Bird C.H.1, Prescott M.1, Harper I.2 and Bird P.I.1 1Department of Biochemistry and Molecular Biology, Monash University, Australia. 2Monash Micro Imaging, Monash University, Australia.

Granzyme B (GrB) is a serine proteinase found in the cytolytic granules of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). Upon target cell engagement, GrB and other granule proteins are released into the intercellular space. It is thought that perforin facilitates the entry of GrB into the target cell cytosol where GrB activates the apoptotic pathway. However, there is currently no direct evidence of GrB release into the cytosol of target cells, and there is increasing evidence that it is involved in extracellular remodelling. Here we describe the generation and characterisation of a GrB/GFP fusion protein which is expressed stably in NK cells and trafficks correctly to the granule. The GrB is inactive so will not induce cell death, but rather act as a tracer. Using this fusion protein we wish to follow GrB during target cell engagement and lymphocyte migration.

INTRACELLULAR TRAFFICKING OF RICIN TOXIN: COMPUTATIONAL MODELLING AND KINEMATICS

Chong D., Walker A. and Skvortsov A. Defence Science and Technology Organisation, 506 Lorimer St., Fishermans Bend, VIC 3207, Australia.

To explain the efficacy of inhibitors against toxins in various cell types, the intracellular trafficking of the inhibitors and indeed, the agent itself must be understood. In this study, live cell 3D/4D confocal microscopy was used to visualise ricin toxin transport in human small airway epithelial cells to develop a computational model of toxin trafficking. In order to predict the rate at which the toxin will reach various subcellular compartments, such as the juxtanuclear Golgi, the trafficking route of the toxin is modelled as a diffusive transport process. An analytic solution of the diffusion equations is then constructed by treating the boundaries of the cell wall and the nucleus as conformally invariant fractals. The driving parameters of the subcellular trafficking of ricin (i.e. mean squared displacement) were derived from confocal microscope image analyses and were used to calibrate the transport model. This work ultimately aims to establish a computational model of the interactions between inhibitor, agent and target cell.

SIDEROPHORE CONJUGATES AS ANTIMICROBIAL AND IRON-OVERLOAD AGENTS

Obando D., Brites L.J., Shi C., Liu J. and Codd R. School of Medical Sciences (Pharmacology), University of Sydney, NSW 2006, Australia.

Under iron-deprived conditions, pathogenic and non-pathogenic bacteria produce low-molecular-weight molecules called siderophores to solubilise Fe(III), which is sparingly soluble under the oxic, aqueous and pH neutral conditions in the environment and in the mammalian host. An avid recognition event between the Fe(III)-loaded siderophore and receptors at the bacterial cell surface is followed by the complex traversing the cell to ultimately deliver iron to the cytoplasm for incorporation into key Fe-containing molecules, such as cytochromes and ribonucleotide reductase. The competition between bacteria for Fe is reflected in the chemical diversity of native siderophores: most bacteria produce a unique siderophore that is recognisable only by its cognate cell surface receptor. Molecules that thwart regular Fe(III)-siderophore mediated uptake present a platform for the design of new narrow spectrum antibiotics. In our group, we have modified the structure of a siderophore native to a non-pathogenic gram positive bacterium to produce a suite of potential antibiotic compounds. Of the four compounds we prepared, one of these prevented growth of our target bacterium at an MIC of 60 nmol/cm2 lawn. The chemical differences in the family of compounds we prepared is subtle, which presents some intrigue with regard to fully understanding the mechanism of action. These results will shed new light on the potential of tackling antibiotic design via iron deprivation and the possibilities of designing siderophore-based compounds against mammalian pathogens, including Pseudomonas aeruginosa, Bordetella pertussis and Mycobacterium tuberculosis. In other work, we have prepared new siderophore conjugates that show promise as new therapeutics for the treatment of iron-overload disease in humans. In this paper, details of each of these programs will be discussed.





STRUCTURAL REARRANGEMENTS OF THE P. FALCIPARUM GAMETOCYTE: AN ESSENTIAL PROCESS FOR PARASITE TRANSMISSION

Dearnley M.K.1, 2, Dixon M.W.A.1, 2, Yeoman J.1, 2, Hanssen E.3 and Tilley L.1, 2 1La Trobe Institute for Molecular Science, La Trobe University, VIC 3086, Australia. 2ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, VIC 3086, Australia. 3Bio21 Institute, University of Melbourne, VIC 3000, Australia.

Transmission of the malaria parasite Plasmodium falciparum depends on the production of the specialised sexual blood stage parasites called gametocytes. Despite the fact that the first forms of the malaria parasite identified were the sexual stages or gametocytes, many questions remain about the fundamental biology of this lifecycle stage. Maturation of the gametocyte to the transmissible stage V form is dependent on the parasites ability to remodel its host RBC. During this remodelling, parasite derived structures are formed within the RBC cytoplasm called Maurer’s clefts which function as protein-sorting organelles for parasite proteins en route to the RBC membrane. In this study we assess the genesis and maintenance of the Maurer’s clefts and the resident Maurer’s cleft protein the Ring Exported Protein 1 (REX1), across gametocyte development. We also investigate the formation and composition of the inner membrane complex (IMC) of the gametocyte across several developmental stages. Previous studies have identified this complex within the gametocyte; however few have drawn parallels to the IMC found in the merozoite. We aim to identify novel constituents of the IMC and investigate its resemblance to the IMC in the asexual merozoite. Purified gametocytes from differing developmental stages were examined by electron and fluorescence microscopy to map the morphology of the developing gametocyte with particular emphasis on the Maurer’s cleft using resident protein REX1 and the IMC protein GAP50. An understanding of the structures and remodelling mediating gametocyte maturation will prove invaluable in the development of novel transmission blocking approaches.

RAB2A PARTICIPATES IN THE TRAFFICKING AND SECRETION OF APOLIPOPROTEIN E

Dinnes D.L.1, Kockx M.1, Jessup W.1 and Kritharides L.1, 2 1Macrophage Biology Group, Centre for Vascular Research, UNSW, Sydney, Australia. 2Concord Repatriation General Hospital, University of Sydney, Sydney, Australia.

Apolipoprotein E (apoE) is a ~34kDa glycoprotein that is secreted from several cells, including macrophages, hepatocytes and astrocytes, with known roles in immunoregulation, protection from atherosclerosis and Alzheimer’s disease. We have previously shown that apoE is transported in vesicles along the microtubular network and is regulated by PKA, PP2B (calcineurin) and intracellular Ca2+ (Kockx M et al, Circ Res (2007) 101:608; Kockx M et al, J Biol Chem (2009) 284:24144). However, the transport proteins regulating its vesicular movement are unknown. The aim of this study was to identify if any proteins of the Rab GTPase family play a role in this process. HEK293 cells stably transfected with human apoE or apoE tagged with GFP (apoE-GFP) were subjected to Rab1A, 2A, 6A, 8A, 10 or 27B siRNA using non-silencing sequences as a control. Silencing of Rab2A inhibited the rate of secretion 5-fold relative to control, whereas silencing of other Rab family members had no effect. Rab2A silencing had no effect on apoE mRNA levels, increased apoE protein levels within cells and pulse chase metabolic labeling confirmed a direct inhibition of apoE secretion. Confocal microscopy demonstrated accumulation of apoE in a perinuclear compartment after Rab2A silencing, and this was supported with a loss of highly sialylated apoE isoforms (determined by 2-dimensional electrophoresis) implying a block of transport through the Golgi network. We have demonstrated for the first time a major role for the GTPase Rab2A in regulating apoE transport and secretion, the manipulation of which may provide new avenues for modulating apoE secretion.

DETERMINING THE INTRACELLULAR LOCALISATION OF ATNHX5 AND ATNHX6 AND THEIR ROLE IN CELLULAR TRAFFICKING AND SALT TOLERANCE

Ford B.A. and Gendall A.R. Depratment of Botany, La trobe University, Victoria, Australia.

AtNHX5 and AtNHX6, like AtSOS1 and AtNHX1-4 are part of the large CPA1 monovalent cation/proton antiporter sub group of the sodium hydrogen exchanger super family. AtNHX5 has been shown to suppress the Na+ sensitive phenotype of the nhx1 yeast mutant and is up-regulated in response to NaCl. Arabidopsis plants over expressing the tomato AtNHX5/6 ortholog LeNHX2 showed increased tolerance to high Na+ concentrations. The intracellular localisation of AtNHX5 and AtNHX6 in Arabidopsis has been determined by studying the expression of a 35S::NHX5ORF:CFP, a 35S::NHX6ORF:YFP and a 35S::YFP:NHX6ORF. Our results show that AtNHX5 and AtNHX6 co-localise to the same intracellular location and are not localised to either the tonoplast or the plasma membrane, and may be localized to endosomes. Endosomal compartments are the main transport vesicles in the secretory and endocytic pathways. It has been proposed that the endosomal localized NHXs regulate endosomal pH and are essential for protein sorting in the endocytic and secretory pathways. Preliminary analysis of root tip cells from the nhx5 nhx6 double mutant shows a disruption to the normal development of lateral roots. It may be the case that in the nhx5 nhx6 double mutant the endosomal traffic required to deliver auxin influx carriers AUX1 and LAX3 to their ultimate destination is disrupted preventing the normal auxin signaling required for lateral root development.

BINDING OF P110 RETINOBLASTOMA PROTEIN INHIBITS NUCLEAR IMPORT OF SV40 LARGE TUMOUR ANTIGEN THROUGH A PHOSPHOREGULATED MECHANISM

Fulcher A.J.1, Dias M.M.1 and Jans D.A.1, 2 1Monash University, Clayton, Victoria, Australia. 2ARC centre of Excellent for Biotechnology and Development.

Nuclear import of the simian virus SV40 large tumour antigen (T-ag) is dependent on its nuclear localisation signal (NLS) within amino acids 126-132 that is recognised by the importin α/β1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction c. 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by further N-terminal sequences (amino acids 102-110) which represent the binding site (BS) for the retinoblastoma (Rb) tumour suppressor protein. Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicate that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110Rb implicate p110Rb binding as being responsible for the reduced nuclear accumulation, with the serine106 phosphorylation site within the RbBS appearing to enhance the inhibitory effect. The involvement of p110Rb in modulating T-ag nuclear transport has implications for the regulation of nuclear import of the other proteins from the various other viruses of medical significance that interact with p110Rb, and how this may relate to transformation.





REGULATORY ROLE OF PRIP IN EXOCYTOSIS THROUGH THE INTERACTION WITH PROTEIN PHOSPHATASE

Gao J., Takeuchi H., Zhang Z. and Hirata M. Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University.

Exocytosis is one of the most basal and important cellular events. The minimal machinery for the final step, membrane fusion, is SNARE complex consists of syntaxin, SNAP-25 and VAMP. Phospho-state of these proteins are known to be involved in the regulation of exocytosis. We have shown that a protein phosphatase-1 anchoring protein, PRIP (phospholipase C-related but catalytically inactive protein), has some inhibitory role in regulating exocytosis. In the present study, we examine the role of PRIP in phospho-dependent regulation of exocytosis using pheochromocytoma cell line, PC12 cells that secrete noradrenalin (NA). Pretreatment of the cells with forskolin enhanced NA secretion and the enhancement was gradually diminished by removing forskolin. Exogenous expression of PRIP accelerated the decreasing process of NA secretion. Correlatively with the NA secretion, SNAP-25 was strongly phosphorylated by forskolin treatment of the cells and dephosphorylated gradually after removing forskolin. In addition, exogenous expression of PRIP accelerated the dephosphorylation process. We also showed that dephosphorylation of SNAP-25 at Thr138, phosphorylated by cAMP-dependent protein kinase, was mainly catalyzed by PP-1. These results suggest that PRIP is involved in phospho-dependent regulation of SNARE proteins through modulating the activity of protein phosphates-1, thus regulating exocytosis. .

ARL5B IS A GOLGI-LOCALISED SMALL G PROTEIN INVOLVED IN REGULATION OF RETROGRADE TRANSPORT

Houghton F.J. and Gleeson P.A. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne.

Small G proteins play an essential role in the regulation of membrane traffic. Small proteins (~20kD) include members of the Rab, Arf and Arl families. The Arf/Arl family members were identified initially by sequence homology (Kahn et al., 2006). In the GTP-bound form, the Arfs/Arls are able to bind specific effectors. Although some Arf-like or Arl family members have been well characterised, such as Arl1 (Lu et al., 2001) and ARFRP1/Arl3 (Panic et al., 2003; Setty et al., 2003), the function of many Arls remains undefined. An important role of small G proteins is in the regulation of membrane traffic at the trans-Golgi network (TGN) (Derby et al, 2004; Lu and Hong, 2003). The cDNA encoding a number of Arls were amplified by RT-PCR from HeLa cells, mutated to the GTP or GDP bound forms by PCR mutagenesis and cloned into GFP vectors. Using confocal microscopy analysis of transfected HeLa cells, we identified Arls that localise to the Golgi. Arl5bWT and Arl5bQ71L localise to the Golgi in close proximity to TGN golgins, and Arl5bQ71L-GFP is observed on tubules associated with the TGN. Functional analysis was conducted using siRNA depletion and membrane transport was studied using antibody internalisation assays. Depletion of Arl5b by RNAi in cultured cells results in disruption of the intracellular localisation of mannose 6-phosphate receptor, and alters the transport dynamics of the membrane cargo TGN38 and the shiga toxin B fragment between endosomes and TGN. Therefore, Arl5b is a trans-Golgi small G protein which regulates endosome-TGN transport. Kahn, R.A., et al. J Cell Biol (2006), Lu,L. and Hong,W. Mol Biol Cell (2003), Derby, M C. et al. J Cell Sci.(2004)., Lu,L., et al. J Cell Sci (2001), Panic,B., et al. Curr Biol (2003), Setty, S.R., et al Curr Biol (2003).

TOXICITY OF AMYLOID TRANSTHYRETINS TO DOWN SYNDROME FIBROBLAST

Annanon S., Kaewmeechai S. and Prapunpoj P. Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

Amyloidosis is a group of human diseases characterized by the intra- or extracellular deposition of aggregated amyloid proteins, leading to progressive disruption of the normal tissue architecture and consequently impairing organ function. Among of these proteins, amyloid β (Aβ) which is the major constituent of amyloid plaques in brain cortex of Alzheimer ‘s disease (AD) and Down syndrome (DS), and transthyretin (TTR) are of interest. Inter-relationship in particular the protective function of TTR on cytotoxicity of Aβ has been demonstrated. Among peripheral tissues, fibroblasts from DS patients were shown carrying Aβ protein precursor (APP) and having endocytic dysfunction similar to neurons in AD. In this study, the cytotoxicity and the protective effect on Aβ of TTR were explored in greater details in normal and DS fibroblasts. Human wild-type TTR and TTR variants including V30M and L55P were synthesized using the heterologous gene expression system of Pichia pastoris. Cell viability and apoptosis were examined and compared. The preliminary results showed that not only Aβ but also amyloidogenic TTR V30M and L55P were toxic to both normal and DS fibroblasts, however with different sensitivity.

LONG-TERM MEMORY AND LEARNING IMPAIRMENT IN NEDD4 HETEROZYGOUS MICE

Bongiorno D.1, 2, Boase N.3, Kumar S.3 and Poronnik P.1, 2 1School of Medical Science. 2Health Innovations Research Insitute (HIRi), RMIT University, Bundoora. 3Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide.

Nedd4 (Neural precursor cell Expressed Developmentally Down-regulated 4) a ubiquitin ligase (E3) has been implicated in neuronal development. Although numerous potential substrates of Nedd4 have been uncovered, the exact physiological function(s) of Nedd4 remain unclear (Yang and Kumar 2010). As Nedd4 knockout mice die at birth due to growth retardation and vascular defects, we investigated the impact of Nedd4 in memory and learning using Nedd4 heterozygous mice. Nedd4 heterozygous (n=13) and wild-type (n=8) littermate controls were assessed for short-term memory using Y-maze. Long-term spatial memory and learning was also assessed using Morris Water Maze (MWM). Time taken to locate a hidden platform corresponds to the learning phase. On the final day of testing, the platform is removed and duration to enter the platform quadrant corresponds to the memory phase. We found that Nedd4 heterozygote mice took significantly longer to find the hidden platform than wild-type mice in MWM test (Day 6, 65.3±9.5 and 27.0±4.6 sec). Furthermore, when the hidden platform was removed, Nedd4 heterozygote mice also took significantly longer to enter the platform quadrant compared to wild-type mice (23.0±5.4 and 6.4±1.3 sec). However, there was no significant difference in time spent in the novel arm of the Y-maze between Nedd4 heterozygote and wild-type mice (150.4±5.4 and 158.3±8.7 sec). These data show that in Nedd4 heterozygous mice, where ~50% of the ubiquitin ligase is removed is sufficient to produce long-term spatial memory and learning deficits, with spared short-term memory. Yang B, Kumar S. 2010. Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death Differ 17:68-77.





THE INVOLVEMENT OF EXOSOMES IN INTERCELLULAR PRION TRANSMISSION

Coleman B.M.1, 2, 3, Hanssen E.3, 5, Masters C.L.4, Lawson V.A.2, 4 and Hill A.F.1, 3, 4 1Department of Biochemistry and Molecular Biology, The University of Melbourne. 2Department of Pathology, The University of Melbourne. 3Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne. 4Mental Health Research Institute, The University of Melbourne. 5Electron Microscopy Unit, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne.

Prion diseases are invariable fatal neurodegenerative disorders, which are associated with an abnormal isoform (PrPSc) of the host encoded cellular prion protein (PrPC). The diseases are also transmissible, with the infectious agent postulated to be composed principally of PrPSc. However, the conditions, cofactors, conformations and aggregates necessary for transmission remain to be defined. PrPSc and prion infectivity are present within exosome preparations. Exosomes are 40-100 nm membrane vesicles of endocytic origin released by most cell types in vitro and recent studies have also identified them in vivo in body fluids. However, due to limitations associated with standard sucrose density gradients it is not clear whether prion infectivity localises within exosomes or with an alternate, potentially unique vesicle. Recent developments in virus purification techniques have enabled the separation of virus particles (such as HIV-1 and influenza) from exosomes and other membrane vesicles. This was previously unobtainable using standard sucrose density gradients. We have demonstrated the application of a novel, rate-zonal gradient separation technique to show that prion infectivity is indeed associated with exosomes; a finding that is further validated with biochemical techniques. A benefit of this technique is that it produces highly purified exosomes without disturbing their native structure, making them amenable to a variety of downstream analytical techniques. Using this technique coupled with transmission electron microscopy we have found that the ultra-structure of exosomes is far more intricate than previously thought. This could potentially provide insight into the currently unknown mechanism by which exosomes interact with cells.

EVALUATION OF SRU BIND LABEL-FREE TECHNOLOGY FOR ASSESSMENT OF KAPPA OPIOID RECEPTOR LIGANDS IN CELL-BASED ASSAYS

Deuis J.1, Vetter I.2, Lewis R.2 and Cabot P.1 1School of Pharmacy, University of Queensland, Australia. 2Institute for Molecular Bioscience, University of Queensland, Australia.

Opioid receptors have long been known to be effective targets for the production of analgesia. Recent interest has developed in targeting the kappa opioid receptor (KOR) subtype, which couples to inhibition of adenylate cyclase through Gαi. Label-free technology is now available to assess ligand-receptor interactions in cell-based assays, replacing more conventional measurement of second messenger signalling. The aim of this study was to assess the ability of BIND label-free technology (SRU Biosystems) to detect the activation of the KOR. HEK-293 cells transfected with the rat KOR were exposed to varying concentrations of U-50488, a KOR agonist, with or without naloxone, an opioid receptor antagonist. Addition of U-50488 caused a naloxone-sensitive, concentration-dependent increase in peak wavelength shift with an EC50 of 2.7 nM, consistent with literature values. Therefore BIND label-free technology appears to be an effective technique to assess activity of KOR agonists in cell-based assays, and may be a useful tool in the future to identify new KOR agonists.

DYNEIN/COPII INTERACTION WITH MUTANT SOD1 IN SOD1G93A TRANSGENIC MICE AND NSC34 CELLS

Farg M.1, Walker A.1, Turner B.2, Horne M.2 and Atkin J.1, 2 11 Department of Biochemistry, La Trobe University | Bundoora, Victoria. 2Howard Florey Institute, University of Melbourne, Australia.

Recent evidence suggests that disruption of the dynein/dynactin transport machinery occurs in ALS. Mutation in the dynactin subunit p150 Glued causes human ALS and mice overexpressing mutant dynein heavy chain or Tgdynamitin mouse display slowly progressive motor neuron degeneration. Also, mutant but not wildtype SOD1 physically interacts with dynein and dynein co-localises with mutant SOD1 inclusions. Impairment of dynein-dynactin function by p50 dynamitin over-expression interferes with endosome trafficking and results in Golgi fragmentation. Fragmentation of the neuronal Golgi apparatus has been observed in both sporadic and SOD-mediated familial ALS as well as in pre-symptomatic transgenic SOD1G93Aanimals. Bidirectional transport between the endoplasmic reticulum (ER) and Golgi apparatus is mediated by the dynein transport machinery. Proteins exiting the ER are transported in vesicles coated by coat protein complex II (COPII). Recently there has been a surge of publications describing the importance of ER stress in ALS. We described induction of the whole unfolded protein response in both transgenic SOD1G93A animals and in human sporadic ALS patients. We hypothesised that disruption in ER to Golgi trafficking may contribute to ER stress in ALS. Anterograde and retrograde transport proteins were examined in transgenic SOD1G93A

mice and in NSC34 cell lines transfected with mutant and wt SOD1. We demonstrated a physical interaction between mutant and not wildtype SOD1 and both COPII and dynein in transgenic SOD1G93A animals at p10, 60 days before the onset of symptoms and before the onset of ER stress. This was confirmed; in NSC34 stable cell lines express human SOD1 A4V/ G85R/ G37R and G93A. Also, COPII/ COPI found to co-localise with mutant SOD1 inclusions. These data suggest that alteration in dynein mediated transport is a very early event in disease and preceeds ER stress in ALS.

ASCENDING GABAERGIC/PEPTIDERGIC CONTROL OF AROUSAL, STRESS AND MOTIVATED BEHAVIOUR: FOCUS ON NUCLEUS INCERTUS AND RELAXIN-3 SIGNALLING

Gundlach A.L.1, Ma S.1, 2, Smith C.M.1, Ryan P.J.1, Hossain M.A.1, Wade J.D.1, Bathgate R.A.D.1, Verberne A.J.M.2, Blasiak A.3 and Olucha-Bordonau F.E.4 1Florey Neuroscience Institutes, The University of Melbourne, Australia. 2Department of Medicine, Austin Health, The University of Melbourne, Australia. 3Institute of Zoology, Jagiellonian University, Krakow, Poland. 4Department of Anatomy, University of València, València, Spain.

The nucleus incertus (NI) in the ventromedial pontine grey is a major source of ascending GABAergic projections that innervate limbic and hypothalamic regions involved in arousal, sleep/wakefulness and related autonomic and neuroendocrine functions. NI neurons and other small midbrain populations express the neuropeptide, relaxin-3; and existing anatomical and functional studies suggest relaxin-3 receptor (RXFP3) signalling should modulate ‘behavioural state’, arousal and responses to stress, and related behaviours. The precise nature of these actions and the circuits and transmitters/peptides involved are not known, however. We have conducted studies in rats and mice that reveal: (i) NI neurons in the rat are activated by behavioural activity and neurogenic stressors, and by CRF and orexin; (ii) relaxin-3 neurons in rat and mouse innervate networks controlling circadian activity and metabolic balance, and the emotional and cognitive circuits of the amygdala and septohippocampal system; (iii) central RXFP3 activation increases feeding in satiated rats; (iv) RXFP3 activation/inhibition in medial septum modulates spatial working memory and hippocampal theta rhythm in the rat; (v) RXFP3 activation in central amygdala can block fear expression and may enhance fear extinction in rats; and (vi) relaxin-3 knockout mice are hypoactive and have altered sleep patterns during the dark/active phase of the circadian cycle. These studies have increased our knowledge of the role of the ascending NI GABA/relaxin-3 network in homeostatic and complex behaviours and identified its potential as a therapeutic target in neuropsychiatric disease.





OXIDATIVE STRESS INDUCES MULTIPLE CASPASE-INDEPENDENT CELL DEATH PATHWAYS IN PRIMARY CORTICAL NEURONS

Higgins G.C.1, Devenish R.J.1, Beart P.M.2 and Nagley P.1 1Department of Biochemistry and Molecular Biology, Monash University, Wellington Rd, Clayton 3800, Victoria, Australia. 2Florey Neuroscience Institutes, University of Melbourne, Parkville, Victoria 3010, Australia.

Neuronal cells can undergo a diverse range of death responses. These death outcomes were previously thought to be limited to either apoptosis (involving caspases and energy-dependent) or unregulated necrosis (independent of both caspases and the need for ATP). We have recently shown that primary cortical neurons from mice exposed to an acute insult of hydrogen peroxide (H2O2) undergo caspase-independent cell death that is highly regulated, predominantly manifesting autophagic cell death and programmed necrosis. In this current work we show these neurons undergo caspase-independent cell death when exposed to a chronic dose of superoxide (provided by exogenous xanthine oxidase in the presence of catalase, which acts as a sink for H2O2). We found that while key mitochondrial intermembrane space proteins (cytochrome c, Smac, AIF and Endonuclease G) are redistributed to cytosol, downstream caspases (-3, -7, -9) were not activated. While this outcome was similar to that of our previous work done with H2O2, we found that knockdown of either Endo G (programmed necrosis) or Atg7 (autophagic cell death) using siRNA was much less potent in blocking cell death. We conclude that superoxide invokes a diverse death response that involves some autophagic cell death and programmed necrosis, but is predominantly unregulated necrosis. This work highlights the significance of the type of oxidative stress exposure, in relation to the specificity of the neuronal cell death outcomes.

THE C-SRC PROTEIN TYROSINE KINASE IS REGULATED BY CALPAIN MEDIATED CLEAVAGE UPON OVER STIMULATION OF GLUTAMATE RECEPTOR IN CULTURED PRIMARY NEURONS

Hossain M.I. and Cheng H.C. Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne.

Glutamate-induced excitotoxicity is one of the main features of ischemic stroke and neurodegenerative diseases. Excess glutamate causes over-stimulation of NMDA receptor leading to excessive influx of extracellular calcium ion into neurons. The excessive influx of calcium ion activates calpains which cleave many functional proteins and in turns enhances neuronal death. c-Src, an important member of non-receptor Src family protein kinases (SFKs), binds to scaffolding proteins in the NMDA receptor to form a complex to regulate activity of the receptor. In this way c-Src plays crucial role in glutamate-induced neuronal death. Herein we report that under glutamate-induced excitotoxicity, c-Src is cleaved by calpain in cultured primary cortical neurons. The same phenomenon was also found when recombinant c-Src was digested with calpain in vitro. Using an N-terminal directed c-Src antibody we mapped the cleavage site to the N-terminal region of c-Src. We also monitored kinase activity of the protein under excitotixic condition using specific Src optimal peptide and phosphospecific antibodies. Kinase activity assay demonstrated that c-Src activity undergoes a biphasic change – it increases shortly after glutamate treatment but decreases once the calpain-mediated cleavage starts. In summary results of our studies demonstrate the involvement of c-Src in excitotoxic neuronal death. We demonstrate for the first time that calpain mediated cleavage of c-Src as regulatory mechanism in neurons. Further investigation on the effect of calpain mediated cleavage of c-Src on its activity and regulation may provide insights into the mechanism of excitotoxic neuronal death.

INVESTIGATING THE ROLE OF COPPER IN THE NERVOUS SYSTEM

Hwang E.C.J. and Burke R. School of Biological Sciences, Monash University, Wellington Rd, Clayton, VIC, 3800, Australia.

Copper is an important trace metal required in balanced amounts for proper cellular function and development. Strict control of copper levels is required as copper dyshomeostasis results in multiple adverse effects, such as cellular damage due to the generation of reactive oxygen species. Proper copper homeostasis is achieved via the action of copper transport proteins which serve to either import, sequester or export copper. While the overall role of copper in the body has been investigated quite well, less is known about the specific role of copper in the nervous system. Previous studies have shown that copper is likely to play a role in both the function and development of the nervous system, however, further study is require to elucidate the specific mechanisms by which this occurs. Using Drosophila Melanogaster as a model, I have created a system within which to study the effects of both copper excess and scarcity on the nervous system. This has been achieved through the targeted misexpression of copper transport proteins in the nervous system via use of the GAL4-UAS system, thereby generating an in vivo model within which to study the effects of copper dyshomeostasis. I have shown that both copper excess and scarcity result in the disruption of nervous system function, and have investigated some of the mechanisms by which this occurs.

THE EXPRESSION OF TNF AND ITS RECEPTORS IN SCHIZOPHRENIA AND MOOD DISORDERS

Jeon W.J.1, 2, Gibbons A.S.1, 2 and Dean B.1, 2 1Rebecca L. Cooper Laboratories, the Mental Health Research Institute, Parkville, Victoria, Australia. 2Department of Psychiatry, the University of Melbourne, Parkville, Victoria, Australia.

Abberant expression of cytokines involved in pro-inflammatory pathways have been proposed to underlie the pathology of schizophrenia and mood disorders. We have recently reported an increase in the level of tumor necrosis factor (TNF) protein in the dorsolateral prefrontal cortex (Brodmann’s area (BA) 46), but not the anterior cingulate cortex (BA 24), from subjects with major depression. We sought to determine whether the mRNA expression of TNF and its receptors is altered in the cortex of post-mortem subjects with schizophrenia and mood disorders. Real-time PCR was used to measure the levels of TNF, TNFR1 and TNFR2 mRNA in BA 24 and BA 46 from subjects with schizophrenia (n=20), major depression (n=10), bipolar disorder (n=10) and matched control subjects. The level of TNF mRNA was significantly increased in BA 24, but not BA 46, from subjects with schizophrenia compared to controls (p<0.05). The level of TNFR1 mRNA was increased in BA 24 (p<0.05) and BA 46 (p<0.0001) in subjects with schizophrenia. TNFR2 mRNA expression was not altered in schizophrenia (p>0.05). Furthermore, there was no change in the mRNA levels of TNF and either receptor in BA 24 and BA 46 from subjects with major depression and bipolar disorder compared to controls (p>0.05). Our findings suggest that abnormal TNF signalling may be involved in the pathology of schizophrenia. Furthermore, the increased TNF protein expression in BA 46 that we previously reported in subjects with major depression does not appear to result from increased gene expression.





TARGETED GENE DELIVERY TO MICROGLIA VIA THE SCAVENGER RECEPTOR CLASS B, TYPE I

Malmevik J.M.K.1, Rogers M.-L.1, Nilsson M.2, Nakanishi Y.3, Rush R.A.1, Sims N.R.1 and Muyderman H.1 1Centre for Neuroscience, Flinders Medical Science and Technology, Flinders University, Australia. 2Institute for Neuroscience, Goteborg University, Sweden. 3Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.

Microglia are the major contributors to inflammatory responses in the brain. Investigations of microglia are limited by the difficulty of selectively targeting these cells within the complex environment of the mature brain. Receptor-mediated gene delivery constitutes a non-viral approach which also avoids immune responses that can be produced even by highly modified viral vectors. In this study, we tested the possibility of selectively targeting microglia in vivo utilising receptor-mediated gene delivery via the scavenger receptor class B type I (SR-BI). SR-BI expression in mixed glial primary cultures and in purified microglial cultures was confined to microglia. Incubations of an antibody targeted at the extra-cellular domain of SR-BI resulted in microglia-specific uptake. This SR-BI antibody was linked to the polycation polyethylenimine (PEI) and bound to a plasmid encoding green fluorescent protein (GFP) to form an ‘immunogene’. GFP expression was seen in microglia following incubations of this construct in mixed glial primary cultures. Intrahippocampal infusions of the ‘immunogene’ resulted in a substantial GFP expression in CD11b- and ED1-positive microglia around the infusion site (n=5). GFP-positive microglia were found up to 4.2 mm from the site of infusion. No colocalisation with astrocytic or neuronal markers was found. Control infusions with PEI and the GFP plasmid alone produced no expression of the reporter gene (n=3). This research demonstrates for the first time the use of a non-viral transfection system to selectively target the microglial cell population in vitro and in vivo.

TOWARDS ISOFORM-SELECTIVE DYNAMIN INHIBITORS

Mariana A.1, Hill T.A.2, McGeachie A.B.1, Chau N.1, Daniel J.1, Gorgani N.N.1, McCluskey A.2 and Robinson P.J.1 1Cell Signalling Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia. 2Department of Chemistry, University of Newcastle, NSW 2308, Australia.

The GTPase enzyme dynamin I is predominant in brain; dynamin II is ubiquitously expressed; and dynamin III is found in brain and testes. Dynamin I and dynamin II are required for synaptic vesicle endocytosis (SVE) and receptor-mediated endocytosis (RME), respectively. Dynamin II has a second function in the abscission that completes mitosis. Therefore, dynamin I-specific inhibitors might be useful in diseases that involve SVE (epilepsy), while dynamin II-specific inhibitors might be effective as anticancer drugs with minimal side effects. Our team has developed various classes of dynamin inhibitors which have been discovered using a malachite green dynamin I-based GTPase assay (dynamin I being activated by liposomes) and dynamin II-mediated RME cell-based assay. We have now developed a dynamin II-based GTPase assay, using dynamin II produced by transient expression in Sf9 insect cells, which retains high specific activity comparable to endogenous brain dynamin I, as well as dynamin I cell-based assay using synaptosome. When we tested our classes of dynamin inhibitor that have been published (MiTMAB, pyrimidynes) in our 4 assays, they appear to be non-selective (PAN inhibitors). We recently published new class of dynamin inhibitors (iminodyns). These drugs inhibit dynamin GTPase activity, as well as endocytosis. By utilising our 4 type of assays, the iminodyns do not show any isoform-selectivity for dynamin I or II GTPase activity. Despite this, the iminodyns include three nanomolar potent dynamin I and II inhibitors, which also block endocytosis. Nevertheless, our screening system allows further development towards isoform-selective dynamin inhibitors from the iminodyns as well as other classes.

CLINICAL SIGNIFICANCE OF URINE SURVIVIN MRNA IN PROSTATE CANCER DETECTION

Almaghrebi M., Kehinde E.O. and Kapila K. Kuwait University - Faculty of Medicine.

Survivin is one of the most tumor-specific molecules, which antagonizes apoptosis and promotes tumor associated angiogenesis. Thus, it comes to no surprise that it is overexpressed in many types of cancers. In prostate cancer, PSA levels alone have a low overall efficiency of accurate diagnosis. Survivin was evaluated as an alternative molecular marker in patient urine samples. Expression levels were measured by quantitative reverse transcription-real time polymerase chain reaction (RT-PCR) and correlated with clinicopathological data in urine samples from patients with BPH, BPH & Prostatitis, and prostate cancer. Survivin levels were significantly elevated in urine from prostate cancer patients compared with healthy controls (P <.001) and with noncancerous prostate disorders: BPH (P <.05) and BPH with Prostatitis (p <0.05). An optimal cutoff value of 25 pg was determined. Accordingly, 21% of patient samples had survivin levels above 25 pg, of which only 3% were healthy individuals. In contrast, 55% of individuals with survivin levels above 25 pg were prostate cancer patients. The results indicate that urine survivin levels are elevated during prostate cancer developmnt, demonstrating its strength as a potential marker for discriminating benign from malignant disease.

IDENTIFYING THE ROLE OF SPECIFIC NON-CONSERVED RESIDUES ON PI3K IN THE DISCOVERY OF ISOFORM-SELECTIVE PI3K INHIBITORS

Amran S.I. Monash Institute of Pharmaceutical Science, 381 Royal Parade, Vic 3052 Australia.

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the biosynthesis of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3 - second messengers that trigger a wide range of downstream signalling cascades involved in cell survival, growth, adhesion and proliferation. The heterodimeric class 1 PI3K proteins are composed of a regulatory subunit (p85) complexed with either one of 4 different isoforms of catalytic subunit (p110α, p110β, p110δ and p110γ). The PI3KCA gene encoding the α-isoform has been found to be frequently mutated in cancers such as breast, prostate, colon, liver and brain. The overall aim of this program is to elucidate the molecular mechanism of binding of isoform selective small molecule PI3K inhibitors. Previous studies have shown that specific regions within the catalytic subunit contain non-conserved residues that contribute to isoform selectivity. In this study mutant P13Kα isoforms were generated by swapping the residues of the α-isoform for corresponding residues of other isoforms using site-directed mutagenesis followed by recombination of the mutant sequences into the baculovirus vector system. The resultant proteins were expressed in insect cells and purified. The purified enzymes have been characterised using western blot analysis and kinase assays. With retained enzyme activity these mutant isoforms can be used to identify the role of non-conserved residues in inhibitor binding and contribute to rational design of novel PI3K inhibitors.





EVASION STRATEGIES OF ECHINOCOCCUS GRANULOSUS TO TH1 HOST PROTECTIVE RESPONSE DURING HUMAN INFECTION

Amri M. and Touil-Boukoffa C. Team: “Cytokines and NOSynthases”, Laboratory of Cellular and Molecular Biology, Faculty of Biological Science, USTHB, PB 32, El-Alia, 16111, Algiers, Algeria.

Background: Human echinococcosis is one of the world’s major zoonotic infections. It usually manifests as unilocular cyst(s) mainly located in the liver. More recently, we have highlighted an evident role of IFN-gamma (Th1 cytokine) in parasite killing by NOS2 (Nitric oxide Synthase2) pathway. Moreover, IL-10 (Treg cytokine) production seems to be an evasive mechanism taken by the parasite to establish in the host by Arginase pathway (Amri et al., 2007 & 2009). Of note, NOS2 and Arginase are known to compete for the common substrate, L-Arginine. Moreover, IL-10 downregulates IFN-gamma production. Indeed, more researches are required to identify factors present in parasite cyst which affect protective Th1 response in Echinococcus granulosus human infection. Method: We investigate the effect of laminated-layer (accelullar layer of hydatic cyst) extract (LLs) on Th1/Treg and NOS2/Arginase balance in culture performed with mononuclear cells (PBMC) of hydatid patients and healthy donors. Furthermore, we have investigated the effect of LLs on parasite viability in PBMC-parasite cocultures. Results: Our results demonstrated that LLs reduced IFN-gamma/NO production and enhanced IL-10 production and Arginase activity. In addition, LLs enhanced parasite survival in vitro. Similar findings are observed in cultures and cocultures performed with PBMC of patients and healthy donors. Moreover, the major antigenic fraction in LLs: the fraction 4 (12kDa, purified by chromatography) has the same effect as LLs. Conclusion: Collectively, the present study provides evidence that Echinococcus granulosus laminated layer impairs Th1 protective response and allow the parasite to survive. Inhibition of these mechanisms seems to be important issue to address during the design of anti-hydatic treatment.

ETHYLENE MEDIATED DEFENCE AGAINST THE ROOT INFECTING FUNGAL PATHOGEN RHIZOCTONIA SOLANI IN MEDICAGO TRUNCATULA

Anderson J.P.1, Lichtenzveig J.1, 2, Oliver R.P.2 and Singh K.1 1CSIRO Plant Industry, Floreat, Western Australia. 2Australian Centre for Necrotrophic Fungal Pathogens, Murdoch, Western Australia.

The fungal necrotrophic pathogen Rhizoctonia solani (Kühn) is a significant constraint to the production of a range of crops as diverse as cereals, canola and legumes, including causing the second largest disease constraint to rice production in China and India. No strong genetic resistance has been identified in these crops despite wide ranging primary and secondary germplasm screens, suggesting alternative strategies to improve resistance are required. In this study we characterise moderate resistance to R. solani AG8 identified in the model legume Medicago truncatula. The activity of an ethylene and jasmonate responsive promoter element was associated with the moderate resistance as was the induction of specific ethylene/jasmonate response transcription factors. Over-expression of some of these transcription factors in transgenic Medicago “hairy roots” increased resistance to R. solani as well as to another destructive root pathogen, the oomycete Phytophthora medicaginis. Over-expression of these genes had no apparent impact on symbiosis with nitrogen fixing bacteria as the transgenic roots formed root nodules at comparable rates to wild type. This suggests that enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and ethylene dependent control of nodule number is distinct from ethylene dependent defenses.

ATRX IS A SERTOLI CELL SURVIVAL FACTOR AND REGULATOR OF SPERMATOGENESIS VIA INTERACTION WITH ANDROGEN RECEPTOR

Bagheri-Fam S.1, Argentaro A.1, Svingen T.2, Combes A.2, Koopman P.2 and Harley V.1 1Prince Henry’s Institute of Medical Research, Melbourne, Victoria, Australia. 2Institute of Molecular Bioscience, Brisbane, Queensland, Australia.

The ATR-X syndrome, (α-thalassemia mental retardation, X-linked) is a developmental disorder affecting males. The testicular abnormalities of ATR-X patients comprise small testes, few seminiferous tubules, and a lack of germ cells. ATRX protein remodels chromatin in vitro though its bona fide target genes are unidentified. To understand the gonadal role of ATRX, we inactivated Atrx specifically in Sertoli cells (ScAtrxKO mice) from embryonic day 14.5. Fluorescence-based three-dimensional modeling at E17.5 revealed that testis cord volume in ScAtrxKO mice is ~30% of wildtype, with discontinuous or isolated testis cords apparent. While Sertoli cell apoptosis is increased by 10-fold when compared to wildtype, no difference in Sertoli cell proliferation was observed. These data suggest that ATRX is required for fetal Sertoli cell survival and, in turn, for elongation of fetal testis cords. Adult ScAtrxKO testes weigh ~25% of wildtype suggesting that seminiferous tubule hypoplasia is primarily established during fetal life. Histological examination showed that a third of tubules contain germ cells arrested in late meiosis or at the round spermatid stage. We found that the Androgen Receptor (AR)-dependent genes, Rhox5 and Claudin3, were significantly down-regulated in Sertoli cells of ScAtrxKO testes. Moreover, we show that ATRX and AR proteins interact in the TM4 Sertoli cell line and co-operatively activate the Rhox5 promoter. In summary, ATRX plays an important role in Sertoli cell survival during fetal testis development and in adult testis function, where ATRX protein interacts with AR to regulate the transcription of AR-dependent genes that control spermatogenesis.

POLYMORPHISM OF SOD2 GENE IN PATIENTS OF BRONCHIAL ASTHMA

Bhadoria D.P.2, Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh S.1, Kumar R.1, Bhadoria P.2, Anand R.2 and Sharma G.L.1 1Institute of Genomics and Integrative Biology, University Campus Mall Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of Science, University of Melbourne, Victoria 3010 Australia.

Superoxide dismutase (SOD2) is an antioxidant protein and polymorphism of its gene may contribute to susceptibility to bronchial asthma. There are no studies on polymorphism in SOD2 gene from the Indian subcontinent. The present study was conducted to investigate association between SOD2 gene polymorphisms and pathogenesis of bronchial asthma in Indian patients. Fifty patients of asthma diagnosed as per ATS guideline 1987 and 50 normal controls were included in this study for drawing blood. Genomic DNA was isolated from peripheral blood leucocytes and used for the amplification of SOD2 gene. PCR product was subjected to sequencing by Applied Biosystems 3730 DNA sequence analyzer. The sequencing data thus obtained was analyzed using SISA and Epi Info statistical softwares. Screening of sequencing data of the subjects in either group identified an intronic mutation at base number 14788 (rs2842980. Twenty eight subjects of asthma (56%) and 9 from the control group (18%) were heterozygous (A/T) for this SNP. One patient as well as one control was found to be homozygous mutant. Wild type allele T was present in 70% of asthmatics and 89% of controls showing the role of A/T genotype susceptibility of asthmatic patients to the disease.The occurrence of T to A mutation at 14788 base position was higher in asthmatics than in controls (p<0.001) indicating its involvement in asthma and it could be an important risk factor the disease.





STATUS OF ANTIOXIDANTS IN BRONCHIAL ASTHMA

Bhadoria P.2, Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh S.1, Kumar R.1, Bhadoria D.P.2, Anand R.2 and Sharma G.L.1 1Institute of Genomics and Integrative Biology, University Campus Mall Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of Science, University of Melbourne, Victoria 3010 Australia.

There is an increased production of reactive oxygen species (ROS) by inflammatory cells in the airways of patients of bronchial asthma, resulting into oxidative stress. In order to combat and neutralize the deleterious effects of ROS, various endogenous antioxidant processes occur in the hosts involving enzymatic and non-enzymatic mechanisms. The present study was undertaken to investigate the status of antioxidant markers in Indian asthmatic patients. Fifty asthmatics diagnosed as per ATS guidelines (1987), and 50 normal controls were recruited for drawing blood. Sera separated from whole blood was used for estimation of antioxidant markers namely, Superoxide dismutase (SOD), Catalase, Ascorbic acid and lipid peroxidation. Total SOD was estimated by the method of Kono et al. (1978). The activity of catalase enzyme was assayed by the method of Beers and Sizer (1952). Ascorbic acid assay was performed by the method of Natelson el al. (1971). The assay for microsomal lipid peroxidation byproduct, Malondialdehyde, was performed by the method of Wright et al 1981. Results revealed that there was marked decrease in total SOD (p<.01) and Catalase (p<.01) in asthmatics as compared to controls. However, there was no significant difference in ascorbic acid levels between patients and controls (p>.05). There was increased lipid peroxidation and antioxidant levels were found to be reduced in asthmatics. It seems that their level was not sufficient to scavenge free radicals produced during oxidative stress and tissue damage.

EFFECT OF ANTIOXIDANTS SUPPLEMENTATIONS IN SALT-INDUCED DYSLIPIDAEMIA IN ALBINO RATS

Bilbis L.S., Saidu Y., Ahmad K., Abbas A.Y., Baba A.U. and Shuaib A.M. Usmanu Danfodiyo University Sokoto, Nigeria PMB 2346 Sokoto, Nigeria.

Cardiovascular disease (CVD) is associated with many risk factors including oxidative stress and dyslipidaemia. The current work evaluated the effects of antioxidants supplementation on salt-induced dyslipidaemia in albino rats. Rats were grouped into 11 groups of 7 rats each. Groups 2-11 were fed 8% salt diets for 4 weeks while group 1 served as control and were fed normal rat feed. Water was provided to all the groups ad libitum. The animals in groups 3-11 were then supplemented with vitamin A; vitamin C; vitamin E; Cu; Mn; Zn; vitamins A,C and E combined; Cu, Mn and Zn combined; and all the vitamins and minerals combined respectively for additional 4 weeks simultaneously with salt loading. Group 2 was not supplemented and served as the negative control. The body weight changes, pulse rate of the rats were monitored. Serum levels of Total cholesterol, triacylglyceride, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, high density lipoprotein cholesterol and glucose were estimated. The results indicated that the vitamins reduced significantly serum lipid profiles and the atherogenic index by up to 80 %. The serum glucose levels of the rats supplemented with antioxidant vitamins and minerals were also significantly (P<0.05) lowered compared with the negative control group. These results suggest that the reduction of serum lipid profile and glucose level may be due to regulation of cholesterol and lipoprotein metabolism and increased insulin sensitivity as a result of the supplementations. It may thus suggest that the antioxidants may provide protection against CVDs and metabolic syndrome in salt induced dyslipidaemia in rats.

TARGETING THE TROPOMYOSIN ISOFORM TM5NM1 IMPAIRS TUMOUR SURVIVAL, PROLIFERATION AND MIGRATION

Bonello T.1, Stehn J.1, Schevzov G.1, Coombes J.1, McCluskey A.2, Haass N.3, Dixon I.4 and Gunning P.1 1Department of Pharmacology, School of Medical Sciences, University of New South Wales, NSW, Australia. 2School of Chemistry, University of Newcastle, NSW, Australia. 3Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, NSW, Australia. 4Genscreen Pty Ltd, Melbourne, VIC, Australia.

The actin cytoskeleton is a fundamental regulator of key cellular functions including proliferation, motility and apoptosis. Aberrations in these processes are hallmarks of tumorigenesis, making the actin cytoskeleton an attractive chemotherapeutic target. The function of actin filament populations is regulated by their association with distinct tropomyosin isoforms. As cells transform they demonstrate an increased reliance on a subset of low molecular weight (LMW) tropomyosins. The LMW isoform, TM5NM1, has been shown in over-expression and knock-down models to impart a proliferative advantage on the cell and regulate directed cell movement. Objective: We have developed a novel class of compounds designed to target TM5NM1 containing filaments, and aim to characterise their effect on cellular function. Summary of Results: The lead compound, TR100, reduced viability in a panel of neuroblastoma and melanoma cell lines (average LC50 ~2-3μM) and inhibited survival and growth in a 3D melanoma model which simulates the tumour microenvironment. To dissect the role of TR100 in cell death, FACS analysis was performed with the human melanoma cell line SK-N-MEL28. Cells treated with 5μM TR100 underwent G0/G1 cell cycle arrest, while programmed cell death was induced at higher concentrations of TR100. Finally, migration towards a directional cue, measured by in vitro scratch wound assay, was significantly impaired at non-lethal concentrations of TR100. Conclusions: We have described a novel class of chemotherapeutic compounds which specifically target an actin filament population required for tumour survival, proliferation and migration.

INVESTIGATION OF MITOCHONDRIAL FUNCTION IN A METHYLMALONIC ACIDURIA MOUSE MODEL

Buck N.E.1, Siddiqui T.1, Laskowski A.2, Frazier A.E.2, Pitt J.J.3, Thorburn D.R.2, 3 and Peters H.L.1 1Cell & Gene Therapy Group, Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville, Victoria, Australia. 2Mitochondrial Research, Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville, Victoria, Australia. 3VCGS Pathology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia. Methylmalonic aciduria (MMA) is an organic acid disorder resulting from a defect in the mitochondrial enzyme methylmalonyl-CoA mutase. We have developed an MMA mouse model that recapitulates key aspects of the intermediate form of MMA: elevated methylmalonic acid levels in urine, blood and various organs and survival into adulthood. We have investigated the impact of deficient methylmalonyl-CoA mutase on mitochondrial enzyme activities. Respiratory chain complexes (I, II, III, IV), citrate synthase and aconitase activities were assayed. There was no difference between Co II, aconitase and citrate synthase activity between MMA and control mice, suggesting that the tissues were not affected by significant oxidative stress. Comparison of MMA to control mouse samples show reduced levels (up to 50%) of the complexes with subunits encoded by mitochondrial DNA (I, III and IV) in the liver and muscle, and Co III, IV in the brain (kidney enzyme activity unchanged). Fluorescent microscopy showed no difference in the number and distribution of mitochondria in kidney, muscle, lung or skin from control and MMA mice. Mitochondrial DNA levels were determined using real-time PCR by comparing Mtco1 to β-actin. The amount of mtDNA present in MMA mouse kidney, heart and muscle were 60±20% the amount present in controls. There was little change in the amount of mitochondrial DNA in MMA mouse liver and brain (85±23% of controls). These data support the concept that pathogenesis of MMA may be mediated by an affect on expression of respiratory chain complexes with subunits encoded by mitochondrial DNA.





THE ROLE OF UBIQUITIN IN THE REPLICATION OF HEPATITIS C VIRUS

Cardeira S.S. and Bishop N.E. Microbiology Department, La Trobe University, Bundoora, Vic, Australia.

Hepatitis C Virus (HCV) infects 170 million people worldwide. Its high prevalence, rate of chronic infection and the major risk of developing liver cirrhosis and hepatocellular carcinoma make hepatitis C a prominent global problem. The HCV genome encodes 10 proteins, four structural proteins -core, E1, E2 and p7- and six non-structural proteins -NS2, NS3, NS4a, NS4b, NS5a and NS5b. Current treatment for hepatitis C is effective in only approximately 50% of cases. By increasing our knowledge regarding the non-structural proteins of HCV, and their role in replication, new targets for antiviral treatments may be revealed. Ubiquitin is a 76 residue protein that becomes covalently bound to specific lysine residues of target proteins. The ubiquitination of proteins is a regulatory post-translational modification, much like protein phosphorylation. Ubiquitin is known to be important for the budding of many enveloped viruses from cellular membranes, a crucial step in viral replication. My work is focused on characterizing the role of ubiquitin in the replication of HCV. The NS5a protein has been implicated in establishment of persistent HCV infections by its ability to modulate intracellular signalling pathways. For example NS5a expression inhibits the degradation of epidermal growth factor receptor, a process highly dependent on ubiquitination, but the exact mechanism is unknown. HCV core protein is also known to be ubiquitinated, which leads to its subsequent degradation. I am screening HCV proteins for interactions with proteins involved in cellular ubiquitination processes. I have preliminary evidence for the direct interaction of ubiquitin with one of the HCV non-structural proteins, and am investigating the wider role for ubiquitin in HCV replication.

THE MITOCHONDRIAL CITRATE CARRIER IS REQUIRED FOR CHROMOSOME INTEGRITY IN DROSOPHILA AND HUMAN CELLS

Carrisi C.1, Morciano P.2, Cestra G.3, De Benedetto G.E.1, Corona D.F.V.4, Musio A.5, Cenci G.2 and Capobianco L.1 1University of Salento. 2University of L’Aquila. 3Sapienza University. 4University of Palermo. 5ITB Pisa.

Chromosomal aberrations are key events in the initiation and progression of cancer. We isolated a P-element induced mutation in Drosophila that causes chromosome fragmentation. We named sea the gene specified by this mutation. The P-element is inserted into the 5’UTR of CG6782 which encodes a protein orthologous to the mammalian citrate carrier SLC25A1. It exports citrate from mitochondria supplying the cytosol with acetyl units. Sea shows biochemical properties similar to those of SLC25A1 indicating a functional conservation of this carrier. We found that Sea is reduced in mitochondria of sea mutants which also exhibit a reduced citrate transport activity and low levels of citrate in cytosolic extracts as revealed by LC/MS. Interestingly, western-blot of nuclear protein extracts and immunostaining of polytene chromosomes with anti-acetylated histone antibodies revealed a reduction of AcH3 and AcH4 in sea mutants. Therefore, the chromosome breakage phenotype observed in sea mutants seems to be a consequence of reduced levels of histone-acetylation. Notably, SLC25A1 siRNA-treated human primary fibroblasts exhibited a phenotype similar to Drosophila sea mutants and low levels of AcH4. These results suggest an unexpected evolutionary conserved role for Sea/SLC25A1 in the chromosome integrity providing an intriguing link between cellular metabolism and epigenetics.

DETECTION OF PROGRP-DERIVED PEPTIDES IN HUMAN COLORECTAL CANCER CELLS AND ASSESSMENT OF THEIR ROLE IN CELLULAR PROLIFERATION.

Patel O.1, Chang M.1, Nordlund M.2, Shulkes A.1 and Baldwin G.1 1Department of Surgery, University of Melbourne, Austin Health, Melbourne, Australia. 2Department of Medical Biochemistry, Radiumhospitalet, Rikshospitalet University Hospital, Oslo.

Oneel Patel1, Mike Chang1, Marianne S. Nordlund2, Arthur Shulkes1 and Graham Baldwin1. Background and aim: Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. We have previously demonstrated that non-amidated peptides derived from the C-terminus of proGRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor [1]. This study investigates the quantities of proGRP-derived peptides Methods: proGRP-derived peptides obtained from boiling water extracts of the human CRC cell lines DLD-1, SW1222, HCT 15, HT-29 and HCT 116 were quantitated by region-specific ELISA. Proliferation of DLD-1 cells after reduction of proGRP-derived peptide concentrations by transfection with proGRP shRNA was measured by [3H]-thymidine incorporation. Results: In CRC cell extracts ELISA assays for proGRP-derived peptides containing residues 48-61, 56-88 or 48-88 detected 3-15, 10-60 and 20-152 fmol/106 cells, respectively. Little or no GRP18-27amide or GRP1-27amide was detected. Stable transfection of DLD-1 cells with proGRP shRNA significantly reduced propresent in a panel of 5 CRC cell lines as well as the effect of such peptides on proliferation in vitro. liferation. Conclusions: These results indicate that non-amidated peptides derived from the C-terminus of proGRP are present in CRC cells and stimulate the proliferation of CRC cells in vitro. Such peptides are attractive targets for novel CRC therapies. Reference: 1. Patel O., Dumesny C., Shulkes A. and Baldwin G.S. (2007) C-terminal fragments of the gastrin-releasing peptide precursor stimulate cell proliferation via a novel receptor. Endocrinology. 148: 1330-1339.

β-ARRESTIN1 PROMOTE CHRONIC MYELOCYTIC LEUKEMIA PROGRESS DEPENDENT ON JNK PATHWAYS †Zhang P.1, Long J.2, Li K.1, Liu H.2, Tan J.1, Tu Z.2 and Zou L.1 1Center for Clinical Molecular Medicine, Children’s Hospital, Chongqing Medical University, Chongqing 400014, China;. 2Department of Clinical Biochemistry and the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education, Chongqing Medical University, Chongqing 400016, China. Arrestins (Arrs) are scaffold proteins consisting of four members: β-arrestin1 (β-Arr1), β-arrestin2 (β-Arr2), x-arrestin, and s-arrestin. Only β-Arr1 and β-Arr2 are ubiquitously expressed. The traditional functions of β-arrestins are to mediate desensitization, sequestration, and recycling of G protein-coupled receptors (GPCRs). Mounting evidence suggests that, in addition to regulation of GPCR signals, β-arrestins also serve as modulators in a number of intracellular signaling pathways, including ERK, JNK and ASK1, which play important roles in the regulation of various cellular functions in both normal and malignant cells. Some reports have showed that β-Arrs participate in tumor-related signaling pathways such as p53/MDM2, TGF-β1, IGF1R pathways, which function vitally as cell antiapoptosis, cell growth, and proliferation in tumor cells. We have previously disclosed that rapid xenograft tumor progression in β-Arr1 transgenic mice by enhancing tumor angiogenesis and PI3K inhibitors suppressed the β-Arr1-elevated MMP9 activity and VEGF. However, little is known about β-Arrs function in leukemia, especially in chronic myelocytic leukemia (CML). Here we demonstrated that the expression of β-Arrs apparently increased in the bone marrow (BM) and periphery blood (PB) samples from leukemia samples compared with benign hematological disease patients. However, there was no significant difference of β-Arr expressions among the diverse subtypes of leukemia (AML, ALL and CML) samples (P>0.05). But the relative expression of β-Arr1 was always higher than β-Arr2 in the same patient of leukemia. We further found that over-expression of β-Arr1 could promote leukemia cells to proliferate, and vice versa in CML K562 cells in vitro. Moreover, K562 cells knocking out β-arrestin1could activate JNK signal pathways, and JNK inhibitors could block the JNK activation by β-arrestin1 in cells. The results from CML xenograft mice further showed that the survival rate was much higher in K562-siRNA-β-arrestin1 mice than in K562-β-arrestin1 mice, which was closely related to JNK activation. Furthermore, JNK inhibitors could affect the tumor growth of K562-siRNA-β-arrestin1 mice. In conclusion, β-arrestin1 could promote chronic myelocytic leukemia progression dependent on JNK signal pathways, which disclosed the pathogenesis mechanism and signal pathways of β-arrestin1 regulating CML. [† This wok was financial supported by Natural Science Fund of China (30871103, 90919013), Chongqing Natural Science Fund CQCBST 20082207 and 2008 New Century Excellent Talent Program from Education Ministry of China.].





THE SEROTONIN 5-HT4, RECEPTOR SPLICE VARIANTS INTERACTS WITH SPECIFIC PDZ DOMAIN PROTEINS VELI 1-3/LIN7A, B, C HOMOLOGUES: MECHANISMS IN RECEPTOR TARGETINGChinkwo K.A., Coupar I.M. and Irving H.R. Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia.

Several disorders of the gastrointestinal tract are associated with abnormal serotonin metabolism and / or signalling such as irritable bowel syndrome (IBS). The largest quantity of 5-HT4 receptors is present in the intestine of mammals and have modulatory and prokinetic functions and form a clinical target for IBS. Alternative splicing results in 11 splice variants of the 5-HT4 receptor, most differing in their C-terminal (Coupar et al., 2007) and the 5-HT4 (a, d, e, f and g) receptor splice variants possess canonical type 1 or type 2 PDZ domains. Mouse 5-HT4a receptor interacts at the C-terminus with Veli3 (Joubert et al, 2004). The Veli or Lin7A, B or C homologue proteins are involved in post synaptic vesicle function and localization (Jo et al., 1999). We hypothesise that the human 5-HT4 receptors containing canonical PDZ domains may interact with Lin7A, B or C homologues and that this interaction may be important in contributing to receptor function (including down-regulation) in the gastrointestinal tract. We have shown that the 5-HT4 a, b, c and d transcripts are expressed relatively strongly in the human sigmoid colon and that the Lin7 A, B and C homologue transcripts are also expressed in the colon (Chetty et al, 2009). To follow up these observations, we are investigating the interaction between 5-HT4a and 5-HT4d receptor splice variants and Lin 7 homologues in COS-7 and colonic cell lines. Here we report on the generation of N-terminal FLAG tagged 5-HT4 receptor and Lin 7 homologues with C-terminal V5, c-Myc and HA constructs and their expression in COS-7 cells. Protein interactions will be detected using immunoprecipitation and immunofluorescence techniques will be used to visualise cellular co-localisation and receptor recycling. Chetty et al., 2009 Neurogastroenterol Motil 21:551–558.e15; Coupar et al., 2007 Curr Neuropharmacol 5: 224-31; Jo et al., 1999 J Neuroscience 19:4186-4199; Joubert et al, 2004 J Cell Sci 117:536-5379.

EFFECTS OF HPV ONCOPROTEINS ON HLA-A2 PROMOTER IN CERVICAL CARCINOMA CELL LINE

Mohd Habib S.H.1, 3, Abdullah A.1, 3, Othman S.1, 3, Karsani S.A.2, 3 and Yusof R.1, 3 1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur Malaysia. 2Biochemistry Division, Institute of Biological science, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 3Drug Design and Development Research Group, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The key transforming proteins of high risk HPV are E6 and E7, which block cell cycle exit in epithelial cells committed to differentiation, thereby allowing viral replication. The major cellular target of E6 and E7 are p53 and retinoblastoma cell proteins respectively, which play a pivotal role in negative regulation of cell growth. E6 and E7 are constitutively expressed in cervical cancer and their continuous expression is required for maintenance of the transformed phenotype. The activities of E6 and E7 are regulated by viral E2, a transcription regulator protein that is characteristically non-functioning in cancer cells. In this study we are prospecting the ability of E2 in mitigating human MHC class 1 gene, specifically on HLA-A2 promoter region using a dual luciferase assay system. We demonstrate that introduction of viral E2 via transient transfection can markedly up regulate the activity of HLA-A2 promoter by as much as 40%. This can be taken as direct evidence that the E6/E7 are responsible for suppressing that activity of HLA-A2, as the ectopic re-introduction of E2 would inhibit the expression of E6/E7.We are currently investigating the underlying mechanisms behind this uptrend and the possible role of E2 in up-regulating the effect of interferon.

A MOLECULAR BASIS FOR THE DIFFERENTIAL EXTRACELLULAR FUNCTION OF PAI-1 AND PAI-2 IN CANCER

Cochran B.J.1, Croucher D.R.2 and Ranson M.1 1Illawarra Health and Medical Research Institute, University of Wollongong. 2Cancer Research Program, Garvan Institute of Medical Research.

Plasminogen activator inhibitors type-1 (PAI-1, SERPINE1) and type-2 (PAI-2, SERPINB2) are potent irreversible inhibitors of the urokinase-type plasminogen activator (uPA). The strong prognostic value of tumour expression of uPA and PAI-1 in metastasis and poor patient outcome is well established. Conversely, tumour expression of PAI-2 is associated with favourable outcome and increased relapse free survival. We have previously demonstrated that the interaction between uPA:PAI-2 and receptors of the low density lipoprotein receptor (LDLR) family is markedly different to that of uPA:PAI-1, offering a molecular basis for the contrasting prognostic data. Herein, we validate this hypothesis by replacing residues of PAI-2 with those corresponding to LDLR binding sites in PAI-1, thereby introducing LDLR binding. This has profound impacts on the physiological functionality of PAI-2; not only increasing the clearance rate of uPA from the cell surface, but altering non-proteolytic functions via the activation of pro-mitogenic cell signalling pathways. This work provides the first definitive explanation of what, on the surface, appears to be paradoxical biological functionalities.

BIOCHEMICAL CHARACTERIZATION OF ABNORMAL FIBRINOGENS CAUSING HEREDITARY DYSFIBRINOGENEMIA

Kotlin R., Suttnar J. and Dyr J.E. Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Praha 2, Czech Republic.

Fibrinogen, a 340 kDa glycoprotein, consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG), clustered on chromosome 4q32.1. The molecule is posttranslationally modified - phosphorylated, glycosylated and sulphated. Fibrinogen is synthesized by hepatocytes and is secreted to the circulation, where it plays a crucial role in the hemostasis, angiogenesis, platelet aggregation, cell migration and inflammation. Inherited defects in fibrinogen are very rare and may cause life threatening complications like thromboses or serious bleeding. Dysfibrinogenemia is a disease characterized by inherited abnormality in the fibrinogen molecule, resulting in functional defects. We have biochemically characterized five new cases of dysfibrinogenemia with abnormal coagulation test results. Genomic DNA was amplified by PCR and dideoxysequencing was performed. Functional examinations were carried out using fibrin polymerization, fibrinolysis, and measurement of kinetics of fibrinopeptide release. Abnormal fibrinogens were studied by SDS-PAGE and mass spectroscopy. We have found two unrelated families with substitution Aα Arg16Cys and two unrelated families with substitution Aα Arg16His. Mutant chains were found to be present in the patient plasmas; abnormal fibrinopeptides release and impaired fibrin polymerization were obtained. Mutations are situated at the site of fibrinopeptide release catalyzed by thrombin. The last case is a novel mutation γ Tyr363Asn impairing fibrin polymerization. Tyr 363 is situated in the fibrinogen polymerization site and substitution γ 363Asn changes the conformation of the interacting site. This work was supported by a grant of the IGA of the Ministry of Health, nr. NS 9636-3/2008; by a grant of The Ministry of Health, nr. 2373601; and by a grant of The Academy of Sciences of the Czech Republic, nr. KAN200670701.





REDUCED FERROPORTIN EXPRESSION EXPLAINS WHY INTESTINAL IRON ABSORPTION IN THE NEONATAL RAT IS HYPORESPONSIVE TO STIMULI THAT REDUCE ABSORPTION IN ADULTS

Darshan D., Wilkins S.J., Frazer D.M. and Anderson G.J. Queensland Institute of Medical Research, Brisbane, Australia.

Neonatal mammals have a particularly high rate of intestinal iron absorption to ensure an adequate supply of iron for growth. In adults, iron absorption responds to a range of systemic signals, but whether the neonatal gut is similarly responsive remains unclear. We thus examined in rats whether elevated body iron (i.p. iron dextran; 0.3mg/g) and inflammation (LPS; 100ng/g), treatments that reduce iron absorption in adults, have similar effects in neonates. Gene expression was measured by qPCR and protein levels by western blotting. Intestinal iron absorption was determined by measuring the retention of an oral dose of 59Fe. Neonatal rats (15 days old) absorbed 93.1±2.0% of an oral iron dose compared to 39.1±2.6% in adults (7 weeks old). In response to iron dextran and LPS, iron absorption in adults decreased by 53% and 72% respectively, whereas the corresponding reductions in suckling animals were only 14% and 9%. Despite this, expression of the iron-regulatory hormone hepcidin was strongly induced by both iron dextran and LPS at both ages. The hepcidin target, ferroportin (Fpn), was expressed at the mRNA level but not at the protein level in the duodenum of suckling animals up to 17 days of age. Expression was robust in adults. The Fpn1A splice variant, which is under iron-dependent translational control, predominated in neonates. In conclusion, iron absorption in the neonate is largely refractory to systemic signals. This reflects the limited expression of Fpn protein in the small intestine at this time and the dominance of an iron-dependent Fpn splice variant. This adaptation helps to ensure maximal iron supply to the growing pup.

CONTRIBUTION OF METABOLIC PERTURBATIONS TO THE NEURODEGENERATIVE EVOLUTION OF ALZHEIMER’S DISEASEDaulatzai M.A. University of Melbourne, Parkville, Vic 3010, Australia.

Mak Adam Daulatzai Sleep Disorders Group/EEE, University of Melbourne, Victoria, Australia Alzheimer’s disease (AD) is relentlessly progressive with significant burden on medical and financial resources. Our longevity is increasing and aging is the most important risk factor. AD is diagnosed only when considerable brain damage is done. The goal of this paper is to address important senescent stigmata, and highlight metabolic disturbances that exacerbate AD neuropathogenesis. The latter is correlated with cholinergic hypofunction and neuronal losses in several key neocortical areas including hippocampus, basal forebrain (BFB), entorhinal cortex (ERC), posterior cingulate cortex (PCC), and retrosplenial parietal cortex (RPC) to name a few. These affected regions possess amyloid plaques and neurofibrillary tangles (NFT). Neuroimaging has shown significant hypoperfusion of BFB and several key brain regions in old rats and elderly humans. These perfusion decreases occur prior to substantial brain atrophy in AD. Furthermore, there is significant evidence of decreased cerebral glucose metabolic rate (CMRglc) in parietal and temporal cortices also years prior to clinical AD diagnosis. Most consistently documented CMRglc hypometabolism occurs in parietal cortex, particularly the most vulnerable RPC. Respiration is essential to life. Upper airway (UA) patency is due to motor activity of dilator muscles, particularly genioglossus innervated by hypoglossal nerve. Nocturnal hypofunction of genioglossus results in variable collapse of UA. Almost all elderly suffer from some kind of sleep-disordered breathing condition including obstructive sleep apnoea which has reached an epidemic proportion. There is well documented involvement of brain stem nuclei including reticular, solitarius, ambiguous, motor nucleus of vagus, and hypoglossal, in ventilatory regulation, genioglossus function and UA patency. Hypofunction/atrophy of the above nuclei in elderly, results in UA collapse, hypoxia, hypoxemia, depressed CMRglc, and enhancement of AD neuropathology. Significant data would be presented to substantiate this thesis.

APOE POLYMORPHISM IN CORONARY HEART DISEASE PATIENTS FROM PUNJAB, INDIA

Dhiman S.R.1, Khanna D.2 and Balgir P.P.2 1Department of Human Biology, Punjabi University, Patiala-147002,Punjab, India. 2Department of Biotechnology, Punjabi University, Patiala-147002, India.

Although there is a decline in several forms of infectious diseases with a general improvement in the health and socio-economic status, there is an increase in various diseases like, hypertension, stroke, diabetes and coronary heart disease due to the changes in life style and dietary habits. Coronary Heart Disease (CHD) contributes significantly to the morbidity and mortality in many countries and is becoming the leading cause of death in developing countries too. It has been shown that there is a high prevalence of CHD, in both, urban and rural populations of India.In the present study, Apolipoprotein E genotypes were determined by Polymerase chain reaction at SNPs rs7412 and rs 429358 amongst 152 Coronary Heart Disease (CHD) patients and compared with 300 normal healthy individuals (Controls) from North Western Indian population of Punjab, India. All the three ApoE alleles - ApoE2, ApoE3 and ApoE4 were observed in the CHD patients and normal controls. The frequency of ApoE3 allele was found to be the highest, followed by ApoE4 and ApoE2 alleles, both, in the CHD patient, as well as, in the Control series. Chi-square analysis reveals statistically non-significant differences between the patient and control series (Chi-square =1.435, d.f. = 2, P = 0.4879). ApoE being a prominent mediator in cholesterol homeostasis pathway has a significant influence on the plasma lipid levels. The influence of ApoE genotypes on plasma lipid and lipoprotein levels will be discussed.

EFFECTS OF RHO GTPASES IN METASTASIS

Dinsdale B., Li S.F. and Parish R.W. LaTrobe University.

Transition from a primary carcinoma to secondary tumours result in higher mortality rates. The spread of cancer, is primarily dependant on cell migration, cell invasion and metastasis. DNA arrays have defined a pattern of gene expression that correlates with the progression to metastasis, and among the genes found to play a role is a member of the RAS-superfamily of small guanosine triphosphates (GTPases), RhoC. RhoC is a known regulator of cellular migration through its effects on the cytoskeleton and cellular adhesion. Changes in expression and activation of RhoC can result in a metastatic phenotype. RhoC is over-expressed in metastatic cells of many cancers, and as such we are interested in varying expression in three sublines from a murine mammary model (derived from spontaneously arising tumour in a Balbc/cfC3 mouse) that demonstrate varied metastatic potential. By ectopically expressing RhoC in different sublines, we have observed distinct morphological changes that are indicative of a significant shift in metastatic potential. We have carried out Matrigel invasion assays to further characterise these transgenic lines, in conjunction with creating RhoC loss-of-function mutants to further investigate its role in metastasis.





SEC14-DEPENDENT SECRETION OF THE FUNGAL INVASIN, PHOSPHOLIPASE B1, IS ESSENTIAL FOR PATHOGENICITY OF CRYPTOCOCCUS NEOFORMANSDjordjevic J.T.1, 2, Chayakulkeeree M.1, 2, 3, Johnston S.4, Bijosono Oei J.1, 2, May R.4, Williamson P.R.5 and Sorrell T.C.1, 2 1Faculty of Medicine, University of Sydney. 2Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Westmead Hospital, Sydney. 3Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. 4School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom. 5National Institute of Allergy and Infectious Diseases, NIH Bethesda, MD, USA.

The secretory pathway of the AIDS-related pathogen, Cryptococcus neoformans, exports phospholipase B1 (Plb1), an enzyme essential for fungal invasion and dissemination throughout the host. Using gene deletion analysis, secretion of Plb1, but not other major virulence determinants, is shown to be dependent on CnSEC14-1, which encodes a putative phosphatidylinositol transfer protein (PITP). Similar to the Plb1 deletion mutant (CnΔplb1), the Plb1 secretion-deficient CnSEC14-1 deletion mutant (CnΔsec14-1) displayed reduced lung and brain burdens and attenuated pathogenicity in an animal model. Furthermore, expulsion of live C. neoformans from macrophages was attenuated in both Plb1 secretion-deficient strains. CnSEC14-1 restored high-temperature growth to an S. cerevisiae SEC14TS mutant confirming it to be the orthologue of the secretion-essential Golgi PITP-encoding gene, ScSEC14. Deletion of CnSEC14-2, a closely-related homologue of CnSEC14-1, and CnSFH5, the more distantly-related SEC fourteen-like homologue, singly or in combination (CnΔsec14-2, CnΔsfh5 and CnΔsec14-2/CnΔsfh5, respectively), abrogated neither Plb1 secretion nor pathogenicity. However, Plb1 secretion and pathogenicity were restored in CnΔsec14-1 following genetic reconstitution with either CnSEC14-1 or CnSEC14-2, despite CnΔSEC14-2 retaining a wildtype phenotype, confirming that CnSEC14-2 over-expression functionally compensates for CnSEC14-1. C. neoformans therefore uses distinct mechanisms to export virulence determinants, with CnSEC14-1 constituting part of a Plb1-specific secretion pathway essential for pathogenicity.

STEAROYL-COA DESATURASE 1 DEFICIENCY REDUCES APOPTOSIS IN THE HEART OF OBESE OB/OB MICE

Dobrzyn P.1, Pyrkowska A.1, Miyazaki M.2, Ntambi J.M.3 and Dobrzyn A.1 1Laboratory of Cell Signaling and Metabolic Disorders, Nencki Institute of Experimental Biology, Warsaw, Poland. 2Divisions of Renal Diseases and Hypertension & Endocrinology, Metabolism and Diabetes, University of Colorado, Denver, CO, USA. 3Departments of Biochemistry and Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA.

Rationale: Stearoyl-CoA desaturase (SCD), the rate limiting enzyme in the synthesis of monounsaturated fatty acids, was recently shown to be involved in regulation of cardiac substrate utilization. Objective: In the present study, using ob/ob;SCD1-/- mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle function in leptin deficiency. Methods and Results: We show that disruption of SCD1 gene significantly improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction and decreasing left ventricle mass and diameter. The improvement is associated with reduced expression of genes involved in fatty acids (FA) transport and lipid synthesis, and reduction in cardiac free FA (by 39%), diacylglycerol (by 20%), TG (by 33%), and ceramide (by 34%) levels. The rate of FA beta-oxidation is also significantly lower in the heart of ob/ob;SCD1-/- mice compared to ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Conclusions: Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be the main mechanism responsible for improved left ventricle function in ob/ob mice caused by SCD1 deficiency. These results suggest that inhibition of SCD could be a potential therapeutic strategy for the treatment of obesity-related cardiomyopathies. This work was supported by Polish Ministry of Science and Higher Education grant N N301 0129 33.

DOES DYSFERLIN DO MORE THAN JUST REPAIR MEMBRANES?

Evesson F.J.1, 2, Peat R.A.1, Lek A.1, 2, Lemckert F.A.1, 2, North K.N.1, 2 and Cooper S.T.1, 2 1Institute for Neuroscience and Muscle Research, The Children’s Hospital at Westmead, Westmead, 2145, NSW, Australia. 2Discipline of Pediatrics and Child Health, The University of Sydney, Sydney, 2000, NSW, Australia.

Dysferlin is a muscle membrane protein which is mutated in a form of late onset muscular dystrophy. Dysferlin belongs to the ferlin family of proteins which have ancient origins in eukaryotic biology, and emerging roles in a variety of vital cellular processes. Deficiency of dysferlin is characterised by an impairment of rapid calcium-activated resealing of damaged muscle fibres, although its precise role in this process remains poorly understood. We have created a panel of dysferlin expression constructs bearing domain deletions or targeted patient mutations for use in cell biology assays studying protein lifetime, plasma membrane expression and trafficking. We have used co-expression models and co-immunoprecipitation to study associations and interactions of dysferlin with other proteins involved in cellular trafficking and membrane repair. Our studies demonstrate that dysferlin is a dynamic transmembrane protein, showing relatively transient expression at the plasma membrane of cultured muscle cells (half-life of ~ 3 hours). This plasma membrane expression is attenuated in domain deletion mutants, and in several patient missense mutants, resulting in a significant acceleration of endocytosis and protein degradation. Our efforts are now focused on defining the constituents and cargo of dysferlin’s endocytic pathway. Recent results demonstrate that syntaxin-4 demarks the dysferlin endocytic route. We are now defining the relationship between dysferlin and syntaxin-4, with potential relevance to the regulated exocytosis of membrane resealing, or the altered immune function often observed in patients with dysferlin muscular dystrophy.

ABILITY OF α-CASEIN IN PREVENTING OF AMYLOID FIBRIL FORMATION OF STRESSED α-LACTALBUMIN IN CROWDED SYSTEM

Ghahghaei A. and Faridi N. Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.

Amyloid fibrils arise from the slow aggregation of intermediately folded protein states. In this study the kinetics of the protein fibril formation of α-lactalbumin and its prevention by αs-casein in the presence and absence of crowding agent, dextran (68 kDa) have been compared by thioflavin T binding assay. It was found that αs-casein, a molecular chaperone found in bovine milk, is a potent in vitro inhibitor of α-lactalbumin fibrillization. The effect of αs-casein in preventing fibril formation was significant, although reduced in comparison with the absence of crowding agent, dextran. Interaction between chaperone and α-lactalbumin and structural change in target protein are also indicated by intrinsic fluorescence intensity, ANS binding assay, CD spectroscopy and size-exclusion HPLC. In summary, α-casein interact with α-lactalbumin and prevent amyloid formation but not as well as in the presence of crowding agent, dextran.





A HIGH-THROUGHPUT METHOD FOR THE DETECTION OF HOMOEOLOGOUS GENE DELETIONS IN HEXAPLOID WHEAT

Fitzgerald T.L.1, Kazan K.1, Li Z.2, 3, Morell M.K.2, 3 and Manners J.M.1 1CSIRO Plant Industry, 306 Carmody Road St Lucia QLD 4067 Australia. 2CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia. 3CSIRO Food Futures National Research Flagship, PO Box 93, North Ryde 1670, NSW, Australia.

Plant lines featuring full or partial gene deletions provide a useful resource for the study of gene function by reverse genetics and also have the potential to be exploited for crop improvement. The use of deletion lines in polyploid plant species has been impeded by the presence of multiple copies of homoeologous genes corresponding to each of the ancestral genomes. Such homoeologous gene copies usually show high DNA sequence homology, and may have either redundant or homoeologue-specific functions. Hexaploid (‘bread’) wheat (Triticum aestivum) is an economically significant polyploid crop species, and is an allopolyploid possessing three progenitor genomes designated as ‘A’, ‘B’, and ‘D’. To facilitate the screening for specific homoeologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput and large-scale detection of homoeologous copies in any gene of interest. To test the system, we chose the TaPFT1 gene, an orthologue of the disease susceptibility gene PFT1 in Arabidopsis, where mutants lacking a functional PFT1 gene have been shown to possess increased resistance to a Fusarium pathogen. We applied this method to the screening of 4500 M2 mutant wheat lines generated by Heavy Ion Irradiation in order to identify knockouts of each homoeologous copy of TaPFT1. We detected multiple lines featuring deletions of each TaPFT1 homoeologue, and confirmed these deletions using a CAPS method. Mutants in TaPFT1 and other wheat disease susceptibility genes are now being further developed for phenotypic testing for potential changes in response to fungal pathogens.

UNTANGLING THE WEB OF HEPATITIS C VIRUS NON-STRUCTURAL PROTEIN 4B FUNCTION

Fitzsimmons N.M. and Bishop N.E. La Trobe University, Bundoora.

Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and/or hepatocellular carcinoma, in an estimated three percent of the human population. HCV is a single-stranded, positive-sense RNA virus, classified in its own genus Hepacivirus within the Flaviviridae family. The virus encodes ten proteins: Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The Core, E1 and E2 proteins are structural proteins, while the remainders are non-structural proteins. My project focuses on the role of the non-structural protein NS4B, which has a poorly-characterized role in viral replication. A number of positive-sense RNA viruses replicate their genome on the surface of intracellular membranes. Some viruses also rearrange the intracellular membranes. The purpose of rearranging membranes is not fully understood however some hypotheses are that they could be used as a physical support for the viral replication complex, allow compartmentalization of viral products, may permit viral RNA tethering, or perhaps protect the RNA from host defenses. For HCV these rearranged membranes are termed membranous webs or membrane-associated foci (MAFs). Evidence to date suggests NS4B plays a role in formation of these MAFs. Current data indicate the membranes rearranged by HCV are derived from both the endoplasmic reticulum and early endosomes. However, both the molecular mechanisms involved, and the impact on host cell function due to the MAF formation, is poorly characterized. I will present my latest data on the characteristics and formation of HCV-induced MAFs, and data on the impact of the membrane rearrangements on normal host cell function. My results are based on findings in cultured cells over-expressing HCV NS4B.

INDUCED SPLICE-SWITCHING TO STUDY DYSTROPHIN ISOFORM FUNCTION AND EXPRESSION

Fletcher S., Adams A., Greer K., Johnsen R.J. and Wilton S.D. University of Western Australia, 35 Stirling Highway, Crawley WA 6009.

Duchenne muscular dystrophy (DMD) is an X-linked, relentlessly progressive muscle wasting disorder with a predictable course and fatal outcome. DMD is caused by mutations in the dystrophin gene that prematurely terminate the protein, and affects 1 in 3500 male births. Although treatment options have been limited, advances in clinical care have doubled the life expectancy of affected boys over the last 2-3 decades, but do not address the primary etiology of DMD, the absence of dystrophin. Biological therapeutics are now becoming available, and antisense oligomer-mediated splice manipulation to exclude selected exons can overcome disease-causing mutations and restore dystrophin expression. We show that peptide-conjugated phosphorodiamidate morpholino oligomer induced splice-switching can also be used to alter isoform expression, or induce a frame-shift and prevent translation of functional products. Exclusion of selected exon blocks to yield in-frame transcripts can allow mapping of functional protein domains, based upon exon boundaries, and permits physiological evaluation of dystrophin isoforms to aid in the design of splice-manipulation therapies. It is also possible to efficiently disrupt the normal dystrophin mRNA reading frame and ablate dystrophin expression in wild-type mouse muscle. Total suppression of dystrophin gene expression was induced by selected exon removal and maintained for several weeks in vivo, resulting in severe dystrophic pathology in diaphragm within 4 weeks of commencing treatment in wild-type neonatal mice. Manipulating expression through altered splicing patterns could be applied to many different genes, offering the opportunity to induce transient mouse models to study the consequences of gene suppression or isoform selection in vivo.

MODULATION OF MHC CLASS I GENE EXPRESSION BY DENGUE VIRUS

Othman S., Abdul Rahman N. and Yusof R. Drug Design and Development Research Group, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Mosquito-borne dengue fever (DF) and dengue haemorrhagic fever (DHF) are caused by dengue viruses belonging to the Flaviviridae family that comprised four closely related but antigenically distinct serotypes, DV1 to DV4. There are over 100 million cases of dengue infections, half a million cases of DHF and 24000 deaths worldwide every year, with more than 2.5 billion people at risk for endemic transmission. Despite aggressive efforts in dengue research, the control of dengue diseases awaits complete elucidation of its complex immunopathogenesis, many theories of which showed participation of both vector and host responses, involving both innate and adaptive components of the human immune response. In the adaptive immune response, the major histocompatibility complex (MHC) class I molecules are critical for antigen-specific immune recognition, expressed on the surface membrane of virtually all mammalian cells to display fragmented pieces of foreign antigen. In contrast to many viruses that escape this host response by suppressing the MHC Class I pathway, infection by other Flaviviruses has been shown to up-regulate the cell surface expression of MHC Class I complex. In dengue infection, the mechanisms by which the virus regulates the MHC Class I remain elusive. Hence, this study elucidates comprehensively and systematically the effect of all serotypes of dengue virus infection on the expression and regulation of transcriptional activities of the human MHC class I HLA-A2 gene. This adds further understanding to the role of dengue virus in manipulating the MHC antigen presentation pathway in this immune-pathogenic disease that has been posing great economic and social burden on affected many nations, Malaysians included.





D-AMINO ACID OXIDASE: PATHOPHYSIOLOGICAL BASIS AND MOLECULAR TARGET FOR SCHIZOPHRENIA

Fukui K., Ono K., Abou El-Magd R.M., Iwana S., Kawazoe T., Chung S.P., Song Y., Shishido Y., Yorita K. and Sakai T. Division of Enzyme Pathophysiology, The Institute for Enzyme Research, The University of Tokushima, Tokushima, JAPAN 770-8503.

D-Amino acid oxidase (DAO) has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia and the modulation of the enzyme activity is expected to be therapeutical. Here we show the inhibitory effect of a classical antipsychotic drug, chlorpromazine, and an atypical drug, risperidone, on human DAO activity. As for chlorpromazine, human DAO was inhibited with Ki value of 0.7 mM. We also found that the oligomerized chlorpromazine produced by irradiation, showed an enhanced inhibition (Ki =7 μM). Risperidone was also shown to have a hyperbolic mixed-type inhibition with Ki value of 41 μM. The effect of risperidone was then tested using rat C6 cells and stable C6 transformant over-expressing mouse DAO (designated as C6/DAO) as well as pig kidney epithelial cell line (LLC-PK1). Risperidone exhibited a protective effect from D-amino acid and oxidative stress induced cell death in both C6/DAO and LLC-PK1 cells. In search of the in vivo pathophysiological role of DAO, we then analyzed the distribution of DAO mRNA and protein in the brain. In rat, the distribution of DAO mRNA was newly detected in choroid plexus (CP) epithelial cells in addition to glial cells of pons, medulla oblongata, and especially Bergmann glia of cerebellum. In agreement with the results in rat, the immunoreactivity for DAO was detected in glial cells of rhombencephalon and in CP in the human brain. Furthermore, higher level of DAO expression was observed in schizophrenic CP epithelial cells than that in non-schizophrenic cases. These results suggest that DAO expression level is altered in schizophrenia and the increase in DAO expression is involved in aberrant D-amino acid metabolism. In particular, gene expression of DAO in CP suggests that DAO may regulate D-amino acid concentration by modulating the cerebrospinal fluid, and may be regarded as a potential therapeutic target for schizophrenia.

IRON HOMEOSTASIS IN WHOLE BODY AND INTESTINE-SPECIFIC HEPHAESTIN KNOCKOUT MICE

Fuqua B.K.1, Wolkow N.2, Bell A.1, Hsu J.1, Nguyen M.P.1, Yu C.C.1, Dunaief J.L.2, Anderson G.J.3 and Vulpe C.D.1 1Department of Nutritional Science and Toxicology, University of California, Berkeley, USA. 2FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, USA. 3Queensland Institute of Medical Research, Brisbane, Australia.

Hephaestin (Heph) is a vertebrate multicopper ferroxidase (MCF) important for the transfer of dietary iron from intestinal cells to the blood. The gene is mutated in the sex-linked anaemia (sla) mouse and results in severe iron deficiency. However, sla mice still retain some hephaestin ferroxidase activity. They survive, breed and their anaemia improves with age. Heph is also expressed at low levels in other tissues, including the brain, pancreas, and placenta, but its role in these tissues is less clear. To gain a better understanding of the role of Heph in iron homeostasis, we used the cre-lox system to generate knockout mouse models with whole body and intestine-specific (villin promoter) ablation of Heph. Both types of mice are small, yet viable, indicating that Heph is not essential and that other mechanisms exist, MCF-dependent or not, to compensate for Heph deficiency. At 8 weeks of age the knockout mice remain small, but their rate of growth is comparable to that of wild-type littermates. Both strains also develop a microcytic, hypochromic anaemia, a picture consistent with severe iron deficiency, confirming that Heph plays an important role in iron acquisition. In addition, mice born to knockout mothers, regardless of pup genotype, exhibit truncal hair loss that improves after weaning, also consistent with iron deficiency. These mouse models will serve as valuable tools to study the role of Heph and associated proteins in iron transport in the small intestine and other tissues.

POLYMORPHISM DEPENDANT SIGNALING BIAS IN THE HUMAN CALCITONIN RECEPTOR

Furness S.G.B., Khreish M., Chrisopoulos A. and Sexton P.M. Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences, Parkville Victoria 3052, Australia.

The calcitonin receptor is a Family-B G protein-coupled receptor that mediates responses to the peptide hormone calcitonin. Calcitonin is involved in calcium homeostasis by inhibition of osteoclast-mediated bone resorbtion and potentiation of renal calcium secretion. Calcitonin also exerts anorectic and analgesic effects in the central nervous system as well as suppressing gastric secretion and intestinal mobility. A number of single nucleotide polymorphisms have been reported for the calcitonin receptor. Of these, a coding polymorphism resulting in either Leucine or Proline at position 447 in the carboxy terminal tail has been reported to be associated with osteoperotic risk. We hypothesized that these polymorphisms may result in altered signaling profiles providing a molecular basis for apparent calcitonin receptor dependant clinical changes in calcium homeostasis. Since both salmon and human calcitonin are used clinically, we also hypothesized that these ligands may show different signaling profiles with respect to this polymorphism. A single previous report using a transient transfection model demonstrated no difference in calcitonin stimulated adenylate cyclase activity at either polymorphic variant of the receptor (Wolfe et. al. Mutat Res. 2003; 522:93-105.). We have used several model cell backgrounds, COS-7, 3T3 and HEK-293, to analyse differences in signaling arising from these polymorphs. We have shown that there are a number of polymorphism-dependant changes in signaling efficacy toward adenylate cyclase, extracellular signal-regulated kinase (ERK) phosphorylation and intracellular calcium release. Further, we have demonstrated that these signaling biases are dependent on cellular background. We have also observed that there are no apparent polymorphism dependent differences in signaling profiles in response to salmon and human calcitonin in these model cell systems.

ANTI-HELICOBACTER PYLORI ACTIVITY OF EDIBLE PLANT FROM NORTHEASTERN THAILAND

Gamnarai P.1, Rojpibulstit P.1, Kangsadalampai S.1, Boonsiri P.2, Daduang J.3 and Vilaichone R.K.1 1Faculty of Medicine, Thammasat University (Rangsit Campus), Pathumthani, 12121. Thailand. 2Faculty of Medicine, Khon Kaen University, Khon. Kaen, 40002, Thailand. 3Faculty of Associated Medical Sciences, Khon Kaen University, Khon. Kaen, 40002, Thailand.

ABSTRACT Helicobacter pylori is a common bacterial pathogen in human. It is found in association with peptic ulcer diseases and gastric cancer. In Thailand, the infection has been reported to be higher in northeastern part than in any other regions of the country. Surprisingly, the prevalence of peptic cancer among people in this region was very low. The explanation for this finding was not clear. It was thought that a variety of local plants in their daily foods may play an indirect role in the cancer protection by reducing the severity of the H. pylori infection and, consequently, lower the rate of peptic cancer. In this study, we assessed the anti-H. pylori activity of 21 edible plants from northeastern Thailand to look into the potential nutraceuticals against the H. pylori ATCC 43504. Minimum inhibitory concentration (MIC) was determined by agar dilution method for ethanol and ethyl acetate plant extracts. Of all 21 plant extracts, ethanol extracts of Polygonum adoratum Lour., Limnocharis flava (L.) Buchen., Limnophila aromatica Merr, Tiliacora triandra Diels, and Spilanthis acmella Murr. (MIC 125 microgram/ml) and ethyl acetate extracts of Polygonum adoratum Lour., Justicia gangetica, Limnophila aromatica Merr, Cratoxylum formosum subsp. pruniflorum (Kurz) Gogel., Cratoxylum formosum (Jack) Dyer ssp, Tiliacora triandra Diels, Ottelia alismoides (L.) Pers., Hydrocharis morsus-ranae Linn., Tacca chantrieri Andre and Spilanthis acmella Murr. (MIC 62.5 to 125 microgram/ml) exhibited the highest anti-H. pylori activity. This information provides more insights into another role of edible plant as an anti-bacterial neutraceutical food.





SUBCELLULAR TRAFFICKING OF VIRAL PROTEINS IS CENTRAL TO VIRAL IMMUNE EVASION AND PATHOGENICITYMoseley G.W.1, Wiltzer L.1, Oksayan S.1, Rowe C.L.1, Marsh G.2, Wang L.F.2, Blondel D.3, Ito N.4 and Jans D.A.1 1Monash University, Clayton, Victoria, Australia. 2AAHL, Geelong, Victoria, Australia. 3Gif-sur-Yvette, France. 4Gifu Univeristy, Gifu, Japan; CNRS.

The principal host response to viral infection is the production of interferon (IFN), which induces a potent antiviral state in host cells. Productive infection therefore requires evasion/subversion of the IFN system, which is achieved by viral proteins with IFN-antagonist activity. These IFN-antagonists often undergo regulated subcellular trafficking via interaction with host cellular transport machinery (nuclear import/export proteins and cytoskeletal factors), enabling the targeting of multiple stages of intracellular IFN signaling pathways in specific subcellular sites. IFN-antagonist trafficking thus represents a potential target for antiviral therapy. To demonstrate the importance of IFN-antagonist trafficking to viral pathogenicity, we have examined the archetypal IFN-antagonist of rabies (P-protein), finding that it undergoes intricately regulated targeting via several nuclear export (NESs) and nuclear localization (NLSs) sequences, as well as sequences for association with microtubules (MTs) and the MT motor, dynein. This appears to be central to P-protein’s targeting and inactivation of the vital IFN signaling factors IRF3 and STAT1 within different cellular compartments. Our recent data indicate that NES-mediated P-protein nuclear export is essential to its inhibition of IFN-dependent STAT1 signaling, and is a key pathogenicity factor for infectious rabies virus, the first such demonstration for any virus. Our data further indicate that P-protein nuclear import makes similar crucial contributions to IFN-antagonist function, and that P-protein-MT interaction provides additional, novel mechanisms of STAT1 inhibition. These data indicate that efficient viral immune evasion requires viral protein interaction with diverse cellular factors in multiple subcellular sites, with implications for the development of therapies for incurable human-pathogenic viruses.

COMPARING THE CHAPERONE ACTIVITY OF GLYCEROL AND β-CASEIN IN PREVENTING AGGREGATION OF PROTEINS IN CROWDED SYSTEM

Ghahghaei A. and Rahmany Asgarabad F. Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.

Aggregation of proteins is a frequent occurrence during their transition from random coil to native structure. The influence of glycerol and β-casein in the refolding of ovotrnasferrin, insulin, α-lactalbumin and catalase under aggregating conditions was studied by visible absorbtion spectroscopy, intrinsic fluorescence, ANS binding assay and HPLC. Both chaperone effectively prevented aggregation of ovotransferrin, and α-lactalbumin during renaturation of proteins in the absence of dextran. As dextran increased the rate of aggregation, β-cacein and glycerol was found less efficient in preventing them. It was observed that dextran accelerated insulin aggregation but it had little effect on the time to the onset of insulin aggregation. β-Casein and glycerol were found equally effective in preventing aggregation of insulin both in the presence and absence of dextran. Glycerol effectively suppressed thermally induced aggregation of catalase whereas β-casein co precipitated of proteins. In conclusion glycerol is a more effective chaperone than β-casein to protect protein from aggregation and precipitation. The more enhanced protein reactivation by glycerol may be due to their more ability to bind to hydrophobic sites in protein folding intermediate(s) followed by their subsequent removal as the protein refolds. In addition structural change in β-casein at higher temperature may explain the different effects of glycerol and β-casein in protecting catalase.

THE EXPRESSION OF TRANSMEMBRANE TUMOUR NECROSIS FACTOR IS INCREASED IN THE ANTERIOR CINGULATE OF SUBJECTS WITH BIPOLAR DISORDER

Gibbons A.S., Tawadros N., Scarr E. and Dean B. Rebecca L. Cooper Laboratories, Mental Health Research Institute of Victoria, Parkville, Victoria 3052, Australia.

Altered pro-inflammatory cytokine signalling has been implicated in the pathologies of mood disorders and schizophrenia. We have previously reported an increase in the expression of the transmembrane isoform of tumour necrosis factor (tmTNF) in Brodmann’s area (BA) 46 but not BA 24 from subjects with major depression, while the expression of soluble isoform of TNF (sTNF) was not altered in either region. To determine whether TNF is involved in other psychiatric disorders, we examined TNF expression in post-mortem subjects with bipolar disorder (n=10) and schizophrenia (n=20) and matched control subjects. tmTNF and sTNF protein expression was measured using Western blotting. Contrasting our previous observations in major depression, the level of tmTNF was significantly increased in BA 24 (mean ratio of internal standard ± SE: CON = 2.78 ± 1.03; BD = 7.15 ± 1.75; P<0.05) but not BA46 (CON = 0.88 ± 0.10; BD = 1.84 ± 0.47; P>0.05) from subjects with bipolar disorder. The level of sTNF protein was not changed in subjects with bipolar disorder in either region examined (BA 24: CON = 1.31 ± 0.17; BD = 1.49 ± 0.26; P>0.05; BA 46: CON = 1.35 ± 0.15; BD = 1.44 ± 0.17; P>0.05). The expression of tmTNF and sTNF protein was not altered in subjects with schizophrenia in BA 24 or BA 46 (P>0.05). While our findings suggest increased tmTNF expression is associated with mood disorders, the affected brain regions differ between bipolar disorder and major depression. Furthermore, aberrant TNF signalling in mood disorders may not involve the classical pro-inflammatory signalling pathways mediated by sTNF.

MITOCHONDRIAL ABNORMALITIES IN THE MECP2TM1TAM MOUSE MODEL OF RETT SYNDROME

Gold W.A.1, Williamson S.L.1, Pelka G.J.2, Tam P.P.L.2, Gibson J.1 and Christodoulou J.1, 3 1NSW Centre for Rett Syndrome Research, Western Sydney Genetics Program, Children’s Hospital at Westmead, Sydney, Australia. 2Embryology Unit, Children’s Medical Research Institute, Sydney, Australia. 3Disciplines of Paediatrics & Child Health and Genetic Medicine, University of Sydney, Australia.

Rett Syndrome (RTT) is a severe childhood neurodevelopmental disorder, primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). RTT occurs predominately in females with an incidence of 1 in 8-10,000. There is considerable phenotypic variability, with ‘classical’ RTT patients exhibiting developmental arrest in infancy, stereotypic hand movements, loss of communicative skills, progressive loss of intellectual functioning, and the deceleration of head growth. Phenotypic similarities between RTT and patients with primary mitochondrial respiratory chain disorders prompted investigations into abnormalities of the respiratory chain function as well as mitochondrial ultrastructure in RTT patients as well as in mouse models. The aim of this study was to investigate the mitochondrial function of the brain and other organs in the Mecp2tm1Tam mouse model, by investigating the individual respiratory chain complexes (COI, COII, COIII, COII+III and COIV) using spectrophotometric techniques. When we compared symptomatic mice to their wild type litter mates we observed an increase in COIII activity (wt=171.3±58.9; null=256.6±75.8, p=0.0302) in the heart, and a decrease in COII+III (wt=111.2±46.59; null=61.19±20.89, p=0.003) and COIV (wt=151.6±53.75;null=79.56±32.49, p=0.0007) activity in skeletal muscle. Interestingly, we found no changes in activity in the brain of symptomatic mice. Furthermore, no differences were observed in mitochondrial activity in pre-symptomatic mice. This data imply that perturbations in mitochondrial function appear to occur only at disease onset and may contribute to the clinical phenotype in RTT patients.





THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN TYPE 1 DIABETES

Gorasia D.G., Dudek N.L., Wee S., Mangum J.E., Hubbard M.J. and Purcell A.W. The University of Melbourne.

Type I Diabetes (T1D) is an autoimmune disease which results from the specific destruction of pancreatic beta cells. Proinsulin is a target of infiltrating T cells but it’s unclear why it’s trageted. A role of misfolded proinsulin in diabetes is seen Akita mice which harbors a mutated proinsulin II (Cys96Tyr) gene and develops diabetes due to endoplasmic reticulum (ER) stress. Recently, protein misfolding and associated cellular stress is emerging as a key factor in beta cell death mediated by apoptosis. Proinsulin folding takes place in the ER but little is known about how ER chaperones guide proinsulin production, particularly under conditions of ER oxidative stress. In this study we have characterized a number of chaperones that assist in the folding of proinsulin. The tools to study proinsulin folding were generated by expressing flag tagged proinsulin in a pancreatic beta cell line (NIT-1). Immunoprecipitation studies were performed using flag antibody. We have identified a number of chaperones such as BiP (GRP 78), PDI and DnaJ homolog subfamily B member 11 (DnaJ11B or HSP 40), that interact with proinsulin. It has been shown that BiP interact with proinsulin, to our knowledge this will be the first study to show that BiP together with Dnaj 11 B interact with proinsulin. Our study on proteomic analysis of islets from NOD mice showed that islets undergo oxidative stress during development of diabetes as chaperones like BiP were upregulated. Further understanding of the role, each identified chaperone plays in proinsulin folding may provide insights into how proinsulin is targeted during development of T1D and how oxidative stress affects folding of proinsulin.

SHARK ANTIBODIES AND NCAMS AS NOVEL DIAGNOSTICS

Griffiths K., Casey J., Parisi K. and Foley M. Biochemistry Department, Latrobe University, Australia.

Variable domains from the shark immunoglobulin new antigen receptor (IgNARs) and homologous domains from human neural cell adhesion molecules (NCAMs) are small, robust molecules which can be engineered to bind antigens of interest with high affinity and specificity. IgNARs have superior physicochemical properties to conventional mammalian antibodies, showing greater stability to extreme temperature, urea concentration, and pH. There is significant potential for their application as diagnostic reagents, particularly in point of care situations with high ambient temperature and humidity. Similarly, NCAMs have widespread potential as therapeutic and diagnostic reagents. We used a phage display approach to select high affinity binders from IgNAR and NCAM libraries, and detail and compare here their various properties.

PROPAGATION OF THE PRION PROTEIN VIA THE EXOSOMAL PATHWAY

Guo B.B.1, 2, Vella L.J.2, 3, Cappai R.2, 3, 4, Bellingham S.A.1, 2, 4 and Hill A.F.1, 2, 4 1Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia. 3Department of Pathology, The University of Melbourne, Victoria 3010, Australia. 4Mental Health Research Institute of Victoria, Melbourne, Victoria, Australia.

Prion diseases are a group of fatal neurodegenerative diseases characterised by conversion of the host encoded prion protein, PrPC, into the infectious isoform, PrPSc. Unlike other neurodegenerative diseases, prion diseases are transmissible. According to the protein only hypothesis, the misfolded variant acts as an infectious agent, instigating the conversion of PrPC to PrPSc. Despite extensive research, the pathway by which this infection is transmitted between cells and tissues remains elusive. We have identified a role for exosomes in transmitting prion infectivity between different cell types. Exosomes are small membranous vesicles released by cells, and are derived from the endosomal system. Both PrPC and PrPSc has been detected in exosomes isolated from two cell models: the rabbit epithelial (RK13) cell line overexpressing mouse PrP; and the mouse hypothalamic GT1-7 cell line expressing endogenous levels of PrP. Exosomes allow efficient intercellular spread of prion infectivity, however the factors that govern the sorting of PrP into exosomes is unknown. A difference between the N-terminal immunoreactivity and glycosylation patterns of exosomal and cellular PrP was observed, prompting the hypothesis that PrP isoforms undergo preferential packaging into exosomes. Previous studies have identified mono-ubiquitination as a tag for exosomal sorting and we aimed to investigate if exosomal PrP is ubiquitinated. This modification could have important implications for intercellular transmission of prion infectivity as it could be a pre-requisite or consequence of exosomal sorting.

FIELD TRIALS OF TRANSGENIC COTTON EXPRESSING A DEFENSIN GENE FOR FUNGAL CONTROL

Hinch J.M.1, Gaspar Y.M.1, McGinness B.S.1, McKenna J.A.1, Anderson M.A.2 and Heath R.L.1 1Hexima Limited c/o School of Botany, The University of Melbourne Vic 3010. 2Dept of Biochemistry, La Trobe University Vic 3086, Australia.

Cotton plants (Gossypium hirsutum) expressing a gene encoding the defensin NaD1 from the female sexual tissues of Nicotiana alata have been tested for enhanced resistance to fungal pathogens in the field. Transgenic cotton plants were tested in Queensland and in New South Wales, Australia in fields naturally infested with Fusarium wilt fungus (Fusarium oxysporum f. sp. vasinfectum) or Verticillium wilt fungus (Verticillium dahliae). Three trials against Fusarium wilt in the growing seasons of 2006-2007, 2007-2008 and 2008-2009 demonstrated that the defensin technology gave very clear advantages to cotton plants, with almost three times the survival rate in a high disease year compared with plants that lack the defensin gene. The surviving plants had higher numbers of bolls per plant and consequently a higher yield per plant. Two trials against Verticillium wilt in 2007-2008 and 2008-2009 also demonstrated a yield increase associated with defensin technology. No adverse phenotypic effects or phytotoxicity were observed in transgenic defensin cotton throughout the trials.





PROTEOMIC STUDY ON RAPAMYCIN TREATED HUMAN CANCER CELL LINE

Rajagopalan S.1, Guha N.1, Reddy Y.1, Joseph S.1, Lateef S.1 and Hennessy T.2 1Agilent Technologies, India Pvt.Ltd, Bangalore, India. 2Agilent Technologies, Singapore Pte Ltd, Yishun, Singapore.

Rapamycin is an immunosuppressant drug that specifically inhibits mTOR activity and cellular hyper-proliferation in many cell types resulting in G1 growth arrest. In the current study, we aim to understand the effect of rapamycin treatment on various biological pathways using genomic, proteomic and metabolomic analysis of rapamycin treated human cancer cell line HEK293. Human cell line HEK293 was treated with rapamycin and cells were harvested at 15min, 45min, 2hrs and 16hrs after rapamycin treatment. Control samples without rapamycin treatment were likewise harvested at similar time points. Proteins were extracted from the cells using PPS silent extraction kit, reduced, alkylated and digested using trypsin. Peptides were analyzed in five replicates on a Q-TOF coupled to an HPLC chip system. Differentially expressed features between the rapamycin treated and non-treated samples were identified. Concentration of rapamycin was optimized with respect to cell toxicity and the optimum concentration was found to be 40nM. Proteins were extracted from one million cells of treated and control samples respectively at the specified time points. Experimental procedures for protein extraction, digestion and LC-MS analysis were optimized. Approximately 3000 features were obtained in each LC-MS analysis. The reproducibility in the replicate LC-MS analyses of the digested protein mixture was tested and found to be good. Statistically significant differentially expressed features were analysed for control and treated samples of corresponding time points and in a subsequent experiment identified using targeted MS/MS analysis and database search.

MINCLE IS A NOVEL REGULATOR OF THE INNATE IMMUNE RESPONSE TO TREHALOSE-6,6-DIMYCOLATE

Hitchens K.J., Manzanero S. and Wells C.A. Eskitis Institute for Cell and Molecular Therapies, Griffith University.

Macrophage inducible C-type lectin, Mincle, plays a non-redundant role in the innate immune systems recognition and response to the opportunistic pathogen Candida albicans. Recently, Mincle has been demonstrated to be a key receptor for Trehalose-6,6-dimycolate (TDM). TDM is a highly inflammatory component of the Mycobacterium tuberculosis cell wall. TDM alone can induce granuloma formation and is a component of the tuberculosis subunit vaccine. Absence of Mincle results in poor induction of T cell immunity to the vaccine, making it important for generating a memory immune response. In this study significant differences were found in the inflammatory profile of Mincle knockout (in comparison to wild type) macrophages responding to TDM. The absence of Mincle resulted in the ablation of some inflammatory responses (i.e. TNFα) or in the partial reduction of others (ie. IL-1β). Surprisingly, the response to TDM’s building blocks, the molecules Trehalose or Mycolate alone, appeared to be independent of Mincle. Our results establish that Mincle works both independently of and in collaboration with other receptors in the macrophage response to TDM, and is only involved the response to the whole TDM molecule. Therefore Mincle plays a key role directing part of the inflammatory response to TDM.

EFFECTS OF MISSENSE MUTATIONS IN PAH ON PROTEIN QUATERNARY STRUCTURE AND ENZYME FUNCTION: IMPLICATIONS FOR DISEASE SEVERITY IN PHENYLKETONURIA

Ho G.1, 2, Reichardt J.3 and Christodoulou J.1, 2 1Genetic Metabolic Disorders Research Unit, Children’s Hospital at Westmead, Sydney, Australia. 2Disciplines of Paediatrics & Child Health and Genetic Medicine, University of Sydney, Sydney, Australia. 3School of Medical Sciences, University of Sydney, Sydney, Australia.

Phenylketonuria (PKU) is a recessive inborn error of phenylalanine metabolism, predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. There is a high level of genetic heterogeneity, with over 500 mutations reported to date. The majority of mutations are missense or non-synonymous amino acid changes, which are postulated to destabilise the structure of the folded protein. In PAH, a homotetrameric protein, slight local changes can reduce the efficiency of catalytic function by decreasing the amount of properly-folded tetramers, the sole functional form. To study the effects of different missense mutations we transfected COS-7 cells with PAH of wild-type human sequence or containing a missense mutation previously identified in patients. Protein lysates were separated by native PAGE and analysed by Western blot. Quaternary structure (tetramer, dimer or monomer) was deduced by size. The amount of intact tetrameric protein roughly correlated with the predicted severity of the missense mutations based on patient phenotype. Missense mutations known to cause the most severe form of PKU (e.g. p.F299C and p.R408W) led to very low or undetectable levels of tetrameric PAH, whereas mutations (p.V245A, p.E390G) causing the mild form of PKU had similar levels of tetrameric PAH compared to wild-type. Mutations of the latter group showed decreased levels of enzyme activity, suggesting that mutations of amino acids involved in catalytic activity in PAH are likely to cause a less severe form of the disease compared to mutations causing structural instability.

G-QUADRUPLEX STABILIZER BMVC4 INDUCES SENESCENCE THROUGH BOTH INHIBITING TELOMERASE ACTIVITY AND REPRESSING OF HTERT EXPRESSION IN CANCER CELLS

Huang F.-C.1, Chang T.-C.2 and Lin J.-J.1 1Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan. 2Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan.

Telomeres consist of tandem repeats of guanine-rich sequences that can fold into a variety of four-stranded structures, G-quadruplexes. Telomerase is the enzyme responsible for telomere replication and represents a promising neoplasia therapeutic target. Inhibition of telomere replication can be achieved by stabilization of G-quadruplex DNA structures. Here we designed and synthesized carbazole derivatives that stabilized G-quadruplex DNA structure. We found 3,6-bis(4 -methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) increased the melting temperature of G-quadruplex and inhibited telomerase activity. The BMVC4-treated cells ceased to divide after a lag period. Hallmarks of senescence including morphological changes, detection of senescence-associated β-galactosidase activity were detected in BMVC4-treated cancer cells. The BMVC4-induced senescent phenotype is accompanied by progressive telomere shortening and partial co-localization of the DNA damage foci and telomere association protein TRF2, indicating that BMVC4 caused telomere uncapping after long-term treatments. Interestingly, we also found that BMVC4 repressed the expression of hTERT, the catalytic subunit of telomerase. Repression of hTERT appeared to be caused by the reduction of c-myc, a major transcription factor, upon BMVC4 treatments. Since disruption of the G-quadruplex-forming sequence located at the promoter of c-myc eliminated the repressing effects of BMVC4, the results indicated that BMVC4 repressed c-myc through stabilization of the G-quadruplex structure located at the promoter of c-myc. All together, our results indicated that BMVC4 induced senescence through both inhibiting telomerase and repressing telomerase expression.





CHARACTERIZATION OF TOXICITY OF ALS-LINKED SOD1 MUTANT PROTEINS

Jang J.Y.1, Yoon E.J.1, Cho H.M.1, Kim Y.M.1, Rhim H.S.2 and Kang S.M.1 1School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. 2Research Institute of Molecular Genetics, Department of Biomedical Sciences, College of Medicine, the Catholic University of Korea, Seoul 137-701, Korea.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the degeneration of motor neurons. The disorder causes muscle weakness and atrophy throughout the body. In many cases there is a family history of the disease, in which case it is referred to as familial ALS (FALS). Of these, 20% are mapped to the Cu/Zn superoxide dismutase (SOD1) gene on chromosome 21. SOD1 is an enzymatic antioxidant found in the cytosol, nucleus, peroxisomes, as well as the mitochondrial intermembrane space of eukaryotic cells and in the periplasmic space of bacteria. The mechanism for mutant SOD1 toxicity remains unknown. However, two main hypotheses are the impairment of the functions of important proteins by interaction of misfolded mutant SOD1 proteins and the toxic effect of mutant SOD1 aggregates. Therefore, an attractive therapeutic approach could be to reduce levels of mutant SOD1 expression in neuronal cells. Previously, we found that intracellular Aβ directly interacted with SOD1. Furthermore, we mapped the SOD1 binding region to Aβ amino acids 26-42. Interestingly, intracellular Aβ bound to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. Using a fragment (a.a. 26-42) of Aβ as a competitive inhibitor, we reduced the interaction between mutant SOD1 and proteins that were reported to bind mutant SOD1. Furthermore, we mapped a domain in SOD1 that interacts with the proteins. We also developed ways to disperse SOD1 mutant aggregates. These studies will contribute to therapeutic approach of ALS.

HEPATOMA-DERIVED GROWTH FACTOR (HDGF) IS CORRELATED WITH MALIGNANCY AND INVASIVENESS OF BREAST CANCER

Chen S.C.1, Yeh M.H.4, Chen H.Y.3 and Tai M.H.1, 2, 3 1Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan. 2Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan. 3Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan. 4Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.

Hepatoma-derived growth factor (HDGF) is a novel growth factor that participates in the pathogenesis of a variety of cancers. However, the role of HDGF in breast cancer progression remains unclear. This study aimed to elucidate the association of HDGF expression with prognosis of breast cancer. A total 93 surgically resected breast cancer specimens were collected for immunohistochemical analysis. It was found that HDGF was significantly elevated in breast cancer tissues compared with non-tumor ones. Moreover, elevated HDGF expression was correlated with tumor grades, stages, Ki-67 proliferation index, and lymph node metastasis. By analysis of HDGF expression in various human breast cancer cell lines, it was found that HDGF was significantly up-regulated at the protein level in the most invasive human breast cancer cell line, MDA-MB-231. Moreover, exogenous supply of recombinant HDGF augmented the invasiveness of MDA-MB-231 cells in a dose-dependent manner. Thus, it is concluded that HDGF over-expression is correlated with malignancy and invasiveness of breast cancer. Besides, HDGF may represent a novel diagnostic and therapeutic target for breast cancer.

THE ROLE OF GLUTAREDOXIN1 IN CELLULAR COPPER HOMEOSTASIS

Ackland S.1, Singleton W.1, McInnes K.1, McKirdy R.1, Winnall W.2, Mercer J.1 and La Fontaine S.1 1Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, VIC. 3125. 2Centre for Reproduction and Development, Monash Institute of Medical Research, Clayton, VIC. 3125.

Copper (Cu) is an essential heavy metal that is required by all organisms for a wide range of critical enzymes that harness the CuII/CuI redox couple. Hence, it is important in many physiological and disease processes. Uncontrolled intracellular Cu levels can lead to the generation of excessive reactive oxygen species (ROS) and oxidative stress. The dire consequences of Cu dysregulation are evident in the inherited Cu transport disorders, Menkes and Wilson diseases, and in neurodegenerative diseases. Cells have developed systems to regulate levels of metal ions and ROS to prevent their toxic accumulation. The mammalian Cu-transporting P-type ATPases, ATP7A and ATP7B regulate the Cu status of the body by transporting Cu into the secretory pathway for incorporation into essential enzymes, and by relocating to the cell periphery to efflux excess Cu. We have identified new key roles for glutathione (GSH) and the thiol oxidoreductase, glutaredoxin 1 (GRX1), in regulating the activity of ATP7A and ATP7B. GSH is a major redox buffer in cells and is the most abundant intracellular antioxidant. GRX1 has a protective role in oxidative stress-related conditions and plays a pivotal role in redox regulation of protein function. The Cu-ATPases are glutathionylated. That is, they are postranslationally modified at their Cu-binding cysteines to form protein-SSG mixed disulfides. When Cu levels are elevated, GRX1 interacts with ATP7A and ATP7B to deglutathionylate them, thus allowing them to bind Cu for subsequent transport. The effects of GRX1 overexpression and knockdown on Cu-ATPase activity, cellular Cu homeostasis and overall cell growth and viability will be discussed.

TWO BAM COMPLEXES IN KLEBSIELLA PNEUMONIAE

Alcock F.H., Clements A. and Lithgow T.J. Host-Pathogen Molecular Biology Unit, Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Australia.

The cell envelope of Gram negative bacteria consists of the cytoplasmic membrane, a peptidoglycan layer and an outer membrane, which contains lipopolysaccharide in the outermost leaflet. The majority of clinical K. pneumoniae isolates also produce a mucoid polysaccharide capsule. These two polysaccharide layers are primarily responsible for the barrier quality of the outer membrane, which provides the cells’ primary defence against fluctuating environmental conditions and toxic small molecules. Different classes of outer membrane proteins are required for uptake of sugars and cofactors essential for growth. Correct assembly of outer membrane proteins, LPS and capsular polysaccharide, and its regulation, is a complex task, mediated by a number of multi-component machines and pathways. The BAM complex is a multimeric machine found in the outer membrane of all Gram-negative bacteria, which functions to assemble β-barrel proteins in the membrane. The central component of this complex is BamA, an essential, 90 kDa β-barrel protein, with homologues in the outer membranes of mitochondria and chloroplasts. We analysed the genome of Klebsiella pneumoniae, a Gram-negative bacterial pathogen, and found that it encodes two BamA-like proteins: the canonical BamA, identified as such by gene synteny, and a second isoform, which we have called BamK. We are using a combination of microbiology, microscopy, cell biology and biochemical techniques to investigate the localisation and protein partners of BamK, and its role in assembly of the K. pneumoniae cell envelope.





INVESTIGATING THE ROLES OF KRüPPEL-LIKE FACTORS IN MEGAKARYOCYTE DIFFERENTIATION

Artuz C.M.1, Mak K.S.1, Funnell A.P.W.1, 2, Pearson R.C.M.1, 2 and Crossley M.1, 2 1School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052. 2School of Molecular Bioscience, University of Sydney, NSW 2006.

The Krüppel-like factors (Klfs) are a family of transcription factors that bind GC rich sites and CACCC-boxes present in the promoters and enhancers of many genes. They have been shown to participate in a diverse range of biological processes including erythropoiesis, adipogenesis and stem cell biology. One of the family’s transcriptional activators, erythroid Klf (Eklf or Klf1) is best known for its roles in red blood cell differentiation. Recently, it has also risen to prominence as an inhibitor of the megakaryocyte program. Erythrocytes and megakaryocytes differentiate from a common progenitor, termed the Megakaryocyte/Erythroid Progenitor (MEP). In order to elucidate the mechanisms by which Klf1 influences differentiation decisions in the MEP, it is important to consider two highly related repressor Klfs, basic Klf (Bklf or Klf3) and Klf8. The three Klfs have been shown to function in a tight regulatory circuit. Klf1 activates both Klf3 and Klf8, and Klf3 represses Klf8. Since these Klfs recognise similar DNA motifs, they may compete or act redundantly. Our aim is to understand the molecular network of gene control through which Klf1, 3 and 8 influence cell fate during differentiation in the MEP, particularly in megakaryopoiesis. Given that Klf1 acts primarily as a transcriptional activator, we are investigating whether it indirectly represses megakaryopoiesis through driving either or both of the repressors Klf3 and Klf8. Our strategy is to establish the molecular contributions that Klf1, Klf3 and Klf8 make to the regulation of megakaryopoiesis, using both cell lines and knockout mouse models.

EZRIN AND NHERF ARE REQUIRED FOR THE RECOMBINANT PODXL ENHANCING ADHESION, MIGRATION AND CELL-CELL INTERACTIONS

Fernandez D.2, Nowakowski A.2, Vilar-Egea M.P.2, Parrilla R.1, 2 and Ayuso M.S.1, 2 1Centre of Biological Research, CSIC, Calle Ramiro de Maeztu 9, 28040 Madrid, Spain. 2CIBER de Enfermedades Raras, ISCII.

Podocalyxin (PODXL) is a type I transmembrane glycoprotein initially described in the epithelial cells of the kidney glomeruli, postulated to exert antiadhesive properties; however, we have previously observed that heterologous expression of recombinant PODXL enhanced the adherence of cells to immobilized ligands, spreading, migration, as well as the interaction with other cells. The enhancing effect of PODXL on cell adhesion and migration decreased significantly in deletion mutants of the cytosolic domain or the DTHL domain. Thus, the integrity of the cytosolic carboxylic domain of PODXL, included the terminal DTHL domain, is required for PODXL to exert its effect on cell adhesion and cell-cell interactions. In the podocytes of kidney glomeruli the DTHL domain of PODXL interacts with ezrin and NHERF to establish a link with the actin cytoskeleton, essential for the function of podocytes. To determine whether the recombinant PODXL expressed in CHO cells was also linked to the cytoskeleton through ezrin and NHERF we performed immunoprecipitation studies of cell extracts. We detected ezrin in anti-PODXL immunoprecipitates and PODXL when the immunoprecipitation was performed with anti-ezrin or with anti-NHERF. Thus, recombinant PODXL expressed in CHO cells enhance the cell adhesion properties even though the signaling to the cytoskeleton are similar to the one reported in cells like podocytes that show anti-adherent properties. These observations suggest the cellular environment might be a crucial factor in defining the functional properties of PODXL.

CELL BIOLOGY OF AUTISM: CHARACTERISATION OF THE DELETED IN AUTISM-1 GENE FAMILY

Aziz A. and Bishop N. Department of Microbiology, La Trobe University, Bundoora.

Autism spectrum disorder (ASD), first described by Leo Kanner in 1943, is among the most heritable of all neurological conditions, with a prevalence of at least 1:100 being commonly accepted. Variation in, or deletion of, many different genes appears causative of ASD, and an emerging common theme is a defect in the secretory pathway, secreted proteins and/or regulators of these. The Deleted In Autism 1 (DIA1) gene was recently identified in a study designed to identify recessive autism genes. The normal cellular function of the DIA1 gene product is unknown. We have carried out detailed bioinformatics-based analyses of DIA1 which has revealed a closely related human gene. We have named this gene DIA1R, for DIA1-related. DIA1R was generated by a gene duplication event preceding the divergence of vertebrates. While deletion of DIA1 is implicated in development of ASD, DIA1R localizes to a region of the X chromosome implicated in mental retardation and syndromes with ASD-like features. We found both gene products to be ubiquitously expressed, including in brain tissue. At the cellular level, both proteins have predicted signal peptides, indicating a role in the secretory pathway. We are currently investigating the subcellular localisation and cellular role of both DIA1 and DIA1R. Characterising the role of genes involved in ASD will lead to a better understanding of the biological basis of ASD, leading to opportunities for improved diagnosis and therapy for those with ASD.

MOLECULAR SIZES OF ACID-INSOLUBLE AND ACID-SOLUBLE GLYCOGEN AND THEIR RESPONSES TO EXERCISE AND RE-FEEDING

Barnes P.D.1, Clode P.2 and Fournier P.A.1 1School of Sport Science, Exercise and Health, The University of Western Australia. 2Centre for Microscopy, Characterisation and Analysis, The University of Western Australia.

Muscle glycogen exists as acid soluble (ASG) and acid insoluble (AIG) forms, with AIG reported to be the most responsive fraction to exercise and re-feeding when glycogen is extracted using homogenisation-free protocols, whereas ASG is the most responsive fraction using homogenisation-dependent protocols. It has been proposed that AIG corresponds to a population of lower molecular weight glycogen species that act as precursors for ASG synthesis. However, the molecular size distributions of AIG and ASG in skeletal muscles and their responses to changes in glycogen levels have never been examined. Using transmission electron microscopy analyses of AIG and ASG from muscles extracted using a homogenisation-free and homogenisation-dependent protocols, we compared the molecular size distributions of AIG and ASG from fed Wistar rats as well as the responses of these fractions to 3-minutes of intense exhausting swimming with or without a subsequent 24-hours re-feeding period in rats previously fasted for 24 hours. Contrary to the interpretations made in the literature, the molecular size distribution of both AIG and ASG were normally distributed, with similar ranges and average molecular sizes irrespective of extraction protocol. Immediately after exercise, the ASG molecular size distribution shifted markedly towards smaller glycogen molecules, and AIG distribution changed relatively little. After 24 hours of recovery, both the AIG and ASG molecular size distributions returned to those found in fed rats. All changes in total glycogen concentrations were accounted for by the ASG fraction. In conclusion, the different responses of AIG and ASG suggest that these are physiologically distinct glycogen populations.





FUNCTIONAL STUDIES OF HOMEDOMAIN-INTERACTING PROTEIN KINASE USING C. ELEGANS AS A MODEL

Berber S. and Nicholas H.R. School of Molecular Bioscience, The University of Sydney, NSW 2006.

The Homeodomain-Interacting Protein Kinases (HIPK) are a family of serine/threonine protein kinases shown to be critical in regulation of many cellular processes including cell survival, proliferation and apoptosis. Since there are four HIPK family members in mammals that show some redundancy, functional studies of this protein family can be simplified with the nematode Caenorhabditis elegans since it expresses a single HIPK protein (HPK-1). Both HIPKs and HPK-1 localise to nuclear speckles suggesting that they are likely be involved in analogous cellular processes. To expand on this knowledge we performed phenotypic analyses on a worm strain carrying a deletion mutation within hpk-1 with the global aim of discovering some of the in vivo functions of worm HPK-1. Additionally, to gain insights into molecular mechanisms of HPK-1 function, microarray analysis was performed on the hpk-1 mutant strain. These analyses have revealed that various cellular processes may be regulated by HPK-1 including ageing, metabolism and expression of collagens.

SUMO MODIFICATION OF THE ZINC-FINGER PROTEIN CTCFL/BORIS

Bernier-Latmani J. and Shaw P. Experimental Pathology Division, Institute of Pathology, University of Lausanne.

Post-translational modification by the small ubiquitin-like SUMO proteins (SUMO-1 and SUMO-2/3) is recognized to play an important role in target protein function by affecting DNA binding, sub-cellular localization, protein stability and co-factor association. CTCFL is a paralog of the ubiquitous genome regulator CTCF, but is expressed solely in the testis and some cancers. Due to high homology in their respective zinc-finger regions it has been proposed that CTCF and CTCFL bind similar DNA sequences but perform different functions. CTCFL has been implicated in the establishment of methylation at the Igf2/H19 imprinting control region and transcriptional regulation of a number of genes including BRCA1, c-myc, and BAG1. Recently CTCF has been shown to be modified by SUMO, whereby enhancing repression of a c-myc promoter reporter construct. In the present work we show that CTCFL is efficiently SUMOylated in vitro and when over-expressed in 293T cells. Endogenous CTCFL is SUMOylated, as determined by immunoprecipitation of SUMO-1 and SUMO-2/3 modified proteins in K562 cells. Both SUMO-1 and SUMO-2/3 are attached at two residues, K181 and K645, as shown by point mutation analysis. Characterization of the functional consequences of CTCFL SUMOylation is ongoing. Alteration of CTCFL sub-cellular localization by SUMOylation is being analyzed by immunofluorescence confocal microscopy. Furthermore, the effect of SUMOylation on CTCFL DNA-binding and gene regulation activity is being assessed using EMSA and luciferase reporter assays. Also, the consequence of SUMOylation on the stability of CTCFL is being investigated. This work will help elucidate basic CTCFL function and potentially provide a basis to understand its role in both testis and cancer.

REGULATION OF COPPER UPTAKE AND EFFLUX IN DROSOPHILA INTESTINAL ENTEROCYTES

Binks T. and Burke R. School of Biological Sciences, Monash University. Wellington Rd Clayton 3800 Victoria, Australia.

All animals require the essential biometal copper as an enzymatic cofactor for processes as diverse as energy production, free radical detoxification and pigmentation. Most copper is absorbed via the polarized enterocytes that line the intestinal lumen. Active transport is required to transfer copper across the apical enterocyte membrane from the lumen into the cells, then again across the opposite basolateral enterocyte membrane and out into the bloodstream for distribution throughout the body. Therefore the intestinal enterocytes are an excellent system to study directional transport of copper through a polarized cell layer. We are studying this process in the Drosophila larval midgut, using targeted over expression and suppression of known copper transport and copper chaperone genes together with two Green Fluorescent Protein (GFP) reporter genes that respond to copper depletion and copper accumulation respectively. We will also present protein localization studies in these midgut cells using GFP fusion proteins created for the major fly copper uptake and efflux proteins. And using our Drosophila enterocytes model, we will demonstrate novel roles in copper homeostasis for genes previously implicated in cellular vesicle trafficking / transport pathways.

CHARACTERIZATION OF MAVI-P35, A BACULOVIRAL MULTIPLE CASPASE INHIBITOR

Brand I.L.1, George C.1, Lovric M.1, Pantaki D.1, Kitevska T.1, Clem R.J.2 and Hawkins C.J.1 1La Trobe University, Department of Biochemistry, Melbourne 3086, Australia . 2Molecular, Cellular and Developmental Biology Program, Arthropod Genomics Center, Division of Biology, Kansas State University, Manhattan, KS 66506, USA.

P35 from Autographa californica (Ac) MNPV was the first identified multiple caspase inhibitor expressed by a baculovirus to prevent the host cell from undergoing infection-induced apoptosis. A number of P35 relatives have since been identified, including some less closely related homologues belonging to the P49 sub-family. Ac-P35 has been studied extensively. It protects against mammalian, nematode and insect cell death and inhibits all caspases tested, with the exception of the initiator caspases DRONC, Sf-caspase-X and caspase-9. In contrast, the best studied P49 sub-family member SlNPV-P49 seems to be a generally less potent caspase inhibitor, but it can inhibit Ac-P35-resistant initiator caspases. A new member of the P35-family has been recently identified by sequencing the genome of the baculovirus Maruca vitrata (Mavi) MNPV. This P35-family member (Mavi-P35) shares 85% sequence homology to Ac-P35, but its active loop possesses a different caspase cleavage sequence, reminiscent of sequences efficiently cleaved by the insect initiator caspase DRONC. This suggested to us that, unlike other members of the P35 sub-family, Mavi-P35 may inhibit initiator caspases such as DRONC. In this study, Mavi-P35 was characterized by testing its ability to inhibit caspase-induced yeast death and apoptosis of mammalian and insect cells. Recombinant Mavi-P35 was tested in vitro against a range of caspases and their susceptibility to inhibition was quantitated. Our results show that Mavi-P35 is able to inhibit an even broader range of caspases than Ac-P35, including the Drosophila melanogaster caspase DRONC.




POS-MON143 POS-TUE-144

THE GENETICS AND CELL BIOLOGY OF COPPER HOMEOSTASIS IN DROSOPHILA

Lye J.1, Binks T.1, Hwang J.1, Camakaris J.2 and Burke R.1 1School of Biological Sciences, Monash University. Wellington Rd Clayton, Victoria 3800. Australia. 2Department of Genetics, University of Melbourne. Parkville, Victoria 3010. Australia.

Copper is an essential micronutrient required as a cofactor for enzymes such as Cytochrome C oxidase, Cu, Zn Superoxide dismutase and Tyrosinase. Disruption of copper homeostasis can result in serious human conditions such as Menkes disease (copper deficiency) and Wilson disease (copper toxicity). The major copper uptake (Ctr1) and efflux (ATP7) genes are highly conserved from yeast through to vertebrates. Drosophila has three Ctr1 orthologues and a single ATP7 orthologue, DmATP7. We are using a combination of targeted gene over expression and gene suppression to examine the in vivo role of the key copper transport genes in various Drosophila tissues. We find that reduction of copper uptake or increase in copper efflux in cuticle-producing cells results in a striking hypopigmentation phenotype due to lack of activity of the Laccase cuproenzyme. Genetically-induced copper deficiency in the Drosophila adult eye results in a severely flattened eye phenotype while increased copper uptake in the eye causes a rough eye phenotype typical of increased cell death. We will present genetic interaction studies showing that the effects of increased copper uptake can be compensated for by increasing copper efflux and are exacerbated by blocking copper efflux. We will also present data showing how the cellular localization of these copper transport proteins fits with their proposed cellular functions, and exciting recent data from the Australian Synchrotron where we have used X ray Fluorescence Microscopy to produce elemental maps of Drosophila tissues and prove directly for the first time, that our genetic manipulations do indeed alter copper levels in a targeted manner in vivo.

PEPTIDASE EXPRESSION IN LYMPHOCYTES

Chen Y., Yao T.W., Chowdhury S., Gorrell M.D. and Yu D.M.T. Centenary Institute, Sydney Medical School, University of Sydney, NSW, Australia.

The dipeptidyl peptidase (DP) IV gene family is specialized in hydrolyzing the prolyl bond, which is useful for degrading many peptide hormones. DPIV (CD26) has roles in lymphocyte activation and proliferation. DPIV and its closest relative, fibroblast activation protein, also have roles in liver disease. We have associated DP8 and DP9 expression with disease pathogenesis [1] and observed DP8 and DP9 expression by leukocytes and B and T cell lines while others have shown a role for DP9 in processing antigenic peptides [Geiss-Friedlander R, et al., JBC 2009]. A greater understanding of DP8 and DP9 in the immune system is needed. This study investigated DP8 and DP9 expression in stimulated lymphocytes and diseased human liver. DP8 and DP9 expression levels were measured by Western blot and quantitative PCR in Jurkat T cells and Raji B cells treated with mitogen, hydrogen peroxide, dithiothreitol, Mitomycin C or serum starvation. DP8 and DP9 protein levels were upregulated in mitogen stimulated Jurkat T cells but unaffected by the other treatments. DP9 protein expression was similar in human primary biliary cirrhosis liver compared with normal liver. The novel finding that DP8 and DP9 expression was upregulated in activated T cells suggests that, like DPIV/CD26, DP8 and DP9 might have roles in T cell activation. Reference: [1] Yu, D.M.T., et al., The in vivo expression of dipeptidyl peptidases 8 and 9. J Histochem Cytochem 2009. 57:1025-1040.

PURINE BIOSYNTHESIS AND ITS REGULATION IN NODULES OF SUB-TROPICAL LEGUMES

Chen C.1, Bussell J.2, 3, Atkins C.2 and Smith P.1 1School of Biological Sciences, The University of Sydney, NSW 2006, Australia. 2School of Plant Biology, The University of Western Australia, WA 6009, Australia. 3Umea Plant Sciences Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU), 901 83 Umea, Sweden.

Sub-tropical legumes such as cowpea and soybean (mainly of the tribe Phaseoleae) export fixed nitrogen as ureides. De novo synthesis of purine nucleotide is the major pathway for assimilation of fixed nitrogen in these legumes and the product, IMP, is oxidised to form ureides that are exported from the nodules. There are 10 enzymatic steps in the purine biosynthetic pathway and the enzymes involved are encoded by nine genes (the pur genes). The role of the products of nitrogen fixation in regulation of these genes was investigated in nodules of cowpea by growing plants with their root systems in an atmosphere devoid of nitrogen (80% Ar: 20% O2). In the absence of N2 (plants grown in Ar:O2) expression of Vupur1, 4, 5, 6, 7 and 8 is at a level required to maintain basic cellular processes only. In air the level of these transcripts increases dramatically after N2 fixation begins, suggesting transcription is regulated by the products of fixation. Other transcripts studied (Vupur2, 3, 9, IMPDH and uricase) showed no change in the level of expression over time, however, the expression of Vupur2 and 9 and IMPDH was reduced in Ar:O2 compared to air while that of Vupur3 and uricase was the same in both treatments. We have investigated promoters of the two genes encoding the fifth enzyme in the pathway, AIR synthetase (Gmpur5-1 and Gmpur5-2), in soybean using GUS/GFP fusions. The Gmpur5-1 promoter drives expression in all cells but the infected cells of the nodule while the Gmpur5-2 promoter drives expression almost exclusively in infected cells. We are currently investigating the regulation of these promoters in response to nitrogen using these constructs. We are also investigating the targeting of the enzymes to organelles in nodules.

AUTOPHOSPHORYLATION OF ATM AT SERINE 1981 MEDIATES THE INTERACTION WITH MDC1 AND STABILIZES ATM AT SITES OF DNA DOUBLE-STRAND BREAKS

Davis A., Zheng C. and Chen D.J. Division of Molecular Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas Texas, USA.

Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11-RAD50-NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Ablation of the autophosphorylation site affects the ability of ATM to phosphorylate its downstream targets after DNA damage especially at later time point after irradiation and confer radioresistance. Biochemical evidence shows that autophosphorylated ATM directly interacts with the FHA domain of MDC1. Knock-down of MDC1 protein recapitulates the effects of S1981A mutation on the retention of ATM at DSBs and phosphorylation of downstream substrates. Moreover, ablation of the ATM autophosphorylation site and MDC1 depletion did not show any additive effect on radiosensitivity. Together, these data illustrate the importance of autophosphorylation at serine 1981 for the interaction of ATM with MDC1 which serves to stabilize ATM at DSBs and thereby promote a full-scale response to DNA damage in human cells.





SAME SIGNAL TRIGGERS DIFFERENT CELL RESPONSES IN VARIOUS CELL LINES

Cheung T.W. and Lam Y.W. City University of Hong Kong.

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine. Andrographis paniculata, is an abundant component of the plant Andrographis that has been commonly used as a folk remedy for alleviation of inflammatory disorders in Asia for millennia. Also, it is known to possess potent anti-cancer activities. According to previous studies, it can trigger most of cell lines to go through G1/S arrest followed by apoptosis. Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events are completed with high fidelity. In addition, checkpoints respond to damage by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. However, a feature of tumor cells is the alteration of appropriate cell-cycle progressions which are closely related apoptotic process. Currently, people mainly uses anti-mitotic drugs which work by perturbing spindle assembly through the activation of the spindle assembly checkpoint for treating cancers. The drugs cause mitotic arrest, and triggers apoptosis. Based on past study, the response of HepG2 cells is different from the other cell lines under Andro treatment. To clarify what the differences are, we use live cell imaging and western blotting approaches in the study. We showed that Andro induces both G2/M and G1 arrest in two tested cell lines. Then, in HeLa cells, it quickly goes into apoptosis. But, HepG2 cells mainly stay long in cell cycle arrests until necrosis occurred. The differences may not relate to DNA damaging or increasing ROS production. And, many M-phase related proteins show some regulation to reason the longer G2/M arrest observation in HepG2 cells, not in HeLa cells.

FUNCTIONAL CHARACTERIZATION OF A NOVEL AURORA-A BINDING PROTEIN, AIBP IN CENTROSOME STRUCTURE AND SPINDLE FORMATION

Chou C.H1, 2, Wu C.H. 2, Huang C.Y. 3, Hsu C.M. 1, Howng S.L.4 and Hong Y.R.1, 2 1Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung,Taiwan. 2Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 3Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan. 4Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

Aurora-A is involved in chromosome alignment, centrosome maturation, mitotic spindle assembly and regards to an oncogene. Aurora-A is also known to bind to several other proteins affecting its up-regulation or down-regulation and localization. However, how these different binding signals work together to regulate Aurora-A is not properly known. To explore more Aurora-A interacting proteins, the low-copy yeast two-hybrid screening using Aurora A as bait protein was performed. One novel gene, AIBp, was demonstrated to associate with Aurora-A in yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization showed that AIBp possessed a binding site at the C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also interacts with hNinein at the N-terminal, which overlaps a previously reported hNinein and GSK3β binding site. In kinase assay, AIBp interacts with the Aurora-A kinase domain functions as a positive regulator, whereas AIBp binding to hNinein appears to block the phosphorylation of hNinein by both Aurora-A and GSK3β. siRNA-mediated elimination of AIBp from HeLa cells, results in a donut-like shape, asymmetrical spindle pole and multiple spindle pole formation. We also demonstrated that both AIBp and Aurora-A are co-overexpressed in various brain tumors. These studies demonstrate that AIBp may not only be required for the dynamic movement of Aurora-A at the centrosomes and spindle apparatus during the cell cycle, and may also be important during brain tumorigenesis.

PROTEOMIC PROFILE OF THE SOYBEAN SYMBIOSOME MEMBRANE

Clarke V.C.1, Loughlin P.C.1, Jacoby R.P.2, Taylor N.L.2, Millar A.H.2, Day D.A.3 and Smith P.M.C.1 1School of Biological Sciences, The University of Sydney, NSW, Australia. 2ARC Center of Excellence in Plant Energy Biology, The University of Western Australia, WA, Australia. 3Flinders University, SA, Australia.

Soybeans are able to form a symbiotic association with soil bacteria, Bradyrhizobium japonicum, whereby atmospheric nitrogen is fixed by the bacteria and made available to the plant in exchange for nutrients. This symbiotic relationship occurs within specialised root structures termed nodules. Free-living Bradyrhizobium bacteria invade the soybean roots and become engulfed within the plant cell, surrounded by a membrane of plant origin known as the symbiosome membrane (SM). It is this membrane that regulates the movement of solutes from plant to bacteroid (the symbiotic form of the rhizobium) and vice versa. The SM is a unique structure containing an array of plant-derived proteins through which the plant can regulate the symbiosis. Previous attempts to characterise the protein complement of this membrane have been hindered by its hydrophobic nature and the absence of a complete soybean genome for reference. In this study, SM was isolated from mature nitrogen-fixing soybean root nodules and analysed using shotgun proteomic techniques. The recent release of the complete soybean genome has allowed for the identification of peptide sequences. Our initial proteomic analysis of soybean SM has identified forty putative SM-localised proteins, including ten previously localised to the SM such as nodulin-26, an aquaporin. Bioinformatic analysis of novel matches has suggested that half may contain membrane-spanning domains, making them candidates for SM transporters. Further in-depth proteomic analysis of the membrane is currently being undertaken and the function and localisation of putative SM proteins will be investigated.

TROPOMYOSIN TM5NM1 MEDIATES ACTIN-DEPENDENT CELLULAR PROLIFERATION

Coombes J.D.1, 2, Schevzov G.1, Stehn J.1, Creed S.3, Desouza M.1, Bach C.T.2, 3, O’Neill G.2, 3, Musgrove E.4 and Gunning P.1 1Oncology Research Unit, School of Medical Sciences, University of New South Wales. 2Sydney Medical School, University of Sydney. 3Kids Research Institute, Children’s Hospital at Westmead. 4Cancer Research Program, Garvan Institute of Medical Research.

Progression through the cell cycle is associated with profound changes in the actin cytoskeleton. Disruption of the actin cytoskeleton with pharmacological agents causes arrest in G1 phase and failure to enter S-phase, indicating that an intact actin cytoskeleton is required for cell cycle progression. The lack of specificity of these agents has hindered the analysis of the mechanisms responsible for this regulation. We have previously identified functionally distinct populations of actin filaments, each containing different isoforms of the actin filament-associated protein, tropomyosin (Gunning, O’Neill, Hardeman, Physiol Revs 2008). In the current study, we hypothesised that a specific population of Tm isoform-containing actin filaments regulates cell proliferation. The role of Tm5NM1-containing actin filaments was investigated in knock-out, knock-down, and overexpression model systems. Our data show that embryonic fibroblasts (MEFs) isolated from Tm5NM1/2-knockout mice have an impaired rate of proliferation in response to growth factor stimulation. This is accompanied by a dysregulation of MAPK signalling (phospho-ERK1/2) and decreased expression of the key G1-phase mediator, Cyclin D1. Neuroblastoma SHEP cells treated with siRNA against Tm5NM1/2 also have reduced proliferation rates and decreased Cyclin D1 expression. In comparison, Tm5NM1-overexpressing neuroblastoma-derived B35 cells display accelerated proliferation, and reduced propensity to withdraw from the cell cycle when treated with differentiation factors. These data suggest that actin-filaments containing Tm5NM1 mediate G1 phase progression, potentially via controlling the fidelity of signalling through the ERK/MAPK pathway. This indicates that Tm5NM1 has potential as a novel anti-proliferative therapeutic target.





DEXAMETHASONE INHIBITS INTESTINAL SECRETORY CELL ER STRESS BY UPREGULATING DEGRADATION OF MISFOLDED PROTEIN ER STRESS (ERAD)

Das I.1, 2, Png C.W.1, Tran T.1, 3, Eri R.1, Lourie R.1, 3, Adams R.1, Oancea I.1, 3, Crane D.1, 2, Florin T.1, 3 and McGuckin M.1, 3 1Mater Medical Research Institute. 2Griffith University. 3University Of Queensland.

Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with the pathogenesis of Inflammatory Bowel Diseases (IBD) which introduces ER stress, the unfolded protein response (UPR) and ER-associated degradation (ERAD) of misfolded proteins as pathways for therapeutic targeting in IBD. Glucocorticosteroids are an effective treatment for IBD, but little is known of their effect on ER stress. This study determined the effect of a glucocorticosteroid (dexamethasone, Dex) on ER stress in cultured LS174T colonic cells and in Winnie mice with intestinal secretory cell ER stress caused by Muc2 mucin misfolding. mRNA expression of ER stress markers GRP78, spliced-XBP1, ATF6 and CHOP; glycoprotein chaperones CNX and CRT; and ERAD genes EDEM1, VCP and SEC61 were measured by qRT-PCR. Histological analysis was used to assess intestinal inflammation and mucin biosynthesis. Upregulation of GRP78 (a key indicator of ER stress, 15.1±1.3 and 14.3±1.0 fold increased by tunicamycin and thapsigargin in LS174T cells, respectively) was completely inhibited by Dex (P<0.001). All other ER stress markers followed the same pattern. Winnie mice showed accumulation of misfolded Muc2 in secretory cells and a 40±3 fold increase in intestinal Grp78 compared to wild-type mice, and elevated expression of all other ER stress/UPR markers. Four weeks treatment with Dex significantly inhibited the increase in ER stress genes (P<0.001) and substantially increased production of correctly-folded Muc2 (P<0.001). The ERAD genes Edem1 and Vcp were the only ER stress-related genes that increased in Dex-treated mice. These results suggest that Dex ameliorates ER stress by enhancing removal and degradation of the mis-folded Muc2 from the secretory cells by up-regulation of proteins involved in the ERAD pathway.

AGED-RELATED INCREASE IN XANTHINE OXIDASE EXPRESSION AND ACTIVITY IN SPLEEN FROM MICE

Vida C.1, Gonzalez E.1, Hernandez O.1, Rodriguez-Teres S.1, Hernanz A.2, Corpas I.1 and De la Fuente M.1 1Department of Physiology. Faculty of Biology. Complutense University of Madrid (Spain). 2Department of Biochemistry. La Paz Hospital. Madrid. Spain.

In mammalian cells, one of the major sources of reactive oxygen species (ROS) is the enzyme xanthine oxidase (XO), which uses hypoxanthine and xanthine as reducing substrates producing ROS, such as superoxide anion and hydrogen peroxide. XO has been implicated in a variety of pathophysiological states and oxidation stress-related diseases. In addition, ageing is based on chronic stress, with an excessive production of ROS and a loss of the antioxidant defences, in which the immune system has an important role. The aim of the present work was to determine the age-related changes in XO expression and activity in mouse spleen. ICR-CD1 adult (6 month-old), mature (13 month-old), old (18 month-old) and long-lived (31 month-old) female mice were used. Animals were sacrificed and spleen was obtained, which was homogeneized in order to determine XO activity using a comercial kit (Invitrogen). The XO protein expression was assayed by western-blot analysis. The results showed that both XO expression and activity increase with ageing. XO activity levels were significantly increased in old (p≤0,001) and long-living (p≤0,001) mice with respect to adult mice. Finally, we analyzed the XO protein expression in this tissue, which showed a significant increase in old (p≤0,05) and long-living mice (p≤0,01). Nevertheless, mature mice show similar XO activity levels and XO protein expression as compared to the adults. In conclusion, the increase in the XO expression and activity in spleen from older mice may have a crucial role in the pathophysiological changes of the ageing process. MICINN(BFU2008-04336); Research-Group-UCM(910379ENEROINN); RETICEF (RD06/0013/0003).

KRüPPEL-LIKE FACTOR 3 (KLF3) AND THE UV-RESPONSE PATHWAY

Dewi V.A., Pearson R.C.M. and Crossley M. School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW, 2052.

Following DNA damage, cells enter either cell cycle arrest or apoptosis depending on the severity of the damage. Upon extensive DNA damage by ultra violet (UV) light, a master regulator of cell cycle and apoptosis, p53, is phosphorylated by the kinase, Homeodomain-interacting protein kinase 2 (Hipk2). This phosphorylation of p53 stabilises the protein and is recognised as a hallmark of apoptosis. The transcriptional co-repressor C-terminal binding protein 2 (Ctbp2) is also phosphorylated by Hipk2 upon DNA damage. In contrast to p53, phosphorylation of Ctbp2 marks it for degradation. Krüppel-like factor 3 (Klf3) is a transcriptional repressor known to recruit Ctbp2 to silence target genes. Using in vitro kinase assays we have shown that Hipk2 can also phosphorylate Klf3. This led us to hypothesise that Klf3 plays a role in the UV-response pathway. Using NIH-3T3 as a model cell line, we conducted time course experiments to study the effect of UV irradiation on the expression of Klf3 and its target genes. We observed downregulation of both Klf3 and its co-repressor Ctbp2 following irradiation. We are currently exploring the hypothesis that changes in expression of Klf3 target genes play an important role in the response to DNA damage.

THE SECRETIN PROTEIN SUPER-FAMILY: NEW STRUCTURES FOR BACTERIAL OUTER MEMBRANE PROTEINS

Dunstan R., Alcock F., Webb C. and Lithgow T. Department of Biochemistry & Molecular Biology, Monash University, Clayton, 3800 Australia.

It was considered for some time that all integral membrane proteins in bacterial outer membranes had an architecture referred to as a β-barrel, and recently it has become clear that these proteins are assembled by the BAM complex. Very recently a new architecture of bacterial outer membrane proteins was discovered: large, multimeric complexes containing amphipathic helical transmembrane domains rather than the conventional β-barrel structures. These “secretins” form pores for the secretion of proteins of the type II and type III secretion systems, formation of type IV pili and the assembly of filamentous bacteriophage. They also form the outer membrane collar of bacterial flagella. The precise mechanism and kinetics of secretin assembly and insertion into the OM is unclear. Using a Hidden Markov Model (HMM) strategy, based on sequences of known secretins, novel candidate secretins have been identified from the enteropathogenic Escherichia coli strain E2348/68. We are tailoring an assay in which the kinetics of assembly of these secretins can be determined and aim to determine the machinery responsible for secretin assembly in bacterial outer membranes.





POLLEN-SPECIFIC DEPOLL GENE EFFECTS THE EXPRESSION OF LTP PROTEINS IN ARABIDOPSIS THALIANA

Duplakova N.1, 2 and Honys D.1, 2 1Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Prague 6, Czech Republic. 2Department of Plant Experimental Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic.

Plant cell wall is highly organized and complex structure consisting of carbohydrates, proteins and aromatic compounds surrounding and separating cells from outer environment. It has been assigned many different functions like regulation of cell volume, protoplast protection, cell shape determination and many others including its key role in morphogenesis. The pollen wall represents its special type as it is composed by two distinct layers - exine and intine. Moreover, pollen wall is covered with unique and specialized lipid pollen coat with functions in pollen dispersal and pollen stigma recognition. The list of cell wall proteins is very extensive and one of these, lipid-transfer proteins (LTPs), are present in pollen wall separable fraction. LTP proteins are basic, soluble, 9-kDa proteins representing up to 4% of total soluble proteins in higher plants. They were found to be secreted and located extracellularly, in the cell wall, although earlier hypotheses considered their function in intracellular lipid dynamics. Several roles for LTPs were suggested in plant growth and development, defense reactions against phytopathogens, symbiosis and the adaptation of plants to various environmental conditions. Here we show that T-DNA insertion in pollen-specific gene depoll encoding 21 kDa (185 aa) C2 protein caused increased expression of numerous LTPs in male gametophyte accompanied by defects in pollen cell wall formation or cell death. Acknowledgment: Authors gratefully acknowledge the financial support from the Czech Science Foundation (grant 522/09/0858) and Ministry of Education, Youth and Sports of the Czech Republic (grants LC06004 and OC10054).

PROLIFERATIVE AND SURVIVAL PATHWAYS IN OESOPHAGEAL CANCER

Esau L.E. and Hendricks D. University of Cape Town, Cape Town, Western Province, South Africa.

Oesophageal cancer is the 7th most common cancer worldwide and available treatment does not significantly enhance patient survival. The epidermal growth factor receptor EGFR is commonly over-expressed in oesophageal cancer and its over-expression correlates with tumour aggression and survival. Reports exists that the insulin-like growth factor receptor 1 IGF-1R plays a role in survival and that interactions exist between EGFR and IGF-1R. The objective of this study is to determine the role of EGFR and IGF-1R in proliferation and survival of oesophageal cancer. EGFR and IGF-1R function in cultured oesophageal cancer cell biology was determined by using western blot analysis, inhibitor to EGFR or IGF-1R shRNA. Receptors were activated with specific ligand. EGFR kinase domain phosphorylation activated IGF-1R as receptors were shown to immunoprecicpitate with each other. Targeting EGFR resulted in decreased proliferation and an IC50 of +/-10uM however increased Akt activity was observed. IGF-1R knockdown resulted in increased pEGFR and pERK1/2 with diminished Akt activity which lead to a 3 fold increased sensitivity to EGFR inhibitor compared to EGFR inhibition alone. EGFR and IGF-1R show potential as drug targets in Oesophageal cancer as combined targeting of these two receptors show increased response than targeting EGF receptor alone.

TARGETS OF RNA-BINDING PROTEIN MUSASHI-1 IN MOUSE GERM CELL DEVELOPMENTFraser B.A.1, 2, Sobinoff A.2, Pye V.J.2, Roman S.D.1, 2, Hime G.R.1, 3, Siddall N.A.1, 3, Koopman P.1, 4 and McLaughlin E.A.1, 2 1ARC Centre of Excellence in Biotechnology & Development. 2Reproductive Science Group School of Environmental & Life Science, University of Newcastle, Callaghan NSW 2308. 3Dpt of Anatomy & Cell Biology, University of Melbourne, Parkville VIC 3010. 4Institute for Molecular Biosciences, University of Queensland, St Lucia QLD 4072.

RNA-binding proteins, such as Musashi-1 (Msi-1), can contribute to posttranscriptional control and are believed to have an important role in the maintenance of germline stem cells and germ cell differentiation. We have previously confirmed that Msi-1 is predominately expressed in gonocytes and mitotic spermatogonia. Protein-RNA pulldown and microarray and bioinformatic analysis has provided us with a number of possible targets of Msi-1 in the testes. Further to this, we aimed: i) to generate mice that over-express Msi-1 in differentiating male germ cells and examine the resultant phenotype; (ii) to use RNA interference to knockdown the expression of Msi-1 in cultured spermatogonial cells; and (iii) to use a Musashi-1 specific antibody to immunoprecipitate target RNA from a spermatogonial cell lysate. Initial phenotypic analysis of the transgenic mice indicated that, whilst these mice produced spermatozoa, they have reduced fertility or are infertile. They have anomalous sperm formation and the sperm have a decreased ability to bind to mature oocytes. Increasing transgene expression resulted in male sterility due to spermatogenic arrest and a total absence of mature sperm. Quantitative gene expression of the putative targets demonstrated significantly increased transcript levels with concomitant increase in Msi-1 transgene expression. The relative expression of the target genes was down-regulated in cultured spermatogonia following shRNA Msi-1 knockdown. Immunoprecipitated mRNA from spermatogonial lysate was significantly enriched with Msi-2 mRNA. In conclusion, our preliminary results indicate that Msi-1 is required for normal spermatogenesis and that it may target a number of mRNAs, particularly Musashi-2, to achieve posttranscriptional control of mouse spermatogonial differentiation.

THE GOLGI-LOCALISED CALCIUM ATPASE IN BREAST CANCER

Grice D.M.1, Vetter I.2, Faddy H.M.1, Kenny P.A.3, Monteith G.R.1 and Roberts-Thomson S.J.1 1School of Pharmacy, The University of Queensland, Brisbane, QLD, 4072, Australia. 2Institute for Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia. 3Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

Golgi-localised calcium ATPases maintain the calcium concentration of the Golgi apparatus and as such are associated with cell signalling, apoptotic signalling and post-translational modification of proteins. We assessed the mRNA expression of one of these Golgi-localized calcium ATPases in 295 breast tumour samples classified into five transcriptional subtypes each associated with a different prognosis and response to therapeutic treatment. The Golgi-localised calcium ATPase was enriched in particular breast cancer subtypes. To characterize the role of this calcium ATPase in breast cancer, its expression was silenced in MDA-MB-231 MCF7 and T-47D breast cancer cell lines using Dharmacon OnTarget plus Smartpool siRNA. We assessed the consequences of ATPase silencing on cell viability, response to induction of endoplasmic reticulum stress and affects on G-protein coupled receptor cytosolic calcium signaling stimulated by trypsin, thrombin and ATP using a high throughput FLIPRTETRA imaging system and the potential regulation of Golgi-resident enzymes. Our results suggest that alterations in the expression of a Golgi-localised calcium ATPase may be a feature of specific breast cancer subtypes, and that inhibition of the expression of these calcium pumps differentially affects pathways important in breast cancer progression.





CONTROL OF CELL GROWTH AND SURVIVAL VIA 14-3-3 (ZETA) AND TYPE 1 INSULIN-LIKE GROWTH FACTOR RECEPTOR

Hadzir S.1, Ramshaw H.2, Krake R.2, Delaine C.1, Guthridge M.2, Wallace J.C.1, Lopez A.F.2 and Forbes B.E.1 1School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide SA,5005. 2Division of Human Immunology, Centre for Cancer Biology, Adelaide, SA, 5000.

We have developed the first isoform specific 14-3-3ζ-/- mouse. 14-3-3ζ-

/- mice exhibit post-natal growth deficiency (P7 onwards) with 20-30% reduction in size compared to wild type littermates. 14-3-3 proteins are intracellular phosphoserine/phosphothreonine binding proteins involved in modulation of crucial biological processes such as proliferation, signal transduction, metabolism, cell cycle and apoptosis. Importantly, 14-3-3ζ binds the type 1 insulin-like growth factor receptor (IGF-1R) and several proteins involved in IGF-1R downstream signalling pathways including IRS-1 and 2, p85, Akt and Raf. These interactions modulate IGF-1R survival, proliferation and transformation activities. Initial observations suggest a perturbation of the GH/IGF-I axis in 14-3-3ζ-/- mice. Preliminary analyses have revealed low pituitary growth hormone (GH) levels and low serum IGF-I (~ 2.7 fold), IGF binding protein-3 (IGFBP-3) and acid labile subunit (ALS) levels. 14-3-3ζ-/- mice growth deficiency is evident before weaning (P7 onwards) whereas GH deficiency models show growth deficiency only at puberty and IGF-I knockout mice are pre and postnatally growth deficient The phenotype suggests it is different to most mouse models of IGF-I deficiency, although it is reminiscent of the hypothalamic IGF-1R knockout phenotype. Interestingly, we have shown a perturbation in the IGF-1R signalling via Akt and Erk1/2 in embryonic fibroblasts derived from the 14-3-3ζ-/- mice. Therefore, our evidence so far suggests that the growth deficiency is driven by abnormalities in GH/IGF-I axis and perturbation in 14-3-3ζ action involving IGF-1R signalling (via Akt and Erk). The mechanism underlying the GH deficiency is still not clear.

OXIDATION AND INFLAMMATION INCREASE WITH AGEING IN IMMUNE CELLS FROM MEN AND WOMEN

Mate I.1, Arranz L.1, Garcia-San Frutos M.V.1, Vida C.1, Hernanz A.2 and De La Fuente M.1 1Department of Physiology. Faculty of Biology. Complutense University of Madrid. 2Biochemistry Department. La Paz Hospital. Madrid. (Spain).

A direct relationship exists between ageing and increasing oxidation and inflammation. The immune cell functions change with ageing and this immunosenescence seems to be a consequence of this age-related oxidative and inflammatory stress. We have proposed a relevant role of the immune cells on oxi-inflamm-ageing. The aim of the present work was to determine the age-related changes in several oxidative and inflammatory parameters as well as in some oxidative stress-related functions in leukocytes from men and women. A total of 334 healthy human volunteers were studied (171 women and 163 men), and divided into three age groups: adult (30-49 years of age), adult-mature (50-59 years) and mature (60-79 years). Blood samples were extracted and the following parameters analyzed in neutrophils and lymphocytes: adherence to tissues, spontaneous mobility (SM), chemotaxis, phagocytosis, proliferation of lymphocytes in response to mitogens, superoxide anion (SA) levels, TNF-α secretion in response to LPS, as well as plasma C-reactive protein (CRP). The results showed statistically significant increases of CRP, SA, TNF-α, adherence and SM in mature groups with respect to adults, whereas the other functions were decreased. These changes were more significant in women than in men. Thus, oxi-inflamm-ageing seems to increase in postmenopausal women as a possible consequence of the loss of the protective role of oestrogens. Financial support: MICINN (BFU2008-04336); Research-Group-UCM (910379ENEROINN); RETICEF (RD06/0013/0003).

ASSEMBLY OF MITOCHONDRIAL PORINS: ADAPTATION TO AN ENDOSYMBIOTIC LIFESTYLE

Hewitt V.L., Gabriel K., Traven A. and Lithgow T.J. Department of Biochemistry and Molecular Biology, Monash University, Melbourne.

The outer membrane of mitochondria is the interface between the organelle and its host. The SAM (sorting and assembly machinery) is embedded in this membrane where it helps insert and assemble vital membrane proteins and protein complexes. The core components of the SAM complex are conserved across eukaryotes but it appears to be able to co-opt additional subunits for some tasks. While previous studies have identified some of these subunits there is still very little known about how the core of the sorting and assembly process really works even for the most abundant proteins, such as porin (VDAC). Though extensively studied in Saccharomyces cerevisiae, understanding the details of the assembly and insertion of porin has been hampered by the instability of the porin complex. Preliminary studies in Candida albicans show porin assembles more quickly and into a more robust complex than in Saccharomyces. Import of radiolabeled proteins into purified mitochondria is used to elucidate the role of the SAM complex in this process. This more stable model system enables a more thorough comparison of the core SAM subunit, Sam50 and its bacterial homologue BamA (Omp85). Understanding the origins, interactions and roles of these components can help reveal how free-living bacteria were assimilated into the cell to become mitochondria.

MARSUPIAL INTERFERON INDUCIBLE TRANSMEMBRANE PROTEINS

Hickford D.E., Shaw G. and Renfree M.B. Zoology Department, The University of Melbourne.

The Interferon Inducible Transmembrane (IFITM) family is a group of cell surface proteins with roles in diverse cellular processes, including promoting homotypic cell adhesion, mediating cell differentiation and acting as a transducer of anti-proliferative signals. In humans, IFITM genes are up-regulated in some cancers and IFITM2 acts as a pro-apoptotic factor. In the mouse and human the IFITM genes are clustered; five Ifitm genes within a 68 kb locus on chromosome 7 in the mouse and four genes within a 26.5 kb locus on chromosome 11 in humans. Sequence comparison suggests that gene duplications at the mouse and human IFITM locus have occurred independently in both species. Despite the broad range of processes the IFITM genes have been implicated in, nothing is known about marsupial IFITMs. We have identified five IFITM homologs in the tammar wallaby Macropus eugenii, four of which are clustered in a 74.5 kb locus. There are four homologs in the opossum Monodelphis domestica, three of which are clustered on chromosome 3. Amino acid identity between the various IFITM proteins is 27-76% in the tammar and 35-64% in the opossum. Sequence analysis suggests that similar to the mouse and human, IFITM genes have been duplicated independently in marsupials. Using RT-PCR, we analysed the expression of IFITM genes in adult organs, embryos and fetuses of the tammar wallaby. Expression of the tammar IFITM genes differs to that in the mouse, suggesting that the roles of the IFITM genes may not be conserved between marsupials and eutherians.





DIFFERENTIAL EXPRESSION OF HEDGEHOG SIGNALING COMPONENTS AND SNAIL/E-CADHERIN IN HUMAN BRAIN TUMORS

Hong Y.R.1, 2 and Chou C.H.1, 2 1Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 2Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.

The Hedgehog (Hh) transcription factor Gli induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin. Epithelial-mesenchymal transition is also a determinants of the progression of tumorigenesis, following downregulation of E-cadherin. However, the role of Hh signaling components and Snail/E-cadherin in brain tumors is not yet fully understood. We analyzed the expression of Hh signaling components and Snail/E-cadherin in 69 brain tumors by reverse transcription–polymerase chain reaction (RT-PCR). The data showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. Our results also indicated that Snail and E-cadherin showed opposing expression in malignant tumors (high grade astrocytoma and metastasis). Snail and E-cadherin showed less correlation in benign brain tumors. We further investigated mutations of Gli2 and Snail by RT-PCR and direct sequencing. No mutation was observed on Gli2 but several sporadic mutations on Snail were found, including S96G, S111L, S111L/S119Y and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants were decreased 15%, 25% and 50%, respectively, whereas Y158* was increased 40% compared to wild type. Furthermore, our data showed that wild type Snail and S96G, S111L, S111L/S119Y translocated into the nucleus, while the Y158* mutant failed to translocate into the nucleus. Taken together, our results demonstrate that Hh signaling components, the expression and mutations of Snail, and the expression of E-cadherin may play an important role in human brain tumorigenesis.

CYTOTOXICITY AND OPTICAL-PHYSICAL STUDIES OF 6-ALKYNYL-COUMARIN DERIVATIVES

Hisao C. and Hong F. Department of Chemistry, National Chung Hsing University, Taiwan.

Many coumarin derivatives are biologically important molecules that possess antifungal, antibacterial, antimicrobial insecticidal, anti HIV and antibiotic activites. The coumarin moiety as chromophore of fluorescent ion indicator is a useful physical property for the bio-imaging technique in biological systems. It was expected that modification on coumarine with conjugated multiple bonds might bring about the desired change in either red-shift of wavelength or intensity of the emission. It is our intention to apply these compounds to biological systems by combining both the cytotoxicity and fluorescence property. Several attempts in the modification of 6-Br-coumarine with substituted alkynes by the Sonogashira reaction were pursued. Indeed, it led to the formation of several coumarine derivatives with expected red-shift of wavelength in fluorescence. It was also found that optical property was affected greatly by slight change the substitutents on the phenyl moiety which is linked to coumarin mainframe through a triple bond. Moreover, we applied these coumarine derivatives to biological systems, especially, on the study of cytotoxicity towards HeLa cells. These newly-made compounds indeed showed higher cytotoxicity towards cells compared with the unmodified one.

IDENTIFICATION OF PROTEIN-PROTEIN INTERACTIONS REGULATING SODIUM-PROTON ANTIPORTER ACTIVITY

Huynh D.1, 2 and Gendall T.1 1Department of Botany, La Trobe University, Bundoora, Victoria 3086, Australia. 2Cuulong Delta Rice Research Institute, Cantho, Vietnam.

In plants, the concentration and ratio of Na+ and K+ is crucial for homeostasis, particularly in stressful conditions, such as salinity, cold and drought. Ion homeostasis in plants is regulated by a large number of ion exchangers that control the movement of ions across the plasma and intracellular membranes. The class of sodium-proton exchangers designated as NHEs in animals or NHXs in plants and yeast, is highly conserved throughout eukaryotes. NHX5 and NHX6 are intracellular-localized proteins that are widely conserved in diverse plants. The C-terminal tail of NHX5 and NHX6 is also well conserved in many plant species suggesting that it may mediate a protein-protein interaction and act as a part of pH sensing mechanism. To investigate the functional significance of this well conserved C-terminal tail in NHX5 and NHX6, a yeast 2-hybrid screen has been performed to identify interacting proteins. The predicted C-terminal tail and the highly conserved regions of both NHX5 and NHX6 have been used as baits to screen existing 2-hybrid libraries. Positive colonies will be identified, and the interaction further will be analyzed using by deletion analysis of both the C-terminal region of NHX5 and NXH6, and the interacting protein(s). These results will be validated by in vitro pull down assays using recombinant proteins. To determine if NHX5 and NHX6 function as homo- or hetero-dimers, interactions between NHX5 and NHX6 have also been investigated by yeast two-hybrid system, bimolecular fluorescence complementation and co-immuno-precipitation.

THE HIJACKING OF MITOCHONDRIAL IMPORT MECHANISMS BY PORB FROM NEISSERIA MENINGITIDIS

Jiang J.-H.1, 2, Davies J.3, Strugnell R.A.2, Lithgow T.1 and Gabriel K.1 1Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia. 2Department of Microbiology and Immunology, the University of Melbourne, Victoria, Australia. 3Department of Microbiology, Monash University, Victoria, Australia.

PorB is a β-barrel protein from the pathogen Neisseria meningitidis that is targeted to host cell mitochondria and modulates apoptosis during bacterial invasion. The sorting and assembly machinery (SAM) is required for eukaryotic β-barrel protein import into mitochondria. To investigate the mechanisms behind PorB import, S35-labeled PorB was in vitro translated and incubated with mitochondria isolated from mouse tissue or import machinery deficient yeast strains. Folded PorB was distinguished from unfolded protein using semi-native gel electrophoresis. The rate of PorB folding in mammalian mitochondria is decreased if the mitochondrial outer membrane is ruptured by hypo-osmotic shock. This treatment releases soluble inter-membrane space (IMS) proteins such as the small Tim(s) and hence this suggests a role for IMS proteins in PorB folding. Mitochondria isolated from Sam50 mutant which are deficient in import of native mitochondrial β-barrel proteins also fold PorB at a slower rate. Taken together, the import of PorB into mitochondria is SAM pathway dependent. The exact localization of PorB in mitochondria and the roles of other SAM components will also be assessed in the future.





EXPRESSION AND REGULATION OF THIOREDOXIN IN CANCER CELLS DURING HYPOXIA

Karlenius T.C.1, 2, Shah F.L.1, 2, Clarke F.M.1, 2 and Tonissen K.F.1, 2 1School of Biomolecular and Physical Sciences, Griffith University, Nathan QLD 4111, Australia. 2Eskitis Institute for Cell and Molecular Therapies, Griffith University.

Redox homeostasis is crucial for cell survival. Too much oxygen in the cell leads to oxidative stress through the production of ROS, which reacts with the cells macromolecules causing cell damage and finally cell death. The cells defend themselves against oxidative stress through the production of antioxidants, which either neutralise ROS or reverse ROS-induced damage. In contrast, low oxygen levels in the cell lead to hypoxia. Under hypoxic conditions a signalling pathway involving a key regulator termed hypoxia-inducible factor (HIF) is switched on. HIF drives the induction of many genes controlling multiple cell functions such as angiogenesis, metabolism and apoptosis/survival. Thus, the level of oxygen in a cell dictates the molecular response of cells through modulation of gene expression. Furthermore, both oxidative stress and hypoxia are common features of solid tumours. Both oxidative stress and hypoxia lead to changes in the cellular redox balance within cancer cells. High levels of antioxidants and redox control systems, especially the Thioredoxin system, are often observed in cancer cells and are believed to play a major role in cancer progression. We are currently investigating the expression and regulation of Thioredoxin in breast cancer cell lines cultured in hypoxic conditions and after re-oxygenation.

PHYSIOLOGICAL SIGNIFICANCE OF D-AMINO ACID DEGRADATIVE ENZYMES IN NEMATODE CAENORHABDITIS ELEGANS

Katane M.1, Saitoh Y.1, Seida Y.1, Kawata T.1, Maeda K.1, Sekine M.1, Furuchi T.1, Kobuna H.2, Sakamoto T.1, Inoue T.3, Arai H. 3, Nakagawa Y.

1 and Homma H. 1

1Department of Pharmaceutical Life Sciences, Kitasato University, Tokyo, Japan. 2School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan. 3Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, D-Asp has been proposed to play important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO and DDO reportedly regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans has at least one active DAO gene and three active DDO genes, and that these enzymes exhibit different and distinctive enzymatic properties. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we examined the localization of these enzymes within the whole body of C. elegans using green fluorescent protein-based gene expression analysis, revealing that the spatiotemporal distributions of these enzymes differ from one another. We also examined several phenotypes of four C. elegans mutants in which each gene for these enzymes is partially deleted and inactivated. We will report the phenotypes of these C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.

CIAP ANTAGONISTS AND IFNγ ACTIVATE CASPASE AND RIPK1 DEPENDANT DEATH PATHWAYS IN CANCER CELLS

Khan N.1, Miasari M.1, Chau D.1, Wong W.W.-L.1, Mckinlay M.2, Chunduru S.K.2, Benetatos C.A.2, Condon S.M.2, Vaux D.L.1 and Silke J.1 1La Trobe University, Melbourne, VIC 3086, Australia. 2TetraLogic Pharmaceuticals 343 Phoenixville Pike Malvern, PA 19355.

Synthetic IAP antagonists have been shown to kill tumour cells as single agents or sensitise them to existing anti-cancer treatments, validating Inhibitor of apoptosis (IAPs) proteins as targets for anti-cancer therapeutics. We have previously shown that a peptide-mimetic IAP antagonist compound has similar effects on cells as the TNF family cytokine, TWEAK. Because some cancer lines can be sensitised to TWEAK cytotoxicity by co-treatment with IFNγ, we hypothesised that IFNγ might synergise with IAP antagonists to kill tumor cells. Consistent with our hypothesis, tumor cells that are sensitive to TWEAK/IFNγ were killed when treated with IFNγ and IAP antagonist while primary untransformed lines were unaffected. JAK/STAT signaling was required for cell death because synergistic killing could be blocked by SOCS1 overexpression, but the TNFR1 pathway was not. Another distinguishing feature of this IAP antagonist/IFNγ death was that it could not be blocked by caspase inhibition alone even though cells displayed classic apoptotic features. Surprisingly, caspase inhibition together with a RIPK1 inhibitor (Nec1) significantly attenuated synergistic killing by Compound A and IFNγ, as did RIPK1 knockdown. Our results suggest that IAP antagonists can activate both apoptotic and non-apoptotic cell death which may extend their clinical use.

INVESTIGATION OF HOW RASACT COOPERATES WITH RHOGEF2 OVER-EXPRESSION AND SCRIBBLE MUTANTS IN TUMOURIGENESIS

Khoo P., Brumby A.M., Humbert P.O. and Richardson H.E. Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria.

scribble (scrib) is a tumour suppressor gene which regulates cell polarity in Drosophila epithelial cells. Expression of activated Ras (RasACT) in scrib- clones in the Drosophila eye disc results in the development of tumours, characterised by decreased cell death, loss of cell polarity, hyper-proliferation and reduced differentiation. RhoGEF2, a gene involved in cell shape changes, also cooperates with RasACT in eye disc clones to result in tumourigenesis. Jun kinase (jnk) activity is both up-regulated and required for tumourigenesis in RhoGEF2 and RasACT tumours and scrib- + RasACT tumours. We compare the development of tumours arising from the co-expression of RhoGEF2 and RasACT in eye disc with scrib- + RasACT tumours and investigate the pathways downstream of RhoGEF2 in its cooperation with RasACT in clones. We show that RhoGEF2 + RasACT clonal tissue over-grows but not to the same extent as in scrib- + RasACT tumours and is not as invasive. Preliminary data indicate that when Rho 1, Rho kinase or Zipper levels are reduced in RhoGEF2 + RafGOF (a downstream effector of Ras) clones using UAS-RNAi transgenes, morphological defects are suppressed and differentiation is restored. These results suggest that Rho1, Rho kinase and Zipper are required for tumourigenesis arising from RhoGEF2 and RafGOF co-expression in clones.





PHYSIOLOGICAL FUNCTION OF A NOVEL NF-κB-REGULATING MOLECULE, NUCLING, IN IMMUNE SYSTEM

Kim S.M., Sakai T., Tran N.H. and Fukui K. Division of Enzyme Pathophysiology, The Institute for Enzyme research, The University of Tokushima.

Nucling is a novel stress-induced protein isolated as a marker for cardiac development. Recent our studies have revealed that Nucling is a regulator of nuclear factor-kappa B (NF-κB) operated by its cytoplasmic retention through the physical interaction with Nucling. Notably, Nucling is considered to be important for the regulation of NF-κB signals in liver. Nucling-KO mice showed several liver dysfunctions including hepatitis and cancer with some defects of NF-κB signal in liver. In addition, a number of kupffer cells, residential macrophages in liver, and hepatic dendritic cells apoptotically decreased in the Nucling-KO mice. Thus we focused on the function of Nucling in immune system. Further analyses revealed that macrophages of the Nucling-KO mice were decreased in lung. On the other hand, Nucling-KO mice were resistant to endotoxin (Lipopolysaccharide) similarly to TNFα-KO mice. It is correspond to our previous report showing the defect of NF-κB signal in Nucling-KO mice. Our recent studies indicated that the spleen of Nucling-KO mice showed abnormally activated germinal centers without immunologic stimulation. Besides, we observed dissimilarity of B cell subset distribution between Nucling-KO mice and wild-type mice. Therefore, we speculate that there are some differences between Nucling-KO mice and wild-type mice concerning the functions of macrophages in the aspect of its quantity or activity. Nucling is considered to play an important role in the immune system with the regulation of NF-κB signal pathway.

REACTIVE OXYGEN SPECIES GENERATED THROUGH NADPH OXIDASE MEDIATE UV-INDUCED GSK-3BETA ACTIVATION

Kim H.S., Kwon Y.H., Kim J.Y. and Sohn J. Department of Biochemistry & Molecular Biology, Korea University College of Medicine, Seoul 136-705, Korea.

Previously, we have reported that UV induced ATR-dependent GSK-3β activation and subsequent proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, we show that reactive oxygen species (ROS) are generated by UV irradiation and GSK-3β activation. ROS generation was detected in the cytosol and mitochondria at 5 min after UV exposure. NADPH oxidase contributed to the ROS generation since chemical inhibitors of NADPH oxidase, DPI and AEBSF, abrogated it. NOX4 mRNA was expressed in MCF-7 cells, and transfection of the cells with NOX4 siRNA prevented UV-induced ROS generation. DPI and apocynin also inhibited UV-induced GSK-3β activation indicating that ROS generated by NADPH oxidase mediated GSK-3β activation. Further supporting redox-sensitive activation of GSK-3β, treatment of cells with hydrogen peroxide activated GSK-3β. ROS scavengers and NOX4 siRNA abolished p21 protein degradation after UV-irradiation, confirming the previous finding that p21 phosphorylation by GSK-3β was required for p21 degradation. To investigate whether changes in the [Ca2+]i mediated NADPH oxidase activation after UV irradiation, the effect of Ca2+ chelators on ROS production was assessed. Indeed, pretreatment with EGTA or BAPTA-AM blocked ROS generation indicating Ca2+-dependent activation of NADPH oxidase. In MCF-7 cells, increase in [Ca2+]i was detected < 5 min after UV irradiation and continued up to 2 h. Since GSK-3β activation by UV was shown to be dependent on ATR, it was next studied whether an increase in [Ca2+]i and ROS generation were upstream to ATR activation. Phosphorylation of ATM/ATR substrates after UV irradiation was observed 15 min after UV irradiation. Pretreatment of cells with BAPTA-AM, Tiron or NAC prevented their phosphorylation indicating that ATR activation was dependent on intracellular Ca2+ and ROS. Taken together, UV mobilizes intracellular Ca2+ and thereby activates NADPH oxidase. ROS generated through NADPH oxidase then activate ATM/ATR and that, in turn, induces GSK-3β activation and p21 degradation.

A ROLE FOR SODIUM/PROTON EXCHANGERS AND INTRACELLULAR HYDROGEN ION CONCENTRATION IN REGULATING VITAMIN C-DRIVEN ELECTRON TRANSPORT ACROSS THE PLASMA MEMBRANELane D.J.R.1, Robinson S.R.2, Czerwinska H.2 and Lawen A.1 1Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University. 2School of Biomedical Sciences and Psychology & Psychiatry, Monash University. Ascorbate is the major electron donor to a transplasma membrane electron transport (tPMET) system that was originally identified in human erythrocytes [1]. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g., non-transferrin-bound iron). Though this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. Here we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose-transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated tPMET by the Na+/H+-exchange inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride, which is diminished by bicarbonate, suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, while it was significantly stimulated by bicarbonate-induced intracellular alkalinisation. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH [2]. Though the identity of the putative enzyme(s) responsible for electron transfer across the plasma membrane has yet to be identified, it has been suggested that members of the ubiquitous cytochrome b561 family (e.g., Dcytb) are likely to be involved. [1] Lane and Lawen (2009) Free Radic. Biol. Med. 47, 485-495. [2] Lane et al. (2010) Biochem. J. 428, in press.

GSK3β INTERACTS WITH AND PHOSPHORYLATES BCL2L12 IN BRAIN CANCER CELLS

Lin C.C., Chen W.J, Lin R.C., Chou C.H., Wu C. and Hong Y.R. Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

A novel GBM oncoprotein, Bcl2-Like 12 (Bcl2L12, a BH2 containing protein), was recently identified which is significantly expressed in the majority of primary GBM tumor specimens and distantly related to canonical Bcl-2 proteins. By using large scale yeast 2-hybrid screening, Bcl2L12 was found as a GSK3β binding partner in testis cDNA library. Our data showed that Bcl2L12 middle fragment (118-240 residues) which locates outside of Bcl2L12 C-terminal BH2 motif is responsible for the binding to GSK3β and also confirmed by far-western blotting experiment. In vitro kinase assay showed that GSK3β phosphorylates Bcl2L12 at S156 but not Bcl2L12A (Bcl2L12 splicing transcript variant), LiCl (GSK3 inhibitors) and phosphatase were used to confirm that GSK3β indeed phosphorylates Bcl2L12 at S156. Interestingly, the S156 phosphorylation site on Bcl2L12 does not fit the GSK3β consensus sequence (S/T-X-X-X-S/Tp or S/T-P). To explore the significance of the interaction between GSK3β with Bcl2L12 and Bcl2L12 S156A for assessing the morphological changes and caspases 2, 3 and 7of apoptosis, we transfected Bcl2L12 and Bcl2L12 S156A into the HeLa and Glioma cell lines (U87MG and GBM 8401).The data showed that Bcl2L12 induced apoptosis in Hela cell, whereas in GBM and U87MG may function as anti-apoptosis. Ongoing studies will combine TMZ (an alkylaing agent against recurrent glioma) to examine how Bcl2L12 gets involved and which may function as a chemotherapy biomarker in brain tumorgenesis.





PLASTIDAL STARCH PHOSPHORYLASE IN SWEET POTATO ROOTS IS PROTEOLYTIDY REGULATED BY THE 20S PROTEASOMELin Y.C., Chou I.M., Chen A.N., Cheng C.H., Chang S.C. and Juang R.H. Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei 106, Taiwan. Starch phosphorylase (SP, EC 2.4.1.1) catalyzes the reversible phosphorolysis of starch and produces glucose 1-phosphate (Glc-1-P) as one of its products. Plants express two isoforms of SP, which are classified as low-affinity (L-form SP, L-SP or Pho1) and high-affinity (H-form SP, H-SP or Pho2) types according to their binding affinities to starch. Although the role of L-SP in starch metabolism is unclear, several studies have found that the gene expression and catalytic activity of L-SP correlate with starch content in plants, indicating that L-SP might be involved in starch biosynthesis. In addition, L-SP in Arabidopsis leaves might play a role in the tolerance of abiotic stress (Zeeman et al., 2004). Previous studies with sweet potato roots have shown that the activity of L-SP may be regulated by proteolysis of the central 78-amino acid peptide (L78). Removal of L78 increased the catalytic activity of L-SP in a phosphorolytic direction (Chen et al., 2002). During the purification of L-SP from sweet potato roots, Chang (1999) observed an unknown high molecular weight complex (HX), whose mobility is significantly slower than the typical L-SP on non-denaturing polyacrylamide gel electrophoresis, also performed L-SP activity. Following this observation, we utilize mass spectrometry, coimmunoprecipitation, agarose-gel based double diffusion, two-dimensional gel electrophoresis, and confocal microscopy as the tools and demonstrate that HX was composed of L-SP and the 20S proteasome. Furthermore, we found that HX was reduced immediately after 45°C heat treatment, and then a stepwise degradation of L-SP was observed in a time-dependent mode which was strongly inhibited by MG132, suggesting that the 20S proteasome was involved in L-SP turnover. In addition, kinetic studies indicated that the proteolytic modification of L-SP might increase the binding affinity of L-SP against soluble starch and subsequently enhance its phosphorolytic activity. This work demonstrates the role of 20S proteasome as a regulator of L-SP activity which might be controlled by environmental heat stress.

P21-ACTIVATED KINASE 1 IS IMPORTANT FOR SURVIVAL OF COLORECTAL CANCER CELLS

Liu H., Huynh N., Baldwin G. and He H. Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia 3084.

Background and Aim: P21-activated kinase 1 (PAK1) functions as a key node in various signalling pathways leading to cell survival, migration and growth. PAK1 is overexpressed and activated in several human tumors including colorectal cancer (CRC), which is the second most common cause of cancer death. PAK1 is also required for vascular endothelial growth factor (VEGF) expression and consequently for angiogenesis, which in turn promotes tumor growth and metastasis. VEGF is a downstream target gene of hypoxia-inducible factor 1α (HIF-1α) which is involved in cellular adaptation to hypoxia and important for cell survival. The aim of this study was to investigate the importance of PAK1 in CRC cell survival. Methods: PAK1 knock-down (KD) clones of the human CRC cell line DLD1 were obtained by stable transfection with shRNA targeting PAK1. Hypoxia was mimicked by treatment with CoCl2 and cell survival was measured by [3H]-Thymidine incorporation. Protein expression was determined by Western Blot and VEGF production was measured by ELISA. Results: After treatment with 150μM CoCl2, cell survival was significantly reduced by 40% in PAK1 KD cells (P<0.05). The expression of HIF-1α and the production of VEGF were both significant lower in PAK1 KD cells compared to control cells. Conclusion: This study demonstrates that PAK1 is required for cell survival and VEGF production of CRC cells. PAK1 may regulate these cellular processes through a HIF-1α-dependent pathway.

PSPC1 IS AN IMPORTIN ALPHA NUCLEOCYTOPLASMIC TRANSPORT CARGO

Major A.T.1, 3, Hogarth C.A.4, Miyamoto Y.2, 3, Young J.C.2, 3, Jans D.A.2,

3 and Loveland K.L.1, 2, 3 1Department of Anatomy and Developmental Biology, Monash University, Australia. 2Department of Biochemistry and Molecular Biology, Monash University, Australia. 3The ARC Centre of Excellence in Biotechnology and Development, Australia. 4School of Molecular Biosciences, Washington State University, USA.

Importin proteins facilitate classical nuclear import by binding nuclear localisation sequences (NLSs) on cargo proteins and translocating them into the nucleus. Through a Y2H screen of an E12.5 mouse testis cDNA library, we identified paraspeckle protein 1 (PSPC1) as an importin α2 cargo. The long isoform of PSPC1 is 523aa, contains two putative NLSs and is highly expressed in the testis. PSPC1 is a marker for paraspeckles, a distinct subnuclear domain formed around the non-coding RNA transcript, NEAT1, and present only in differentiated cells. Paraspeckles are rich in RNA transcripts and two other core paraspeckle proteins, PSF and NONO. Recent research suggests that paraspeckles form a structural base by which A to I edited RNA transcripts may be retained within the nucleus. We addressed the hypothesis that trafficking of PSPC1 into the nucleus and its subsequent localisation into paraspeckles is mediated specifically by importin α2. Binding between PSPC1 and both importin α2 and α6 was confirmed using recombinant proteins in an ELISA based binding assay. The extent of PSPC1 nuclear import was quantitated following transient transfection of HeLa cells with constructs that over-express GFP-tagged full length and truncated (dominant negative) constructs encoding several importins. Data are collected following co-transfection of DsRed2-tagged PSPC1 or assessed following endogenous PSPC1 detection via immunostaining. Dominant negative importin α2 appears able to reduce the number of detectable paraspeckles, indicating the importance of regulated importin synthesis to cell function.

REGULATION OF EMBRYONIC GLOBINS BY THE KRüPPEL-LIKE FACTORS

Mak K.S.1, Funnell A.P.W.1, Pelka G.J.2, Radziewic T.2, Power M.2, Tam P.P.2, Pearson R.C.M.2 and Crossley M.1 1School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052. 2Embryology Research Unit, Children’s Medical Research Institute, Westmead, NSW, Australia.

Basic Krüppel-like Factor (Bklf/Klf3) is one of 17 members of the Krüppel-like family of transcription factors that regulate biological processes such as cell proliferation, differentiation and apoptosis. Bklf is highly expressed in erythroid tissues, consistent with its direct activation by Erythorid Krüppel-like Factor (Eklf), an erythroid-specific transcriptional activator. Bklf knockout mice exhibit a mild deficiency in the erythroid population Chromatin immunoprecipitation experiments have shown that Bklf directly binds to the Klf8 gene which is up-regulated in the Bklf-null erythroid tissues. Klf8, like Bklf, functions primarily as a transcriptional repressor, recognising similar DNA sequences and repressing genes by recruiting the same co-repressor, Ctbp. Given the similarities between Bklf and Klf8, we hypothesize that Klf8 might functionally compensate for the absence of Bklf and that this may account for the mild phenotype of the Bklf knockout mouse. We have intercrossed Bklf and Klf8 mutant mice and purified erythroid cells from compound mutants of various genotypes. Affymetrix arrays have been performed to study the differences in gene expression resulting from the disruption of Bklf or Klf8 or both. Eliminating Klf8 activity in erythroid cells should reveal not only the full biological function of Bklf, but also distinguish target genes that are shared between or specific to Bklf and Klf8.





HEPATOCYTE GROWTH FACTOR (HGF) ACUTELY REGULATES THE EPITHELIAL JUNCTIONAL CYTOSKELETON AND ZONULA ADHERENS BY CONTROLLING JUNCTIONAL MYOSIN VI

Mangold S. Institute for Molecular Bioscience, 306 Carmody Rd., Brisbane 4072, QLD.

HGF plays an important role in various morphogenetic events during embryonic development, as well as being implicated in tumor invasion and metastasis. Many studies have investigated the long-term effect of HGF treatment on epithelial cells. HGF binds with high affinity to the receptor tyrosine kinase c-Met. Activation of c-Met leads to recruitment of adaptor proteins and in turn to activation of a multitude of down stream signalling molecules, including PI3-Kinase, Src and Ras/MAPK. The resulting signalling cascades initiate a broad range of cellular responses such as proliferation, migration, invasion and even complex events like branching morphogenesis in 3D matrices. All of these effects occur after long-term treatment (several hours to days) with HGF. Our lab is interested in the formation and function of E-cadherin based cell-cell adhesions and their significance both under physiological conditions and in disease. To allow morphogenetic changes or processes like cell migration, the zonula adherens and the cytoskeleton must be able to respond quickly and in an orchestrated fashion to the initiating HGF signal. In this study we investigate the acute effects of early HGF treatment on the epithelial junctional cytoskeleton and the zonula adherens in CaCo2 cells. We report a dramatic re-organization of the apical actin ring within the first 15min of HGF treatment, which is accompanied by a loss of Myosin VI from the adherens junctions. Myosin VI is an unconventional actin binding motor protein that has previously been shown to play a role in the stabilization of adherens junctions and the regulation of F-actin organization. We identify Myosin VI as a novel down stream target of HGF signalling and elucidate that Calcium signalling plays a crucial role in the HGF induced regulation of junctional Myosin VI.

ACTIVATION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) BY DNA-DEPENDENT PROTEIN KINASE (DNA-PK) OR ATAXIA TELANGIECTASIA MUTATED (ATM) IS NOT NECESSARY FOR ITS PROAPOPTOTIC FUNCTIONS

Marzec K.A., Martin J.L. and Baxter R.C. Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia.

Chemotherapeutic drugs such as doxorubicin, which induce apoptosis by causing DNA double strand breaks (DSB), are opposed by the phosphoinositide-3-kinase-related protein kinases, DNA-PK and ATM, which promote DSB repair. Our laboratory has previously described the phosphorylation by DNA-PK of IGFBP-3, which was reported by others to be required for the pro-apoptotic function of IGFBP-3. The aim of this study was to investigate the dichotomy where DNA-PK inhibition enhances apoptosis by preventing DSB repair, yet opposes apoptosis by inhibiting IGFBP-3 phosphorylation. ATM phosphorylates similar sites to DNA-PK and may also have a role in activating IGFBP-3. We assessed the response of phenotypically normal breast cells (MCF-10A) and breast cancer cells (Hs578T, MDA-MB-231 and MDA-MB-468) to doxorubicin, alone or with the ATM inhibitor KU55933 or the DNA-PK inhibitor NU7441 (KuDOS Pharmaceuticals), monitoring caspase-3 activity in cell lysates as a marker of apoptosis. Apoptosis induced over 24-72 h by 0.3-1 μM doxorubicin was significantly enhanced in all cells treated with 1 μM NU7441 or 10 μM KU55933. To investigate its role in this response, endogenous IGFBP-3 was downregulated in Hs578T cells ≥80% with small interfering RNA. Doxorubicin-induced apoptosis was significantly reduced in IGFBP-3-silenced cells compared to control cells, confirmed by decreased cleavage of the caspase-3 substrate, poly(ADP-ribose) polymerase. These data suggest that endogenous IGFBP-3 potentiates doxorubicin-induced apoptosis, and this activity is not prevented by DNA-PK or ATM inhibition.

GENETIC TRANSFORMATION IN COLLETOTRICHUM TRUNCATUM ASSOCIATED WITH ANTHRACNOSE DISEASE OF CHILI PEPPER

Auyong A.S.M., Ford R. and Taylor P.W.J. BioMarka/Plant Health Centre, Department of Agriculture and Food Systems, 3010 Parkville, The University of Melbourne, Australia.

Colletotrichum truncatum is the causal agent of chili pepper anthracnose causing severe economic loss through reduced yield and marketability of infected fruit. To this extent an efficient and reliable transformation system of the pathogen is required to prove the function of isolated pathogenicity genes through gene silencing or mutagenesis. Agrobacterium tumefaciens-mediated transformation (ATMT) system was developed for C. truncatum. A. tumefaciens carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene was used to transform the conidiospores of two C. truncatum pathotypes (F8-3B and BRIP26974). Optimum transformation efficiency was obtained when equal volume of A. tumefaciens strain AGL1 carrying either pJF1 or pPK2 binary vector was transformed with 1000000 C. truncatum conidiospores/ml and co-cultivated at 24°C for three days. Southern blot analysis and TAIL-PCR indicated that most of the transformants contained randomly inserted, single copies of the T-DNA. Infection and colonization of chili pepper fruit at the mature red stage with F8-3B-GFP and BRIP26974-GFP confirmed the virulence of these transformed pathotypes. Analysis by fluorescent microscopy showed that colonization of parenchyma cells of fruit pericarp tissue occurred by subcuticular intramural infection. In the susceptible Capsicum annuum genotype, fungal hyphae was detected between the cell walls of parenchyma cells up to 1 cm in advance of the visible lesion, indicating that C. truncatum entered a short endophytic stage in its disease cycle before becoming necrotrophic.

PROTEOMIC STUDIES ON A HIGH AMYLOLYTIC NIGERIAN MAIZE CULTIVAR

Awoyinka O.A.1, 2, Adebawo O.O. 1, 2, Daini O.A.2 and Dipanker C.3 1Babcock University, Ilisan-Remo, Nigeria. 2Olabisi Onabanjo University, Ago-Iwoye, Nigeria. 3Indian Institute of Science, Bangalore India.

This study sought a high amylolytic Nigerian maize cultivar. It also established the amino acid sequence that forms the primary structure of the β amylase found in the maize cultivar. Purification steps comprising of fractional precipitation by ammonium sulphate, gel filtration and anion exchange chromatography was used respectively to purify β amylase from the malt of TZEE*TZEE-W*DEMARSCUS*TZEE-W before determination of the primary structure of the β-amylase with the aid of Matrix assisted laser desorption ionization time- of- flight (MALDI- ToF) mass spectrometry. Results obtained showed that An apparent 60 KDa monomeric protein was detected on a single dimensional 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Well complete primary structure deduced showed a signature of a highly conserved ubiquitous not yet reported β-amylase comprising of 505 amino acid sequence. However, mass spectrometry analysis further carried out on concomitant proteins eluted with the β-amylase during partial purification gives a hypothetical insight into the metabolic activities of the high amylolytic maize cultivar during malting. This could be explored in quest of improving the diastatic power of the Nigerian maize cultivar. Keywords: β-amylase, Malting, Maize, Mass spectrometry and Proteomics.





NON-DESTRUCTIVE HIGH-THROUGHPUT PHENOTYPING IN THE PLANT ACCELERATOR TO STUDY PLANTS UNDER SALT AND WATER STRESS CONDITIONS

Berger B.1, 2, 3 and Tester M.1, 2, 3 1The Plant Accelerator. 2Australian Center for Plant Functional Genomics. 3University of Adelaide, Waite Campus, Urrbrae SA, 5064.

Salinity and drought have a major impact on agricultural productivity, likely to increase with climate change. To secure global food production, crop plants with increased yields under these stress conditions are urgently needed. Whereas gene and marker discovery have become faster than ever, it is now the phenotyping process that is increasingly limiting the generation of more tolerant crop plants (Finkel, 2009; Furbank, 2009; Tester and Langridge, 2010). Both salinity and drought trigger dynamic physiological responses that require continuous phenotypic measurements to be able to dissect overall tolerance mechanisms into individual traits. The Plant AcceleratorTM combines automated plant handling and high-throughput imaging to alleviate the phenotyping bottleneck. Water stress regimes are controlled and recorded with automated watering and weighing stations. At the same time, various phenotypic traits can be assessed non-destructively by digital imaging in different wavebands. Colour images can be used to estimate biomass production and to measure growth rates and the degree of senescence. Temperature differences between control and stressed plants, commonly used as a surrogate for stomatal conductance, are determined with infrared cameras. Furthermore, changes in leaf water content over time are monitored using near infrared imaging. The Plant AcceleratorTM, therefore, enables researchers to monitor important aspects of the physiological changes occurring under salt and water stress conditions. The high-throughput capacity provides the opportunity to screen large populations, making possible forward genetics approaches to the study of many relevant aspects of plant responses to salt and drought stress. Finkel, E. (2009) Science 325, 380-381. Furbank, R.T. (2009) Functional Plant Biology, 36, v-vi. Tester, M. and Langridge, P. (2010) Science 327, 818-822.

MOLECULAR ANALYSIS OF PHOSPHITE INDUCED RESPONSES IN ARABIDOPSIS

Berkowitz O.1, Jost R.2, Hardy G.S.E.J.1 and O’Brien P.A.1 1Murdoch University, School of Biological Sciences and Biotechnology. 2The University of Western Australia, School of Plant Biology.

Phosphite (H2PO3-) is a phosphate analog widely used to protect

plants from oomycete pathogens such as Phytophthora and Phytium. Phytopthora species are prominent pathogens in agriculture, e.g. Phytopthora infestans being the causing agent of potato blight (Irish potato femine). Phytopthora cinnamomi has devastating effects (“dieback disease”) on native ecosystems with over 2000 plant species at risk in Western Australia alone. Phosphite is the only known protectant of plants and exhibits a complex mode of action. At elevated concentrations it directly inhibits the pathogen’s growth by interference with its phosphate-dependent metabolism which is paralleled in plants grown on high phosphite concentrations. At the same time it also inhibits the plant’s phosphate starvation response, e.g. the up-regulation of high-affinity phosphate transporters, and thus has constrictive effects on plant growth under low phosphate supply. In addition to these direct effects phosphite also induces some of the plant’s defence responses, e.g. treatment of plants leads to increased expression of defence genes. However, the underlying mechanism of this indirect effect is not understood. We have started to characterise the impact of phosphite on plant defence responses by analyses of gene expression and metabolic pathways. Transgenic plants have been generated that express a microbial phosphite dehydrogenase which converts phosphite into phosphate. These plants are a valuable tool to dissect direct from indirect phosphite effects.

GENE EXPRESSION OF PONCIRUS TRIFOLIATA, CITRUS SUNKI AND THEIR HYBRIDS UNDER INFECTION OF PHYTOPHTHORA PARASITICA USING REAL-TIME QUANTITATIVE PCR

Boava L.P., Cristofani-Yaly M., Mafra V.S., Kubo K.S., Stuart R.M. and Machado M.A. Centro APTA Citros Sylvio Moreira, CP4, 13490-970, Cordeiropolis-SP, Brazil.

Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) is an important oomycete causing damage in Citrus nurseries and orchards and around the world. To integrate breeding and genomics programs to provide resistance against P. parasitica, the identification of genes involved in disease response is required. In this study, we proposed that genes differentially expressed between resistant and susceptible hybrids of P. trifoliata and C. sunki (resistant and susceptible, respectively) may provide key candidates to identify transcripts involved in disease resistance. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison to their parents 48 hours after P. parasitica inoculation using real-time quantitative PCR. Our analysis searched up regulated genes (fold ≥ 2 and p-value ≤ 0.05) in the resistant genotypes relative to the susceptible, found in previous microarray study. These genes were selected due to the biological interest based on their function according to CitEST database, among them, genes that encode enzymes participating in defense-related metabolic pathways such as the biosynthesis of phenylpropanoids and antimicrobial compounds such as phytoalexins, flavonoid biosynthesis and resistance genes such as CC-NBS-LRR and TIR-NBS-LRR. Our results showed that all analyzed transcripts were up regulated in P. trifoliata relative to C. sunki and in the resistant pool relative to C. sunki and to susceptible pool. This suggests that a number of defense strategies are activated following the recognition event encoded by Phytophthora resistance genes. The genes that we have identified as up regulated across the resistant genotypes will be valuable for ongoing work in eQTL mapping. Financial support: FAPESP-INCT/CNPq.

AUXINS: CRUCIAL TO GRAPE BERRY RIPENING

Bottcher C.1, Harvey K.1, Forde C.2, Boss P.K.1 and Davies C.1 1CSIRO Plant Industry, PO Box 350, Glen Osmond SA 5064, Australia. 2Nestle S.A., 1800 Vevey, Switzerland.

Fruit ripening is a complex process which seems to be largely regulated by plant hormones. In contrast to climacteric fruit, such as bananas and tomatoes, the ripening of non-climacteric fruit, e.g. strawberries and grapes, seems to be less dependent on ethylene and might be controlled by several other hormones. We are investigating the role of hormones during grape berry development and are testing their ability to manipulate ripening. While the application of some hormones, e.g. absicic acid and brassinosteroids, can advance the onset of berry ripening (veraison) others, e.g. auxins, delay it. The work presented here focuses on the ripening-delaying effects of auxins that have been reported for a number of climacteric and non-climacteric fruit. Of particular interest is the ability of synthetic auxins like 1-naphthaleneacetic acid (NAA) to retard grape berry ripening. This ability to delay ripening, and therefore harvest, has potential advantages for the wine industry. NAA applied to berries approximately two weeks prior to veraison delayed ripening by about ten days as measured by reduced sugar levels and skin colouration. A decrease in levels of indole-3-acetic acid (IAA), the most abundant auxin in plants, occurs during the development of a number of different fruit. This decrease also occurs in grapes and the potential for IAA to act as an endogenous inhibitor of grape berry ripening is a current research focus. In this context the role of IAA-conjugation in the initiation of berry ripening is examined.





IN VIVO ROLE FOR FLAVONOIDS IN AUXIN TRANSPORT AND GRAVITY RESPONSES

Buer C.S. and Djordjevic M.A. ARC Centre of Excellence for Integrative Legume Research; Research School of Biology; College of Medicine, Biology, and Environment; The Australian National University; Canberra ACT; Australia.

Flavonoids are bioactive plant secondary metabolites with a myriad of important functions (Buer et al., 2007, 2009, 2010). The well-characterised Arabidopsis thaliana transparent testa (tt) mutants are compromised in various flavonoid pathway structural enzymes and regulatory genes (Buer et al., 2007, 2008). Several lesions result in differential root flavonoid accumulation when compared to wild-type roots (Buer et al., 2010). This differential accumulation allowed testing whether certain flavonoids controlled auxin transport and subsequent root gravity responses. The greatest changes in gravity response, root elongation, and auxin transport were in the mutants accumulating quercetin (tt3, tt8, tt10, and ttg1). Increased quercetin accumulation caused rapid gravitropic curvature, faster root elongation rates, and higher auxin transport inhibition compared to the wild type. Those mutants with no or low quercetin accumulation (tt4, tt5, and tt6) were inhibited in gravity responses and had increased auxin transport rates confirming earlier experiments with tt4 (Buer and Muday, 2004). These in vivo assays extend earlier in vitro assays (Jacobs and Rubery, 1988) showing that flavonoids inhibit auxin transport with quercetin showing the greatest effects and that flavonoids are important for normal auxin flux and plant development. Buer C, Muday G, Djordjevic M (2007) Plant Physiology 145: 478-490. Buer C, Muday G, Djordjevic M (2008) Plant Signalling and Behavior 3: 415-417. Buer C, Djordjevic M (2009) Journal Experimental Botany 60: 751-763. Buer C, Imin N, Djordjevic M (2010) Journal of Integrative Plant Biology 52: 98-111. Buer C and Muday G (2004) Plant Cell 16: 1191-1205. Jacobs M and Rubery P (1988). Science 241: 346-349.

SPECTRAL EXTENSION OF PHOTOSYNTHESIS

Chen M. University of Sydney.

Chlorophyll a is typically the major photosynthetic pigment in all oxygenic photosynthetic organisms (plants, algae and Cyanobacteria) with Chl b and c playing only an accessory light-harvesting function. The newly discovered cyanobacterium Acaryochloris marina contains Chl d as its major pigment. Chl d in Acaryochloris is involved in both the accessory light-harvesting processes and as the primary donor pair in Photosystem I and Photosystem II. This is the first known example where a chlorophyll other than Chl a is involved in the primary chemical reaction in a reaction center as is the case in all other oxygenic photosynthetic organisms. The unique optical properties of chlorophyll d fill in the absorption gap between chlorophylls (Chl a/b) and bacteriochlorophylls which has shattered the old scheme that Chl a is the only major chlorophyll of photosynthesis in oxygenic organisms, and has broadened the potential to extend the range of photosynthesis into a new spectral region. Chl d is the only Chl known so far that can replace all roles of Chl a in oxygenic photosynthesis. Here, I wish to report the discovery of the biochemical steps, at gene and protein levels, leading to the formation of the unique pigment chlorophyll d, which will lead us to the possibility for genetic engineering of these important chlorophylls. A potential new biotechnology application has been developed. We are seeking the application in improving the crop spectral properties, by extending their light absorption properties into a new spectral region. Recently, we have discovered an unknown chlorophyll from stromatolites pigment extractions. Using various spectral analyses, we confirmed that it is a new type of chlorophyll molecule, which showed a even further red-shift absorption Qy peak. This discovery challenges the limitation of spectral wavelength region for oxygenic photosynthesis, which may open new bioenergy applications.

RNAI-MEDIATED MANIPULATION OF NITROGEN DEFENCES IN PLANTS USING PYRIDINE ALKALOID BIOSYNTHESIS IN NICOTIANA AS AN EXPERIMENTAL SYSTEM

Dalton H.L., DeBoer K.D., Neale A.D. and Hamill J.D. School of Biological Sciences, Monash University, VIC, Australia.

Alkaloid synthesis in plants usually involves the diversion of amino acid precursors from primary into to secondary metabolism. A good example is the production of toxic pyridine alkaloids, which are a feature of all species in the genus Nicotiana and some other genera in the Solanaceae family. Alkaloids are recognised as being important defensive compounds, the synthesis of which can increase in response to wounding in order to provide protection from predators in native habitats. Wound- or jasmonate-induced production of nicotine in Nicotiana tabacum is correlated with elevated transcript and enhanced activity of a number of key alkaloid biosynthetic enzymes such as ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). Our previous research has indicated that antisense-mediated down regulation of ADC transcript levels has very little effect upon capacity of transgenic N. tabacum to produce nicotine. In contrast, RNAi-mediated down regulation of ODC transcript levels in N. tabacum produced a substantial decrease in nicotine levels which was accompanied by a marked increase in concentrations of anatabine. Treatment of ODC-RNAi hairy root cultures and regenerated plants with methyl-jasmonate did not restore the plants capacity to produce normal amounts of nicotine and anatabine. These results suggest that ODC has an important role in providing the putrescine used in alkaloid biosynthesis in N. tabacum, particularly in the wound response. Ongoing work is examining the wider effects of these RNAi manipulations upon activity of related genes and enzymes in primary and secondary metabolism and the capacity of these transgenic Nicotiana tissues to redirect nitrogen from secondary metabolism back into primary metabolism and growth – particularly in response to abiotic and biotic stresses.

CHARACTERISATION OF TWO NECROSIS AND ETHYLENE INDUCING PROTEIN-LIKE (NLP) GENES FROM SCLEROTINIA SCLEROTIORUM

Dinsdale A.J.1, Van Den Elsen F.2, Barnett R.1 and Plummer K.1 1La Trobe University, Bundoora, Victoria, Australia. 2Wageningen UR Plant Breeding, AJ Wageningen, The Netherlands.

The necrotrophic phytopathogen, Sclerotinia sclerotiorum, induces plant cell death in order to colonise host plants and release nutrients. This pathogen is known to secrete various compounds, including oxalic acid and lytic enzymes during infection. Infection is often facilitated in other necrotrophs (such as Fusarium oxysporum and Botrytis cinerea) by the secretion of small, phytotoxic effector molecules (necrosis and ethylene inducing peptides, NEPs). We have cloned two genes from Sclerotinia sclerotiorum with significant similarity to NEPs called NEP-like1 (Nlp1) and Nlp2. Both NLPs appear to induce necrosis when transiently expressed in Nicotiana tobaccum and N. benthamiana. Multiple 35S fusion constructs, designed to investigate the effect of in planta NLP protein expression (with or without signal peptides) have also been completed and transformations are currently underway. Both genes have also been expressed in Pichia pastoris resulting in purified NLP1 and NLP2 that will be used for further transient assays to assess the specific response induced in host plants when presented with one or both proteins. Multiple Nlp1RNAi mutant lines have been generated which display inhibited growth rates both in vitro and in planta; further characterisation of these will be presented. Expression localisation studies are also underway using Nlp promoter:GFP fusion constructs.





CHLAMYDOMONAS REINHARDTII - A MODEL ALGA FOR THE STUDY OF TRIACYLGLYCEROL BIOSYNTHESIS AND LIPID BODY BIOGENESIS

Djordjevic M.A., Chen H.-C., Hillier W., Hocart C. and James G. Plant Science Division, Research School of Biology, The Australian National University, Canberra, Australia.

C. reinhardtii is an emerging model algal system to study triacylglycerol biosynthesis and storage in lipid bodies (Wang et al., Eukaryot. Cell, 2009; 8: 1856-1868; Moellering et al., Eukaryot. Cell, 2010; 9: 97-106; Yanto et al., Metabolic Engineering, doi:10.1016/j.ymben 2010.02.002; Dean et al., Bioresource Technology, 2010; 12: 4499-4507). Normally, C. reinhardtii, when cultivated under nutrient limited conditions, stores carbon predominantly as starch with only low levels as triacylglycerols. Mutations in ADP-glucose pyrophosphorylase inhibit starch synthesis and concomitantly redirect carbon into lipid biosynthesis and storage of triacylglycerols in lipid bodies when nitrogen deprived. We have found C. reinhardtii can store 40% of dry cell weight as lipid under nitrogen starvation at low light (100 µmol m-2 s-1). Fourier transform infrared (FTIR) spectroscopy was used to develop high throughput methods for semi-quantitative measurements of cellular protein, carbohydrate and lipid content. Targeted transcriptional profiling by quantitative real-time PCR was performed to investigate the temporal relationships of key genes involved in the induction of lipid biosynthesis and those associated with lipid body formation. Proteomic analysis on isolated lipid bodies identified a key lipid body associated protein. We conclude that C. reinhardtii is an alga that can be manipulated to produce high levels of triacylglycerols making it a novel model to study triacylglycerol biosynthesis and lipid body biogenesis.

EVALUATION OF GROWTH, NODULE DEVELOPMENT AND THE PRESENCE OF SAT1 HOMOLOGS IN THE AUSTRALIAN WILD SOYBEAN RELATIVE GLYCINE CANESCENS

Ebrahimie E., Mazurkiewicz D., Mohammadidehcheshmeh M., Chiasson D. and Kaiser B. School of Agriculture Food and Wine, The University of Adelaide, Australia.

SAT1 is a basic helix-loop-helix transcription factor located on the peribacteroid membrane in soybean nodules (Kaiser et al 1998). Loss of SAT1 activity in soybean limits nodule development and nitrogen fixation producing a typical nod plus, fix minus nodule phenotype (Loughlin et al unpublished results). We are interested in the role of this protein in symbiotic nitrogen fixation and the signaling cascades it participates in. We have identified SAT1 homologs in all sequenced plants (legumes and non-legumes) often present in multiple gene families. As part of this examination we have looked for the presence of SAT1 in other Glycine species. The perennial soybean, Glycine canescens F.J.Herm. is widely distributed throughout inland regions across Australia. G. canesscens is a wild relative of G. max however it is often found growing in extreme environments where both water and nutrients are often limiting. Very little is known about the genetic and physiological properties of G. canesscens. In this study we examined the physiological properties of G. canescens growth and nodule development and evaluated the level of conservation in nodule expressed SAT1 genes. G. canescens was found to contain both determinate (young) and indeterminate nodules when inoculated with Bradyrhizobium japonicum (CC1601a). The nodules were capable of nitrogen fixation as plants grew adequately in the absence of external nitrogen. We identified four SAT1 homologs in G. canescens, which are related to those found in G. max. A summation of our findings and a bioinformatic analysis of the SAT1 clones will presented.

THE IMPACT OF CHLOROPLAST NUMBER ON PHOTOSYNTHETIC PROPERTIES OF TOBACCO

Evans J.R.1, Tazoe Y.1, Pengelly J.J.L.1, Nugent G.D.2 and Von Caemmerer S.1 1Research School of Biology, Australian National University. 2RMIT.

The mesophyll conductance to CO2 diffusion between intercellular airspaces and chloroplast stroma depends on the surface area of chloroplasts exposed to intercellular airspace. We characterised chloroplast division mutants in tobacco to investigate the effects of chloroplast number on mesophyll conductance to CO2 diffusion. We analysed four different lines: two lines which had only one to two chloroplasts per cell, one line which had more chloroplasts compared to control and control plants. Tobacco plants were grown under full sunlight in a greenhouse. Gas exchange was measured concurrently with carbon isotope discrimination to assess photosynthetic properties and the drawdown in CO2 concentration between intercellular airspaces and chloroplasts. Leaves were subsequently sampled for Rubisco and chlorophyll content and quantitative anatomy. Mesophyll cells in leaves from plants with 1-2 chloroplasts per cell were distorted. The large chloroplast covered most of the cell wall area, but was not always in close contact with the cell wall, buckling away in places. Despite large differences in chloroplast numbers between different lines, CO2 assimilation rates, mesophyll conductance, Rubisco content and leaf anatomy were similar. The relationship between Rubisco content and CO2 assimilation rate at a given CO2 concentration in the chloroplast will be assessed.

THE MODIFICATION OF FLOWER COLOUR IN DIANTHUS CARYOPHYLLUS

Fiorito S.D.1, Parish R.W.1, Brugliera F.2 and Gendall A.R.1 1Department of Botany, La Trobe University, Bundoora, Victoria, Australia, 3086. 2Florigene PTY LTD, 1 Park Drive, Bundoora, Victoria, Australia, 3086.

Flower colour is an important evolutionary aspect for many plants, attracting a variety of pollinators such as insects, birds and small mammals. The interaction of pigments, co-pigments, metal ions, inter- and intramolecular staking and vacuolar pH can contribute to an array of colours in petals, which is valuable to the floricultural industry. Anthocyanin compounds, synthesized via the flavonoid biosynthetic pathway, are the most abundant group of pigments associated with flower colours, occurring in the majority of plant species. Recombinant DNA technology has been utilised by the floricultural company Florigene, to create novel flower colours, with a particular emphasis on the generation of blue petal colour. Florigene were the first company to release the purple coloured carnation (Dianthus caryophyllus) varieties known as the FLORIGENE Moon series TM. Various transcription factors and putative pH regulators genes will be identified in carnation using a degenerate primer approach. A rapid transient system has been applied to carnation floral tissues to generate transient down regulation of known genes within the flavonoid biosynthetic pathway and putative pH regulators. Constructs have been generated and the transient system tested; with GUS expression optimised. A selection of constructs will be stably transformed into carnation to confirm if the technology is functional in carnation. Once these genes have been identified amiRNA or RNAi constructs will be generated and transient and/or stable down regulation applied.





PROTEOMIC ANALYSIS OF THE RESPONSE OF WHEAT CULTIVARS THAT DIFFER IN THEIR CAPACITY TO WITHSTAND DROUGHT

Ford K.L., Cassin A. and Bacic A. Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne.

Using a series of multiplexed (iTRAQ) experiments we have studied the changes in protein abundance of three Australian bread wheat cultivars (Triticum aestivum). The three cultivars differ in their ability to maintain grain yield under drought conditions. Plants were grown in the glasshouse with cyclic drought treatment that mimics conditions in Southern Australia’s wheat growing regions. Buffer soluble proteins were isolated from the leaves of the wheat plants and the iTRAQ system with multiple rounds of chromatography was used to follow the changes in protein abundance, a pooled internal standard was used to allow cross experiment comparison. The study contributed to a wheat database of 2,183 proteins, as well as identifying 110 leaf proteins that changed in abundance in response to drought. The number of significant changes in the cultivars at the different time points reflected their differing response to drought. In general we observed an increase in proteins involved in oxidative stress metabolism and a down regulation of proteins involved in photosynthesis and the Calvin cycle. Drought responsive proteins, including dehydrins and LEA proteins were also significantly upregulated.

THE AFFECTS OF LIGHT ON CYANOGENESIS IN FORAGE SORGHUM

Fromhold S.M.1, O’Donnell N.H.1, Blomstedt C.K.1, Stuart P.N.2, Gleadow R.M.1, Hamill J.D.1 and Neale A.D.1 1School of Biological Sciences, Monash University, VIC, AUS. 2Pacific Seeds, QLD, Australia

Sorghum is a C4, drought resistant plant species, which is also cyanogenic in all tissues except the mature seeds. The cyanogenesis pathway in sorghum produces the stable cyanogenic glucoside, dhurrin. When the plant tissue is disrupted the dhurrin is hydrolysed to release hydrogen cyanide (HCN). At low concentrations HCN can be metabolised safely by mammals but at higher concentrations it is toxic as it causes metabolic asphyxiation. The HCN potential (HCNp) of sorghum is important in the cattle industry as forage sorghum is used as an alternate feed crop over summer. Environmental factors, such as drought, are known to increase the HCNp of sorghum but little is known regarding the affects of other conditions, including light levels. Analysis of the putative promoter regions of the three biosynthetic genes in the cyanogenesis pathway; CYP79A1, CYP71E1 and UGT85B1, showed that in all three sequences there are potential light and circadian response elements. It has been postulated that sorghum may also go through a circadian or diurnal rhythm in relation to its cyanogenesis pathway. An experiment has been conducted to compare different light treatments on forage sorghum that were hydroponically grown and placed in cabinets with controlled temperature and nutrients. The plants subjected to three light treatments; total light, total darkness and 14/10 light/dark cycle, and plants were harvested every four hours for 60 hours. This experiment was designed to determine if there is a circadian or diurnal effect on the cyanogenesis pathway in forage sorghum and the results will be discussed.

A MODEL DESICCATION-TOLERANT GRASS, SPOROBOLUS STAPFIANUS GANDOGER

Gaff D.F.1, Blomstedt C.K.1, Neale A.D.1, Hamill J.D.1, Ghasempour H.R.2 and Sutaryono Y.A.3 1School of Biological Sciences, Monash University, Clayton 3800, Australia. 2Department of Biology, Razi University, Taghbostan, Kermanshah, Iran 6. 3Lembaga Penelitian Universitas Mataram, Jl.Pendidikan No. 37, Mataram, 83125 NTB, Indonesia.

Of the ~40 grasses that can recover from dehydration to air-dryness, Sporobolus stapfianus is a most versatile tool for research into desiccation tolerance in vegetative grass tissue. This African tropical-subtropical perennial grass is easily propagated by subdivision or from seed. A number of comparisons of desiccation-tolerant and desiccation-sensitive leaves can be made, eg. leaves become desiccation-tolerant while they dry attached to intact plants in the light, whereas leaves detached before drying remain desiccation-sensitive, - this permits comparison of leaves with identical genotypes. Many desiccation-sensitive Sporobolus species and 6 desiccation-tolerant ones are available for comparison with S. stapfianus. S. stapfianus is salt tolerant and its roots adapt to withstand waterlogging if they grow into water. Of 7 desiccation-tolerant grass species tested in preliminary field trials in the Northern Territory, S. stapfianus, a C4 grass, produced the greatest dry matter increment per unit basal area. S. stapfianus has favorable digestibility, and satisfactory protein and phosphate levels. S. stapfianus exemplifies an advanced stage of an evolutionary trend in desiccation tolerant plants toward increased importance of the dehydration phase for induction of desiccation tolerance accompanied by synthesis of protectants and extensive proteomic changes. In the resurrection monocot Borya contracta and dicot Craterostigma plantagineum, the induction of desiccation tolerance during drying is controlled by the phytohormone abscisic acid (ABA), the induction in S. stapfianus is ABA-independent. S. stapfianus offers a valuable resource for future research on desiccation tolerance in a most important family economically.

SCLEROTIAL FORMATION IN SCLEROTINIA

Greenhill A.1, Gendall A.1, Benny U.2, Rollins J.2, Porter I.3 and Plummer K.1 1La Trobe University, Victoria, Australia. 2University of Florida, Florida, USA. 3Victorian Department of Primary Industries, Victoria, Australia.

Sclerotinia diseases cause over $100M loss to vegetable crops annually in Australia, with significant losses worldwide. Control of these diseases is severely impeded by the pathogen’s ability to produce sclerotia; highly melanized structures crucial to the pathogen’s propagation, reproduction and survival. T-DNA mutants have been created via Agrobacterium-mediated transformation. In this process a short DNA sequence (T-DNA) is inserted randomly into the fungal genome. As the sequence of this T-DNA tag is known, the gene or region into which it has been inserted can be identified. Approximately 1200 T- DNA mutants were created and screened for altered sclerotium phenotypes, with approximately 40 of these presenting a sclerotia-minus, or aberrant sclerotia phenotype. T-DNA insertion points have been identified in a number of these mutants and work is underway to characterise the interrupted genes. In addition to this genetic study the role of melanin in sclerotial formation is also being examined. Vectors to knock out or silence melanin biosynthesis genes have been constructed. Additionally, chemical melanin inhibitors have been tested against Sclerotinia in vitro. This work is supported by funding from Horticulture Australia Limited and an Australian Postgraduate Award.





EXPRESSING RED RUBISCOS IN GREEN PLANTS

Gunn L.H. and Whitney S.M. Plant Sciences Division, Research School of Biology, The Australian National University, Canberra A.C.T. 0200, Australia.

The CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has a pervasive influence on photosynthetic carbon assimilation in plants. In higher plants Rubisco is comprised of eight catalytic large subunits (L, rbcL gene, plastome encoded) and eight small subunits, essential for maximal activity (S, rbcS gene, nuclear encoded). Despite millions of years of evolution, Rubisco is an inefficient enzyme as O2 competes with CO2 as a substrate producing a product (2-phosphoglycolate) whose dissipation via photorespiration consumes energy. In nature, Rubisco shows considerable catalytic diversity with some variants in red algae having properties that surpass those in higher plants. Replacement of higher plant Rubisco with that of some red-algae (in theory) has the potential to improve photosynthetic C-assimilation, however such endeavours have been thwarted by the finding that the folding and assembly properties of Galdieria sulphuraria Rubisco (a red algae) could not be met by tobacco plastids. Here we expand on this work to introduce L- and S-subunits from the red algae Griffithsia monilis and a “red-like” Rubisco from the bacterium Rhodobacter sphaeroides into inverted repeat regions of a plastome transforming tobacco-master line (cmtrL1). To evaluate whether the putative molecular chaperone CbbX (coded by the cbbX gene located 3’ to the rbcL-S genes in red algae) influences assembly of G.sulphuraria Rubisco in tobacco, the rbcL-S-X operon have also been introduced into cmtrL1. The current status of this research will be presented.

MANIPULATION OF TOXIC NITROGEN SECONDARY METABOLISM IN PLANTS AND OPPORTUNITIES IN PLANT BIOTECHNOLOGY

Hamill J.D., Blomstedt C.K., Dalton H.L., DeBoer K.D., DeGuzman G., Gleadow R.M., Ling H.Y., O’Donnell N.H., Neale A.D. and Walmsley A.M. School of Biological Sciences, Monash University, Melbourne, Victoria 3800.

Many plants produce high levels of toxic nitrogen-containing specialized metabolites, such as alkaloids and cyanogenic glycosides, which generally are regarded as offering environmental protection against predators. Using Nicotiana (dicot - alkaloids) and Sorghum (monocot - cyanogenic glycosides) as experimental species, we are studying the interplay between primary and secondary metabolism and the extent to which movement of nitrogen can be experimentally redirected in vivo. A screening program to identify mutations in key genes necessary for cyanide production in Sorghum is well underway. We have also produced transgenic lines of Nicotiana with markedly altered patterns of pyridine alkaloids, both qualitatively and quantitatively. These studies have relevance to biotechnology, in relation to the redirection of metabolites to produce plants with altered growth potential. They also have relevance to other ongoing research interests involving the production of plant-made vaccines in plants without concomitant negative connotations that may be associated with the co-production of toxic nitrogen-based secondary metabolites.

IDENTIFICATION OF FLAGELLAR MASTIGONEME PROTEINS FROM PHYTOPHTHORA

Blackman L.M.1, Arikawa M.2, 3, Yamada S.2, Suzaki T.2 and Hardham A.R.1 1Plant Sciences Division, Research School of Biology, Australian National University, Canberra, ACT 2601, Australia. 2Department of Biology, Graduate School of Sciences, Kobe Univesity, Nada-ku, Kobe 657-8501, Japan. 3Department of Cardiovascular Control, Kochi Medical School, Nankoku, Kochi 783-8505, Japan.

Motile, flagellated zoospores of Phytophthora and Pythium species play a key role in pathogen dissemination and the initiation of infection of host plants. The diseases these pathogens cause are highly destructive and result in extensive losses in agriculture and natural ecosystems worldwide. Tripartite tubular hairs called mastigonemes on the anterior flagellum of Phytophthora and Pythium and other protists in the Stramenopile taxon are responsible for reversing the thrust of flagellar beat and for cell motility. Immunoprecipitation experiments using antibodies directed towards mastigonemes on the flagella of zoospores of Phytophthora nicotianae have facilitated the cloning of a gene encoding a mastigoneme shaft protein in this pathogen. Expression of the gene, designated PnMas2, is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Analysis of the sequence of the PnMas2 protein has revealed that, like other Stramenopile mastigoneme proteins, PnMas2 has an N-terminal secretion signal and contains four cysteine-rich epidermal growth factor (EGF)-like domains. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Bioinformatic analysis has revealed that Phytophthora species typically have three or four putative mastigoneme proteins containing the four EGF-like domains. These proteins are similar in sequence to mastigoneme proteins in other Stramenopile protists including the algae Ochromonas danica, Aureococcus anophagefferens and Scytosiphon lomentaria and the diatoms Thalassiosira pseudonana and T. weissflogii.

MOLECULAR ANALYSIS OF THE EARLY STAGES OF SOYBEAN NODULATION

Hayashi S.1, Reid D.1, Lorenc M.2, Stiller J.2, Edwards D.2, Ferguson B.J.1 and Gresshoff P.M.1 1ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St. Lucia, QLD 4072, Australia. 2Australian Centre for Plant Functional Genomics, School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, Qld 4072, Australia.

Nitrogen is typically a limiting factor for plant growth. Legumes can form ‘nitrogen-fixing root nodules’ de novo through a symbiotic association with soil-living bacteria called rhizobia. This provides them with nitrogen, thus enabling them to grow in nitrogen-poor environments. Nodule formation is initiated via a chemical exchange between the plant and the bacteria. This is followed by a series of signalling cascades in the root, which induces cell proliferations leading to the formation of nodule primordia. In recent decades, mutagenesis programs using several legume species have identified numerous components required for nodule development. These studies also indicated that root epidermal and cortical/pericycle cells must tightly coordinate their development during the early stages of nodulation in order to establish functional nodules. To further identify genes that are activated during the early stages of nodulation, we conducted a root transcriptome analysis using high-throughput deep-sequencing technology (Illumina’s GAIIx) in soybean (Glycine max). Soybean root tissue was harvested from the zone of nodulation to specifically focus on nodulation-expressed genes. The tissue was harvested at various times following rhizobia inoculation. Over 6,000 differentially expressed genes were identified, including known, and many previously unknown, nodulation-dependent genes. In addition, we identified a number of novel molecular pathways (i.e., hormone biosynthesis and signalling) that are differentially regulated during nodulation. Findings from these studies will be presented.





SILENCING OF THE PHOSPHOLIPASE C GENE IN PROTOPLASTS FROM COFFEA ARABICA L. SUSPENSION CELLS

Poot-Poot W.A., De Los Santos-Briones C., Munoz- Sanchez J.A. and Hernandez-Sotomayor S.M.T. Centro de investigacion Cientifica de Yucatan.

Phospholipase C (PLC) is an enzyme involved in signal transduction pathways. PLC catalizes the hydrolysis of phosphatidylinositol 4,5 bisphosphate generating two second messengers: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) which is immediately phosphorylated to phosphatidic acid (PA) by diacylglycerol kinase (DGK) in plant cells. Likewise, PLC has been observed that is involved in the response to different types of stress such as drought, salinity, cold, pathogens and metals such as Al3+. RNA interference (RNAi) technology is a widely used tool to analyze gene function in different organisms. In order to obtain a protocol for silencing PLC from protoplasts of C. arabica suspension-cell, cells were treated with a mixture of hydrolytic enzymes. To determine the concentration of polyethylene glycol (PEG) required for the transformation, we made a curve to different concentrations of PEG and viable protoplasts were quantified and the optimal concentration of PEG to use for transformation was 20%. Protoplasts were exposure to 20 % (PEG) and the plasmid for RNAi which contains the catalytic fragment of PLC from C. arabica plus the red fluorescent protein. Protoplasts were observed with fluorescence microscopy at 12, 24, 48, 72 hours. Our results showed greater transformation with of 20% PEG and the suitable time to analyze the RNAi-PLC of 72 hours. These results allow us to obtain information in a short time on the role of the PLC to different stimuli. Acknowledgments: Scholarship to WPP CONACYT (157993) and a CONACYT grant (98352) to SMTHS.

SEQUENCE DYNAMICS OF AN ARABIDOPSIS THALIANA GENOMIC LOCUS UNDER HIGHLIGHT (HL) STRESS

Ichim M.C.1, Milcamps A.2, Papazova N.3, Depicker A.4, De Loose M.3 and Van Den Eede G.2 1Stejarul Research Centre for Biological Sciences. 2Joint Research Centre. 3Institute for Agricultural and Fisheries Research. 4Flanders Interuniversity Institute for Biotechnology.

The integration of the T-DNA into the plant genome is reported to be often accompanied by simple or complex rearrangements at the integration genomic locus. It is suspected that transgenic events, instability can arise upon plant exposure to abiotic stresses. The stability of the transgene and the sequences flanking the T-DNA is an important aspect for the approval of GMPs. The structural stability of transgenes and their integration locuses is a concern for risk assessment, food labeling, traceability and post-release monitoring. We have performed sequence analysis of a genomic locus, corresponding to a T-DNA insertion point from an Arabidopsis thaliana transgenic line. The analyses of the target locus will allow defining if and what kinds of rearrangements are specifically induced under highlight (HL) stress (PPFD of 600 μmole/m2/s), known to be a mutagenic oxidative stress due to the generation of ROS molecules. In order to be able to detect even the smallest mutations possible (SNPs) we have tested and optimized the Single Strand Conformational Polymorphism analysis coupled with Capillary Electrophoresis (SSCP-CE). The results indicate that in wt plants, both those grown in standard conditions and those exposed to highlight stress, the sequence considered does not undergo any major or minor re-arrangement. Moreover, as method for mutation detection, the SSCP-CE has proved to have the potential to be used in the field of GMO research for stability analyses and post-release monitoring purposes.

THE ARABIDOPSIS PHYTOSULFOKINE RECEPTOR CONTAINS A FUNCTIONAL GUANYLYL CYCLASE DOMAIN ENABLING CGMP-DEPENDENT DOWNSTREAM SIGNALLING IN VIVO

Kwezi L.1, 2, Ruzvidzo O.2, Wheeler J.I.1, Govender K.2, Iacuonne S.1, Thompson P.E.1, Gehring C.2, 3 and Irving H.R.1 1Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia. 2University of the Western Cape, Bellville 7535, South Africa. 3Computational Bioscience Research Centre, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.

Phytosulfokines (PSKs) are sulphated pentapeptides that stimulate cell growth and differentiation in plants. The PSK receptor (PSKR1) is a leucine rich repeat receptor like kinase but its role in mediating PSK signals is undefined at a biochemical level. We identified a putative guanylate cyclase (GC) catalytic centre in PSKR1 that is encapsulated within the kinase domain. This is a different architecture to known animal receptor GCs where a linker domain separates the GC catalytic centre from the kinase homology domain. Hence we tested the hypothesis that the GC worked in conjunction with the kinase to signal downstream events in PSK signalling. We expressed the recombinant complete kinase domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 protein. This same recombinant protein also has GC activity in vitro that is significantly enhanced when calcium is present. Overexpression of the full length AtPSKR1 receptor in freshly isolated protoplasts from Arabidopsis thaliana leaves results in the endogenous basal cGMP levels being raised over 20-fold indicating that the receptor has GC activity in vivo. In addition, we demonstrate that PSK α itself induces small and rapid increases in cGMP levels and that the relatively inactive non-sulphated peptide backbone n-PSK does not. Together these results indicate that the AtPSKR1 contains dual functioning GC and kinase catalytic activity and represents a novel enzymatic class of kinases with overlapping catalytic domains.

A COMPARATIVE PROTEOMICS APPROACH FOR IDENTIFICATION OF EFFECTORS IN VENTURIA INAEQUALIS

Jones D.1, 2, Mesarich C.3, 4, Hill G.3, 4, Shiller J.2, Bowen J.3, Templeton M.3 and Plummer K.1, 2 1Cooperative Research Center for National Plant Biosecurity. 2La Trobe University. 3Plant and Food Research, New Zealand. 4The University of Auckland, New Zealand.

Venturia inaequalis and V. pirina are the causative agents of apple and pear scab (respectively). This project aims to identify and characterise effectors in these species. Effectors are pathogen proteins involved in infection. Effectors can also be recognised as foreign by plant receptors, which then initiate a signal transduction cascade that results in plant resistance. Breaking of resistance can occur if either the plant receptor gene or the effector gene is lost, or if the receptor is unable to recognise the effector due to a mutation. Due to this evolutionary pressure, effectors often vary across species and races. Effectors are therefore of interest for their direct role in infection, for their role in activating plant resistance, and for use in developing molecular tests that can differentiate strains of V. inaequalis and/or species of Venturia. We are developing a PCR-based test using a putative effector, a fungal hydrophobin; this test would be of use in surveillance, particularly in Western Australia, which is currently free of V. inaequalis but requires ongoing surveillance to verify area freedom. Using 2-D DIGE, we intend to compare proteins from Venturia sp. grown on cellophane/PDA culture against proteins on PDA culture, since V. inaequalis grows infection structures (stroma) on cellophane but not PDA. Therefore, proteins present only in cellophane culture may be involved in infection structures. Isolated proteins can be identified either by de novo sequencing, or by Peptide Mass Fingerprinting. To this end, the genomes of V. inaequalis and V. pirina are being sequenced using pyrosequencing.





DOES PHOSPHITE MIMIC PHOSPHATE IN REGULATING THE PHOSPHATE-STARVATION RESPONSE IN PLANTS?

Jost R.1, Pharmawati M.2, Berkowitz O.3, Lambers H.1 and Finnegan P.M.1 1School of Plant Biology, The University of Western Australia, Crawley WA 6009, Australia. 2Biology Department, Faculty of Mathematics and Natural Sciences, Udayana University Campus Bukit, Jimbaran, Bali. 3School of Biological Sciences and Biotechnology, Murdoch University, Murdoch WA 6150, Australia.

Phosphate is one of the most important macronutrients for plant growth and elaborate mechanisms have evolved to sense and maintain phosphorus homeostasis. Phosphite, a reduced form of phosphate, is considered to be metabolically inert when taken up by plants, and is in wide use as an effective fungicide against a wide range of devastating plant diseases caused by oomycetes, particularly Phytophthora species, e.g. P. infestans, the causative agent of the potato famine in 18th century Europe, and P. cinnamomi, the dieback pathogen that currently threatens the long-term survival of many Australian native ecosystems. The effects on plant productivity upon phosphite treatment in the field vary and depend on the presence of phosphite-oxidizing microorganisms in the soil, as well as the internal phosphorus status of the plant. Phosphite has been demonstrated to suppress phosphate starvation responses in plants with low phosphorus status, but to date there is very little understanding of how phosphite interferes with the plant phosphorus signalling networks and how/if this is linked to the long-term resistance to P. cinnamomi observed in plants in the field after aerial spraying of the host canopy. Our project aims to characterize the molecular effects of phosphite on the phosphate-sensing machinery within an Arabidopsis thaliana model.

MODIFICATION OF THE INTERCELLULAR CONTACTS IN PLANT SUSPENSION CELLS THROUGH ADAPTATION TO ENVIRONMENTAL STRESS

Kasprowicz A.K.1, Michalak M.J.1, Baluska F.2 and Wojtaszek P.1 1Department of Molecular and Cellular Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan. 2Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany.

The cell wall-plasma membrane-cytoskeleton continuum is responsible for mechanical integration of plant cells. Long-term water deprivation leading to osmotic stress activates adaptory mechanisms which enable cell to function more or less normally. During stepwise adaptation of tobacco BY-2 suspension cells to growth in the presence of various osmotically active agents, cell lines diverged into independent, osmoticum type-specific lines. The changes in growth patterns were accompanied by the alterations in the composition of cell walls. It was determined that cell walls of cells grown in the presence of ionic agents were homogenous, while longitudinal walls and cross-walls in cells adapted to nonionic agents were significantly different. In plants, cross-walls within cell files of axial organs exhibit specific properties that allow them to act as domains of contact and intense intercellular communication e.g. via endocytosis, and the sites of anchorage of cytoskeleton. Similar properties were also observed in cross-walls connecting cells grown in the presence of nonionic osmoticum. The presented results were revealed with immunolocalization, live confocal microscopy and proteomics approaches. This research was founded by the Polish Ministry of Science and Higher Education grants: PBZ-KBN-110/P04/2004 and 2944/B/P01/2008/34 to PW and grant of Dean of Biology Faculty for PhD students and scholarship from European Social Fund Programme for best PhD students from Wielkopolska to AK.

EVOLUTION OF A CIS-ELEMENT THAT REGULATES PETAL LOSS EXPRESSION IN THE PERIANTH OF THE ARABIDOPSIS FLOWER

Kilinc A. and Smyth D.R. School of Biological Sciences, Monash University, Melbourne, Vic 3800.

An outstanding issue in biology is the evolution of complex traits, and how changes in regulatory networks drive this process. Flower structure in the Brassicaceae is remarkably conserved. The outermost whorl consists of four sepals, and the second whorl contains four petals arising between them, giving a distinctive tetrameric appearance. The PETAL LOSS (PTL) gene is a transcription factor of the trihelix family with 29 members in Arabidopsis, a member of the Brassicaceae. The ptl mutant phenotype is characterised by a loss of petals and aberrant sepal development. PTL is expressed between developing sepal primordia (termed the Inter Sepal Zone, ISZ) where it functions to suppress sepal enlargement and to promote petal initiation in nearby internal spaces. We have identified a specific 16 bp regulatory enhancer (RE) within the sole intron of PTL which is required to activate its expression in the ISZ. This element is highly conserved within orthologous genes in tetrameric species of the related Brassicaceae, Cleomaceae and Capparaceae families tested, but surprisingly, was not detected in more distantly related pentameric families within the order Brassicales. The ISZ RE shows close homology to a sequence within the LTRs of some gypsy-like retrotransposons, although the latter sequences are 2 bp longer. Deletion of these 2 nucleotides is sufficient to activate ISZ expression. Evolution of the distinct tetrameric perianth pattern of Brassicaceae may have arisen through insertion of a retroelement into the intron of the PTL ancestral gene, followed by simple mutational change in the LTR sequence to create an active ISZ RE and thus recruit PTL function to this region.

NOVEL TRANSIENT GENE-REPLACEMENT SYSTEM TO INVESTIGATE RNA PROCESSING ACTIVITIES OF DICER-LIKE ENZYMES IN PLANTS

Kim K.W.1, Eamens A.L.1, 2, Fusaro A.1, 2 and Waterhouse P.M.1, 2 1University of Sydney NSW 2006. 2CSIRO Enquiries Locked Bag 10 Clayton South VIC 3169 Australia.

Since their discovery, RNA silencing pathways have been the focus of intense research in both plants and animals. The use of HELA cells and Drosophila embryo lysates have allowed animal RNA silencing pathways to be studied rapidly in vitro, whereas in plants, RNA silencing pathways have been studied mostly in vivo, within different stable mutants of Arabidopsis thaliana. A.thaliana, has been used as the model plant for studying plant RNA silencing pathways because of its short generation time, complete annotated genome and its capacity to be transformed by a non-tissue culture, floral dip method with stable transgenes. A rapid alternative to stable transformation is the use of transient Agrobacterium-mediated transformation (agro-infiltration). However, this technique is only compatible with an Australian native, Nicotiana benthamiana, whoes genome remains to be fully sequenced. In an attempt to combine the benefits of both systems, we developed a novel transient gene-replacement strategy to investigate the RNA processing activities of Arabidopsis Dicer-like proteins in N. benthamiana. This involved the transient knock-down of endogenous N. benthamiana DCL1 protein expression using an artificial miRNA (amiRNA) that is specific to the endogenous NbDcl1 gene, followed by the transient over-expression of functional exogenous Arabidopsis DCL1 protein via agro-infiltration. This resulted in the successful complementation of DCL1-dependent processing of miRNA-precursors, and therefore demonstrated that the concept of transient gene-replacement was achievable for studying specific components of the miRNA silencing pathway.





STRUCTURAL BASIS OF DISEASE RESISTANCE IN FLAX AGAINST FLAX RUST

Ve T.1, Williams S.1, 2, Valkov E.1, Stamp A.1, Sornaraj P.2, De Courcy-Ireland E.2, Ellis J.G.3, Dodds P.N.3, Anderson P.A.2 and Kobe B.1 1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia. 2School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia. 3CSIRO Plant Industry, Canberra, Australia.

Plant diseases significantly affect economically important crops. Plant immunity is triggered by the recognition of a pathogen effector protein by a plant resistance (R) protein, leading to the activation of plant defences and a localized cell death response. The effectors usually have roles in virulence and are structurally diverse, while R proteins generally fall into a few conserved families. The effector-R recognition event is poorly understood at molecular and structural levels. We have used the fungal pathogen flax rust interaction with flax as a model system to characterize this process. The flax R proteins consist of a core nucleotide-binding, N-terminal Toll-interleukin-like (TIR), and C-terminal leucine-rich repeat (LRR) domain. Previously, we have shown the direct interaction of the effector proteins AvrL567 and AvrM with R proteins L6 and M, respectively (1,2). We also determined the crystal structure of AvrL567 (3). Here, we report the first crystal structure of a TIR domain from a plant R protein (L6) at 2.3 Å resolution. The structure reveals important differences from the structures of mammalian TIR domains, and highlights three separate functionally important protein surfaces, involved in dimerisation, interaction with a downstream signalling partner, and regulatory intramolecular interactions, respectively. We also determined the crystal structure of flax rust effector protein AvrM, which has no significant sequence similarity with proteins of known structure. The 2.7 Å resolution structure reveals a novel L-shaped helical fold, with two chains forming a dimer with an unusual non-globular shape. Our results bring us a step closer to understanding the molecular basis for the disease resistance process and the ability to engineer novel resistance specificities. 1. Dodds et al (2006) Proc Natl Acad Sci USA 103: 8888. 2. Catanzariti et al (2010) Mol Plant Microbe Interact 23: 49. 3. Wang et al (2007) Plant Cell 19: 2898.

IDENTIFICATION OF PLANT HOST PROTEINS TARGETED BY FLAX RUST EFFECTOR PROTEINS

Koeck M.1, 2, Dodds P.N.2, Jones D.A.1, Ellis J.G.2 and Hardham A.R.1 1Australian National University, CMBE, RSB, PS. 2CSIRO Plant Industry, Black Mountain, Canberra.

Secreted rust fungal proteins found inside the plant cell during infection are thought to manipulate host metabolism and defence responses. Flax (Linum usitatissimum) and its rust pathogen (Melampsora lini) is a useful model system for this infectious disease, and several secreted effectors have been identified from flax rust, including AvrL567. The aim of this study is to identify proteins that interact with AvrL567, which may represent such host targets. Using a Yeast-2-Hybrid screen with cDNA from infected flax leaves, three interactors of AvrL567 could be identified, namely LuCKX1, C2MP and SIA2. Interaction between AvrL567 and these three targets was confirmed by a Bimolecular Fluorescence Complementation assay in planta. LuCKX1 (Linum usitatissimum cytokinin oxidase 1) is a cytosolic plant gene closely related to AtCKX7, one of seven genes in Arabidobsis thaliana responsible for irreversible degradation of cytokinin. These plant hormones are involved in cell development such as lateral root formation and their cellular concentration changes upon pathogen infection. Homologues of the plant gene C2MP (C2-domain containing membrane-targeting protein) have been reported to be involved in pathogen responses in rice. A.thaliana lines expressing these rice genes were more resistant to Pseudomonas syringae infection compared to the wild type plants. The third interactor, SIA2 (Secreted Interactor of AvrL567a), is another secreted fungal protein that may be a co-factor for AvrL567. SIA2 also shows binding to two other rust effector proteins (AvrM and AvrP), suggesting it may have an important general role in protein translocation and infection. Additionally, homologous of SIA2 are associated with pathogenicity in powdery mildew and rice blast fungi.

DE NOVO ASSEMBLY OF THE NICOTIANA ALATA POLLEN TRANSCRIPTOME USING NEXT-GENERATION SEQUENCING

Koh P.1, Cassin A.M.2, Edwards D.3, Bacic A.1, 2 and Newbigin E.1 1Plant Cell Biology Research Centre, School of Botany, University of Melbourne, VIC 3010, Australia. 2Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, VIC 3010, Australia. 3Australian Centre for Plant Functional Genomics and School of Land Crop and Food Sciences, University of Queensland, Brisbane, Australia.

We report the sequencing of the Nicotiana alata pollen grain transcriptome using a Next-Generation or deep-sequencing approach followed by de novo sequence assembly. Next-Generation sequencing is increasingly being used to profile transcriptomes although to date this approach has mainly been used with model organisms where a sequenced genome is used to align the transcriptome sequence reads. De novo transcriptome assembly offers a way of using this powerful technology in non-model organisms such as N. alata, for which there are few genomic resources. In de novo assembly, long RNA transcripts are first converted into a library of short cDNA fragments that are then sequenced in a high-throughput manner to obtain small lengths of sequence information from one end of each cDNA fragment. Computer programs then reconstruct full-length and partial transcripts from this vast amount of sequence information. Here we describe the workflow used to de novo assemble the N. alata pollen transcriptome, some of the features of this transcriptome, and RT-PCR validation of selected cDNAs.

CHARACTERISATION OF LEGUME ANGIOGENIC ACTIVE METABOLITES

Kordbacheh F.1, Hocart C.1, Du Fall L.1, Oakes M.1, Bezos A.2, Parish C.1, 2 and Djordjevic M.1 1ARC Centre of Excellence for Integrative Legume Research, Plant Science Division, Research School of Biology, GPO Box 475, Australian National University, Canberra ACT 2601, Australia. 2Department of Immunology and Genetics, The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, ACT, 2601 Australia.

Legumes have many beneficial health effects in humans and animals due to their nutritional properties and they are a known source of bioactive metabolites that affect plant, animal and bacterial cells (Buer et al, Journal of Integrative Plant Biology, 2010, 52: 98-111; Dixon and Sumner, Plant Physiology 2003; 131: 878-885; Banunas and Kinghorn 2005 Life Sciences 78: 431-441). We have isolated, identified and partially characterised metabolites from legumes that modulate angiogenesis. Angiogenesis is the formation of new blood capillaries, veins and arteries from pre-existing blood vessels to supply blood and nutrients and remove metabolic wastes from the tissues. Size filtration and high performance liquid chromatography (HPLC) were used to purify and isolate the bioactive metabolites from the crude extract. The bioactive components were identified using activity-guided assays. An in vitro angiogenesis bioassay based on in vivo blood vessel formation (Parish et al, Cancer Research 1999; 59: 3433-3441) was used to discover at least three different molecules that possess pro-angiogenic activity. Mass spectrometry strategies including gas and liquid chromatography (GC/MS, LC/MS) were used to determine the molecular masses of the compounds, establish elemental formulas and determine structural details of the bioactive compounds. These compounds may find utility in cardiovascular disease and wound healing.





GENOME-WIDE TRANSCRIPTOME ANALYSIS OF CITRUS HYBRIDS PLANTS RESISTANT OR SUSCEPTIBLE TO CITRUS LEPROSIS C (CILV-C)

Kubo K.S.1, 2, Cristofani-Yaly M.1, Ribeiro-Alves M.3, Boava L.P.1, Costa F.M.1, 5, Kishi L.1, Mafra V.S.1, 2, Freitas-Astua J.1, Bastianel M.1 and Machado M.A.1 1Centro de Citricultura Sylvio Moreira/IAC, CP4, 13490-970, Cordeiropolis-SP, Brazil. 2Institute of Biology, CP 6109, State University of Campinas – UNICAMP, 13083-970, Campinas, SP, Brazil. 3Centro de Desenvolvimento Tecnologico em Saude (CDTS), Fundacao Oswaldo Cruz (Fiocruz), 21040-900. Rio de Janeiro, RJ, Brazil. 4Embrapa Cassava and Tropical Fruits, 44380-000, Cruz das Almas, BA, Brazil. 5Department of Agronomy, Universidade Federal Rural de Pernambuco, 52171-900, Recife, PE, Brazil.

The molecular response of citrus plants against Citrus leprosis C (CiLV-C) is still unknown. To identify differentially expressed genes in response to CiLV-C, pools of seven highly tolerant and seven highly susceptible hybrids from Murcott tangor (Citrus sinensis x C. reticulata) and Pera sweet orange (C. sinensis) were evaluated by microarray analysis. In order to define susceptible and tolerant hybrids, they were infested by viruliferous mites in 2002 and the symptoms were evaluated during six years. Symptomless leaves of each hybrid were collected for RNA extraction. The experiment was designed in ten experimental blocks, the leaves were collected from four blocks, each being a biological repetition and each repetition containing seven individuals. The microarray chip contained 32,121, 18,873 and 12,873 unigenes from Pera sweet orange, Ponkan mandarin (C. reticulata) and Poncirus trifoliata, respectively. These unigenes were obtained from the Citrus ESTs database (CitEST) of Centro de Citricultura Sylvio Moreira/IAC. The microarrays were performed by Roche Nimblegen and Bayesian moderated T-test yielded 466 diferentially expressed genes (p.val ≤ 0.05 and fold-change ≥ 2). From these, the majority was related to metabolism, cell rescue, defense and virulence, and protein fate. Financial support FAPESP 2007/00117-4/INCT/Cnpq.

FORMATION OF SECOND WHORL ORGANS IN THE ARABIDOPSIS FLOWER INVOLVES AUXIN INFLUX AND IS SENSITIVE TO DISTORTION OF THE FLOWER MERISTEM SHAPE

Lampugnani E.R., Kilinc A. and Smyth D.R. School of Biological Sciences, Monash University, Melbourne, Vic 3800.

We are interested in how organ primordia are initiated within flowers, and in identifying the genetic factors and pathways involved in this process. Flowers of mutants of the PETAL LOSS (PTL) gene of Arabidopsis show a progressive loss in the ability to generate petals, and the sepals are wider, closer together and sometimes fused. PTL encodes a trihelix transcription factor that is expressed between sepal primordia and in sepal margins during early stages of flower development. Measurements of the numbers of cells that express PTL in this region indicate that there are more when PTL function is lost. Thus PTL may have a role in the suppression of growth between sepals and in sepal margins. Disruption of petal initiation in ptl mutants may be the consequence of disruption of a signal required for nearby petal initiation. It seems likely that auxin provides such a signal because disruption of auxin influx in aux1 ptl double mutants further compromises petal initiation. This is supported by observations on further disruptions to petal number in mutant combinations of ptl, and aux1, with other genes, including cuc, and eep1, which further disrupt flower meristem shape when their function is lost. Changes in flower meristem shape apparently cause disruptions to auxin signalling, and the initiation of second whorl primordia is especially sensitive to variation in its strength.

TAPETUM AND POLLEN DEVELOPMENT REGULATED BY ATMYB103 AND ITS APPLICATION IN HYBRID SEED PRODUCTION

Li S.F., Phan H., Iacuone S., Avdic A., Xu Y., Pham H. and Parish R.W. Botany Department, School of Life Sciences, La Trobe University, Melbourne, Vic. 3086, Australia.

The Arabidopsis AtMYB103 gene codes for a R2R3 MYB domain protein whose expression is restricted to the tapetum of developing anthers and to trichomes. We have previously shown that blocking the function of the AtMYB103 gene using either an insertion mutant or an AtMYB103EAR chimeric repressor construct results in complete male sterility and failure to set seed. These plants exhibit similar abnormalities in tapetum and pollen development, with the tapetum and pollen degenerating prematurely. We are now studying the molecular network regulated by AtMYB103 protein and developing male sterility systems for hybrid seed production in crop plants. AtMYB103 regulates the expression of genes that are involved in tapetum and pollen development including the release of microspores from tetrads. These putative target genes of AtMYB103 protein are being characterized and they will provide clues to the AtMYB103 molecular network. To develop a reversible male sterility system and an inducible male sterility system for hybrid seed production, several AtMYB103 homologues were cloned from crop plants. They are highly conserved in their deduced amino acid sequences and their promoters are only active in developing anthers. These homologues are the functional equivalents of AtMYB103 in regulating tapetum and pollen development as they were able to restore male fertility of a male sterile atmyb103 mutant. The male sterility systems using chimeric MYB103EAR repressors for hybrid seed production are discussed.

INVESTIGATING CELLULAR SIGNALLING PATHWAYS IN CANCER CELL LINES TREATED WITH A NOVEL COMPUTATIONALLY DESIGNED MYXOMA VIRUS T5 PEPTIDE ANALOGUE

Almansour N.M.1, Pirogova E.2 and Istivan T.1 1School of Applied Sciences, Health Innovations Research Institute, RMIT University, Bundoora West, Australia. 2School of Electrical and Computer Engineering, Health Innovations Research Institute, RMIT University, Melbourne, Australia.

M-T5 is a myxoma virus ankyrin-repeat host range protein that is shown to be required for virus replication in rabbit lymphocytes and the majority of human tumor cells. We investigated the cytotoxic effects of RRM-MVT5, a short peptide analogue for this protein, on melanoma cell lines as a future cancer therapeutic candidate. This novel computationally designed bioactive peptide has significant apoptotic/ necrotic effects on cancer cell lines and a negligible toxic effect on normal cells. Akt activation is essential for normal cell processes such as programmed cell death, proliferation and angiogenesis. Yet, the regulation of Akt activation is impaired in cancer cells. It was previously shown that viral M-T5 binds with phospho-Akt to regulate Akt signalling in some human cancer cell lines. In this study we investigated the effect of RRM-MVT5 treatment on Akt activation in skin cancer cell lines to detect the possible cellular pathway that is affected by this peptide. The cellular expressions of total Akt and phspho-Akt in cell cultures of the malignant melanoma cell line, MM96L, the human squamous carcinoma cell line COLO-16 and B16-F0 murine melanoma were detected using western blotting. The levels of phospho-Akt expression were assessed in cell cultures treated with RRM-MVT5 peptide analogue, in the presence or absence of the PI3 kinase inhibitor (LY294002) Our preliminary results indicated that the cytotoxic effects of RRM-MVT5 are not targeted towards this pathway as the expression of both total and phospho-Akt in cancer cells were not affected by this peptide analogue.





ANALYSIS OF SUBUNIT INTERACTIONS WITHIN THE NUCLEOSOME REMODELLING AND DEACETYLASE (NURD) COMPLEX

Alqarni S., Thong S., Josep F. and Mackay J. School of Molecular Bioscience, University of Sydney, NSW, Australia.

Our lab is interested in the structure and function of the Nucleosome Remodelling and Deacetylase (NuRD) complex, which is conserved across a wide range of eukaryotes and expressed broadly in mammalian tissues. This ~2-MDa complex appears to comprise at least ten polypeptides, including Mi-2/CHD-family proteins, HDAC1/2, RbAp46, RbAp48, MTA-family proteins (MTA1, MTA2 or MTA3), MBD2 or MBD3, p66α and p66β. Despite its importance, almost nothing is known about the structure, assembly or biochemical mechanism of action of this complex. RbAp46/RbAp48 and MTA1/MTA2 are core members of the complex and have been shown previously to interact with each other. To identify the residues of MTA proteins that are critical for mediating RbAp46/48 interactions, we generated a series of constructs encoding truncations and mutants of MTA1 and MTA2. These constructs were used to generate in vitro translated 35S-labeled proteins. Using pulldown experiments, we tested the interactions between RbAp46/RbAp48 and different truncations of MTA1 and MTA2. Our data indicate that RbAp46/RbAp48 proteins interact with MTA1 constructs in the same manner and we have identified a short motif in MTA1 that appears to be necessary for the interaction with RbAp48. We have used a range of RbAp48 mutants to delineate the MTA1-binding surface of RbAp48. Together, these data provide the first detailed information on the inter-subunit interactions that facilitate the assembly of the NuRD complex, and also provide insight into the manner by which NuRD interacts with promoter-bound proteins to regulate gene expression.

A MODULAR BAM COMPLEX IN THE OUTER MEMBRANE OF THE α-PROTEOBACTERIUM CAULOBACTER CRESCENTUS

Anwari K.1, Poggio S.2, Jacobs-Wagner C.2 and Lithgow T.J.1 1Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia. 2Department of Molecular, Cellular and Developmental Biology and Microbial Pathogenesis Section, Yale University, New Haven, Connecticut, United States of America.

The BAM (β-barrel assembly machinery) complex is a multi-subunit protein complex present in the outer membrane of all Gram-negative bacteria. It comprises of a core β-barrel protein, BamA and associated lipoproteins that collectively participate in the folding and insertion of β-barrel proteins. In this study, we demonstrate that the BAM complex possesses modular characteristics and contains BamA and three outer membrane lipoproteins (BamB, BamD and BamE) in the α-proteobacterium, Caulobacter crescentus. In addition to the three known lipoproteins, we identify an essential subunit of the BAM complex referred to as peptidoglycan-associated lipoprotein (Pal). We propose that Pal is a protein that anchors the BAM complex to the peptidoglycan layer and promotes proximity to the inner membrane Sec machinery for efficient outer membrane protein assembly.

STRUCTURE OF DIHYDRODIPICOLINATE SYNTHASE FROM BACTERIA AND PLANTS: INSIGHTS INTO REGULATION AND INHIBITION OF A VALID DRUG TARGET

Atkinson S.C., Dogovski C., Dobson R.C.J. and Perugini M.A. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.

Agrobacterium tumefaciens is a Gram-negative bacterium and the causative agent of Crown Gall disease, which is characterised by the formation of tumours at sites of environmental damage in plants. The disease is common to stone fruit, including grape vines, causing millions of dollars of damage each year to the wine industry worldwide. Despite its widespread occurrence, there is currently no treatment for Crown Gall disease. Accordingly, there is an urgent need to discover, characterise and validate new antimicrobial targets in this organism. One such target is dihydrodipicolinate synthase (DHDPS), which is an essential bacterial enzyme that catalyses the condensation of aspartate semialdehyde and pyruvate in the diaminopimelate (DAP) pathway, ultimately yielding the amino acid lysine. Previous studies in Gram negative bacteria and plants demonstrate that the downstream product of the DAP pathway, lysine, can feedback inhibit DHDPS allosterically This project aims to characterise the structure, regulation and inhibition of DHDPS from both Agrobacterium tumefaciens (AgT) and the common grape vine, Vitis Vinifera (Vv). Both AgT-DHDPS and Vv-DHDPS have been cloned, expressed and purified to homogeneity. The recombinant enzymes are enzymatically active, folded and form tetramers in solution. We have solved the crystal structure of AgT-DHDPS in the apo, pyruvate-bound and pyruvate & lysine bound forms and have used the apo structure to conduct an in silico screen using a virtual library comprised of more than 4 million drug-like compounds. The screen has yielded 100 potential inhibitors, targeting either the active or allosteric sites. 10 of these compounds show greater than 10% inhibition in vitro at a concentration of 25 μM. Inhibition studies using the Vitis vinifera form of the enzyme as a control reveal that several of these hits show specificity towards AgT-DHDPS and do not inhibit the plant enzyme in vitro. We are currently in the process of further validating these hits both in vitro and in vivo.

MECHANISTIC PERSPECTIVE FOR POLYMERIZATION OF ADH AND ROLE OF α-CYCLODEXTRIN AS AN ARTIFICIAL CHAPERONE FOR SOLUBILIZATION OF LARGER AMORPHOUS NANOAGGREGATES

Barzegar A.1, 2 and Moosavi-Movahedi A.A.2 1Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran. 2Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

A crucial problem of protein-based agents in biotechnology and biopharmaceuticals is their long-term stability or so-called shelf-life. The shelf-life of biotechnological potent enzymes is limited by thermal stress because of self-assembly of proteins into nanoaggregates such as nanoensembles or nanofilaments. Since biotechnological application of mesophilic alcohol dehydrogenases (ADH) in the pharmaceutical industry is limited because of easily polymerization during thermal process. We have evaluated the mechanism of ADH aggregation with a view to establish of thermal stabilization in order to improve the shelf-life of the ADH enzymes. The fluorescence, circular dichroism, UV-Vis spectrophotometry, dynamic light scattering (DLS) technique, enzymatic activity assay, molecular dynamic and molecular docking methods have been used for the mentioned aim. Based on the findings we have proposed a new model of self-assembly for ADH enzymes that construction of nuclei and growing to formless nanoaggregates without enzymes denaturation and unfolding. Enzymes quaternary structural changes delocalization of subunits lead to enzymes polymerization without unfolding. Alpha cyclodextrin (α-CyD) caused two types thermal stabilization of ADH enzymes including kinetic and thermodynamic stabilities by masking delocalized regions of ADH chains. Delocalization of ADH subunits, which causes the exposure of hydrophobic domains, takes part in the enzyme polymerization and has proven to be beneficial for aggregation inhibition and solubility enhancement within the host α-CyD-nanocavity. The thermostabilization of ADH against aggregation proposed for its biotechnological applications.





ANALYSIS OF THE CHROMATIN BINDING PROPERTIES OF CHD4

Bendak K., Mansfield R.E., Burdach J., Crossley M. and Mackay J. School of Molecular Bioscience, University of Sydney, NSW 2006, Australia.

Chromosome helicase DNA–binding protein 4 (CHD4) is a major component of the nucleosome remodeling and deacetylase (NuRD) complex. The NuRD complex contains at least 10 subunits and plays an important role in transcription regulation and development throughout a wide range of eukaryotic species. Mammalian CHD4 contains two plant homeodomain (PHD) fingers. Recent results from our laboratory show that these fingers are capable of binding in vitro to the amino-terminus of histone H3. This interaction is enhanced by acetylation or methylation of H3Lys9, whereas H3Lys4 methylation or H3Ala1 acetylation inhibit the interaction. To examine the functional significance of this interaction, we have sought to determine whether CHD4 can bind to chromatin in vivo. Mutations have been introduced into the PHD domains of full-length CHD4 that compromise binding to H3 and chromatin immunoprecipitation (ChIP) experiments have been used to assess the ability of the mutant CHD4 to colocalize in cells with H3 bearing trimethylated Lys9. Additionally immunoprecipitation experiments have been carried out, to establish recruitment of other NuRD complex components through CHD4.

UNRAVELLING THE ENZYMES OF THE TARTARIC ACID BIOSYNTHETIC PATHWAY IN VITIS VINIFERA

Burbidge C.A.1, Soole K.L.1, Hayes M.A.2 and Ford C.M.2 1School of Biological Science, Flinders University, GPO Box 2100, SA 5001, Australia. 2School of Agriculture, Food, and Wine, University of Adelaide, Adelaide, SA 5005, Australia.

Tartaric (TA) and malic acid (MA) account for 90% of total acid in the developing grape berry. While the majority of MA is metabolised before harvest, TA levels remain constant throughout development. Little work has been conducted into the synthesis of TA due to its rarity in higher plants. Previous work has identified ascorbic acid (Asc, Vitamin C) as the principle precursor of this acid, with additional evidence for a minor synthetic route via D-gluconic acid. To date only one enzyme, L-idonate dehydrogenase has been identified as having an active role in TA production. Bacteria including Erwinia spp and Escherichia coli possess enzymes capable of catalysing reactions identical to those involved in synthesising TA in V. vinifera. Comparative analysis of the E. coli and V. vinifera genomes has identified candidate genes that may encode previously unidentified enzymes of the TA biosynthetic pathways. Candidates were selected based on criteria including the presence of cofactor binding sites and conserved bases. Cloning and expression of enzymes pertaining to two separate steps of the biosynthetic pathway has resulted in high amounts of soluble protein. Analysis of these enzymes indicates activity towards their respective substrates. Determination of TA levels, enzyme activity and gene expression over berry development were used to confirm an enzymatic role for these enzymes within the TA biosynthetic pathway.

AP180 BINDING TO CLATHRIN IN ENDOCYTOSIS: A NEW INSIGHT

Chan L.S., Robinson P. and Graham M. Children’s Medical Research Institute, The University of Sydney.

The assembly protein AP180 is involved in membrane nucleation, which is the first step of synaptic vesicle endocytosis (SVE). AP180 controls the uniformity of synaptic vesicle size and shape. It has two known binding partners – the adaptor protein AP-2, which recruits accessory endocytic proteins to the budding membrane, and clathrin heavy chain, which is the main coat component of the budding vesicle. Similar to other major SVE proteins, AP180 is dephosphorylated when the synapse is stimulated (depolarization). Our aim was to investigate the binding of clathrin to different domains of AP180 and to determine if clathrin binding is phopsho-regulated. We made domain-specific constructs for AP180, and found a new clathrin binding site in AP180 that challenges previous assumptions about clathrin binding. We have also completed a phosphorylation map of AP180 and found that phosphorylation regulates clathrin binding to AP180. The location of the new clathrin binding site and the regulatory phosphorylation sites suggests a staged interaction between clathrin and AP180. This has profound implications on the mechanism of clathrin recruitment during stimulus-dependent SVE.

EXPRESSION AND PURIFICATION OF HUMAN G72 AND DAO, TWO SCHIZOPHRENIA-RELATED PROTEINS

Chen S.Y.1, 2, Chen Y.J.3, Wang C.M.1, Lane H.Y.4 and Chang H.T.1, 4 1Graduate Institute of Molecular Systems Biomedicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. 2Master Program of Life Sciences, National Chung Hsin University, No. 250, Kuo Kuang Road, Taichung 402, Taiwan R.O.C. 3Department of Public Health, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. 4Graduate Institute of Clinical Medical Science, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C.

Schizophrenia, a mental disease, is caused by multigene dysfunction or neurontransmitter dysregulation such as N-methyl D-aspartate (NMDA) receptor agonist decreases and dopamine deficits. Previous studies showed that patients with schizophrenia might have abnormal regulations of D-amino acid oxidase (DAO) and G72. DAO is responsible for D-amino acid oxidation to pyruvates and G72 is defined as a DAO activator. However, the regulatory mechanisms of G72 and DAO are still unclear. Thus, how G72 regulates DAO is the main theme of this study. In order to characterize the interaction between G72 and DAO, we produed and purified recombinant DAO-6H, G72-6H, MBP-DAO and GST-G72 from E. coli BL21(DE3)pLysS cells. The yield was obtained by growing the cells overnight at 25 °C under induction with 0.5 mM IPTG during the exponential phase of growth. We have succeeded to express DAO-6H, G72-6H, and MBP-DAO. However, in which only MBP-DAO was purified from soluble fraction. Initial testing showed that the enzymatic activity of MBP-DAO is not high. It may be due to the MBP tag obstructing the DAO activie sites. Thus the MBP tag will be removed to increase DAO activities. Furthermore, for production of soluble G72-6H, BL21(DE3) containing pG-KJE6 chaperone plasmids will be used. DAO is a key enzyme for oxidizing D-amino acids in brain and its enzymatic activities are thought highly correlated with schizophrenia. We expect to verify DAO abnormally regulated by G72 in vitro and in vivo.





A POTASSIUM SWITCH OF ATP-INDUCED GROEL CONFORMATIONAL CHANGES

Chen J.1, Makabe K.1, 2 and Kuwajima K.1, 2 1Okazaki Institute for Integrative Bioscience and Institute for Molecular Science. 2Department of Functional Molecular Science, the Graduate University for Advanced Studies (Sokendai).

Bacterial chaperonin GroEL utilizes the co-chaperone GroES to assist folding of cellular proteins in an ATP-dependent manner. An ATP-induced allosteric communication through GroEL double rings is prerequisite for the overall chaperonin cycle. It has been shown by Horovitz et.al. (1995) that ATP-induced allosteric transitions of GroEL consist of two phases with one at relatively low ATP concentrations (≤ 100 μM) and the second at higher concentrations of ATP with a midpoint in the range of 16 to 160 μM. Here we studied the effect of essential metal cofactor, potassium ion, on the ATP-driven allosteric transitions of GroEL by using a tryptophan mutated GroEL. Within the test range of potassium concentration, two distinct patterns of ATP-induced allosteric transitions were observed. We showed that at 50 mM potassium concentration, a remarkable increasing second-phase of ATP-induced allosteric transition was observed which is contrary to the decreasing phase at 10 mM potassium concentration. References [1] Yifrach O, Horovitz A. Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL. Biochemistry. 1995, 34(16):5303-8. [2] Inobe T, Kuwajima K. Phi value analysis of an allosteric transition of GroEL based on a single-pathway model. J. Mol. Biol. 2004, 339(1): 199-205. [3] Poso D, Clarke AR, Burston SG. A kinetic analysis of the nucleotide-induced allosteric transitions in a single-ring mutant of GroEL. J. Mol. Biol. 2004, 338(5):969-77.

STRUCTURE-ACTIVITY ANALYSIS OF CAROCIN S2

Chuang D.Y. and Wu J.L. Department of Chemistry, National Chung-Hsing University.

Carocin S2, which contains CaroS2K and CaroS2I, is the low-molecular-weight bacteriocin produced by Pecctobacterium carotovorium 3F-3. CaroS2K is an 85kDa protein that consists of a membrane-tranlocation domain, a receptor-binding domain, and a catalytic domain-in their primary sequences. These phytopathogenic cells also produce inhibitor protein CaroS2I (Mw=10kDa) located downstream of the caroS2K gene in the carocin operon. Fruthermore, in order to minimize potential activity of carocin within the producing cell, the association of the immunity protein with its cognate CaroS2K is rapid, specific, and high affinity. Our results demonstrate clearly that the minimal inhibitory concentration of CaroS2K is closed to 4μg/ml with indicator strain SP33. Here, we use several bioinformatics tools to predict the structure and functional properties of CaroS2K. The analysis accompany with site-directed mutagenesis reveal that the catalytic pocket centred around His696, the general base of the cyclization step of the reaction. The nucleophilic attack participates in the abstraction of a proton from the 2’-hydroxy group of the substrate ribose and in the protonation of the 5’-oxy group of a leaving substrate. The negatively charged pentacovalent transition state can than be stabilized by nearby positively charged residues (Lys692 and Lys695). Other mutant bacteriocin affecting the main residues forming the central groove (Phe760, Ser762, Trp764) or its loop (Tyr734) for stacking on the RNA base are also not active.

STRUCTURE AND FUNCTION OF THE TIMA PROTEIN FROM CAULOBACTER CRESCENTUS

Civciristov S.1, 2, Clements A.1, Purcell A.2, Williamson N.2 and Lithgow T.1 1Department of Biochemistry, Monash University, Clayton. 2Bio21 Institute, The University of Melbourne, Parkville.

Mitochondria have evolved from a bacterial endosymbiont which was probably related to present day α-proteobacteria. They possess complex molecular machines that are found in both mitochondrial membranes. An example is the TIM23 protein translocase of the inner mitochondrial membrane that is responsible for the final step of translocation of the proteins destined to the mitochondrial matrix. The key components of this complex are the Tim23 translocation channel, the mitochondrial motor Hsp70 and the Tim44 protein which is required for docking to the channel. C.crescentus is an α-proteobacterium which has a protein which we named TimA and it’s a protein homologue of the yeast protein Tim44. The aim of my study is to identify if the TimA protein interacts with any other proteins as part of an inner membrane protein complex in C.crescentus and to investigate its function. We found that TimA is a part of a complex in the inner membrane of Caulobacter by BN-PAGE. Immunoprecipitation with the TimA antibody followed by LC-MS/MS revealed that TimA has two partner proteins: FtsH and HflC. These two proteins are part of a complex in E.coli together with a third protein-HflK. The function of this complex in E.coli is in inner membrane quality control of misfolded proteins or jammed translocation machines such as SecY and YidC. HflK/C were shown to negatively regulate the activity of FtsH protease in E.coli. Having in mind that TimA proteins are only found in α-proteobacteria, we are investigating the function of TimA in Caulobacter as a novel component of the FtsH complex as well as its involvement in inner and outer membrane remodeling.

BIOSYNTHESIS OF CIRCULAR PROTEINS IN PLANTS

Conlan B.1, Gillon A.D.1, Barbeta B.L. 1, Colgrave M.3, Craik D.J.2, and Anderson M.A.1 1Department of Biochemistry, La Trobe University, Melbourne VIC 3086. 2Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072. 3CSIRO Livestock Industries, Brisbane, QLD 4067.

Plant cyclotides are a large family of naturally occurring circular proteins that are produced from linear precursors and are thought to be processed by asparaginyl endopeptidases to produce the circular peptide. The production of the circular peptide through excision of the cyclotide domains and ligation of the free N- and C-termini occurs through a little studied mechanism. Within the plant Oldenlandia affinis, the prototypic cyclotide producing plant, the linear precursor Oak1 is cleaved at two sites releasing the kalata B1 domain that is then cyclised resulting in the mature cyclotide. Previous work in our lab has shown that O. affinis produces only the cyclic form of the kalata B1 peptide from the Oak1 precursor, whilst transgenic expression of the same precursor in plants that do not normally produce cyclotides results in the formation of both linear and circular forms of the peptide. We have studied the importance of the residues surrounding the processing site of the cyclotide domain by site-directed mutagenesis, which revealed a number of residues and motifs essential for cyclisation. The substitution of a highly conserved asparagine residue at the C-terminal processing site of the cyclotide domain with an alanine completely inhibited cyclization. Similarly, when the conserved C-terminal tri-peptide motif was truncated no cyclic product was detected. The mutagenesis of a leucine in the C-terminal tri-peptide motif produced a unique processing product with implications for a novel mechanism of cyclisation. The observed processing may be the result of enzyme activity never before documented in planta.



POS-MON-229 POS-TUE230


THE USE OF CELL PERMEABLE PEPTIDES TO INHIBIT SPRY DOMAIN-CONTAINING SOCS BOX PROTEIN 2 (SPSB2) REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE (INOS)

D’Cruz A., Lewis R.S., Kuang Z., Norton R.S., Babon J.J. and Nicholson S.E. The Walter and Eliza Hall Institute of Medical Research.

The production of nitric oxide (NO) and related reactive nitrogen species is important for the killing of intracellular pathogens. iNOS/NOS2 (inducible nitric oxide synthase) is rapidly expressed in response to infection and catalyses the high-output production of NO. We have demonstrated that SPSB2 is a negative regulator of iNOS in Poly(I:C) and LPS stimulated macrophages, targeting iNOS for poly-ubiquitination and proteasomal degradation. The SPSB2 SPRY domain interacts with high affinity with a short linear sequence in the N-terminal region of iNOS. We were interested in investigating whether intracellular delivery of an iNOS peptide could disrupt the endogenous SPSB2/iNOS interaction resulting in enhanced NO production. The highly cationic Arginine-8 (R8) and HIV-1 derived TAT sequences are known to facilitate the entry of covalently attached cargo molecules into cells via macropinocytosis. We utilized the R8 and TAT sequences to facilitate entry of iNOS peptide into macrophages. While the R8-iNOS and TAT-iNOS peptides bound the SPSB2 SPRY domain with high affinity in vitro, much of the fluorescently labelled peptide was trapped in endosomes. We then utilized the influenza virus-derived HA2 sequence to enhance endosomal escape of fluorescently labelled R8-iNOS and TAT-iNOS. Despite observing cytoplasmic localization of the labelled peptides, we saw no differences in the level of iNOS protein expressed in response to stimuli, highlighting the difficulties of using cell permeable peptides to interfere with biological interactions. More experiments need to be conducted to determine if the iNOS peptides remain biologically available upon delivery to the cytoplasm.

MECHANISMS OF TRANSTHYRETIN AMYLOID FORMATION

D’Souza D.G.1, Altland K.3, Richardson S.J.1, 2 and Pattenden L.K.1, 2 1School of Medical Sciences, RMIT University, Bundoora, Australia. 2Health Innovations Research institute, RMIT University, Bundoora, Australia. 3Department of Human Genetics, University of Giessen, Giessen, Germany.

Transthyretin (TTR) is a β-sheet rich homo-tetrameric protein that transports retinol-binding protein and thyroid hormones. For unknown reasons, TTR has the potential to dissociate to dimers, then monomers. Through protein misfolding, the monomers can form insoluble fibrils (amyloids) that disrupt normal cellular functions and result in disease. Preliminary data has shown human TTR forms insoluble amyloid fibrils under mild acidic conditions similar to those found in vivo, however wallaby TTR under the same conditions remains as stable dimers. Each human TTR monomer contains His31, Ser46, and His90, close to the dimer interface where they help stabilise the complex. Wallaby TTR has different amino acid residues in these key positions; Lys31, Ala46, Tyr90. Our work seeks to understand why human TTR dimers are unstable compared to wallaby TTR dimers. We hypothesise that protonation of key Histidine residues in human TTR, absent in wallaby TTR, results in destabilisation of the dimer, facilitating TTR amyloid formation. We produced human, wallaby, and seven cross-species mutant TTRs. SDS-PAGE and Western analysis proved the recombinant TTR subunits were the correct size. The proteins were purified via ion-exchange chromatography. Stability assays were then performed under mild acidic conditions (pH 7.4-6.4). Fibril formation experiments of human TTR, wallaby TTR and their variants were performed at pH 7.4 (physiological), pH 6.5 (inflammation), pH 4.6 (standard non-physiological pH used to make amyloid fibrils). The application of evolutionary comparative biology to produce single amino acid variant TTRs provides a great opportunity to study structure/function relationships and understand molecular mechanisms underlying disease states.

ROLE OF THE BRCA1 BINDING PROTEIN BRAP2 IN THE TESTIS

Fatima S.1, 3, Davies R.G.1, Fulcher A.J.1, Wagstaff K.M.1, Whiley P.2, 3, Loveland K.L.2, 3 and Jans D.A.2, 3 1Nuclear Signaling Laboratory,. 2Testis and Germ Cell Development Laboratory, Department of Biochemistry and Molecular Biology. 3Australian Research Council Centre of Excellence for Biotechnology and Development, Monash University, Clayton, Victoria, Australia.

BRAP (BRCA1 binding protein) 2 is a phosphorylation-regulated negative regulator of nuclear import (NRNI) of specific proteins. It is highly expressed in testis, including a testis specific isoform, consistent with a specific role in testicular development. In situ hybridization analysis indicates BRAP2 expression in spermatogonia and spermatocytes, and to a lesser extent in round spermatids, essentially at the same stages when molecules responsible for nuclear protein import (Importins - IMPs) are highly expressed. In cotransfection experiments, we determined that BRAP2 amino acid 442-592 is sufficient to inhibit nuclear import of IMPα/β-recognised cargoes from testis. In an attempt to determine the interactome of BRAP2 in testis, we screened a yeast two-hybrid library from human testis using a C-terminal fragment of BRAP2 (343-592) as bait. Among various clones identified, several appeared to interact with proteins binding to the actin cytoskeleton, or involved in mitotic spindle function. Future work will involve documenting the apparent link between nucleus and cytoskeleton in mammalian testicular development and the role therein of BRAP2 and its interactors. Keywords: BRAP2, Testis, Negative regulator of Nuclear Import (NRNI).

STRUCTURAL STUDIES WITH CROTOXIN B FROM CROTALUS DURISSUS SUBSPECIES VENOM

Fernandes C.A.H.1, Salvador G.H.M.1, Colombi D.2, Soares A.M.3 and Fontes M.R.M.1 1Departamento de Física e Biofísica, Univ Estadual Paulista, UNESP, Botucatu-SP, Brazil. 2Departamento de Parasitologia, Univ Estadual Paulista, UNESP, Botucatu-SP, Brazil. 3Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto-SP, Brazil.

Crotoxin B is a basic phospholipase A2 found in the venom of several Crotalus durissus ssp. rattlesnakes and is one of the subunits that constitutes crotoxin, the mains component of the venom of these snakes. This heterodimeric toxin is related to important envenomation effects such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, the first structural data only become available two years ago by our research group, with the crystallization of crotoxin B from Crotalus durissus terrificus venom. However, this structure reveled an ambiguous result for the biological assembly, which could be dimeric or tetrameric. In this work, we present the crystal structure of crotoxin B from Crotalus durissus colillineatus venom, an unambiguously dimeric complex formed by two crotoxin B isoforms (CB1 and CB2). A structural comparison between these crotoxin B structures reveals differences in salt bridges that are important to oligomerization and significant Cα deviation of 54-72 region. These structural data combined with SAXS and CD experiments with inhibitors and site-directed mutagenesis studies, currently in progress, may give relevant insights into the understanding of its neurotoxic mechanism of action.





CHARACTERIZATION OF ANTI-STAPHYLOCOCCAL PROTEIN (P128) DERIVED FROM BACTERIOPHAGE FROM IN PROCESS TO PURIFICATION USING MICRO-FLUIDIC BASED LC SYSTEM COUPLED TO AN ADVANCED QTOF-MS

Gudihal R.1, Sundaram P.M.2, Madhavi H.N.2, Asrani J.2 and Fouracre C.G.3 1Agilent Technologies India Pvt. Ltd, Bangalore, India. 2GangaGen Biotechnologies Pvt. Ltd, Bangalore, India. 3Agilent Technologies, Sydney, Australia.

P128 is recombinantly expressed in E.coli. P128 has potent bactericidal activity against methycillin resistant Staphylococcus aureus (MRSA) and is currently under early development for use in humans. Liquid chromatography/Mass spectrometry (LC/MS) technology is a powerful and sensitive technique for characterization and identification of proteins. Chemical modifications and degradation that can potentially occur during manufacturing, formulation, and storage of proteins, necessitate reliable and sensitive methods for the characterization of purity and structural integrity. If suboptimal characteristics of biologics are discovered after initiation of clinical trials can lead to serious and costly delays in time to market. Here we present accurate molecular mass measurement, impurity profiling and peptide mapping for P128 which are some of the biophysical characterization for biopharmaceutical selection. Micro-fluidic based HPLC-Chip which integrates sample enrichment, chromatographic separation and nano-electrospray formation for efficient and high sensitivity nanospray LC/MS was used for the study. Pure P128 as well as in process samples were analyzed using C8 packed micro-fluidic HPLC Chip. Flow from the chip to the accurate-mass QTOF MS based system was 600nL/min and occurred through a nanospray tip etched into the polyamide material of the chip. While for peptide mapping, purified P128 protein was subjected to trypsin digestion followed by peptide separation and mass determination on C18 HPLC Chip at flow rate of 600nL/min coupled to QTOF-MS system. Further the MS/MS experiments were performed so as to confirm the peptide sequence.

EXPRESSION AND PURIFICATION OF RECOMBINANT BENZALDEHYDE DEHYDROGENASE AND BENZOATE DIOXYGENASE FROM RHODOCOCCUS RUBER UKMP-5M

Hamzah A., Rabu A. and Tvakoli A. School of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Bangi 43600 Selangor, Malaysia.

Rhodococcus ruber UKMP-5M strain utilizing aromatic compounds such as toluene and biphenyl was isolated from oil contaminated soils in Malaysia. The bacterium was able to growth in 0.5 and 1 mM toluene.The enzyme activity for toluene degradation was determined with Horseradish peroxidase (HRP) and Indole-indigo method. The catechol production was determined spectrophotometrically by monitoring the activity of catechol 1,2 dioxygenase (260 nm) and catechol 2,3 dioxygenase (375 nm), respectively. The enzyme activity for catechol 1,2 dioxygenase was 0.0142 U/ml and for catechol 2,3 dioxygenase was 0.007 U/ml. These two enzymes, benzaldehyde dehydrogenase (xylC) which oxidized benzaldehyde to benzoic acid and benzoate dioxygenase (benA) which involved in the catechol production were identified from upper (hydrocarbon-carboxylic acid) and lower (carboxylic acid-tricarboxylic acid cycle) pathways. The selected genes were amplified by polymerase chain reaction and cloned into E. coli expression system. The proteins were expressed at 37°C and 22°C with 1mM and 0.5 mM IPTG (isopropyl β-D-thiogalactoside), respectively. Benzaldehyde dehydrogenase (xylC) is a 24 KD protein with pI of 5.22 and benzoate dioxygenase is 25 KD with pI of 10.38. The recombinant proteins were purified by ion exchange chromatography on DEAE-Sephacel, and SP sepharose for xylC and benA.

BEYOND PERMEABILISATION - THE ROLE OF INTRACELLULAR TARGETS IN THE ACTION OF AN ANTIFUNGAL PEPTIDE

Hayes B., Conlan B., Anderson M. and Van Der Weerden N. La Trobe University, Department of Biochemistry, Plenty Road, Bundoora 3086.

Fungal disease is an ever increasing burden in the fields of medicine and agriculture. Fungal pathogens are responsible for damage and loss of many important agricultural crops as well as human illnesses that are often life-threatening, particularly in immunocompromised individuals. With the appearance of resistance to current treatments, novel approaches are required to treat and prevent fungal disease. Antimicrobial peptides have been the focus of intense research in recent years for their use as antibacterial agents and this focus is now turning to include their use as antifungal agents. Plants naturally express a vast array of antimicrobial peptides as part of their innate immune system. One such protein, the plant defensin NaD1, from Nicotiana alata, has potent antifungal activity and is being trialed in the protection of transgenic crops. Its mechanism of action involves entry into the cytoplasm of fungal hyphae where it potentially interacts with an intracellular target. We are using various fluorescence based techniques to elucidate how NaD1 traverses the cell wall and plasma membrane with the ultimate aim of identifying the intracellular target.

PAS-DOMAIN INTERACTIONS OF DROSOPHILA AND MOUSE PERIOD CLOCK PROTEINS

Hennig S.1, Strauss H.2, Arens J.1, Schulze S.3, Yildiz Ö3, Theiss C.1 and Wolf E.1 1Max-Planck Institut für molekulare Physiologie, Dortmund, Germany. 2Max-Planck Institut für Biophysik, Frankfurt, Germany. 3Nanolytics Gesellschaft für Kolloidanalytik mbH, Potsdam, Germany.

Most organisms exhibit a day-night activity cycle of approximately 24 hours due to a circadian pacemaker (lat. circa: about; dies: a day) which is operated by autoregulatory translational and transcriptional feedback loops. The function of PERIOD proteins, as central components of the circadian clock, is controlled by synthesis, cellular localization, phosphorylation, degradation as well as specific interactions with other clock components. Furthermore PERIOD is able to form homodimers via its tandemly organized PAS (PER-ARNT-SIM) domains. Here we solved crystal structures of PAS (PER-ARNT-SIM) domain fragments of Drosophila PERIOD (dPER) and mouse PERIOD2 (mPER2) to get insights into the regulatory role in the circadian clock at an atomic level. In the case of Drosophila PERIOD we were able to show the importance of both the hydrophobic Trp482 and a C-terminal helix for homo dimer stabilization. The mPER2 crystal structure represents the first three dimensional structure of a mammalian clock protein. In contrast to the Drosophila protein, the mouse homolog shows a homodimer stabilized solely by interactions of the Trp419 (Trp482 in dPER) with the PAS-B-β-sheet surface. By solving the PAS-domain structure of both the Drosophila PERIOD and mammalian PERIOD2 and due to our biochemical experiments, we were able to give the first insights into the molecular mechanisms of their homo dimerization.





THE THREE-DIMENSIONAL STRUCTURE OF THE BIFUNCTIONAL FAD SYNTHETASE FROM CORYNEBACTERIUM AMMONIAGENES REVEALS OLIGOMERIC ASSEMBLIES

Herguedas B.1, 2, Martinez-Julvez M.1, Frago S.1, Medina M.1 and Hermoso J.A.2 1Deparmento de Bioquimica, Biologia Molecular y Celular / Institute for Biocomputation and Physics of Complex Systems (BIFI). University of Zaragoza. Pedro Cerbuna 12, 50009-Zaragoza (Spain). 2Crystallography Deparment. Instituto de Quimica-Fisica Rocasolano. Spanish National Research Council (CSIC). Serrano 119, 28006-Madrid (Spain).

FMN and FAD are essential cofactors in flavoproteins, a large group of proteins involved in many vital processes including photosynthesis, fatty acid metabolism, mitochondrial electron transfer chain, apoptosis and DNA repair. FMN and FAD are syntesized in vivo from Riboflavin (RF, Vitamin B2) in two sequential steps, the first one is related with a Riboflavin Kinase (RFK) activity, and the second with an FMN adenylyltransferase (FMNAT) one. In prokaryotes, both activities reside in a bifunctional enzyme, FAD synthetase (FADS), whereas in eukaryotes two independent enzymes catalyze each reaction. Here we present the crystal structure of the bifunctional FADS from Corynebacterium ammoniagenes (CaFADS) solved at 1.95Å. The structure of the protomer reveals two independent modules with the catalytic sites placed 40Å away. In the protomer, the product of the first reaction –FMN- must be released from the protein surface and subsequently bind in the catalytic site of the second reaction, where it is a substrate. Crystal packing interactions also reveal an hexameric assembly formed by the interaction of two trimers. In the oligomer, both distance and orientation of the active sites from different protomers are compatible with the direct transfer of FMN between modules during the FAD synthesis. These results provide evidences, at molecular level, on the mechanism of synthesis of FMN and FAD in prokaryotes.

UNDERSTANDING HOW FLIPS INHIBIT DEATH RECEPTOR-MEDIATED APOPTOSIS

Mirams R.E.1, Vajjhala P.R.1, Stojanovski S.1, Lambert L.K.2 and Hill J.M.1 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072. 2Centre for Advanced Imaging, The University of Queensland, Brisbane QLD 4072.

Upon stimulation, death receptors such as Fas/CD95 recruit the adaptor protein FADD and procaspase-8 into the death-inducing signalling complex (DISC). Assembly of the DISC promotes the dimerisation and activation of procaspase-8 via an induced proximity mechanism. This process can be inhibited by a family of cellular and viral proteins known as FLIPs. cFLIP exists as long (cFLIPL) and short (cFLIPS and cFLIPR) splice variants, all capable of protecting cells from apoptosis by blocking procaspase-8 activation at the DISC. Several herpesviruses and poxviruses also express FLIPs to suppress apoptosis and promote their survival in host cells. The hallmark of FLIPs is the presence of tandem death effector domains (DEDs) that interact with the complementary DED of FADD and prodomain of caspase-8 to hinder caspase recruitment and activation. However, the underlying mechanisms remain unclear. At present structural information on the assembly and regulation of the DISC is relatively limited and DED complexes have remained elusive. To further characterise the molecular basis of FLIP-mediated inhibition of apoptosis, we have optimised the production of FADD for structural studies and successfully formed a stable FADD-FLIP complex. Here we present a detailed structural and biochemical analysis of the FADD-FLIP complex. Our results offer new insights into the mechanism by which FLIPs subvert death receptor signalling.

THE STRUCTURE OF BOO/DIVA REVEALS A DIVERGENT BCL-2 PROTEIN

Hinds M.G.1, Day C.L.2 and Rautureau G.J.P.1 1Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia. 2Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand.

Intrinsic cell death is mediated by interaction between pro-apoptotic and pro-survival proteins of the B-cell lymphoma-2 (Bcl-2) family of proteins. To initiate apoptosis the α-helix formed by the BH3 domain of BH3-only proteins interacts with the hydrophobic groove of a pro-survival protein. Binding events leading to pro-survival neutralization depend upon a combination of conserved and variable contacts and are typically of high affinity. We present the structure, binding properties and biological implications of Bcl-2 protein Boo. Boo has a structure that is highly homologous to other multi-domain Bcl-2 proteins where seven amphipathic helices surround a central hydrophobic helix that forms the base of a surface exposed hydrophobic groove. Although structurally homologous to other Bcl-2 family proteins, Boo has significant sequence differences in the groove, and it lacks affinity for the binding domain of pro-apoptotic proteins. In addition the canonical BH3 domain present in all pro-apoptotic proteins is absent. These observations suggest that Boo may function in a distinct manner to modulate apoptotic signalling pathways.

BACTERIAL DIAMINOPIMELATE EPIMERASE FORMS AN ACTIVE DIMER

Hor L.1, 2, 3, Dobson R.C.J.2, 3, Dogovski C.2, 3, Hutton C.A.1, 3 and Perugini M.A.2, 3 1School of Chemistry, University of Melbourne, Parkville, VIC, Australia. 2Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia. 3Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.

Given the rise in antibiotic resistance, it is has become necessary to identify novel antibacterial drug targets. One potential candidate is diaminopimelate (DAP) epimerase, which is an enzyme in the lysine biosynthesis pathway of bacteria and plants. DAP epimerase catalyses the stereoinversion of LL-DAP to meso-DAP, which is a crucial component of bacterial cell walls. This study has cloned, expressed and purified DAP epimerase from E. coli. The recombinant enzyme is folded, as determined by CD spectroscopy, and is active in solution. Quaternary structure analysis employing analytical ultracentrifugation indicates that DAP epimerase is dimeric in solution. In addition, we have recently solved the crystal structure of E. coli DAP epimerase (2.0 Å) in its open, active conformation. The crystal structure crystallised as a dimer, which is consistent with solution study results. We anticipate the crystal structure will aid in the discovery of novel inhibitors of this enzyme.





SELF-INTERACTION OF THE SULPHATE TRANSPORTER, SHST1

Leves F.P.1, Tierney M.L.2, Price G.D.1 and Howitt S.M.1 1Research School of Biology, Australian National University, ACT 0200. 2John Curtin Sxhool of Medical Research, Australian National University, ACT 0200.

Many transporter proteins form higher order structures, involving either self interaction or interaction with other proteins. The SulP (SLC26) family is a diverse anion transporter family found in all domains of life. Genetic evidence suggests that members of this family may interact with other members and therefore that these transporters may act as oligomers, rather than monomers. We investigated the ability of the sulphate transporter, SHST1, to interact with itself using the split-ubiquitin yeast two hybrid system. We identified an interaction between SHST1 molecules suggesting that in vivo, this transporter does form oligomers. Transporters of the SulP family contain an intracellular, C-terminal domain, the STAS domain, which is homologous to anti-sigma factors These proteins have a role in protein/protein interaction making the STAS domain a good candidate for an interaction domain in SHST1. Deletion experiments were used to test whether the interaction between SHST1 molecules was due to the STAS domain. It appears that interaction is mediated at least partly by the STAS domain, but a successful interaction requires that this domain be correctly oriented with respect to the membrane. Mutagenesis of conserved residues within the STAS domain has been used to investigate the role of particular residues on both sulphate transporter function and on self-interaction. Some of these mutations affect both transport function and interaction, confirming a role for the STAS domain in interaction between SHST1 molecules.

VISUALIZING DIFFERENCE OF CONFORMATIONS OF C-SRC IN LIVE CELLS USING TETRACYSTEINE TAGS AND REASH REACTIVITY

Irtegun S., Schweiggert J., Mulhern T. and Hatters D. Bio21, The University of Melbourne.30 Flemington Road.The University of Melbourne. Victoria 3010 Australia.

c-Src is the prototypic member of the Src family kinases and its activity is upregulated in a wide variety of human cancers. While the structural basis of c-Src catalytic regulation is well understood, the temporal and spatial features of SFK regulation in cells remain unclear. The activity of all SFKs depends on whether their conformation is “open” or “closed”. In the open active conformation, the protein has an extended configuration; while in the inactive closed conformation the protein is more globular and compact. Because many cancers involve dysregulated Src function, it remains critical to establish new approaches to monitor where, within cells, the active and inactive forms accrue and change conformation in a dynamic fashion. Here we have developed novel reporters of c-Src conformation which enable us to distinguish between the “open” active and “closed” inactive states of c-Src in live cells in real time. The localization of c-Src has been determined using Green Fluorescent Protein (GFP) fused to the C-terminus of the protein, while the conformation of c-Src has been monitored by the presence of a genetically encoded tetracysteine (TC) tag, which binds the biarsenical dye ReAsH in a conformation-sensitive manner. We have recently validated that our c-Src reporters work independently of intrinsic kinase activity, subcellular localization and cell morphological differences attributed to Src activation.

DISSECTING THE MECHANISM OF BINDING OF ISOFORM-SELECTIVE PI 3-KINASE SMALL MOLECULE INHIBITORS

Jennings I.G.1, Zheng J.1, Imran S.I.1, Pinson J.1, Schmidt-Kittler O.2, Kinzler K.W.2, Vogelstein B.2 and Thompson P.E.1 1Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia. 2Johns Hopkins University, Sidney Kimmel Cancer Centre, Baltimore, USA.

A number of small molecule inhibitors of Class 1 phosphatidyl 3-kinases (PI3K) are now in Phase 1 clinical trials with potential to be used in the treatment of a range of diseases. However there has been little progress in the development of isoform-selective inhibitors against the highly conserved class 1 PI3K isoforms, p110α, β, γ & δ. Selective inhibitors have the potential to reduce or eliminate unwanted side effects of these treatments. We are interested in developing an α-specific inhibitor as this isoform as it has been shown to be mutated in some cancers resulting in increased enzyme activity. In an attempt to elucidate the structural mechanism of selectivity we have examined the 3-dimensional crystal structure of the PI3K α and γ isoforms and identified 2 regions of non-conserved amino acids within the PI3K catalytic site which we postulate will confer isoform selectivity towards small molecule PI3K inhibitors which bind at or near the ATP binding site. We have produced a range of in vitro mutants of these amino acids in p110α. These mutants have been co-expressed with the regulatory subunit, p85, in insect cells with the resulting complex purified. Results will be presented which correlate a change in inhibitor isoform selectivity with a mutation as a means to identify amino acids critical for isoform selectivity. In addition several chemical classes of inhibitors have been assessed for their isoform selectivity against the four isoforms of PI3K. This has been followed by tuning of the α-selectivity by the addition of substituents to the original scaffold.

DEVELOPMENT OF FLUORESCENT CHEMOSENSOR SUBSTRATES FOR HIGH THROUGHPUT SCREENING OF THERAPEUTIC INHIBITORS AND ASSAY OF ONCOGENIC PROTEIN TYROSINE KINASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA CELLS

Kamaruddin M.A.1, 2, Hossain M.I.1, Jarasrassamee B.1, Thompson P.2, Scanlon D.1, Cheng H.C.1 and Graham B.2 1Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne. 2Department of Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University.

The Src family of protein tyrosine kinases (SFKs) are the most extensively studied protein tyrosine kinases involved in the formation and progression of many types of cancers such as leukemia, colon and breast carcinoma. Recently, aberrant activation and over-expression of the SFK members of Lyn and Hck kinases were found to induce development of drug resistant to chronic myelogenous leukemia (CML). For this reason, development of small molecule compounds capable of inhibition of SFKs are urgently needed for the treatment of patients suffering from cancer. To facilitate the search and development of these therapeutic small molecular SFK inhibitors, we have designed and synthesized fluorescent chemosensor peptides suitable for high throughput assay for SFKs activity. In our design, we combine state of the art solid phase peptide technology and the flexibility of the click chemistry to develop a library of fluorescent chemosensor peptide substrates for SFKs. Among them, some can be used to monitor SFK activity in CML cells with high selectivity and sensitivity. The availability of these chemosensor peptides permit high throughput screening of potential inhibitors of SFKs.





A PROTEOMIC APPROACH TO INVESTIGATE THE MECHANISMS OF SODIUM EXCLUSION IN PHYSCOMITRELLA PATENS

Kelly L., Ford K. and Bacic A. Australian Centre For Plant Functional Genomics, School of Botany, The University of Melbourne, VIC, Australia.

Reduced productivity from agricultural land caused by increasing levels of soil salinity is a growing problem. Plants have evolved a variety of protective mechanisms to adapt to these conditions. These mechanisms in general involve exclusion from the most sensitive tissue, compartmentalisation in the vacuole where elevated salinity is less detrimental to cellular processes, and the production of compatible osmolytes to compensate for the associated osmotic effects. Physcomitrella patens has been shown to be tolerant to a variety of abiotic stresses including salinity. This species is able to survive in sodium chloride concentration up to 600mM, if exposure is gradual. Uniquely, among plants, Physcomitrella patens and the related liverwort Marchantia Polymorpha possess Na+-ATPase (ENA) pumps capable of Na+ extrusion, in addition to the Na+/H+ antiporters utilised by higher plants to transport sodium across the plasma membrane. Much remains unknown about this addition mechanism for sodium exclusion, absent from higher plants and how this process is regulated. Both the Na+/H+ antiporter and the ENA proteins have been identified by tryptic digest and mass spectrometry (MS) in the membrane fraction from Physcomitrella patens treated with 100 mM NaCl. The current work describes the application of subcellular and posttranslational specific enrichment strategies combined with analysis by MS to Physcomitrella patens grown under various salt conditions to shed light on this recently discovered mechanism for salinity tolerance.

CHARACTERISING A CASPASE-2 CLEAVAGE SITE CONSENSUS AND IDENTIFYING ITS SUBSTRATES

Kitevska T.1, Roberts S.J.1, Scott F.L.2 and Hawkins C.J.1 1Biochemistry, La Trobe University, Bundoora, VIC, Australia. 2Burnham Institute for Medical Research, La Jolla, CA, United States.

Apoptosis is a vital pathway which maintains cellular homeostasis. This pathway activates cysteine-aspartate proteases known as caspases. To date, thirteen mammalian caspases have been identified and most have been well characterised. However, the role of some remains enigmatic, including that of caspase 2. Substrates of caspases appropriately illustrate their function. This study hypothesises that the identification of novel caspase 2 substrates may well explain the recently attributed activity of caspase-2 in cell cycle control and tumour suppression. The aim of this study is to determine the minimal substrate specificity of caspase-2 and use that to screen for caspase-2 substrates using a bioinformatics approach. The P5-P1’ minimal substrate specificity of caspase 2 was determined using a unique transcriptional reporter system in the yeast, Saccharomyces cerevisiae. Based on this hexa-peptide profile, a screen for proteins complying with this consensus was carried out using the program, Probability of Protease Specificity (PoPS). The candidate substrates revealed by this screen are now being tested for caspase 2-mediated proteolysis in vitro. Substrates shown to be susceptible to cleavage in vitro will then be further characterised.

EXPLORING MOLECULAR DETERMINANTS OF PROTEIN-CARBOHYDRATE INTERACTIONS IN THE CARBOHYDRATE BINDING MODULE ISOFORMS OF AMPK

Koay A.1, Bieri M.1, Petrie E.J.1, Bailey M.F.1, Park K.2, Gooley P.R.1 and Stapleton D.1 1Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia. 2Biology, University of Incheon, Incheon, Korea.

The AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme essential in sensing and regulating metabolic processes. Two mammalian β-subunit isoforms exist, each containing a central carbohydrate-binding module (CBM). Using NMR and fluorescence spectroscopy, we show that the ubiquitous β1-CBM and muscle specific β2-CBM possess different affinities and specificity toward glycogen mimetics. We find that the β2-CBM binds carbohydrate up to ten-fold more tightly than the β1-CBM counterpart with an 80% sequence identity. Additionally, we observe that β2-CBM binds optimally to α->1,6 glucosyl maltoheptaose, a mimetic of single-glucose branched portions in glycogen that will be exposed during glycogen degradation by glycogen debranching enzyme. A three-fold increase in affinity for glucosyl-maltoheptaose was observed in β1-CBM when a threonine is inserted into position 102. This threonine is naturally present in β2-CBM (Thr101) but not in β1-CBM. Conversely, deletion of Thr101 in β2-CBM saw a three-fold loss in affinity for glucosyl maltoheptaose. Thermodynamic and NMR studies reveal more favourable enthalpy change and diminished chemical shift perturbations in β2-CBM over β1-CBM when titrated with ligand, suggesting an optimized carbohydrate-binding site in β2-CBM. Using structural and biochemical approaches, we aim to define the molecular determinants governing specificity and affinity toward carbohydrates in the CBMs,ultimately contributing to our knowledge on the biological significance of isoform-specific roles in AMPK.

MOLECULAR DESIGN AND TARGETED CELLULAR DELIVERY OF ENGINEERED HUMAN DEOXYCYTIDINE KINASE TO ENHANCE METABOLIC ACTIVATION OF NUCLEOSIDE ANALOG PRODRUGS AND IMPROVE ANTICANCER THERAPYKonrad M.1, Ort S.1, Mcsorley T.1 and Lavie A.2 1Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany. 2University of Illinois at Chicago, Dept. of Biochemistry and Molecular Biology, Chicago, IL 60607, USA.

Nucleoside analogs (NA) are widely used in chemotherapy of cancer and viral infections. These compounds are commonly administered in their unphosphorylated form (prodrug). NAs cross the cell membrane via nucleoside transporters and are then converted to their 5’-triphosphorylated states (NA-TP) by nucleoside and nucleotide kinases. The intracellular NA-TPs can efficiently terminate synthesis of nucleic acids in viral replication and switch on the apoptotic cascade in cancer cells. Poor activation and off-target cytotoxicity often limit the efficacy of these prodrugs. NAs, such as AZT (3’-azido-3’-deoxythymidine) for the treatment of HIV infection, the guanosine analogs acyclovir (ACV) and ganciclovir (GCV) used against Herpes virus, or the anticancer compounds AraC (cytosine arabinoside) and gemcitabine (2’-deoxy-2’,2’-difluorocytidine), are phosphorylated by different kinases. The rate-limiting reaction in metabolic activation often is the first, and in some cases the second phosphorylation step. Our aim is to improve enzyme-NA systems that are of potential use in the treatment of certain cancers. We engineered the human enzyme deoxycytidine kinase (dCK) such that it phosphorylates the prodrugs AraC and gemcitabine more efficiently than does the wildtype enzyme. Remarkably, this enzyme is highly active in phosphorylating non-physiological L-enantiomers of NAs that are less toxic in vivo and biologically more potent than the corresponding D-forms. The modified dCK is delivered to tumour cells via Epidermal Growth Factor Receptor (EGFR1 and 2)-specific single chain antibodies (scFv) or affibodies, and induces apoptosis upon administration of NA. Thus, our work highlights the success of a novel Antibody-Directed Enzyme Prodrug Therapy (ADEPT) concept to significantly improve NA-dependent cancer chemotherapy.- Ref.: Sabini, E. et al. (2003) Nat.Struct.Biol. 10, 513-9. Sabini,E. et al. (2007) J.Med.Chem. 50, 3004-14. Sabini, E. et al. (2008) J. Mol. Biol. 378, 607-21. Hazra, S. et al. (2009) Biochemistry 48, 1256-63.





CHANNELLING SCENT: NEW APPROACHES TO UNDERSTAND THE STRUCTURAL BASIS OF INSECT ODORANT RECEPTION

Kralicek A.V.1, Law A.L.M.1, 2, Carraher C.1, 2, Christie D.L.2 and Newcomb R.D.1, 2 1The New Zealand Institute for Plant & Food Research Limited, 120 Mount Albert Rd, Auckland 1142, New Zealand. 2School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.

In animals the sense of smell is mediated by large families of membrane proteins known as odorant receptors (ORs). We are one of the first groups to show that insect ORs are not G protein-coupled receptors, as mammalian Ors are, but have a distinct membrane topology and ability to function in the presence of G protein signalling inhibitors1. Instead they comprise a novel family of seven transmembrane ligand-gated ion channels2,3. As a major step towards understanding insect OR structure/function we have overexpressed and reconstituted ORs into artificial membranes. This offers a unique opportunity to investigate the stoichiometry and structural nature of the interaction between the ligand-binding and ion channel subunits of the receptor complex. Additionally we are using a comparative approach to understand where odor recognition is encoded. We have identified orthologues of the Drosophila receptor, Or22a, that differ in their response to 2-heptanone using cell-based assays4 (D. melanogaster EC50 = 1.8x10-7 M, D. mauritiana EC50 = 5.4 x10-10 M) and determined EC50 values for a series of chimeric receptors to locate a region necessary for ligand binding. This work is contributing to the design and development of real-time olfactory biosensors using insect ORs and the design of compounds for pest control. 1) Smart et al. (2008) Insect Biochem. and Molec. Biol. 38: 770-780, 2) Sato K et al. (2008) Nature 452: 1002-1006, 3) Wicher et al. (2008) Nature 452: 1007-1011, 4) Kiely et al. (2007) J. Neurosci. Methods 159: 180-194.

STRUCTURAL BASIS FOR THE INHIBITION OF APOPTOSIS BY EPSTEIN-BARR VIRUS BHRF1

Kvansakul M.1, 2, Wei A.1, Fletcher J.I.1, Willis S.N.1, Chen L.1, Colman P.M.1 and Huang D.C.S.1 1La Trobe Institute of Molecular Sciences, La Trobe University, Kingsbury Drive, Bundoora, Victoria 3086, Australia. 2The Walter & Eliza Hall Institute of Medical Research, 1 G Royal Parade, Parkville, Victoria 3050, Australia.

Viruses must evade host apoptotic defences to ensure their own survival. Despite the complexity of mammalian cell death processes, viruses have evolved successful mechanisms for subverting the apoptotic machinery, including homologs of the mammalian pro-survival protein Bcl-2. The ubiquitous Epstein-Barr virus (EBV), a member of the γ-herpesviruses, infects the epithelium of the oropharynx and resting B cells. Acute infection manifests as infectious mononucleosis or glandular fever, whereas chronic EBV-associated transformation is associated with Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma. EBV BHRF1 is a sequence, structural and functional homologue of Bcl-2, however its mechanism of action remained unclear. Previous structural studies indicated that BHRF1 lacks an accessible BH3 binding groove, and shows only weak affinity for BH3 ligands. We show that BHRF1 is a potent inhibitor of apoptosis, and confers chemoresistance in mouse lymphoma models similar to mammalian Bcl-2. Next, we determined the crystal structures of BHRF1 in complex with Bim and Bak BH3 peptides and show that in contrast to previous predictions, BHRF1 interacts with these proteins in a manner similar to its mammalian counterparts. Structure-based mutagenesis enabled us to address the molecular mechanisms underlying BHRF1 activity. We demonstrate that BHRF1 can prevent Bak activation by direct interaction, but prevents Bax activation indirectly by sequestering the BH3-only proteins Bim, Puma and tBid. Unlike mammalian pro-survival proteins, BHRF1 does not interact with the selective/sensitizer BH3-only proteins. These studies indicate that BHRF1 might be targeted by small molecule mimetics of BH3-only proteins.

THE NATURAL HISTORY OF MITSUGUMIN-53 AND THE MEMBRANE REPAIR COMPLEX

Lemckert F.1, Waddell L.1, Tran J.1, Zheng F.1, Evesson F.1, Chen A.1, Clarke N.1, Ma J.2, North K.1 and Cooper S.1 1Institute for Neuroscience and Muscle Research, the Children’s Hospital at Westmead & the University of Sydney, Australia. 2Robert Wood Johnson Medical School, the University of Medicine and Dentistry of New Jersey, Piscataway, USA.

Defects in the muscle membrane repair pathway cause muscular dystrophy. Dyferlin, responsible for limb-girdle muscular dystrophy 2B and Miyoshi Myopathy, was the first muscle membrane repair protein identified. Caveolin-3 and annexinA1 have since been implicated in this pathway; mutations in caveolin-3 cause human muscle disease. A new protein, mitsugumin-53 (MG53), interacts with dysferlin and is involved in muscle membrane repair. MG53 accumulates at sites of membrane damage in normal muscle fibres, and MG53 knockout mice display a progressive muscular dystrophy and impaired resealing of isolated muscle fibres. We sequenced MG53 in 50 patients with muscular dystrophy of unknown genetic basis in which all other common causes of muscular dystrophy had been excluded. Although no primary mutations were identified, we still consider it a feasible disease gene candidate. Little is known about MG53’s role in skeletal muscle and how this and other membrane repair proteins may be intrinsically modulated in patients with muscle disease. We characterised the expression of MG53 and other membrane repair proteins in control and diseased muscle by Western blot and immunohistochemistry, to determine if levels and localisation of these proteins (the membrane repair complex) change with age or in response to disease, and if any correlation exists between intrinsic modulation of membrane repair pathways and disease severity. We show that dysferlin, MG53, caveolin-3 and annexinA1 are markedly upregulated in muscular dystrophy, and by varying levels in different forms of dystrophy. We assessed the degree of protein upregulation through quantitative Western blot and examined the effects of primary dysferlin, dystrophin and caveolin-3 protein deficiencies on modulation of the other membrane repair components.

ANALYSIS OF HOMOTYPIC AND HETEROTYPIC INTERACTIONS OF ARENAVIRUS Z MATRIX PROTEIN

Loureiro M.E., Levingston MacLeod J.M. and Lopez N. Instituto de Ciencia y Teconologia Dr Cesar Milstein (CEVAN), CONICET. Saladilo 2468 (C1440FFX). Buenos Aires, Argentina.

Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome which encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L) and a RING finger protein (Z). TacV-Z is a multifunctional protein that has a key role on virus morphogenesis. In addition, Z is able to self-associate, and exhibits an inhibitory effect on viral RNA replication and transcription through its interaction with the L polymerase (J.Virol. 77:10383-93, 2003). We have previously shown that the region comprised between the residues G36 and R85 of Z is sufficient to maintain Z-L interactions and Z inhibitory functions. To learn more about the roles of individual amino acids in the different interactions of Z, a panel of point mutants of TacV-Z and JunV-Z was created by in vitro mutagenesis. The interaction between Z mutants and L protein was analyzed by a coimmunoprecipitation assay and a minireplicon system was used to examine the effect of mutations on viral RNA synthesis. The capacity of Z mutants to self-associate was also evaluated by coinmunoprecipitation and crosslinking essays. Our results show that single amino acid changes selectively interfere with Z-Z or Z-L interactions.





DIFFERENT DOMAINS OF THE DROSOPHILA INSULATOR PROTEINS INTERACT WITH CP190 AND E(Y)2 PROTEINS MEDIATING VARIOUS FUNCTIONS OF DROSOPHILA INSULATORS

Maksimenko O., Bonchuk A., Stakhov V. and Georgiev P. Institute of Gene Biology RAS, Moscow, Russian Federation.

Insulators affect interactions between promoters and enhancers or silencers and function as barriers for spreading of repressive chromatin. A core of insulator’s protein part is highly specific DNA-binding protein which binds DNA sequence of insulator. Such proteins responsible for activity of the Drosophila insulators are dCTCF, Su(Hw) and Zw5 proteins. It contain multi-zinc-finger DNA-binding domain connected with other poorly studied domains. Recently centrosomal protein CP190 has been identified as functional component of Su(Hw) and dCTCF-dependent insulators. CP190 co-precipitates insulator proteins but details of that interaction remain unknown. In this work we found that CP190 is a major protein which precipitates with purified GST-tagged C-terminal part of dCTCF, middle part of Zw5 from S2 cells crude extract. Using GST pull-down assay we confirmed that CP190 and dCTCF, Zw5 directly interacts in vitro. We mapped region of CP190 sufficient for interaction with dCTCF, Su(Hw) and Zw5 insulator proteins in GST pull-down assay. Surprisingly such a region (308-470aa) overlaps with a microtubule-interacting domain of CP190 and is independent of its zinc-finger domain. Then we have studied interactions with another insulator component - E(y)2/Sus1. Earlier we demonstrated the specific role of the E(y)2 protein in the barrier activity of Su(Hw) insulators. In this work we found that E(y)2 protein interacts with dCTCF in the yeast two-hybrid assay. Using GST pull-down assay we confirmed that the dCTCF and Su(Hw) zinc fingers were essential for interaction with E(y)2. E(y)2 co-precipitates insulator proteins dCTCF and Su(Hw) from S2 cells. Thus we suggest that insulator complexes containing different DNA-anchor proteins have general functional activities owing to the same protein partners responsible for enhancer-blocking and boundary activities of Drosophila insulators.

CATHEPSIN B AS A TARGET FOR CONTROL OF BLOOD-FEEDING PARASITES

Jilkova A.1, Horn M.1, Rezacova P.1, Kopacek P.2, Caffrey C.R.3 and Mares M.1 1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Praha, Czech Republic. 2Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, 37005 Ceske Budejovice, Czech Republic. 3Sandler Center for Drug Discovery, QB3, University of California San Francisco, San Francisco, CA 94158, USA.

Hemoglobin digestion is an essential process for blood-feeding parasites. In blood flukes and ticks, hemoglobin degradation is orchestrated by a network of aspartic and cysteine peptidases operating at acidic pH. Our work focuses on cathepsin B, a cysteine peptidase functioning as both an exo- and endopeptidase. In the hard tick, Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis, we applied functional proteomic approaches to demonstrate that cathepsin B (IrCB) is the most abundant digestive enzyme critical for hydrolysis of hemoglobin fragments. Accordingly, IrCB represents a candidate antigen for developing novel anti-tick vaccines. In the human blood fluke, Schistosoma mansoni, cathepsin B1 (SmCB1) is a therapeutic target for the treatment of schistosomiasis. We have solved the crystal structure of SmCB1 and analyzed the inhibitory regulation of the enzyme active site. The identified structural relationships represent a potential tool for the development of antischistosomal drugs. (This work was supported by grants P207/10/2183 (GACR) and IAA600960910 (GAASCR), research projects Z40550506 and Z60220518, and the Sandler Foundation.).

THE N-TERMINAL EXTENSION OF HOLOCARBOXYLASE SYNTHETASE IS REQUIRED FOR RECOGNITION OF PROTEIN SUBSTRATES

Mayende L., Swift R.D., Bailey L.M., Wallace J.C., Booker G.W. and Polyak S.W. Discipline of Biochemistry, University of Adelaide, Adelaide, 5005.

Protein biotinylation is an example of a protein:protein interaction with exquisite specificity. Biotin protein ligase (BPL) catalyses the activation of biotin-dependent enzymes, through the attachment of the prosthetic group biotin. The catalytic region of all BPLs is contained in the conserved C-terminal region. Human BPL or holocarboxylase synthetase (HCS), contains a long N-terminal extension that is not present in bacterial BPLs. The structure and function of the N-terminal region is poorly understood. In order to delineate the role of this extension the domain structure of HCS was mapped using limited proteolysis. Two protease-sensitive linker regions were identified, one between residues 151-153, the other at residue 314. The domain containing residues 159-314 was implicated in the reaction mechanism despite being distal to the C-terminally located active site. Mutations within this domain give rise to the metabolic disorder multiple carboxylase deficiency (MCD), which is poorly responsive to current therapies. Novel mutations in this N-terminal region were generated using ‘error prone’ PCR and isolated through a genetic screen in bacteria. Residues mutated in these isolates were identified by DNA sequencing and found to be those normally highly conserved between species. Using yeast-two hybrid analysis we present the first evidence for an interaction between the N-terminal and C-terminal halves of HCS. The region of N terminal domain required for interaction was mapped to the domain encompassing residues 159-314. We show that mutations in the N-terminal region compromise the interaction of HCS with its protein substrate, but not the intramolecular interaction between the two halves. This provides a new mechanism for protein biotinylation as well as a molecular explaination for MCD.

CHARACTERIZATION OF ASSEMBLY FACTORS INVOLVED IN COMPLEX I BIOGENESIS AND DISEASE

McKenzie M.1, Lazarou M.1, Thorburn D.R.2 and Ryan M.T.1 11Department of Biochemistry, La Trobe University, Melbourne, AUSTRALIA. 22Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, AUSTRALIA.

Mitochondrial Complex I (NADH: ubiquinone oxidoreductase) is an ~980 kDa multimeric enzyme composed of 45 subunits, with 7 subunits encoded by mtDNA and the remainder by nuclear genes. The assembly of human Complex I is not well understood, complicated by its large size and its regulation by two genomes. In recent years a number of proteins have been described which aid the assembly of Complex I, however the exact functions of these ‘assembly factors’ remains unclear. We have now defined the roles of various assembly factors which act at either early, mid or late stages of Complex I assembly. C20orf7 and C8orf38 are important during early stages, acting as potential transcriptional activators of the mtDNA-encoded Complex I subunit ND1. Pathogenic mutations in the genes of either of these assembly factors result in a translation defect and/or the rapid turnover of ND1, leading to Complex I deficiency and patient death. The assembly factor C6orf66 (NDUFAF4) also acts at early stages of assembly, however it does not interact with any mtDNA-encoded subunits, instead aiding the assembly of early matrix arm intermediates. During the middle stages of Complex I biogenesis the assembly factors CIA30 (NDUFAF1) and ECSIT both directly interact with the subunit ND2, suggesting roles for both as molecular chaperones. At later stages, CIA30, ECSIT, and the assembly factor B17.2L (NDUFAF2) are found in larger Complex I assembly intermediates of ~830 kDa. However, B17.2L only co-precipitated with ECSIT, suggesting the existence of different ~830 kDa species which contain 1) CIA30 and ECSIT after assembling from smaller intermediates or 2) B17.2L and ECSIT following the release of CIA30. Our studies have helped to define the function of different assembly factors during various stages of Complex I assembly, and have also provided insights into how defects in these proteins disrupt Complex I biogenesis and lead to mitochondria disease.





ELUCIDATING THE MOLECULAR DETAILS OF RSSB-MEDIATED TURNOVER OF SIGMAS BY THE AAA+ PROTEASE, CLPXP

Micevski D., Spall S.K., Truscott K.N. and Dougan D. La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne 3086, AUSTRALIA.

In bacteria, promoter recognition and transcription initiation requires both the RNA polymerase core enzyme and a sigma factor. There are seven different sigma factors in Escherichia coli, each of which are able to recognise a specific subset of genes. The SigmaS (also known as RpoS) regulon comprises ~10% of all E. coli genes, and is crucial for the cells response to a variety of different stresses including glucose starvation and DNA damage. As the master regulator of what is often referred to as the “general” stress response it is a key factor in bacterial adaptation. Given this crucial role in bacterial adaptation, the cellular concentration of SigmaS is tightly controlled, not only at the transcriptional and translational levels, but also most importantly by proteolysis. The turnover of SigmaS is an ATP-dependent process, which is carried out by the AAA+ protease, ClpXP. Interestingly an additional factor - RssB (Regulator of SigmaS B) - is also required for SigmaS turnover, however to date the mechanism by which the adaptor protein RssB prepares or delivers SigmaS for ClpXP-mediated degradation remains unclear. One model suggests that RssB delivers SigmaS to the unfoldase ClpX via a direct interaction between the C-terminal tail of RssB and the N-terminal domain of ClpX. To dissect the role of RssB, in ClpXP-mediated SigmaS turnover, we have performed a range of different experiments including site-directed mutagenesis, half-life and co-immunoprecipitation experiments. Collectively our data favours a direct delivery model.

A MECHANISM OF DNA HOMOLOGY SEARCH, BASED ON THE STRUCTURE OF DNA BOUND TO PROTEINS THAT PROMOTE HOMOLOGOUS RECOMBINATION

Masuda T.1, Ito Y.2, Terada T.3, Shibata T.1 and Mikawa T.1, 4 1Cllular & Molecular Biology Unit, RIKEN Advanced Science Institute. 2Department of Chemistry, Tokyo Metropolitan University. 3Graduate School of Agricultural and Life Sciences, The University of Tokyo. 4Biometal Science Laboratory, RIKEN SPring-8 Center.

Homologous recombination (HR) plays a critical role in genetic diversity. The RecA/Rad51 family of proteins is well known for promoting HR in an ATP-dependent manner. Recently, the crystal structure of the RecA-ssDNA complex has been determined. In addition, ATP-independent HR proteins with significantly different structures to that of RecA/Rad51 proteins have been identified. However, the mechanism of HR remains unclear. In this study, we demonstrate a base rotation mechanism for HR based on the structure of ssDNA bound to HR proteins. We determined the structure of ssDNA bound to five evolutionarily distinct HR proteins and found that these proteins induced a common ssDNA structure characterized by long inter-base distances and reduced DNA base stacking. Furthermore, the molecular dynamics simulations of the RecA-ssDNA complex indicated that the bases in RecA filament rotate significantly, providing a mechanism for the homology search process of HR. Interestingly, RNA was unable to adopt this extended conformation. Our results suggest that the extended structure of ssDNA is a critical determinant of HR, and have provided DNA with an important evolutionary advantage over RNA for the development of genomic diversity.

DIHYDRODIPICOLINATE SYNTHASE: O-AMINOBENZALDEHYDE ASSAY FOR HIGH-THROUGHPUT CHEMICAL SCREEN AND CHARACTERISATION OF CHROMOPHORE

Mitsakos V.1, 2, 3, Hutton C.A.1, 2 and Perugini M.A.2, 3 1School of Chemistry, The University of Melbourne, Parkville, VIC, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia. 3Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia.

In search of a new class of antibacterial agents, the exploration of compounds to target the enzyme dihydrodipicolinate synthase (DHDPS) is described in this study. The colourmetric o-aminobenzaldehyde (o-ABA) assay is an assay suitable for a high-throughput chemical screen. Optimisation studies in cuvette format and subsequently 96-well format have allowed the derivatisation of kinetic data using the o-ABA assay, with values matching those shown from coupled assay studies. In addition, the purple chromophore of the o-ABA reaction has been analysed by UV/Vis-spectrophotometry, NMR and MS and found to be a diazaanthracene. The characterisation of the product has provided confidence that the assay is suitable for quantitative work. A high-throughput screen (88,000 compounds) against DHDPS from the pathogen B. anthracis has shown several hits in the micromolar range, which will allow the design of analogues with potent inhibition (nano-picomolar range) against DHDPS.

EBITEIN1, A NOVEL ERK1/2 BINDING PROTEIN

Miura K. and Imaki J. Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.

EBITEIN1 is a novel protein that binds with ERK1/2 that is one of the classical MAP kinases. EBITEIN1 was found abundantly in round spermatids. During spermatogenesis, EBITEIN1 was first translated after meiosis when cells became haploid. Then, the amount of EBITEIN1 protein gradually increased, reaching a maximum at Oakberg’s stage 9. The level of EBITEIN1 decreased such that it was undetectable when the flagellum of the spermatozoon was generated. EBITEIN1 was localized to the cytoplasm on a subcellular level. Binding experiments using various deletion mutants identified a 40-amino acid minimal sequence for binding ERK2. Furthermore, binding experiments using substitution mutants indicated the crucial role of arginine residues in this sequence. Based on empirical and bioinformatic analyses, we proposed two domains in EBITEIN1. One is EB domain, the minimal sequence for binding ERK2, and the other is ECT domain, the EBITEIN1 C-terminal domain. EBITEIN1 bound to nonphosphorylated and phosphorylated forms of ERK1 and ERK2, respectively. Phosphorylation and dephosphorylation experiments indicated that EBITEIN1 is usually phosphorylated in vivo and that it is a substrate of ERK2. The ERK2-binding domain was required for phosphorylation of EBITEIN1. Based on these results, it is suggested that EBITEIN1 is a phosphoprotein and a downstream interactor of ERK2 that participates in the intracellular signal transduction pathway, especially in the morphogenetic development of round spermatids into spermatozoa. Although EBITEIN1 cDNA was originally cloned from a mouse brains cDNA library, the functions of EBITEIN1 in the brains remain to be identified.





ANALYSIS OF THE CONSERVED CX3C MOTIF IN TIM9 AND TIM10 PROTEINS OF THE MITOCHONDRIAL INTERMEMBRANE SPACE

Mooga V.P., Baker M.J., Stojanovski D. and Ryan M.T. La Trobe University.

About ~99% of mitochondrial proteins are encoded by nuclear genes, synthesised in the cytosol and are imported into the organelle. Specific translocases have evolved in the outer and inner mitochondrial membranes to sort nuclear encoded proteins to one of four sub compartments; the outer membrane, the intermembrane space, the inner membrane and the matrix. The Tim9-Tim10 complex of the mitochondria acts to chaperone the movement of selected hydrophobic precursor proteins through the intermembrane space. The crystal structure of the yeast Tim9-Tim10 complex revealed that it is structurally similar to its human counterpart. The conserved dual Cx3C motif in Tim9 and Tim10 allows the formation of two intramolecular disulfide bonds, which stabilise the helix loop helix topology. To analyse the significance of the disulfide bonds in the biogenesis of Tim9 and Tim10 in vivo, yeast with Tim9 and Tim10 cysteine mutants were generated. Removal of both disulfide bonds in either Tim9 or Tim10 resulted in a lethal yeast phenotype. However, deletion of the inner disulfide bond in Tim9 or Tim10 (tim9C39S/C55S, tim10C44S/C61S) caused only a temperature sensitive phenotype, while removal of the outer disulfide bond (tim9C39S/C55S, tim10C40S/C65S) resulted in minor growth defects. Substrate import and assembly studies suggest that a single disulfide bond is sufficient for the biogenesis and function of Tim9 and Tim10, with the inner disulfide bond appearing to be more important than the outer disulfide bond.

PROTEOMIC ANALYSIS OF RICE ROOT CULTURES (ORYZA SATIVA L. SUB. INDICA) DURING SODIUM CHLORIDE STRESS

Bhorncharoennop C.1, Phaonakrop N.2, Jaresitthikunchai J.2, Uyen U.1, Atikankun S.1, Roytrakul S.2 and Wetprasit N.1 1Department of Biotechnology, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand. 2Genome Institute, National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand.

Saline soil is one of the major problems that limit rice production in Thailand. In salt stress response in rice, root is the first organ that perceives salinity. In order to improve the salt-tolerant ability in rice cultivars, the differentially expressed proteins in rice root during salt stress are necessary to investigate. The roots of 2-month old salt-sensitive (Pathumthani 1) and salt-tolerant (Hom-Jan) rice seedlings were cultured in sugar-free liquid MS (Murashige & Skoog) media before applying the salt stress (513 mM NaCl). The roots were then collected at different exposure times as 0, 3, 6, 9 12, 24, and 48 h. Total proteins were extracted and subjected to SDS-PAGE separation, proteins from systematically sectioned gel lanes were identified by nano LC-MS/MS of tryptic peptides. The LC-MS data were analyzed using DeCyderMS 2.0 followed by MASCOT software. Eight proteins were observed as differentially expressed following sodium chloride stress in both varieties and 4 proteins were identified to be the functional proteins. These proteins involved in oxidative stress, photosynthesis system and cell wall organization. Of these 8 proteins showed down-regulation in root culture of the salt tolerant Homjan rice while 7 proteins exhibited up-regulation in root culture of salt sensitive Pathumthani 1. Only 1 protein in Pathumthani 1 root performed an increasing at first and then gradually decreasing expression pattern. These results indicated the extent of the salinity response in rice root cells and suggested a number of key regulatory proteins and pathways that are involved in modulating the response of rice root cells to salinity. These will lead to gain more understanding about salt stress response that could be useful for crop selection and improvement of salt-tolerant in rice.

COLD ADAPTATION OF PROTEINS: A BIOPHYSICAL STUDY OF A PSYCHROPHILIC ALPHA-AMYLASE AND ITS STABILIZED MUTANTS

Cipolla, A.C., Damico, S.D. and Feller, F.G. Laboratory of Biochemistry, Center of protein Engineering, Institute of Chemistry B6a, University of Liege, B-4000 Liege, Belgium.

Permanently cold environments, like polar regions, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently at low temperatures. We have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium Pseudoalteromonas haloplanktis. Two stabilized multiple-mutants (Mut5 and Mut5CC) have been constructed. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic homolog from pig pancreas, PPA. The three enzymes AHA, Mut5 and Mut5CC have been analyzed by differential scanning calorimetry, thermal and chemical denaturation. The flexibility has been studied by acrylamide-induced fluorescence quenching. In order to investigate the kinetic origin of the gain in stability, the kinetics of unfolding and refolding in GdmCl have been monitored at 15°C. The newly introduced weak interactions stabilized the mutants, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. In addition, the two multiple-mutants exhibit an increased optimum temperature for activity. The first results of kinetic studies show a similar refolding phase but differences between the three amylases in the unfolding phase. These results unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures.

COMPARATIVE STUDY OF HIV-1 TAT TRANSIENTLY AND TRANSGENICALLY EXPRESSED IN TOMATO

Cueno M.E., Yamato H., Hibi Y. and Okamoto T. Nagoya City University, Nagoya City, Aichi, Japan.

Plant-based protein production has grown to be an ideal method for transient protein production since it is free from animal pathogens and has lower cost of production. Current methods of protein production in plants include transient expression and transgenic plant development. In this study, we noted the advantages and disadvantages of Tat protein transiently expressed in tomato calli with Tat protein expressed in a transgenic tomato plant. Plant-optimized gene constructs was synthesized and introduced into leaf calli through bombardment. One set of bombarded calli was maintained in zeatin-containing medium to allow Tat transient expression. Another set of bombarded calli was maintained in growth medium to allow Tat-expressing transgenic tomatoes. Immunological assays determined the presence of Tat in both sets of tomatoes. Effects of Tat expression on tomato were visually determined and confirmed by measuring cytokinin levels. Likewise, production time and thermostability of Tat from both tomato sets were compared. Ultimately, Tat produced from both sets were introduced intradermally to Balb/c mice and immunogenic responses were observed through ELISA and ELISPOT. Both tomato sets showed preferential fusion protein expression and thermostable production in room temperature. Transgenically expressed Tat impaired plant development by differentially expressing cytokinin oxidase resulting to lower cytokinin levels. Transiently expressed Tat was found to be secreted and absorbed from bombarded and naive tomato calli, respectively. Both humoral and cellular immune responses were induced using either tomato. Interestingly, higher cellular immune response was induced using transiently expressed Tat as compared to transgenically expressed Tat. Our results show more advantages in transiently expressing Tat in plant calli than developing transgenic tomatoes expressing Tat.





EVALUATION OF ALTERNATIVE MECHANISMS FOR THE EFFICIENT PRODUCTION OF A PLANT-MADE VACCINE

De Guzman G.M., Walmsley A.M., Webster D. and Hamill J.D. Monash University, Clayton, VIC, Australia 3800.

Using plants as an alternative vaccine production system to current systems has shown promise with a range of vaccines produced in a range of plant systems. Plants provide possible advantages for oral delivery, reduced cost, large scale production and storage. In this study, hairy root cultures were utilized for their rapid growth, high genetic stability, potential for rhizosecretion, and their ability to be regenerated into whole plants. The B-subunit of the heat-labile toxin from E.coli was expressed in hairy root cultures of Tobacco, Tomato and Petunia to evaluate LTB production in and between each species. The hairy root cultures of Tobacco and Petunia expressed LTB antigen at high concentrations of LTB ~70ug/g FW tissue on average, while tomato produced low amounts of antigen (~2ug/g FW). Throughout the root growth cycle, LTB was shown to peak after 22 days however, a marked growth impairment was observed correlating to high expression of LTB, interestingly petunia growth was shown to be least affected by high levels of LTB. The ability of hairy roots to secrete LTB into the supplied media was also confirmed in each species; however improvement of secretion methods is required for effectively testing secretion patterns. The ability of hairy roots to regenerate into whole plants and retain their high antigen production was also shown and the ability for cultures to be re-established form these plants has been also shown This research demonstrates hairy roots as a viable system for vaccine production, and in particular roots derived from Petunia where high production coupled with low growth inhibition, secretion potential and regeneration ability allow for a very promising production system.

DIFFERENTIAL ANALYSIS OF VITAMIN D TREATED U937 MYELOID LEUKEMIC CELLS

Durand F.M.1, 2, Levina V.1, 2, Cooke I.1, 2, Coley A.1, 2, Hoogenraad N.2 and Talbo G.H.1, 2 1CRC for Biomarker Translation, La Trobe University, Melbourne, Australia. 2:La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.

Unlike normal progenitor cells, haemopoietic cells from patients with acute myeloid leukemia have an apparent inability to differentiate and terminally mature. U937 myeloid leukemic cells can be induced to differentiate into macrophages by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Growth arrest of these cells has been shown to be blocked at G1 in the cell cycle, happening concomitantly with an upregulation of the cell surface membrane proteinaceous monocyte/macrophage-specific markers, CD14 and CD11b. Granulocyte-macrophage colony-stimulating factor (GM-CSF), when used in conjunction with 1α,25(OH)2D3 has been shown to have a synergistic effect on U937 cells resulting in an increased upregulation of these cell surface markers. Three batches of U937 cells were grown for 96 hours in media plus vehicle, media plus 1α,25(OH)2D3 and media plus 1α,25(OH)2D3 and GM-CSF. Aliquots of cells were subjected to our differential proteomics protocol. In short, the glyco moiety of the outer membrane proteins were oxidised by NaIO4, the cells were lysed and the oxidised carbohydrate covalently bound to hydrazide coupled beads. The covalent binding of the proteins facilitates extensive washing to remove membrane lipids and other debris. The bead bound proteins were reduced, alkylated and digested by trypsin. The residual tryptic glycopeptides were released by PNGase F and analysed by LC-ESI-microTofQ-MS/MS as seen in the poster titled: “Analytical Prerequisites for Differential and Quantitative Proteomics”. The known cell surface proteinaceous marker CD11b was shown to be upregulated on the 1α,25(OH)2D3 as well as the 1α,25(OH)2D3 and GM-CSF treated cells, while the marker CD14 upregulated on the 1α,25(OH)2D3 and GM-CSF treated cells, only.

NOVEL BIOACTIVE SECONDARY METABOLITES FROM AUSTRALIAN MEDICINAL PLANT ENDOPHYTES

Fazendin Y.K.1, Lim K.F.2, Cahill D.1, Rookes J.1, Conlan X.1 and Barnett N.W.1 1Deakin University, Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Vic, Australia 3217. 2Deakin University, Life and Environmental Sciences, 221 Burwood Highway, Burwood, Vic, Australia 3125.

Plant endophytes are producers of biologically active secondary metabolites that have provided new antibiotics, antiparasitics, antimalarial, and anticancer agents. There has been limited investigation of endophytic microorganisms associated with Australian traditional medicinal plants. A range of plant species, incorporating four diverse families and seven genera, were collected from State and National Parks around Victoria, Australia. Of the eight plant species selected, endophytic fungi and bacteria were found in association with the leaves and stems of Psilotum nudum, Ajuga australis, and Phebalium squameum. The fungal endophytes were isolated on potato dextrose agar (PDA), 10% V8 agar, and purified on malt agar. The ITS-5.8S-ITS2 rDNA region of each of the endophytic fungi was PCR amplified using universal primers, and gene sequenced. A BLAST search was conducted to identify the fungal isolates to genus. A preliminary co-culture screen of the fungal endophytes against several fungal plant pathogens showed antifungal activity. A large-scale culture of an endophytic fungus in PDA broth (10 x 500 mL, 5 L) at room temperature (20–25 ° C) was undertaken, followed by extraction of mycelia and growth media with ethyl acetate. The extract is currently being screened for activity against four bacterial strains, including an antibiotic resistant strain of E. coli), and antioxidant activity using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity test and an acidic potassium permanganate (KMnO4) chemiluminescence method. Anticancer activity will also be tested on several cancer cell lines. Bioassay-guided fractionation, and a range of spectroscopic techniques will be used to isolate and characterise the bioactive constituent(s).

OXIDATION OF TETRAHYDROPTERINES BY QUINONES

Garcia Molina F.1, Muñoz Muñoz J.L.1, Martinez Ortiz F.2, Varon R.3, Tudela J.1, García Canovas F.1 and Rodriguez-Lopez J.N.1 1GENZ: Grupo Investigación Enzimologia.Deparatamento de Bioquímica y Biologia Molecular A. Facultad de Veterinaria. Campus de Espinardo. E-30100, Murcia (Spain). 2Grupo de Investigación de Electroquímica Teórica y Aplicada.Departamento de Química Física. Facultad de Química.Universidad de murcia. A. Correos E-30100. Murcia, Spain. 3Departamento de Química-Física. Escuela de Ingenieros Industriales de Albacete. Universidad de Castilla la Mancha. Avda. España s/n. Campus Universitario, E-02071, Albacete, Spain.

Tetrahydrobiopterine (6BH4) can diminish the oxidative stress of keratinocytes and melanocytes by reducing the o-quinones generated by the oxidation of the corresponding o-diphenols. In this work, we demonstrate that 6BH4 and its analogues methyltetrahydropterine (MBH4) and dimethyltetrahydropterine (DMBH4) can reduce all the o-quinones studied, including 1,2 benzoquinone but not p-benzoquinone. The formal potentials of different quinone/diphenol pairs obtained by square wave voltammograms indicate that the o-quinones with withdrawing groups are more potent oxidants than those with donating groups. This work has been partially supported by grants from several Spanish organizations. Ministerio de Educación y Ciencia (Madrid, Spain) Project BIO2009-12956, from the Fundación Séneca (CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08, from the Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004 and from the Consejería de Salud y Bienestar Social de la Junta de Comunidades de Castilla La Mancha, Project FISCAM PI-2007/53. JLMM has a fellowship from the Ministerio de Educación y Ciencia (Madrid, Spain) Reference AP2005-4721. FGM has a fellowship from Fundación Caja Murcia (Murcia, Spain).





CONSERVED BLOCK7 SEQUENCE OF BACILLUS THURINGIENSIS CRY TOXINS. -THE INNOVATIONAL PEPTIDE-TAG FOR EFFICIENT PROTEIN PRODUCTION-

Hayakawa T., Shimizu Y., Ishida T. and Sakai H. Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.

We have developed a novel system to prepare proteins effectively by using peptide-tag derived from the mosquitocidal Cry4Aa toxin of a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of crystal-like inclusion bodies of glutathione S-transferase in Escherichia coli without loosing the enzyme activity. In this study, we analyzed functional motif, in particular Block7, which may work for formation of crystal-like inclusion bodies. Block7 is a stretch of amino acid sequence conserved among Cry toxins. We selected fourteen Cry toxins and synthesized the genes encoding their Block7s. The Block7 peptides were expressed as GST fusions and formation of crystal-like inclusion bodies in E. coli was analyzed. As a result, among the 14 tested Block7 peptides, those from Cry5Ba, Cry32Aa, and Cry48Aa formed crystal-like inclusion bodies as well as that of Cry4Aa. But no inclusion observed for Cry47Aa Block7. Formation efficiency of crystal-like inclusion body for the remaining 10 of the tested Block7 peptides was ranging from 39 to 66%. Thus, the conserved Block7 may be one of factors responsible for crystallization of Cry toxins, but the role of Block7 may vary with type of Cry toxin. In addition, our results suggested the possibility to design a shorter peptide-tag based on Block7 which form protein crystal efficiently. Results of detailed mutational analyses will also be presented.

DEVELOPMENT OF ANALYTICAL METHODS FOR SEQUENCING OLIGONUCLEOTIDES USING MALDI-TOFMS

Minohata T.1, Hewetson J.W.2, Yamazaki Y.1 and Yamada M.1 1Applications Development Center, Analytical Applications Department, Shimadzu Corporation, Kyoto, Japan. 2Shimadzu Scientific instruments (Oceania) Pty Ltd., Rydalmere, Australia.

Studies of oligonucleotides containing both RNA and DNA are of great interest in relation to the development of oligonucleotide therapeutics and fundamental research. It has been recognized that various sequences of oligonulcleotides indicate antisense effect or RNA interference, which are responsible for effective and specific gene suppressions to regulate expressions of unwanted proteins in living system. Although demand for a precise sequencing method of short length oligos (20-30 mer) is increasing to study biological function, only a few analytical methods have been reported, such as acid-hydrolysis and nuclease treatment in conjunction with mass spectrometry. On the other hand, MALDI-TOFMS has been mainly applied to the detection of oligonucleotide molecular weight. We will report studies of two sequencing methods using MALDI-TOFMS, including on-plate acid-hydrolysis and in-source decay (ISD) on MALDI-TOFMS. We modified a previous report of acid-hydrolysis for DNA sequencing and investigated favorable conditions for the reaction of sequencing 19 mer RNA. Hydrolysis was conducted directly on the MALDI plate with a mixture of matrix, strong acid and RNA, and then analyzed by MALDI-TOFMS. Conditions were optimized to generate many ladder signals, between which mass differences represent constituent units of ribonucleotides, thus the sequence of the RNA was clearly observed. Notably, it was possible to apply these conditions to the analysis of 2-O-methylation of siRNA. Whilst ISD, which is a degradation reaction inside the ion source of MALDI, has been applied to protein sequencing, this method has been rarely applied to RNA analysis. We will report successful results of the modified siRNA by using ISD, and introduce newly developed software for the interpretation of results.

EFFICIENT PRODUCTION OF THE RECOMBINANT CYSTATIN C USING INNOVATIONAL PEPTIDE-TAG DERIVED FROM BACILLUS THURINGIENSIS CRY TOXIN

Iwamoto S.1, Fan K.2, Sato S.1, 3, Hayakawa T.3, Sudo S.1 and Sakai H.3 1Japan Lamb CO.,LTD, Department of Bioscience, Development Division, Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama 700-0082, Japan. 2JSR Corp., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305-0841, Japan. 3Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.

Cystatin C is a potent inhibitor of lysosomal proteinases and produced by all human cells with a nucleus. Since Cystatin C is a biomarker of kidney function, the recombinant protein is highly demanded in the market of diagnosis agent. In this study, we present an innovational production system of the recombinant Cystatin C using a peptide-tag derived from Bacillus thuringiensis Cry toxin. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of alkali soluble protein inclusion in Escherichia coli. The recombinant Cystatin C fused with 4AaCter at the N-terminal end was successfully expressed and accumulated as protein inclusion in E. coli. The yield of 4AaCter-Cystatin C was 40mg/L culture and was five fold higher than that of Cystatin C without the tag. The inclusion of 4AaCter-Cystatin C was solubilized in denaturing buffer and purified by tag-based affinity chromatography. The purity of the final product was more than 90% and the final yield was 8mg/L culture. Western blotting revealed that the recombinant protein reacted with anti-Cystatin C antibody as well as that of native Cystatin C. In addition, latex agglutination assay showed that the reactivity of the recombinant Cystatin C by our method was higher than that of the native one. The novel system we have developed using 4AaCter is very simple and enables efficient production of the recombinant Cystatin C with superior quality in low cost.

PROTEIN STRUCTURE DETERMINATION FROM PSEUDOCONTACT SHIFT

Schmitz C.1, Vernon R.2, Otting G.3, Baker D.2 and Huber T.3 1Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands. 2Department of Biochemistry, University of Washington, Seattle, USA. 3Research School of Chemistry, Australian National University, Australia.

The pseudocontact shift (PCS) effect, induced by a bound paramagnetic lanthanide ion, is becoming widely used in protein nuclear magnetic resonance (NMR) spectroscopy as it yields a complementary combination of orientational and (long range) distance restraints. This versatile effect can be accurately determined with highly sensitive NMR experiments and has been successfully used (i) to automatically assign NMR resonances, (ii) to determine the structure of protein-protein complexes and protein-ligand complexes, and (iii) to refine NMR structures. However, it has been speculated whether or not PCS data as the only experimental restraints are sufficient for de novo structure determination of a protein. Here we show that 3D structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. We present results from a statistically meaningful number of proteins with different folds ranging in size from 56 to 186 amino acids and show that PCS restraints implemented in the fragment assembly step of the ROSETTA software is highly efficient in biasing the sampling of the conformational space towards the correct target structure. We further show that the best structures computed have a backbone RMSD from the native structure as low as 1.0 Ångstrom. Finally, the question whether we are performing structure prediction assisted by experimental data, or truly de novo structure determination is discussed.





INNOVATIVE STRATEGY FOR EFFICIENT PROTEIN PRODUCTION USING A NOVEL PEPTIDE TAG DERIVED FROM CRY TOXIN

Sakai H.1, Sato S.1, 2, Iwamoto S.2, Sudo S.2, Sakamoto Y.1, Uchida M.1, Matsushima K.1, Kashino Y.1 and Hayakawa T.1 1Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan. 2Japan Lamb CO., LTD, Department of Bioscience, Development Division, Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama 700-0082, Japan.

We are often confronted with difficulties that some proteins cannot be efficiently produced by ordinary recombinant DNA techniques based on cloned genes. The difficulties may be because the recombinant protein products are unstable or have deleterious effects on the host cell. To overcome the difficulties in producing these difficult-to-produce proteins, some innovative recombinant DNA techniques for heterologous protein production are eagerly desired. We developed an innovative procedure to produce proteins efficiently by using a novel peptide tag derived from the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of crystal-like inclusion bodies of glutathione S-transferase in Escherichia coli without loosing the enzyme activity. For all the target proteins tested to date, it was proved that the recombinant proteins were accumulated in the crystal-like inclusion bodies and their biological activities were also maintained. Application of 4AaCter to the production of syphilis antigens, TpN15, TpN17 and TpN47 from Treponema pallidum, yielded excellent results, including a dramatic increase in the production level, simplification of the product purification and high reactivity with syphilis antibody. The use of 4AaCter may provide an innovational strategy for the efficient production of difficult-to-produce proteins.

FUNCTIONAL MICROPATTERNS TO CONTROL CELL BEHAVIORS

Ho C., Kumar G. and Co C. University of Cincinnati.

Microfabrication techniques are widely used in the electronic industry to generate small features with size between 1-100 μm. This size range is on the same order of a single cell, thus, these microsystems are well suited for studying cell behaviors. This talk will demonstrate some examples of using micropatterned surfaces to control local environment around the cell. Micropatterned surfaces were used to control the cell size and shape. Endothelial cells patterned within the 20 µm grooves formed capillary tube-like structure containing a central lumen. Capillary networks embedded in other tissue specific cell types can be formed on the biomaterials for creating vascularized tissue. Patterned biomaterials can be applied to guide neurons to extend axon and neurite for creating neuronal networks. We have also devised a completely novel microarray-based technique to amplify the natural directional persistence of migrating cells (MANDIP). Using MANDIP, we can amplify this directional persistence to coerce the migration of cells indefinitely along arbitrary paths in one preset direction without chemoattractants, gradients in substrate adhesiveness, or external fields. Potential applications of MANDIP include cell-sorting, drug screening, tissue engineering, wound healing, and mechanistic studies of cell migration, cell-cell interactions, and other cellular processes requiring temporal and spatial regulation. We have devised and are seeking to commercialize a simple in vitro migration assay that offers significant improvements in reliability and ease of implementation compared to traditional “wound healing” assays. Instead of mechanically wounding cells, which leads to interferences caused by dead cell debris that block cell movement and molecules released by wounded cells that alter artificially the rate of cell migration, confluent groups of cells, initially confined within patterns of cell-resistant polyelectrolyte, are released by electrostatic adsorption of a second, cell adhesive polyelectrolyte.

CHLAMYDOMONAS REINHARDTII. - AN ALGAL FEEDSTOCK FOR LIQUID BIOFUELS

James G., Hocart C., Hillier W. and Djordjevic M. Plant Science Division, Research School of Biology, The Australian National University, Canberra, Australia.

C. reinhardtii is a model algal system of intense research focus as a feedstock for the production of biodiesel and the processing of hydrocarbons to jet fuel (Wang et al., Eukaryot. Cell, Dec 2009; 8: 1856-1868, Moellering et al., Eukaryot. Cell, Jan 2010; 9: 97-106, Yanto et al., Metabolic Engineering, doi:10.1016/j.ymben.2010.02.002,. C. reinhardtii usually stores excess carbon as starch when nutrient deprived with only limited production of neutral lipids (triacylglycerols). Mutations in the starch biosynthetic pathway cause carbon to be redirected into lipid biosynthesis and storage triacylglycerols that are suitable for liquid biofuels. The fatty acid profiles of wild-type and starch mutants were quantified by gas chromatography mass spectrometry. We found C. reinhardtii starch mutants produce significantly elevated levels of 16:0, 18:1 and 18:3 fatty acids that are suited for producing biofuels. Fluorescent spectroscopy and fluorescent activated cell sorting was used to develop high-throughput methods for screening neutral lipid accumulation in cells. Global transcriptional profiling by RNA-sequencing is underway on the Illumina platform to identify a putative gene set associated with the lipid biosynthetic pathway and to understand the molecular basis for how cells switch to lipid synthesis. We conclude that C. reinhardtii is an alga that can be manipulated to produce high levels of triacylglycerols.

FLUOROMETRIC APPROACH TO SELECTIVITY SCREENING OF MODIFIED OLIGONUCLEOTIDE HYBRIDIZATION PROBES

Kabilov M.R. and Pyshnyi D.V. Institute of Chemical Biology and Fundamental Medicine SB RAS.

Today various approaches to the detection of point mutations in DNA are used. A number of them are based on principles of allele-specific hybridization (ASH). To improve the efficiency in discrimination of imperfect duplexes, there is a reason to look for oligonucleotide analogues and derivatives with higher hybridization efficiency as compared to their native precursors. In most studies, however, there are no detail characteristics of the influence of modified probes on the selectivity of the analysis because this problem requires the in-depth thermodynamic analysis. Properly speaking, the efficiency of mismatch discrimination should be characterized by comparable thermodynamic parameters (enthalpy, entropy, and free energy) for the formation of perfect and mismatched complexes. Just these values determine the formation efficiency (the extent of association) of duplexes composed by an oligonucleotide probe under given conditions. The use of the standard real-time PCR detection system allows one to detect the change of the fluorescence level when heating samples. The advantage of this system is the opportunity of the parallel analysis of a large number of probes at rather low concentrations. We have considered various options of the fluorescent labeling of both probes and DNA templates, which should ensure the optimal level of the signal. We attempted to design a system, which could answer the question “is the modified probe more selective than native one?” by the analysis of melting curves. The parallel examination of the same oligonucleotide probes using the UV melting technique confirmed our results. The results demonstrate a possibility of the massive parallel analysis of selectivity of modified oligonucleotide probes based on the thermal denaturation of fluorescent DNA complexes.





ROLE OF CARBOHYDRATE BINDING DOMAIN ON EXPRESSION, ACTIVITY AND STABILITY OF XYLANASE C OF CLOSTRIDIUM THERMOCELLUM

Khan M.I.M., Sajjad M. and Akhtar M.W. School of Biological Sciences, University of the Punjab, Lahore-54590, Pakistan.

Xylanase (XynC), a major component of Clostridium thermocellum cellulosome, was cloned and expressed with and without carbohydrate binding domain. Four constructs with the gene encoding only the catalytic domain (xynCCD), the catalytic domain with binding domain at N-terminus (xynCCBD-CD), the catalytic domain with binding domain at C-terminus (xynCCD-CBD) and the catalytic domain with binding domains at both termini of the catalytic domain were produced (xynCCBD-CD-CBD). The XynCCD, XynCCBD-CD, XynCCD-CBD and XynCCBD-CD-CBD expressed at levels ~45%, ~30%, ~30% and ~33% of the total E. coli cell proteins after induction with lactose, as analyzed by photodensitrometric scanning of SDS-PAGE gels. The overall activities produced by XynCCD, XynCCBD-CD, XynCCD-CBD and XynCCBD-CD-CBD were 1,488, 3,489, 3,860 and 5,732 U l-1 OD600

-1 against oat spelt xylan, respectively. All these enzymes with and without non-catalytic domains were found to be quite stable over a broad pH range (pH 4 - 9). XynCCBD-CD was more thermostable as it retained 70% activity on incubation at 70ºC for 2 hrs whereas XynCCD lost total activity under these conditions. XynCCD-CBD retained 56% and XynCCBD-

CD-CBD retained 80% of its activity under these conditions. Km values for XynCCD, XynCCBD-CD, XynCCD-CBD, and XynCCBD-CD-CBD as determined on soluble xylan as substrate were 3.12, 3.57, 3.12 and 1.64 mg ml-1, respectively. Thus presence of carbohydrate binding domain on both terminals leads to the 4 fold increase in specific activity as compared to the native enzyme.

IMMUNOLOGICAL MEASUREMENT OF TROPONIN I IN SERA FROM THE PATIENTS WITH HEART DAMAGE DISEASES

Kim H.J.1, Kim T.K.1, Wang H.1, Oh S.W.2 and Choi E.Y.1 1Department of Biomedical Science, Hallym University, Chuncheon, South Korea 200-702. 2Department of Biology Education, Institute of Fusion Science and Science Education, Chonbuk National University, Jeonju, South Korea, 561-756.

A cardiac-specific iso-form of Troponin I (cTnI) has been known as a marker of heart damage for more than 20 years. At present cTnI is considered to be one of the most specific and sensitive markers of myocardial cell death. For the preparation of antigen, we produced a human cardiac troponin I recombinant protein. The cardiac toponin I clones were transformed into BL21 cell for expression and the recombinant cTn I protein was purified by affinity chromatography. The cTnI protein was confirmed to be an expected size on an SDS-PAGE gel. To generate monoclonal antibodies against the protein, recombinant cTnI was injected into BALB/C mice. From the fusion experiments, several hybridomas were selected by Western blotting and further screened by ELISA method. With the monoclonal antibodies, we developed a POCT type (point of care of test) immunoassay system and conducted performance evaluation for measuring cTnI in human serum. The linearity fell in the range 0-10ng/ml of cTnI and the analytical detection limit was 0.5ng/ml of cTnI. The precision of intra- and inter-assay was CVs<6% and CVs<8%. Also, we found that the amount of cTnI protein mass in the Heart damage patients was much higher level than that in normal population. The performance test result indicated that newly developed immunoassay system using fluorescence bead and lateral-flow chromatography is a simple, fast method for quantifying the cTnI concentration in human blood. Currently we development of an alternative method enables to measure the low amount of cTnI more accurately.

CHARACTERISATION OF THE GRAIN SPECIFIC HOMEODOMAIN TRANSCRIPTION FACTORS FROM WHEAT

Kovalchuk N., Wu W., Bazanova N., Singh R., Ismagul A., Eliby S., Johnson A., Hrmova M., Langridge P. and Lopato S. Australian Centre for Plant Functional Genomics, Hartley Grove, Urrbrae, South Australia 5064, Australia.

Two HDZip class IV genes, designated Triticum aestivum GLABRA like 7 and 9 (TaGL7 and TaGL9), were isolated using Y1H to screen a cDNA library from developing wheat grains (0-6 DAP) with the palindromic repeat -CATTAAATG- as bait. A predicted molecular model of TaGL9 suggested that at least five independently folded domains could be present, although their spatial distribution is hypothetical at this stage. The identified protein domains are currently being used as baits in the Y2H screen of a wheat endosperm library. The 3’UTR of TaGL7 and TaGL9 were used as probes to isolate genomic clones of orthologues/homologues from a Triticum durum BAC library. Full length genes containing 3 kb-long promoter regions were designated TdGL7 and TdGL9. Spatial and temporal expression patterns of TdGL7 and TdGL9 were examined in transgenic wheat, barley and rice expressing promoter-GUS fusion constructs. Whole-mount and histochemical GUS staining patterns were analyzed in large numbers of independent transgenic lines. Although very low activity of the TdGL7 promoter was detected in most plant tissues, much stronger GUS expression was observed in the female gametophyte before fertilization, and later in the syncytial and starchy endosperm. The TdGL9 promoter was active only in grain. GUS activity was initially observed on the 5th day after pollination and persisted in the embryo until grain harvest. Small differences were observed in the spatial patterns of GUS expression in wheat, barley and rice. Wheat transgenic plants with constitutive overexpression of TaGL7 and also with antisense RNA expressed under the TdGL7 promoter were generated and are currently being analysed.

TELOMERASE ACTIVITY AND ITS USE IN MONITORING BLADDER CANCER

Lalla M.1, Dezfouli S.1, Mendez R.1, Tian P.2, Elwood N.2 and Macreadie I.1 1Sienna Cancer Diagnostics Ltd., Bio21 Institute, VIC 3010, Australia. 2Cord Blood Stem Cell Research Institute, Murdoch Children’s Research Institute, VIC 3052, Australia.

The shortening of chromosomes is a normal part of the aging process, however, cancer cells are immortal and overcome lethal chromosome shortening. Most cancer cells maintain chromosomes through the activity of telomerase, a DNA polymerase that extends the ends of chromosomes through the addition of (TTAGGG) repeats. The activity of telomerase is therefore a key biomarker in monitoring cancer. Assays to monitor telomerase activity include the in vitro extension of an oligonucleotide substrate with (TTAGGG) repeats. Typically such assays involve the generation of a few attomoles of telomeric extension products (TEPs) which are amplified by the polymerase chain reaction (PCR) and then analysed on gels. Such PCR reactions are highly unusual since they generate products even without TEPs. A characteristic to be looked for in the telomeric repeat amplification protocol (TRAP) is a telomeric ladder on gels. Further refinements include measurements using real time PCR with amplifluors, however, once again products are generated without TEPs. Because of the high signal in telomerase negative controls, it is considered that PCR-based assays have limitations that may prohibit their utility for clinical assays. Work at Sienna is focused on the development of non-PCR procedures to measure telomerase activity in bladder cancer using exfoliated cells in urine so as to avoid these effects. Significant challenges are the low cell numbers in urine, the harsh environment and non-epithelial cell types. Our aim is to assay for telomerase activity in around 100 cells, each of which may have only 20 molecules of telomerase.





ANALYTICAL PREREQUISITES FOR DIFFERENTIAL AND QUANTITATIVE PROTEOMICSLevina V.1, 2, Cooke I.1, 2, Durand F.M.1, 2, Boyd S.E.2, 3, O’Connor L.2, 3, Hoogenraad N.2 and Talbo G.H.1, 2 1CRC for Biomarker Translation, La Trobe University, Melbourne, VIC, Australia. 2La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia. 3AgriBio, the Centre for AgriBioscience, Melbourne, VIC, Australia. The molecular composition of the cell surface plasma membrane and its dynamic changes determine how a cell interacts with its environment. As cell surface proteins confer specific cellular functions, and are accessible, these are the proteins of greatest interest to modern diagnostic methodology and specifically as biomarkers. Many membrane surface proteins are glycosylated and the chemically distinct properties of carbohydrates from those of proteins, have led to the development of bead based protein isolation approaches, which target cell surface glycoproteins. Plasma membrane proteins isolated by this approach are identified by tandem mass spectrometry. Despite some success most cell surface proteins remain undetected due to the quality of the methodologies currently available for the isolation of cell surface glycoproteins. This is highlighted by the relatively small number, approximately 340, of cluster determinant (CD) cell surface protein markers1 that have been identified to date, compared with the number of predicted human transmembrane proteins being greater than 13,0002. Potentially, even more important than the identification of the plasma membrane glycoproteins is determination of the relative abundance of those proteins in diseased compared with normal states of the cell. Therefore, we set out to develop a protocol encompassing a quantitative workflow from “Cell to Protein Identification and Relative Protein Amount.” In short, the glyco moiety of the outer membrane proteins was oxidised by NaIO4, the cells were lysed and the oxidised carbohydrate covalently bound to hydrazide coupled beads. The covalent binding of the proteins of interest facilitates the extensive washing required to remove membrane and other debris. The bead bound proteins were reduced, alkylated and digested by trypsin followed by washing. The residual tryptic glycopeptide was released by PNGase F and analysed by LC-ESI-microTofQ-MS/MS and MALDI-tof/tof-MS. The results were reproducible; peak intensities greater than 2000 showed less than 12 % CV across 30 replicate samples. 1) Nicholson, IC, Mavrangelos, C, Fung, K, Ayhan, M, Levichkin, I, Johnston, A, Zloa, H and Hoogenraad, NJ. J.Immunol. Methods, 305, 84-93, 2005 2) Zola and Swart, 2003.Trends Immunol.24,353, 2003.

RAPID RESPONSE OF THE HUMAN NUCLEOLAR PROTEOME TO SERUM STIMULATIONLi Z.F.1, Liang Y.M.1, Wang X.2 and So L.K.Y.3 , Lam Y.W.11Dept. of Biology and Chemistry, City University of Hong Kong. 2College of Medicine, Zhejiang University, Hangzhou, China. 3Dept. of Biochemistry, Hong Kong University.

The nucleolus is the most prominent organelle in the cell nucleus. It is responsible for synthesizing and assembling 60S and 40S ribosome subunits, and plays a crucial role in the regulation of cell proliferation and growth. The responses of the nucleolus upon external stimulations is therefore of high interest. The high density and structural stability of the nucleolus makes it possible to purify this nuclear organelle, and to analyse the dynamics of the nucleolar proteomes. However, the existing nucleolar isolation method, developed in 1963, involved relatively tedious procedures that tend to obscure momentous biochemical changes in this organelle. Here we developed a new and simplified nucleolar isolation method in which cells were rapidly harvested and lysed, thus the nucleolar proteome could be “frozen” at precisely controlled time points. The purity of nucleoli obtained using this protocol was comparable to those using the classical method, as judged by electron microscopy and Western blotting. The complete proteomes of nucleoli prepared by these two methods were also compared using SILAC-based quantitative proteomics.To further demonstrate the applications of this new method, we performed a time-lapse nucleolar proteomics after serum stimulation. HeLa cells were serum starved for 24 hours and then serum re-stimulated, and the nucleolar proteome dynamics was followed in the first 10 minutes of serum replenishment. This approach reveals for the first time that some nucleolar proteins responded to serum stimulation within as short as the first 5 min. Subsets of small and large ribosomal proteins, histones and heterogeneous nuclear ribonucleoproteins family increased 20% to even 100% within 10 mins. Interestingly, the eukaryotic translation initiation factor 6 (EIF6) and the proliferation marker Ki-67 were found to be quickly accumulated in the nucleolus within 10 mins. To the best of our knowledge, this is the first study demonstrating these “quick triggering proteins” in the nucleolus. The underlying mechanisms and the roles of these very early events in cell growth will be discussed.

DISCOVERY OF NOVEL PHAGE PROTEINS FOR BIOTECHNOLOGY BY HIGH THROUGHPUT SEQUENCING OF PHAGE DNA FROM SOUTH AFRICAN DEEP GOLD MINES

Mabizela N.B. and Litthauer D. Metagenomics Platform, Department of Microbial Biochemical and Food Biotechnology, University of the Free State. Nelson Mandela Drive, Bloemfontein, South Africa.

Shotgun sequencing of metagenome libraries from South African mines revealed untapped phage communities in the deep subsurface. The majority of the clones from four mines shared no similarity to known proteins. Pyrosequencing was used to assess the metagenomic diversity of phage DNA from Beatrix Mine in South Africa. Annotated data showed that approximately 75% of the proteins had no homology to any known proteins in public databases. About 40% of the proteins that were assigned to a specific function were of viral origin. Most of the hits were from the Enterobacteria phages and Acanthamoeba polyphaga mimivirus. Seven prophage regions of bigger than 4 kb were identified using Prophage Finder. Novel viral proteins with biotechnological functions were identified. In this study a phage DNA ligase protein was identified among the hundreds of genes obtained. Sequence analysis indicated that the protein is novel showing 46% similarity to its closest relative. In addition the second, most conserved motif, SLRFPRFIRIR was not detected; only that containing the catalytic lysine. The gene encoding this protein was cloned and expressed in E. coli. The protein is 41 kDa in size, is ATP-dependent and ligated cohesive and blunt ended λ-DNA fragments, the former being ligated more efficiently. Maximal ligation of the blunt ends was achieved with added polyethelene glycol (10%). The enzyme is active at 4 ºC, 16 ºC and 22 ºC. BamHI cut pUC19 plasmid could be ligated at 30, 40, 50, 60 and 70 ºC.

SIGNIFICANT IMPROVEMENT OF THE THERMO-STABILITY AND OPTIMUM PH RANGE OF CATALYTIC ACTIVITY OF A LACTONE-SPECIFIC ESTERASE BY RANDOM MUTAGENESI

Ishizuka M.1, Tsukimura W.1, Namiki K.1, Akanuma G.1 and Ushio K.2 1Department of Applied Chemistry, Chuo University, Tokyo, Japan. 2Department of Applied Chemistry and Biotechnology, Niihama National College of Technology, Niihama, Japan.

Stable and high stereo-selective lipase (EC 3.1.1.3) and esterase (EC 3.1.1.1) have attracted much attention from viewpoints of industrial usage. Pseudomonas-like bacterium isolated in our laboratory from wastewater sample produces a thermo stable lipase by the addition of fatty alcohols (stearyl alcohol and palmityl alcohol) as the most effective super-inducers. The addition of fatty alcohols brought about more than several hundred-fold enhancement of the lipase activity compared to the case with no additive. This means several dozen-fold enhancement of lipase activity compared with olive oil grown case. Gram per litter lipase production has been capable. On the other hand, carboxylic ester esterase is not induced by the addition of fatty alcohols. In this study, we report a lactone-specific esterase expression in Escherichia coli and significant improvement of the thermo stability and wide optimum pH range of catalytic activity of the mesophilic esterase by random mutagenesis and evolutionary engineering. Thermal stability of a purified mutant esterase (R29C, Q82L, R96H, N212D, R286H) obtained by the error-pron PCR (EP-PCR), DNA shuffling, and thermal selection (three times) rises 12 °C in temperature (10 fold long in half-life period) compared with the stability of the purified wild-type esterase. As a result of site-specific mutagenesis, Contribution of R96H and N212D for thermal stability are 5 °C and 4 °C, respectively. Additionally, wide optimum pH range (7-9) of the mutant catalytic activity and stability were obtained without the loss of the lactone-specificity (γ-Butyrolacton, δ-Valerolactone, and ε-Caprolactone).





EFFECTIVENESS OF PEER DISCUSSION FOR STUDENT UNDERSTANDING OF ACID-BASE EQUILIBRIA

Watters D.J. and Love C.A. School of Biomolecular and Biophysical Sciences, Griffith University, Nathan Campus, Brisbane, Australia 4111.

Students frequently have difficulty in understanding the central concepts of acid-base chemistry and buffers. The term, troublesome knowledge, as defined by Meyer and Land, accurately describes this problem since students are able to perform superficial tasks such as titrations, and apply the Henderson Hasselbalch equation without understanding the basic concepts (ritual knowledge). They also fail to transfer their understanding of simple acids and bases to the structure and function of proteins or other complex biological molecules. Thus pH could fit into the category of a threshold concept which is core to the understanding of many aspects of chemistry, cell biology, physiology and biochemistry. We investigated the use of concept questions in class with the use of clickers to identify student misconceptions, as well as peer discussion, in an effort to improve student learning of these topics. Peer discussion has previously been shown to have a positive effect on student learning. Our results show that, in the case of threshold concepts such as acid-base equilibria, peer discussion is not effective and merely propagates misconceptions. These findings suggest that substantial groundwork needs to be done to enable students to gain the necessary understanding of the fundamental concepts of acid-base chemistry.

ACQUISITION OF SCIENTIFIC WRITING SKILLS THROUGH ACTIVITIES EMBEDDED INTO BIOLOGICAL LABORATORY COURSES

Tonissen K.F.1, Lee S.E.1, Woods K.J.1 and Osborne S.A.1, 2 1School of Biomolecular and Physical Sciences, Griffith University, Nathan, Qld, Australia 4111. 2CSIRO Livestock Industries, St Lucia, Qld, Australia, 4067.

Scientific writing skills are important for a science career, yet specific training can be difficult to integrate effectively into a University program. Rather than design specific courses solely focused on writing, we embedded writing activities into two project style third year biological science laboratory courses where students wrote about their own data. Students were expected to complete the writing exercises during breaks in their experimental procedures and were given feedback during the laboratory session. These activities were focussed on preparing figures, tables, figure legends, and writing results and discussion paragraphs. These exercises provided practice and a model to assist students in writing the remainder of the report. We probed student opinions regarding scientific writing and the use of the exercises by anonymous pre- and post-course surveys using a combination of closed and open questions. In the first course student confidence towards scientific writing and performing simple writing tasks significantly improved after experiencing the writing activities. Therefore students commenced the second course with a higher confidence level. They related that undertaking writing activities in more than one class helped them consolidate their writing skills and challenged them to improve further. Students also commented that they thought the activities helped them develop skills relevant to future scientific careers. Independent assessors evaluated the standard of students’ written reports that originated from the same course held in years before and after writing activities were incorporated into the curriculum. There was a significant improvement in scientific writing quality that correlated with the increase in student confidence and attitudes towards writing.

HOW RESEARCH INFORMS AND ENHANCES LEARNING USING STUDENT PROJECT CASES IN THE MONASH MBBS PROGRAMME

Samarawickrema N.A. and Macaulay J.O. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800.

There is a strong synergistic connection between teaching and research because (1) research fertilises teaching with new topics and methods (2) research provides teachers with personal engagement and (3) academic staff research guarantees connections with developments in the research arena. In turn, students perceive their courses to be up to date and intellectually stimulating. Here we report the results of a study of undergraduate student engagement in a research tutored curriculum based on the current research of the tutor. We used Student Project Cases (SPC) offered in second year of the Monash University medical curriculum, which comprises a teamwork activity that emphasises interdisciplinary learning, where the students research and present a medical disease. The co-topics required for this SPC were specified and the students were required to research into these and prepare a written and an oral presentation as part of the SPC assessment process. At the end of the SPC, students completed a questionnaire which examined their exposure to research and research culture, their experiences on the preparation of this SPC and participated in a Focus group. The students stated that the knowledge that the SPC topic being a current research interest of an academic staff member involved with the SPC process had a positive impact on their attitude towards the topic. They stated that being aware that the research was happening now made it exciting to study about the topic and therefore a motivational factor.

CONSTRUCTING A GENERALIST BIOCHEMISTRY COURSE AROUND CORE CONCEPTS: BUILDING THE BIG PICTURE FOR A LARGE MIXED-LEARNER COHORT

Rowland S.L., Gillam E., Hamilton S., Harrinson J., Wright T. and Ward L. University of Queensland.

The University of Queensland has recently revised the BSc, and consequently we now offer a single, second-year, undergraduate biochemistry course. This course, BIOC2000, must service over 500 students from almost 20 different programs of study. The students have many different backgrounds, goals, and abilities. This is a radical departure from our smaller, more “science-centric” cohorts of previous years. In its first year of delivery, BIOC2000 uncovered serious problems in understanding of basic chemistry concepts for many of the cohort as evidenced by the answers to a chemical concept inventory test that we delivered to the students, and also by the final exam results. We decided to rebuild the course around core chemical and biochemical concepts, rather than around content, and use a “Backwards design” approach to the new curriculum. Developing student understanding of fundamental chemistry concepts and their application to the molecules and structures of life is the primary learning objective of the new curriculum. We surveyed over 80 working scientists to determine the “fundamental concepts” of biochemistry. We will present our data regarding (i) what these concepts are, (ii) how we determined if the students understood them before the course started, (iii) how we integrated these concepts into learning and assessment activities, and (iv) how student understanding of these concepts changed during the course. This project provides a roadmap for generalist course design around the fundamental guiding principles of a discipline, rather than around content. The presentation should be useful to all course designers.





IMI HO’OLA POST-BACCALAUREATE PROGRAM AT THE UNIVERSITY OF HAWAII SCHOOL OF MEDICINE

Ha C., Lee W.M. and Judd N. University of Hawaii School of Medicine, 651 Ilalo Street, MEB306, Honolulu, HI 96813, USA.

Imi Ho’ola (Hawaiian meaning “Those who seek to heal”) is a post baccalaureate program established in 1972 to prepare students from socially, educationally, or financially disadvantaged backgrounds for the entry into the rigors of medical school education. Each year 10 students are selected to participate in the one-year program and upon matriculation students are guaranteed acceptance into the University of Hawaii School of Medicine as regular first year medical students. The curriculum of the Imi Ho’ola program spans over 12 months and consists of three phases: 1. Summer orientation and assessment phase to obtain base-line data on students’ knowledge in the sciences, reading, and learning skills, Phase 2. Enrichment phase to improve students’ critical thinking skills in the content areas of biology, medical biochemistry, scientific basis of medicine, and speech and ethics in healthcare, Phase 3. Prematriculation phase to help students’ smooth transition into regular medical school curriculum with introduction of clinical skills by shadowing physicians. For the past 30 years, Imi Ho’ola program has provided educational opportunities to disadvantaged minority students pursuing a career in medicine. Out of 204 graduates of the program and medical school, 96% provide health services to disadvantaged or underserved populations in the state of Hawaii and the Pacific islands and 72% provide primary care services such as internal medicine, family medicine, and pediatrics. Imi Ho’ola program is a successful educational model that contributes to the development of a diverse healthcare workforce that provides services for the underserved communities of Hawaii and the Pacific region.

STUDENT ENGAGEMENT IN 1ST YEAR BIOSCIENCES

Nott M.W. and Poronnik P. RMIT University, School of Medical Sciences, Melbourne.

Students typically fail to fully realise the relevance of core preliminary subjects, and in particular the generic skills they need to develop in 1st year bioscience studies: chemistry, mathematics and statistics, and even biology. In Pharmaceutical Sciences at RMIT University we have devised a series of interventions that require 1st year students to draw on and integrate their learning in each core subject. For the first intervention, staff from each discipline agreed on the common theme: Malaria and its Drug Treatment. The theme fulfilled criteria aimed to enhance student engagement and their awareness of relevance of each subject. Thus the theme is contemporary, given that many of our students or their families come from malaria-endemic countries; media-rich, given that malaria (and dengue) is increasingly in the news because of global warming and the need for new strategies for its treatment (eg, artemisinins) and eradication (eg, genetically engineered flightless mosquitoes); and research-relevant, given that Melbourne is a centre for immune studies approaches. The theme allowed seamless integration of learning materials in chemistry (which stressed molecular structures of antimalarials and their binding to protein targets); maths and statistics (which stressed epidemiology and vector population studies) and biology (which stressed drug-susceptible stages of the life cycle of plasmodia). Students worked in teams to create and defend posters, thus honing their visual and oral presentation skills as they worked on 9 topics around the central theme. In doing so, students were able to integrate their first year studies as well as get an early start on developing professional capabilities according to RMIT University’s Graduate Attributes: 1) work ready; 2) environmentally aware and responsive; 3) global in outlook and competence; 4) culturally and socially aware; 5) life-long learners; 6) active learners; 7) innovative.

GENE EXPRESSION PROFILING IN CIRCULATING CELLS OF BREAST CARCINOMA PATIENTS

Zima T1, Mikulova V1, Tesarova P1, Kolostova K2, Kubecova M2, Rusnakova V3 and Kubista M3 1General University Hospital and First Faculty of Medicine, Charles University in Prague. 2 General University Hospital Královské Vinohrady and Third Faculty of Medicine, Charles University in Prague. 3 Institute of Biotechnology, Academy of Sciences, Czech Republic.

Background: Tumor cell dissemination is an early process in breast cancer (BC) and circulating tumor cells (CTCs) are considered potential surrogate marker for the detection and characterization of minimal residual disease. Here we monitored hematogenous micrometastasis in BC patients by gene expression profiling of CTCs based on CTC-abundance in blood and expression of 35 oncomarkers at the mRNA level by multimarker quantitative PCR. Methods: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study. Immunomagnetic enrichment of CTCs from the 5ml of whole blood followed by cells characterization using gene expression analysis for the presence of tumor associated genes HER2, MUC-1 and GA 733-2 were processed using AdnaTest BreastCancer® kit. If possible bone marrow samples were obtained for disseminated tumor cells detection using Epimet® Kit. RNA from FFPE tumor tissue (n=85) has been isolated. All obtained cDNA molecules have been gene-specifically pre-amplified for multimarker qPCR analysis measured on Biomark® microfluidic chip. Results: 286 CTC samples have been analyzed in total. The analysis has shown that the gene expression profiles of CTCs in primary breast cancer patients correlated to those measured on the primary tumor, while CTCs of metastatic BC patients had significantly different gene expression profiles. Analyzing the gene expression data from CTC-positive patients in comparison to CTC negative patients and FFPE samples we have revealed several genes that were differentially expressed (p<0,05) (e.g. CK19, GA7332, AURKA, MLF1IP, SATB1, PTEN).

CHARACTERISATION OF PUTATIVE PLASMODESMATA PROTEINS OF THE ARABIDOPSIS CALNEXIN FAMILY

Liu D.Y.T.1, Smith P.M.C.1, Day D.A.1, 2 and Overall R.L.1 1School of Biological Sciences, The University of Sydney, Sydney NSW 2006 Australia. 2Present address: Flinders University, Adelaide SA 5042 Australia.

Plasmodesmata are plasma membrane-lined channels spanning the cell wall, connecting the cytoplasm and endoplasmic reticulum (ER) of adjacent cells. Plant survival depends upon the proper function and regulation of plasmodesmata, since developmental signals, nutrients, and even viruses move through these channels. However, only a few protein constituents of plasmodesmata have been discovered. In this study, we extend a comparative proteomics analysis of Chara (Faulkner et al., Proteomics 5: 2866) by identifying and characterising Arabidopsis proteins with sequence similarity to characean peptides isolated from plasmodesmata-rich cell fractions. These proteins were screened for plasmodesmatal localisation in Arabidopsis and Nicotiana benthamiana using green fluorescent protein (GFP) tagging. Calnexin, one of the proteins identified, is a type I membrane protein localised in the ER with the main catalytic domain lying within the ER lumen where it may function as a chaperone. The Arabidopsis calnexin protein family consists of two members, AtCNX1 and AtCNX2, with 83% amino acid identity. Both proteins colocalise with aniline blue-induced fluorescence of callose at plasmodesmata, although colocalisation is reduced upon deletion of the signal peptide. No significant morphological differences were observed in single or double homozygous Arabidopsis T-DNA knock-out mutants of AtCNX1 and AtCNX2. However, cell-to-cell diffusion of GFP as well as deposition of callose at plasmodesmata were affected in mutants, suggesting some role for calnexin in plasmodesmatal physiology. Quantitative real-time PCR data suggested that in knock-out mutants other chaperones may provide redundancy for CNX, including calreticulin, an ER-lumenal homolog of calnexin. The role of calnexin at plasmodesmata will be discussed.





CRITICAL ROLE OF C-ZYME ON P53IG-MEDIATED APOPTOSIS

Kang M.Y. Department of Bio-Materials Engineering, Graduate School and DNA Repair Research Center, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Korea.

A number of genes involved in control of the cell cycle and apoptosis are regulated by p53-induced genes (p53IGs). A series of p53IG have been identified that are predicted to encode proteins that could generate or respond to oxidative stress. C-zyme, an anti-oxidative enzyme is an important scavenger of ROS related to p53IG activation. We found that purified p53IG coimmunoprecipitated recombinant C-zyme suggesting that p53IG directly binds to C-zyme. In vivo IP analysis also revealed that endogenous p53IG bound C-zyme. A confocal microscopy examination showed both proteins were co-localized after treatment with UV. Purified picogram of p53IG protein significantly suppressed C-zyme activity in a dose-dependent manner. We observed that various cell lines transfected with p53IG exhibited elevation of ROS production. Accordingly, C-zyme activity were significantly decreased. p53IG overexpressed cells infected with Ad C-zyme was resistant to apoptosis. These data demonstrate that after DNA damage, p53IG is involved in triggering apoptosis by repressing C-zyme function. Although the exact role of p53IG in p53 pathway is not yet elucidated, p53IG may be one of the factors involved in p53-induced apoptosis through ROS generation.

PHOSPHORYLATION OF PTP-PEST AT SER-39 IS REGULATED THROUGH THE BINDING OF PP1 ALPHA

Palmer H.E.F., Nakamura K. and Mashima K. Department of Life Science, Rikkyo University.

PTP-PEST is expressed in a wide variety of cell types and is an efficient regulator of integrin-medicated signaling in adherent cells and antigen receptor-mediated signaling in lymphocytes. PTP-PEST-associating molecules are important in elucidating the function of PTP-PEST. The major phosphorylation site of PTP-PEST at Ser-39, regulates the PTP activity. Herein, we have identified protein phosphatase 1α (PP1α) as a novel PTP-PEST binding protein, and tried to elucidate the mechanism of Ser-39 dephosphorylation through this molecule. HEK 293 cells overexpressing exogenous PTP-PEST, were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39 PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated that the phosphorylation at Ser39, detected by anti-pS39-PEST Ab, was dependent on TPA treatment corresponding with previous reports. Moreover, we discovered a significant inverse correlation between the PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive band intensity quantitatively. PP1α wild-type induced dephosphorylation of PTP-PEST at Ser39, where as, PP1α dominant-negative mutant had no effect. TPA-induced Ser39 phosphorylation was not abrogated by PP1α wild-type when cells were expressed with PTP-PEST without the non-catalytic segment, indicating that the dephosphoylating action of PP1α could possibly occur through binding at non-catalytic segment of PTP-PEST. In conclusion, PP1α associates with the non-catalytic segment of PTP-PEST and regulates PTP activity via dephosphorylation of phospho-Ser39. We further seek to specify the binding site of PP1α to PTP-PEST and clarify the cellular function of phosphorylation/dephosphorylation of PTP-PEST at Ser39.

CD317/TETHERIN AND LIPID RAFT ORGANISATION

Billcliff P.1, Rollason R.1, Prior I.A.2 and Banting G.1 1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. 2Division of Physiology, University of Liverpool, Crown St, Liverpool L69 3BX, UK.

CD317 (Bst2, tetherin) has been implicated in preventing the spread of HIV-1 virions from infected cells by tethering them to the cell surface. The cytosolic domain of CD317 interacts indirectly with the actin cytoskeleton, and its knockdown in polarised epithelial cells leads to a loss of the sub-apical actin network. Whilst the N-terminus of CD317 interacts with the actin cytoskeleton, the glycophosphatidylinositiol (GPI)-anchored C-terminus is localised to lipid rafts; this unusual topology of CD317 could enable it to functionally unite the actin cytoskeleton and lipid rafts. Moreover, the extracellular region of CD317 forms a parallel coiled-coil; this rigid structure might therefore limit teh free diffusion of proteins into and out of lipid rafts, thereby organising rafts. We present preliminary evidence that CD317 is indeed involved in the organisation of lipid rafts. CD317 has previously been shown to activate the NF-κB pathway and, in line with this, increased expression of CD317 upregulated the activity of an NF-κB-activated luciferase reporter, denoting that CD317 might aggregate signalling complexes from initally disparate rafts, and thus augment signal transduction events. In addition, using a GPI-YFP reporter plasmid (a lipid raft marker) in concert with fluorescence recovery after photobleaching (FRAP) assays, the lateral diffusion mobility of GPI-YFP was increased in cells where CD317 was knocked down - a result consistent with CD317 playing a role in lipid raft organisation. Further, we use electron microscopy on prepared membrane sheets to investigate the importance of CD317 in the formation and clustering of lipid rafts, using the raft marker GFP-tH.

A NOVEL CONTROL POINT IN CHOLESTEROL BIOSYNTHESIS

Gill S., Stevenson J., Kristiana I. and Brown A.J. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

Cholesterol homeostasis is maintained in part through control of its biosynthesis. Study has focussed on 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), billed as the ‘rate-limiting’ enzyme in the pathway and target of the blockbuster statin class of drugs. In contrast, relatively little is known about the more than twenty other enzymes involved in the synthesis of the essential cholesterol molecule, including squalene monooxygenase (SM). SM performs the first oxygenation step in cholesterol synthesis, prior to cyclisation of its product into the steroid backbone structure. We examined the regulation of SM using a mammalian cell culture system, particularly Chinese hamster ovary (CHO) cells. Surprisingly, cholesterol treatment caused squalene to accumulate, indicating that SM may serve as a ‘rate-limiting’ enzyme beyond HMGCR. This accumulation was also seen in mutant CHO cells lacking sterol regulated transcription, implying that cholesterol regulates SM post-transcriptionally. Indeed, we found that SM protein was degraded within hours of cholesterol addition. Proteasomal inhibition blocked this degradation and reversed the squalene accumulation, suggesting that the cholesterol-induced degradation of SM is a flux control point in cholesterol synthesis. Regulated degradation required the N-terminal domain of SM conserved in higher organisms, but not required for activity. This region also conferred cholesterol-regulated turnover on heterologous fusion proteins. We have identified a significant novel control point in the feedback regulation of cholesterol biosynthesis.





DIVERSITY AND BIOSYNTHESIS OF SANDALWOOD OIL: SCENTS FROM SANTALUM

Plummer J.A.1, Jones C.G.1, 2, Moniodis J.1, Zulak K.G.1, 2, Barbour E.L.1, 3, Bohlmann J.2 and Ghizalberti E.L.4 1Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009 Australia. 2Michael Smith Laboratories, University of British Columbia, 301 - 2185 East Mall, Vancouver BC V6T 1Z4, Canada. 3Forest Products Commission of Western Australia, 117 Great Eastern Hwy. 4Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009 Australia.

Sandalwood oil is arguably the worlds most valuable fragrance ingredient. The heartwood of mature sandalwood trees (Santalum spp.) contains a complex mixture of sesquiterpenes, with a composition profile reflecting differences between and sometimes within species. Plantations relieve harvest pressure on wild stands, however silviculture is complicated and heartwood oil yields are often low. Our research seeks to understand factors which control sesquiterpene biosynthesis in sandalwood. A PCR-based cloning strategy was used to isolate cDNAs encoding several terpene synthase genes responsible for biosynthesis of key components of sandalwood oil from three phylogenetically divergent species (S. album, S. spicatum and S. austocaledonicum). The encoded proteins have been functionally characterised by over-expression in E. coli and incubated with the universal sesquiterpene precursor FPP. The diversity of compounds produced by these enzymes accounts for the natural chemical diversity found across the genus. Genomic structure of these genes is conserved, exemplifying the evolutionary importance of sesquiterpene production in this genus. This research has applications for oil yield improvement in this expanding industry.

DELETIONS, REPEAT-INDUCED POINT MUTATIONS AND AMINO ACID SUBSTITUTIONS DRIVE EVOLUTION OF LINKED EFFECTORS IN A FUNGAL PLANT PATHOGEN

van de Wouw A.P.1, Cozijnsen A.J.1, Hane J.2, Brunner P.C.3, McDonald B.A.3, Oliver R.P.2 and Howlett B.J.1 1School of Botany, the University of Melbourne, Vic, 3010. 2Australian Centre for Necrotrophic Fungal Pathogens, Murdoch University, WA, 6150. 3Institute of Integrative Biology, ETH Zurich, Switzerland.

Pathogen effectors, molecules that include small secreted proteins such as avirulence proteins, facilitate infection or induce defence responses by plants. We report the first large scale study of evolutionary processes affecting linked effector-like genes in a fungal plant pathogen. Mutations affecting seven genes and four single copy non-coding regions located in a 520 kb repetitive element-rich region of Leptosphaeria maculans, a pathogen of Brassica napus (canola) are described. Two genes are avirulence genes, AvrLm1 and AvrLm6, which are complementary to B. napus resistance genes, Rlm1 and Rlm6. Analyses of 295 Australian isolates showed that deletions, Repeat-Induced Point (RIP) mutations and/or non-RIP derived amino acid substitutions account for rapid evolution of four small secreted proteins. RIP was confined to three genes and two other single copy regions and appeared to have ‘leaked’ from flanking repetitive sequences. The RIP alleles were monophyletic and present only in isolates collected after 2004, the year when canola cultivars with resistance conferred by Rlm1 suffered severe yield losses. This co-incided with a large increase in the frequency of isolates with virulence alleles of AvrLm1 and AvrLm6, even though the canola cultivars lacked Rlm6. Evolution of these two effectors thus appears to be influenced both by the genomic environment (flanking repetitive elements) and by selection pressure from extensive sowing of crop varieties with resistance genes complementary to the avirulence effector gene.

SODIUM EXCLUSION GENES INCREASE SALINITY TOLERANCE IN DURUM WHEAT

Munns R. and James R. CSIRO Plant Industry, Canberra.

In a project aimed to increase the salt tolerance of durum wheat to match that of bread wheat, two major genes for Na exclusion named Nax1 and Nax2 were discovered. These lower the Na concentration in the leaf blade by removing Na from the transpiration stream as it flows from roots to shoots. Both genes belong to the HKT transporter family. Nax2 (HKT1;5-A) is expressed in roots; Nax1 (HKT1;4-A2) is expressed in both roots and shoots. Glasshouse studies showed that the Nax genes keep leaves alive for longer, by reducing the rate at which Na accumulates to toxic levels. This allows more photosynthate to reach the developing grain and so increase the grain yield. The Nax genes originated from Tricitum monocoocum and are not present in modern wheat. When crossed into Australian durum wheat and bread wheat cultivars, they reduced the Na+ accumulation in leaves. Field trials validated the function of these genes in Na exclusion. Na concentrations in the flag leaf were reduced over 100 times, e.g. from 250 to 1 umol per g DW. These showed that the presence of Nax2 increased yield of durum wheat in saline soil by 25%.

FLOWERING LOCUS H (FLH) AND THE VERNALISATION PATHWAY

Seedat N. and Gendall A. LaTrobe University Bundoora.

In wild and cultivated annual plant species flowering time is an important trait which coordinates the plants life cycle with the local environmental conditions. The genetic network of flowering time genes in the model species Arabidopsis thaliana has been widely studied revealing a complex genetic network of flowering time genes that are able to detect the external environmental and internal signals to coordinate reproductive development, and ensure it occurs at an optimum time for reproductive success. FLOWERING LOCUS H (FLH) is a novel flowering locus which enhances the vernalisation response resulting in earlier flowering time. This study is aimed at determining FLH’s interaction within the vernalisation pathway. Pervious work undertake in your lab has revealed that FLH has the ability to accelerate flowering in many of the vernalisation pathway mutants such as VERNALISATIN 5, VERNALISATION 1, VERNALISATION INSENSITIVE 3 and FLOWERING LOCUS C. FLH has shown to have an additive affect on these mutations, enabling these late flowering phenotypes to become over-come returning flowering time to an early flowering phenotype yet not as early as wild-type. FLH has also demonstrated the ability to accelerate flowering time in mutations occurring in meristem identity genes in the vernalisation pathway such as FLOWERING LOCUS T (FT), CAULIFLOWER, APETALA1 and SUPPRESSOR OF OVEREXPRESSION OF CO 1. However in the absence of FLOWERING LOCUS D (FD), FLH was unable to over-come the late flowering phenotype. As FT and FD interact to promote flowering, these results suggest that that FLH is able to substitute for the loss of FT but not the loss of FD.





CHARACTERIZATION OF STRAWBERRY PLANTS OVER-EXPRESSING THE ANTHOCYANIN-REGULATING GENE, MYB10

Lin-Wang K.1, McGhie T.K.2, Espley R.V.1, Hellens R.P.1 and Allan A.C.1 1The New Zealand Institute for Plant & Food Research Ltd, Mt Albert Research Centre, Auckland, New Zealand. 2Plant & Food Research, Palmerston North, New Zealand.

Strawberry has become an ideal model for functional genomics research in Rosaceae, because of its relatively small genome size, ease of transformation, and genome sequence resources. Anthocyanins are secondary metabolites found in higher plants that contribute to the colours of flowers, fruits and seeds. The anthocyanin pigment pathway is regulated by transcription factors that include MYB, bHLH and WD-repeat proteins. Anthocyanin-upregulating MYBs from Fragaria ananassa and Fragaria vesca have been successfully isolated (Lin-Wang et al., 2010). Strawberry transgenic lines of both species over-expressing 35S:FaMYB10 and 35S:FvMYB10 had significant increases of anthocyanin in roots, leaves and fruits. The expression analysis of wild-type and over-expressing lines suggests a strong correlation between the expression of the anthocyanin biosynthetic pathway gene CHS, DFR, F3H and UFGT and the expression of these MYB transcription factors. HPLC analysis of fruit showed up to 2- to 4-fold increases in anthocyanin content in 35S:FaMYB10 and 35S:FvMYB10 lines compared with the wild-type strawberry fruit.

ARTIFICIAL MICRORNA-MEDIATED DOWNREGULATION OF STARCH BRANCHING ENZYME IIB EXPRESSION IN RICE

Butardo V.M.1, 2, 5, Fitzgerald M.A.4, Bird T.3, Resurreccion A.P.4, Larroque O.1, 2, Gidley M.J.5, Morell M.K.1, 2 and Rahman S.1, 2 1CSIRO Food Futures Flagship, GPO Box 93, North Ryde, NSW 1670, Australia. 2CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia. 3CSIRO Food and Nutritional Sciences, PO BOX 10041, Adelaide SA 5000, Australia. 4Grain Quality, Nutrition and Postharvest Centre, International Rice Research Institute, Los Baños, Laguna 4031, Philippines. 5Centre for Nutrition and Food Sciences, University of Queensland, Brisbane, Qld 4072, Australia.

The amylose extender (ae) mutation in rice and maize is associated with a significant reduction in the expression of starch branching enzyme IIb (SBEIIb). In this study, we have generated transgenic Nipponbare rice plants containing RNA silencing constructs aimed at specifically down-regulating the expression of SBEIIb in the rice endosperm using artificial microRNA (amiRNA) and hairpin RNA (hp-RNA). By comparing the grain and starch properties of generated transgenic lines, we demonstrate that the ae phenotype can be obtained by endosperm-specific silencing of SBEIIb gene expression. Using artificial microRNA led to somewhat greater changes in starch structure than the hairpin-RNA when compared to wild type and ae rice starch. This study demonstrates that down-regulation of starch enzyme expression in cereal endosperm can be achieved using long hairpin-RNA but also by the short artificial microRNA technique, thereby demonstrating the value of using this RNA silencing technique in modifying starch structure, and therefore functionality, in rice and potentially other cereals.

GROWTH, PROLINE AND ION ACCUMULATION IN SAFFLOWER CALLUS CULTURES UNDER DROUGHT-INDUCED OSMOTIC STRESS

Karimi N., Ghasempour H.R. and Soheilikhah Z. Department of Biology, Science Faculty, Razi University, Kermanshah, Iran.

Safflower (Carthamus tinctorius L.) is s a highly branched, herbaceous crop in Iran that its production affected by several biotic and abiotic stress factors. The selection of bean cultivars adapted to salinity, drought and other abiotic stresses is a main goal of the breeders. This will contribute to understand the resistance mechanisms involved in different safflower genotypes with special reference to drought. Cell cultures of different genotypes (G1: LRV-51-51, G2: Lesaf, G3: Gila, G4: Kino-76, G5: Isfahan) of Carthamus tinctorius were established from callus tissues inoculated in MS liquid medium supplemented with 0.5 mg/L NAA (naphthalene acetic acid), 0.5 mg/L BAP (6- banzeyl aminopurine) and 0.5 mg/L 2,4 D. Mannitol was added to the medium to induce water deficit. Relative growth rate, proline, minerals and callus water content were determined at the end of stress and relief periods. After the stress period, calli derived from five cultivars showed a decrease in RGR, but at lesser extent in G1 than other genotypes. Same tendency was recorded in the callus water content under mannitol induced osmotic stress. Moreover, the G1 genotype accumulated more K+, Na+ and N but less Ca++ and P than the other four genotypes. The proline level showed a positive correlation with the degree of tolerance to water stress (with marked accumulation in G1), which suggests that proline accumulation accompanies survival and growth in drought environment. The results indicate that the response of the callus of G1 Cultivar is more resistance to drought stress in comparison to that of other genotypes.

STRUCTURAL AND FUNCTIONAL CHANGES INDUCED BY TYROSINE NITRATION IN CYTOCHROME C, A BI-FUNCTIONAL PROTEIN.

Diaz-Moreno I.1, Garcia-Heredia J.M.1, Nieto P.M.2, Orzaez M.3, Teixeira M.4, Perez-Paya E.3, Diaz-Quintana A.1 and De La Rosa M.A.1 1Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla - CSIC, Spain. 2Instituto de Investigaciones Químicas. Universidad de Sevilla - CSIC, Spain. 3Centro de Investigación Príncipe Felipe. Valencia, Spain. 4Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa, Portugal.

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Respiratory cytochrome c, with 4-6 tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain and, on the other hand, it serves as a cytoplasmic apoptosis triggering agent. First, we have analyzed the nitration-induced changes in secondary structure, thermal stability, heme environment, alkaline transition and molecular dynamics of the five monotyrosine mutants of human cytochrome c - which have all their tyrosine residues but one replaced by phenylalanines. The resulting data, along with the functional analyses of the mutants, suggests that the specific nitration of Tyr46 and Tyr48 - which are both close to the heme propionate groups - and that of the solvent-exposed Tyr74 impairs the electron transfer to (horse) cytochrome c oxidase, enhances the peroxidase activity of cytochrome c and blocks its ability to activate caspase-9. - See García-Heredia et al. (2010) BBA Bioenergetics, in press.





APPLICATION OF LENTIL LECTIN IN THE GLYCOPROTEOMIC PROFILING OF SERUM SAMPLES FROM PATIENTS WITH NASOPHARYNGEAL CARCINOMA

Seriramalu R.1, Pang W.W.2, Abdul-Rahman P.S.1, 2, Bustam A.Z.2, 3, Khoo A.S.B.4 and Hashim O.H.1, 2 1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. 2University of Malaya Centre for Proteomics Research, University of Malaya, Kuala Lumpur, Malaysia. 3Clinical Oncology Unit, University of Malaya Medical Centre, Kuala Lumpur, Malaysia. 4Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia.

The lentil lectin from Lens culinaris showed high specificity and affinity for the alpha-mannose and branched fucose residues of the N-glycans of glycoproteins. In this targeted proteomics study, we have isolated glycoproteins from pooled serum samples from patients with nasopharyngeal carcinoma WHO type III using immobilised Lens culinaris lectin affinity chromatography. More than twenty clusters of serum proteins were detected when the captured proteins were eluted and subjected to 2-dimensional gel electrophoretic profiling. The protein profile obtained was then compared with that similarly generated from pooled serum samples of normal individuals and identities of the resolved proteins were confirmed using mass spectrometry.

STRUCTURAL DECAY OF FISH MYOGLOBIN THROUGH THERMAL TREATMENT

Ochiai Y. Univ. Tokyo, Japan.

The effect of thermal treatment was examined on myoglobin (Mb) purified from the skeletal muscle of tuna by ammonium sulfate fractionation and gel filtration chromatography. The temperature-dependent changes of molar ellipticity or helical content obtained by circular dichroism spectrometry suggest that Mb performed a three-step denaturation, where small structural changes were initiated even below 20oC. On the other hand, major structural changes took place at around 60oC and 70oC. Differential scanning calorimetry on the thermal denaturation of this Mb demonstrated somewhat different thermal denaturation patterns: namely, four peaks were obtained. The largest denaturation took place at around 73oC, while slight denaturation proceeded at around 28oC. All these results suggest that the structure of fish Mb is subjected to perturbation even at lower temperature compared to mammalian Mbs. Some essential differences were recognized in the modeled tertiary structures of these Mbs, especially in the loop region and cavity volume.

STRUCTURE-ACTIVITY RELATIONSHIP IN HUMAN ANTIQUITIN

Fong W.P., Wong C.P. and Chan K.L. Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.

Antiquitin (ALDH7) is a member of the aldehyde dehydrogenase (ALDH) superfamily which oxidizes various aldehydes to form the corresponding acids. Human antiquitin (ALDH7A1) is believed to play a role in detoxification, osmoregulation and in lysine metabolism. Clinically, mutation on antiquitin is found to be related with pyridoxine-dependent seizures which occur as an indirect result of the failure in oxidizing α-aminoadipic semialdehyde (α-AASA) in lysine metabolism. In the present study, the structural basis of human antiquitin in oxidizing α-AASA was studied. Sequence comparisons between different ALDH families show specifically the presence of two charged amino acid residues at the entrance of the substrate binding pocket in antiquitin. These two amino acid residues, Glu-121 and Arg-301 in the human enzyme, may have charge-charge interaction with the α-amino and α-carboxylate groups of α-AASA. To confirm this hypothesis, site-directed mutagenesis was carried out. The mutants E121A and R301A were purified and kinetically characterized using both the specific α-AASA and the non-specific acetaldehyde as substrate. When compared with the wild-type enzyme, the E121A and R301A substitutions resulted in 16- and 31-fold, respectively, increase in the Km value of α-AASA. In contrast, these substitutions caused only minor changes (< 2-fold) in the Km value of acetaldehyde. For kcat values, the substitutions also caused a much more significant decrease in the oxidation of α-AASA (8.7-fold for E121A and 69-fold for R301A) than that of acetaldehyde (1.6-fold for E121A and 7.3-fold for R301A). To conclude, Glu-121 and Arg-301 are important in determining the substrate specificity in human antiquitin. (This work was supported by a grant from RGC of the HKSAR, Project No. 464407).

ELECTROPHORETIC ANALYSIS OF ISOENZYMES IN THREE CULTIVARS OF DATE PALM (PHOENIX DACTYLIFERA L.) LEAFLETS AND ROOTS FROM AL-AHSA AND AL-QATIF IN SAUDI ARABIA

Al-Issa A.M.1, Al-Helal A.A.2 and Al-Saad F.A.2 1University of Dammam. 2King Saud University.

Date palm trees (Phoenix dactylifera L.) are widely distributed in the Eastern Province of Kingdom of Saudi Arabia. There are more than 70 cultivars that have been grown there for ages, Three cultivars, namely “khalas”, “Shaishi” and “Ruzaiz” have been selected from each of the two localities (Al-Ahsa and Al- Qatif) and subjected to isoenzymes electrophoretic analysis. Leaflets and roots of the three cultivars were analyzed using the PAGE techniques for the occurrence of the isoenzymes EST, GOT, SOD, GDH and LAP. The results show that the three cultivars differed in their isoenzymes pattern within the same location and between different locations. The obtained results also signified that the isoenzymes patterns could be used as genetic expression markers for the cultivars and their interactions with the environmental factors in the two locations.





SOLUTION STRUCTURE AND DYNAMICS OF THE RING FINGER DOMAIN FROM THE HUMAN SPLICING-ASSOCIATED PROTEIN RBBP6 DETERMINED USING HETERONUCLEAR NMR SPECTROSCOPY

Kappo M.A.1, 2, Atkinson R.A.3, Ab E.4, Rees D.J.G.1 and Pugh D.J.R.1 1Department of Biotechnology, University of the Western Cape, South Africa. 2Department of Genetics, University of Stellenbosch, South Africa. 3Randall Division of Cell and Molecular Biophysics, Kings College London. 4Department of Chemistry, University of Leiden, Netherlands.

RBBP6 is a 250-kDa multi-domain protein that interacts with both p53 and pRb and has been implicated in mRNA splicing. The protein contains an atypical RING finger with a C4C4 motif rather than the more common C3HC4 motif. We report here on the solution structure and metal exchange properties of the RING domain from human RBBP6, using heteronuclear NMR spectroscopy. 15N- and 13C-labelled protein samples were generated as GST-fusion by growing bacteria in minimal media. A complete set of heteronuclear NMR data was collected at 600 MHz from which almost complete assignment of the backbone, side-chain and aromatic resonances was achieved. Exchange of Zn2+ with 113Cd2+ confirmed the domain binds two Zn2+ ions, which were coordinated in the expected cross-brace manner as common with RING fingers. Structural data in the form of 2D-NOESY, 15N-separated NOESY and 13C-separated NOESY spectra were recorded and used to determine the structure using restrained molecular dynamics on CYANA platform. Our results further showed the structure of the RING domain closely resembling that of the U-boxes, particularly the U-box from CHIP (C-terminal of Hsp70-Interacting Protein). The domain homodimerises across the same interface as in U-boxes, and features the same hydrophobic groove forming the binding site for E2 enzymes. The structural similarities between the RBBP6 RING finger and the U-box family led us to conclude that RBBP6 may, like CHIP, play a role in protein quality control.

CRYSTAL STRUCTURE OF THE ENTIRE ECTODOMAIN OF GP130: INSIGHTS INTO THE MOLECULAR ASSEMBLY OF CYTOKINE RECEPTOR COMPLEXES

Xu Y., Kershaw N., Luo C.S., Soo P., Pocock M.J., Czabotar P.E., Hilton D.J., Nicola N.A., Garrett T.P.J. and Zhang J.-G. The Walter + Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia.

The cell surface receptor gp130 is a shared signalling receptor subunit for the interleukin 6 (IL-6)-like cytokine family, which includes, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF). This family of cytokines is involved in inflammatory and immune responses and also plays crucial roles in hematopoiesis, liver and neuronal regeneration, embryonic development, and fertility. Dysregulation of signalling contributes to diseases such as inflammatory bowel disease, osteoporosis, multiple sclerosis, multiple myeloma, and prostate cancer. Previous X-ray structures of ligand-receptor complexes of this family lacked the C-terminal domains required for effective signal transduction. I will present the first crystal structure of the entire extracellular portion of human gp130 (D1-D6) at 3.6-Å resolution. This represents the first atomic resolution structure of the complete ectodomain of any “tall” cytokine receptor. The structure shows that, other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. It also reveals that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire “tall” receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C-termini of the membrane-proximal FNIII domains of the tall cytokine receptors in close proximity within the transmembrane complex, favourable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.

TARGETED PEPTIDOMIC APPROACHES TO STUDY IMMUNE RECOGNITION

Dudek N.L.1, 2, Tan C.T.1, 2, Croft N.P.1, 2, Scull K.E.1, 2, Corbett A.J.1, 2, Webb A.I.1, 2, Barr D.P.1, 2, Reilly C.B.1, 2, Williamson N.A.2 and Purcell A.W.1, 2 1Biochemistry and Molecular Biology, University of Melbourne. 2Bio21 Molecular Science and Biotechnology Institute.

The immune system has evolved to recognise foreign antigen in a highly sensitive and specific manner. Until recently functional assays using clonal populations of immune effector cells have been required to aid in the discovery of new targets of immunity and to study antigen presentation. With recent advances in mass spectrometry, and in particular targeted approaches such as precursor scanning and multiple reaction monitoring, the requirement for biological readouts has been replaced by robust, relatively cheap and high-throughput assays. We will present new strategies in the analysis of peptides involved in immune recognition that will ultimately replace many conventional functional cellular immunoassays.

VARIABLE ROLE OF NON-CATALYTIC DOMAINS ON ACTIVITIES OF CELLULASES AND XYLANASES

Sajjad M., Mahmood I. and Akhtar M.W. School of Biological Sciences, University of the Punjab, Lahore, Pakistan.

Whereas carbohydrate binding domains (CBD) are widely associated with cellulases and xylanases, the requirement of CBD seems unnecessary for enhancing catalytic activity of some enzymes, which occur in nature as CBD-catalytic domain complex. Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridium thermocellum cellulosome were cloned and expressed with and without non-catalytic domains in E. coli. In the case of XynC the binding domains seemed to enhance activities as well as stability of the enzyme. These effects were more pronounced when the binding domains were present at both the termini of the catalytic domain (pXynC-BCB). However, for XynZ the deletion of the binding domain not only enhanced expression level but also showed increase in specific activity as well as thermostability significantly. The overall increase in activity was ~9-fold higher for XynZ-C as compared to that of XynZ-BDC. The major cellulosomal endoglucanase, CelA lacks a binding domain in the native form. The gene encoding this enzyme was inserted into pET22b to produce the construct pCelA-C. Binding domain of family 3a was added at its N- and C-termini to produce the constructs pCelA-BC and pCelA-CB, respectively. The overall endoglucanase activity expressed in E. coli in the case of the catalytic domain only was almost twice that of those having a binding domain. However, fusion of the binding domain at C-terminal enhanced thermostability of the enzyme. It is possible that the change in the physical environment from that of the native state, and in the 3-D structure of the protein has varying effects on their activity and stability.





EFFECT OF GENOTYPE, COLD PRETREATMENT AND CULTURE MEDIUM COMPOSITION ON ANTHER CULTURE RESPONSE OF BARELY (HORDEUM VULGARE L.)

Ghasempour H.R.1, Kahrizi D.2 and Abdi P.1 1Biology and Biotechnology Dept., Razi University, Baghabrisham, 67149-67346, Kermanshah, Iran. 2Biotechnology Research Institute, Agricultural Faculty, Razi University, Kermanshah, Iran.

In this study the effects of genotype, cold pretreatment of anthers and different 2,4-D concentrations added to the FHG barely anther culture medium were examined. The callus induction and plant regeneration in anther culture of two barely genotypes (G1, G2) were investigated in FHG induction medium with different levels of 2,4-D (1,2,3 mg/l) after cold pretreatment of spikes for 0, 7, 14, 21, 28 days at 4ْ C. Callus induction was accomplished on FHG induction medium with different levels of 2,4-D (1,2,3 mg/l) in combination with kinetin (0.1 mg/l). Then Calli of 2-3 mm were transferred onto MS regeneration medium containing 1 mg/l 2,4-D and 5 mg/l BAP. Plantlets that were regenerated from calli transferred onto tubes with same regeneration medium but without any growth regulator. Plants of 10 cm length were transferred to pots. Analysis of variance showed highly significant difference between genotypes, cold pretreatment and 2,4-D concentration on all of the androgenic parameters (Callus, Green Plant, Albino Plant, Total Plant). Generally three ways interacted factors showed non- significant effects. Some of interaction effects were important for androgenesis. Results revealed that the response of genotype to various 2,4-D concentration in media for callus induction were significant. The green plant regeneration was genotype dependent. Overall G1 androgenic responses were higher than G2. FHG medium with 3 mg/l 2,4-D was most efficient in callus induction. The optimum green plant regeneration was observed in FHG medium containing 1mg/l 2,4-D. The optimum cold pretreatment period for androgenic response was 14 days. Key words: anther culture, Hordeum vulgare, Plant regeneration, Androgeneis, Cold pretreatment.

DIAGNOSTIC ASSESSMENT AND REMEDIATION STRATEGIES IN INTRODUCTORY MOLECULAR LIFE SCIENCES COURSES

Wright T.1, Hamilton S.1, Rafter M.1, Howitt S.2, Anderson T.3 and Costa M.4 1The Uiversity of Queensland, St Lucia. 2Australian National University, Canberra. 3University of KwaZulu-Natal, South Africa. 44University of Minho, Portugal.

A Molecular Life Science Concept Inventory has been completed recently and can now be used as a diagnostic tool in introductory biochemistry courses. The inventory contains questions drawing on physical, chemical and biological concepts and set in molecular life sciences contexts. The test gives the instructor information on specific concepts that are poorly understood, as well as alternate conceptions or misconceptions that students may have. Results from extensive testing of the inventory, which has been carried out at three universities in Australia and in South Africa, show that different cohorts of students are responding very similarly, and that there are areas where misconceptions are common. This paper will firstly examine a selection of results from the testing of the inventory. It will then propose some strategies to remediate student difficulties identified through the inventory. These are presented as a module of “teaching objects” which target one specific topic in a typical introductory biochemistry course (protein folding), but which we wish to present as a more general model for use in course design. In developing the module, we have considered how to use the topic to (i) remediate student difficulties effectively around key concepts such as bond rotation, free energy, dynamic equilibrium and hydrophobicity; and at the same time (ii) engage students in some of the big conceptual ideas associated with protein folding, for example self assembly and molecular evolution, which can be developed at increasing levels of sophistication in advanced courses.

TIME DEPENDENT EVALUATION OF THE CYTOTOXICITY OF PETROLEUM HYDROCARBON BIOREMEDIATED SOIL

Ho D.H.T.1, Sanderson B.J.1, Ball A.S.2 and Schmidt L.1 1Medical Biotechnology, Flinders University School of Medicine. 2School of Biological Sciences, Flinders University.

Bioremediation is a process which aims to reduce the amount of chemical contaminants in an environmental sample. One goal of bioremediation is to reduce the hazard posed by the contaminated sample. However, the toxicity of the sample may vary during this process due to the breakdown of various contaminants into their metabolites. Two in vitro cell viability assays, the MTT assay and the Crystal Violet assay were used to evaluate the cytotoxicity of petroleum hydrocarbon contaminated soil extracts before, during and after bioremediation. Bioremediation was performed by a combination of bioaugmentation and biostimulation over a period of 0-12 weeks. Chemical analysis of the extracts was carried out using GCMS analysis. Results will be presented comparing changes in cell viability to changes in chemical composition. This will show if there is any relationship between toxicity and the nature of contamination and/or breakdown products of the contaminating material during the bioremediation process.

BORON TOXICITY TOLERANCE IN BARLEY THROUGH REDUCED EXPRESSION OF THE MULTIFUNCTIONAL AQUAPORIN HVNIP2;1

Hayes J.1, Schnurbusch T.3, Hrmova M.1, Baumann U.1, Ramesh S.A.2, Tyerman S.D.2, Langridge P.1 and Sutton T.1 11Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond SA 5064, Australia. 2School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond SA 5064, Australia. 3Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Genebank Department, Corrensstr. 3, D-06466 Gatersleben, Germany.

Boron (B) toxicity is a significant limitation to cereal crop production in a number of regions worldwide, and although the problem has been recognised for many years, cultivated barleys are not well adapted. Here we describe the cloning of a gene from barley, underlying the chromosome 6H B toxicity tolerance quantitative trait locus. It is the second B toxicity tolerance gene identified in barley. Previously, we identified the gene HvBot1 which functions as an efflux transporter in B toxicity tolerant barley to move B out of the plant. The gene identified in this work encodes HvNIP2;1, an aquaporin from the NIP subfamily which was recently described as a silicon influx transporter (Lsi1) in barley and rice. Here we show that a rice mutant for this gene also shows reduced B accumulation in leaf blades compared to wild type and that the mutant protein alters growth of yeast under high B. HvNIP2;1 facilitates significant transport of B when expressed in Xenopus oocytes compared to controls and to another NIP (NOD26), and also in yeast plasma membranes which appear to have relatively high B permeability. We propose that tolerance to high soil B is mediated by reduced expression of HvNIP2;1 to limit B uptake as well as by increased expression of HvBot1 to remove B from roots and sensitive tissues. Together with HvBot1, the multifunctional aquaporin HvNIP2;1 is an important determinant of B toxicity tolerance in barley.





A FAMILY OF PLANT RECEPTOR-LIKE KINASES WITH DUAL FUNCTIONING INTRACELLULAR SIGNALING DOMAINS

Donaldson L.1, Meier S.1, Kwezi L.2, Wheeler J.2, Gehring C.1 and Irving H.2 14700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia. 2Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia.

The presence of the second messenger guanosine 3’5’-cyclic monophosphate (cGMP) in plants has been unequivocally established however the guanylyl cyclase (GC) enzymes responsible for cGMP synthesis remain elusive. We designed a search motif using consensus residues present in the catalytic domain of GCs from lower eukaryotes and animals to identify putative GCs in Arabidopsis. The search returned 36 putative GCs, the majority of which are annonated as receptor-like kinases (RLKs). We have demonstrated that a number of these RLKs function as both GCs and kinases in vitro and in vivo. These include WALL ASSOCIATED KINASE LIKE 10 (WAKL10), BRASSINOSTEROID INSENSITIVE 1 (BRI1), PHYTOSULFOKINE RECEPTOR 1 (PSKR1) and PEP1 RECEPTOR 1 (PEPR1), known for their crucial roles in plant development and stress responses. Moreover, for BRI1 and PSKR1, the natural ligands of these receptors were shown to stimulate increases in cGMP in planta. Interestingly these plant receptor GCs differ from their animal counterparts in that the GC domain overlaps the kinase domain in plants whereas these domains remain distinct in animals. This raises the possibility that the plant receptors might switch between downstream cGMP-mediated or kinase-mediated signaling to elicit desired outputs to particular stimuli. The challenge now lies in understanding the interaction between the GC and kinase domains and how these receptors utilize their dual functionality in planta. The large family of RLKs with both GC and kinase domains implies that these dual functionalities have coevolved due to the importance of both enzyme activities in plant development and stress responses.

ETHANOLIC EXTRACT OF ZINGIBER OFFICINALIS (GINGER) SUPPRESSES HEPATIC NFκB ACTIVITY

Li X.H.1, McGrath K.C.2, Heather A.K.2, 3 and Roufogalis B.D.1 1Herbal Medicines Research and Education Centre, Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia. 2The Heart Research Institute, Sydney, NSW 2042, Australia. 3Medical and Molecular Biosciences, University of Technology Sydney, NSW 2007, Australia.

Objective - Lifestyle changes have resulted in an obesity epidemic associated with increased incidence of type 2 diabetes, hypertension and non-alcoholic fatty liver disease (NAFLD). Hepatic inflammation drives the pathogenesis of these chronic diseases. Nuclear factor κB (NFκB) is the master regulator of the hepatic inflammatory response. Active hepatic IKKα/IκBα/NFκB pathway can drive the onset of insulin resistance, a prominent feature of metabolic syndrome and type 2 diabetes. Previously, we have demonstrated that an ethanolic extract of ginger has protects high fat-fed rats against the development of insulin resistance. We now explore whether this ginger extract protects against insulin resistance by inhibiting hepatic activation of the classical NFκB signaling Bκpathway. Methods - NFκB activity was tested using HuH7 cells ransfected an NF luciferase reporter vector and then exposed to the ethanolic ginger extract for 24 hrs. Cells were then activated with interleukin-1β (IL-1β for 3 hrs before luciferase activity was measured. Real-time PCR (mRNA) and Western blot (protein) were used to measure NFκB target gene and NFκB inhibitor protein (IκBα) expression levels. Results-Ethanolic ginger extract significantly suppressed NFκB activity (IL-1β: 157±17 vs IL-1β+ginger:120±10, p<0.05) and prevented the degradation of IκBα. Ethanolic ginger extract decreased expression of NFκB target genes including serum amyloid A1 (SAA1), interleukin 6 (IL-6) and interleukin 8 (IL-8) in a dose-dependent manner. Conclusions - Ethanolic ginger extract containing gingerol and shogaol components suppresses NFκB activity and downstream expression of genes that orchestrate the inflammatory response in cultured human liver cells. This finding highlights the potential mechanism that may underlie the protective effects of ethanolic extract of ginger on insulin resistance in vivo. (supported by the National Institute of Complementary Medicine (NICM) grant to BDR).

SCHWANN CELLS INTERACT WITH GLIAL CELLS AND NEURONS VIA MOTILE LAMELLIPODIAL WAVES

Ekberg J.A.K., Windus L., Scott S., Lineburg K., Cornock J., Mackay-Sim A. and St John J.A. National Centre for Adult Stem Cell Research, Griffith University, Nathan 4111, Brisbane, Queensland, Australia.

Implantation of olfactory ensheathing cells (OECs) or Schwann cells (SCs) into damaged CNS tracts have led to axonal regeneration but the results are not optimal and the favorable glial type may vary depending on the site of injury. Therefore it is crucial to determine how these glial cells interact with neurons so that treatments can be optimised. We have previously shown that OECs display highly motile peripheral membrane protrusions termed lamellipodial waves, which mediate OEC-OEC contacts as well as OEC-axon contacts. Using time lapse imaging of fluorescently labeled cells, we now have investigated the presence of dynamic lamellipodial waves in SCs. The waves were similar to those in OECs in terms of number of waves/cell and wave area but travelled ~2-fold faster. The number of waves and wave area of SCs correlated positively with whole-cell migration rate. Both OECs and SCs interacted directly with DRG axons via lamellipodial waves and selectively migrated along DRG axons rather than on the surrounding substrate. Contact with axons increased the migration rate of glial cells ~2-fold. Finally, both SCs and OECs significantly promoted survival of DRG neurons with survival after 3 days in a nutrient-free medium increasing from 40 % to 67 % by SCs and to 77 % by OECs. In summary, we have demonstrated that SCs exhibit lamellipodial waves, and that contact with axons leads to increased wave activity and increased migration. Both OECs and SCs promoted neuronal survival, suggesting that transplantation of both SCs and OECs may promote regeneration of damaged sensory neurons.

ASSOCIATION OF SPECTRIN TO BRAIN GAMMA TUBULIN

Shashikala S. and Sengupta S. Rajiv Gandhi Centre for Biotechnology, Trivandrum, INDIA.

γ-tubulin, the newer member of the tubulin superfamily, is known to mediate microtubule nucleation from the centrosome of eukaryotic cells.The major amount of γ-tubulin is believed to be located in the centrosome. However, a considerable amount has been found in the cytoplasm in the form of a ring complex (γ-TuRC) along with some other proteins, whose function is not well known. In our earlier studies by immunoprecipitation and immunoaffinity, we found that the purified brain cytoplasmic γ-tubulin complex contains alpha and beta non-erythroid spectrin, which were not reported in other tissues. Spectrin is a membrane bound protein which forms a flexible scaffold like network. Spectrin has also been reported to interact with centractin which is a centrosomal actin like protein. In this study, we wanted to check the role of spectrin in γ-tubulin complex. In vitro, our tubulin polymerization studies show that the presence of spectrin in γ-TuRC inhibits nucleation and polymerization of tubulin. But when spectrin is complexed with anti-spectrin antibody, tubulin polymerization is restored. With a goal to extend the study in vivo, we initially studied the association of spectrin and γ-tubulin in brain tissue and brain derived cell types at different conditions. Here we report the co-localization of γ-tubulin and spectrin in tissue sections from the cortical region of the brain by immunohistochemistry. Co-localization was also observed by immunofluorescence experiments in both neuroblastoma and gliablastoma cells in the extranuclear parts including the centrosomes which showed intense labelling. Purified centrosomes from goat brains also showed the presence of spectrin along with γ-tubulin and centrin (a centrosomal marker). We therefore conclude that spectrin is associated to γ-tubulin complex in the centrosomes of both neuronal and glial cells and has important role in the function of the complex.





MOLECULAR MECHANISM OF RICE BRAN PHYTIC ACID AS ANTICANCER IN HUMAN COLORECTAL ADENOCARCINOMA CELLS

Shafie, N.H.1, Mohd-Esa, N.1, 2, Ithnin, H.1, 2, Md-Akim, A.2 and Saad, N.1 1Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 2Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.

Phytic acid (inositol hexaphosphate or IP6) is one of bioactive compound that is present in cereals, nuts and legumes. Phytic acid (IP6) is naturally occurring polyphosphorylated carbohydrate recognized to posses various significant health benefits including a potential as anticancer compound. Several in vivo and in vitro studies provide convincing evidence for the anticarcinogenic properties of IP6 in wheat bran whilst the underlying mechanisms by which IP6 exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the growth inhibitory effects of rice bran IP6 on human colorectal cancer cell line, HT-29. IP6 induced marked growth inhibition in HT-29, in a dose and time dependent manner. Flow cytometry was performed for the analysis of cell cycle and apoptosis. Treatment of HT-29 with IP6 resulted in cell cycle arrest. In addition, induction of early and late apoptotic cell death in a dose- and time dependent manner was confirmed using an Annexin V-based assay. PCR analyses indicated that IP6 upregulated proapoptotic genes and downregulated antiapoptotic genes. IP6 is expected to exert anticarcinogenic activity through induction of apoptosis and disruption of cell cycle progression. Our study suggests that rice bran IP6 can be developed as a chemopreventive agent for human colorectal cancer.

SUBCELLULAR LOCALIZATION AND ARGININE METHYLATION OF SERBP1Lee Y.J.1, Hsieh W.Y.2, Chen L.Y.1 and Li C.2 1Institute of Biochemistry and Biotechnology. 2Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan.

Protein arginine methylation catalyzed by protein arginine N-methyltransferases (PRMTs) can regulate several cellular processes. Methylarginines are mostly found in arginine and glycine rich (GAR) motifs or arginine rich domains in proteins. According to the GAR motif, we identified an open reading frame (accession number AL080119) now designated as SERBP1 (SERPINE1 mRNA binding protein 1). SERBP1 contains an arg-rich domain, an RG-rich domain, and an RGG box domain that might be methylated by PRMTs, and a nuclear localization signal (NLS) overlapping the RG-rich domain. In order to analyze the subcellular localization of SERBP1, expression of GFP-fused full-length or truncated SERBP1 in HeLa cells was analyzed by fluorescent microscopy. Full-length SERBP1 localized mainly in the cytoplasm while proteins containing the middle RG-rich domain with N-terminal arg-rich or C-terminal RGG box deletions localized mainly in the nucleus with granular structures. However, the RG-rich domain deleted protein is mostly expressed in the cytoplasm. Treatment of a methylation inhibitor AdOx did not change the localization of all SERBP1 proteins. Furthermore, methylarginine levels of FLAG-fused SERBP1 proteins expressed in HeLa cells were determined by an asymmetric dimethylarginine-specific antibody. Methylarginine signals were detected in purified full-length or truncated FLAG-SERBP1 proteins containing RG-rich or RGG box. AdOx treatment significantly reduced the methylation signals in these proteins. Moreover, the most predominant type I protein arginine methyltransferase PRMT1 was co-immunoprecipitated with SERBP1 and bound PRMT1 decreased upon the addition of AdOx. In conclusion, the SERBP1 protein is localized in the cytoplasm in spite of the NLS in RG-rich region. The N- and C-terminal domains appear to be important for the cytoplasmic localization. The protein is methylated in the RG-rich and RGG box but arginine methylation is not related to the subcellular localization. The presence of asymmetric dimethylarginine and interaction with PRMT1 indicate that SERBP1 is specifically methylated by PRMT1.

QUANTITATIVE ANALYSIS OF ANDROGENIC INFLUENCE ON ZEB-1 GENE EXPRESSION

Anose B.M. and Benjamin D.E. Bethel University, 3900 Bethel Drive, St. Paul, MN 55112, U.S.A.

Roughly one in six men will be diagnosed with prostate cancer during his lifetime. Localized prostate cancer occurs when a tumor is contained within the prostate; this prognosis is quite favorable because the tumor can be removed surgically. The prognosis for metastatic prostate cancer is not nearly as promising because the cancer can be life threatening once it spreads into other tissues and disrupts normal biological function. There is currently no diagnostic tool available to distinguish between localized and metastatic prostate cancer. However, it has recently been discovered that Zinc Finger E-Box Binding Homeobox-1 (ZEB-1) could putatively be used as a biomarker for prostate cancer metastasis. Previous research has shown that changes in the expression of ZEB-1, a transcription factor, correlate with prostate cancer progression. To gain further knowledge about this potential diagnostic gene, human prostate cancer cells from the cell line 22Rv1 were treated with dehydroandrosterone (an androgen) and flutamide (an anti-androgen) to determine their effect on the expression of ZEB-1. After treatment, RNA was isolated and reverse transcribed into cDNA for its use in quantitative real time-polymerase chain reaction (qRT-PCR). qRT-PCR revealed that dehydroandrosterone and flutamide do not significantly influence ZEB-1 expression.

TEACHING EXPERIMENTAL, ETHICAL, AND QUANTITATIVE (TEEQ) BIOLOGY USING CALIBRATED PEER REVIEW™ (CPR)

Pelaez N.J.1, Dasgupta A.P.1, Gundlach E.1 and Russell A.A.2 1Purdue University Department of Biological Sciences, West Lafayette, IN 47907-2054, USA. 2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-1569, USA.

CPR™ was used for Teaching Experimental, Ethical, and Quantitative (TEEQ) Biology in a first-year course to help undergraduate students connect what they learn in lecture to both current and historical research endeavors. CPR is a Web-based program that enables frequent writing assignments even in large classes. CPR was used to help students learn to gauge their own ability and monitor their own thinking about experimental design in primary research publications. This study examined improvements in abilities to (1) identify the treatments in a biological experiment; (2) present a completely randomized design to address a research question; (3) recognize the benefit of limiting sources of variability; and (4) describe the limitations to the scope of inference for a biological investigation. A previously validated statistics item was used as a measure. Students had the most difficulty with the concept of randomization and replication while treatment identification was easier for students. Interestingly, their textbook (Freeman, 2008; Biological Science) illustrates each experiment without replications. Students who had taken a statistics course in their past performed significantly better on the pre-test than those who had no statistics training. On the post-test, scores for students in a biology lecture course where they used CPR were better than scores on the pre-test (including for those who had taken statistics). In summary, CPR made it possible to teach experimental thinking in the context of a large enrollment biology lecture class.





ARABINOXYLAN ARABINOFURANOHYDROLASE (AXAH) GENES IN CEREALS

Laidlaw H.K.C. and Jobling S.A. CSIRO Plant Industry and Food Futures Flagship High Fibre Grains Research Cluster, GPO Box 1600, Canberra ACT 2601 Australia.

Arabinoxylan (AX) is a key component of cereal cell walls and thought to be involved in the remodelling of the cell wall during growth and development. Several studies have shown changes in the structure of AX during development and between different plant tissues. Arabinoxylan arabinofuranosidase enzymes (AXAH; glycosyl hydrolase family 51) are implicated in the removal of the α-L-arabinofuranosyl moieties from the xylose backbone of AX, and are encoded by a complex and variable gene family in cereals. The members of the AXAH gene family have been isolated from wheat and barley and compared to their homologues from rice, sorghum and Brachypodium. Differences in transcript levels of the genes during grain development and between plant tissues have been observed. Biochemical characterisation of selected AXAH enzymes has also demonstrated activity against wheat AX.

TISSUE-SPECIFIC EXPRESSION IN TRANSGENIC TOMATO FRUIT OF GUS GENE DRIVEN BY THE 5’ REGULATORY SEQUENCES OF DESATURASE MESOCARP SPECIFIC OIL PALM (ELAEIS OLEIFERA) GENE

Ismail I. 1, 2, Saed Taha R.1, 2 and Abdullah N.3 1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia. 2Centre for Plant Biotechnology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia. 3Department of Agriculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

Gene promoters are sequences of DNA that drive and regulate gene expression. Some promoters are constitutive in that they allow for continual transcription of associated genes whereas many others are specific that they drive gene expression in particular space and time. In this study the oil palm (Elaeis oleifera) mesocarp desaturase (Des) gene promoter specificity and strength was evaluated and compared to cauliflower mosaic virus (CaMV) 35S promoter. Five deletions were purposely generated by PCR and cloned into pCAMBIA 1301. The ability of these fragments to direct mesocarp-specific expression was studied by fusing them to the β-glucuronidase (GUS) reporter gene. Cotyledons and hypocotyls of tomato (Lycopersicon esculentum) were transformed with Agrobacterium tumefaciens LBA4404 cultures carrying Des promoter and its deletions fused to GUS in pCAMBIA 1301 plasmids. The specificity of GUS expression directed by Des promoters was performed by histochemical GUS staining of fruit and leaf tissues from T0 plants. The results showed that all the deletions of Des promoter directed gus gene expression in tomato mesocarp tissue, whereas no activity was observed in any other vegetative tissues indicating the specificity of Des promoter. The quantitative analysis of GUS expression by flourometric assay has demonstrated much higher activity of the longest Des promoter (~1000 bp) with an average value of 1386 pmol µg-1 min-1 compared to CAMV 35S promoter with an average value of 347pmol µg-1 min-1. Further analysis on the strength of the Des promoter has shown the highest GUS activity by ~ 600 bp Des promoter, thus suggesting the possible presence of silencing motifs upstream of the sequence.

A GENE EXPRESSION SIGNATURE FOR INSULIN RESISTANCE MEDIATED BY EXCESS FAT

Hayward B.1, Konstantopoulos N.1, Molero-Navajas J.1, Jowett J.2 and Walder K.1 1Metabolic Research Unit, Deakin University, Geelong, VIC 3217. 2Baker IDI Heart and Diabetes Institute, Melbourne, VIC 3004.

Background: A key feature of type 2 diabetes is insulin resistance. Multiple pathways are implicated in insulin resistance, such as hyperlipidemia, elevated levels of pro-inflammatory cytokines and/or induction of oxidative or ER stress. The complexity of insulin resistance hinders effective characterisation and treatment of type 2 diabetes. We propose that small sets of genes known as gene expression signatures (GES) that reflect the insulin resistant state can be identified. The GES provides an unbiased transcriptional snapshot of the integrated cellular response to an insult. Methods: Hyperlipidemia was modelled in FAO liver cells using palmitate (PA), and global gene expression profiling was performed using microarrays. This was followed by bioinformatics analyses to identify the GES whose expression levels most significantly discriminated between the insulin resistant and insulin sensitive states. Insulin sensitivity was assessed by glucose production as the functional endpoint. Results: In vehicle-treated cells, insulin (0.1nM, 24h) decreased glucose production by 30±1% (P < 0.0001, n=3) indicating an insulin sensitive state. PA treatment (75μM, 48h) impaired the ability of insulin to decrease glucose production by ~20% (P < 0.04, n=3) indicating a state of insulin resistance. The inhibitory effect of PA on glucose production was reversed by the addition of metformin (0.25mM) and sodium salicylate (2mM) in the final 24h of the PA treatment (P < 0.0001, n=3). Conclusion: This study will identify a set of genes whose expression best defines the difference between insulin sensitivity and PA-induced insulin resistance in FAO liver cells.

TBC1D13: A RAB10/RAB1 BINDING PROTEIN INVOLVED IN INSULIN-STIMULATED GLUT4 TRANSLOCATION

Davey J.R.1, Larance M.1, Junutula J.R.2, James D.E.1 and Stoeckli J.1 1The Garvan Institute of Medical Research, 384 Victoria St, Sydney, NSW 2010. 2Genentech Inc., 1 DNA Way, South San Francisco, CA 94080.

In adipocytes the small GTPase Rab10 plays an important role in the regulation of GLUT4 trafficking. Here we demonstrate that Rab10 forms a stable complex with TBC1D13, a member of the Rab GTPase activating protein (RabGAP) family. This adds to the complexity of Rab10 mediated trafficking because two other RabGAPs, TBC1D1 and TBC1D4, also regulate Rab10. The Rab10/TBC1D13 interaction was GTP-dependent and specific, in that other members of the TBC family did not bind to Rab10. Immunoprecipitation of endogenous TBC1D13 from adipocytes resulted in co-precipitation of Rab10 and Rab1. RNAi ablation of TBC1D13 expression by siRNA reduced insulin-stimulated glucose uptake in adipocytes and overexpression of TBC1D13 completely inhibited insulin-stimulated GLUT4 translocation to the plasma membrane (PM). This was specific to GLUT4 trafficking as TBC1D13 overexpression did not affect transferrin receptor recycling or tsVSV-G trafficking. TBC1D13 did not display any detectable in vitro GAP activity on Rab1 or Rab10 and the catalytically inactive mutant of TBC1D13 did not abolish the inhibitory effect on GLUT4 translocation. These studies reveal a role for TBC1D13 in GLUT4 trafficking in adipocytes that is independent of it’s putative GAP ability. Based upon the Rab binding specificity of this GAP we suggest it might regulate a trafficking step between the trans-Golgi cisternae and endosomes.





ANALYSIS OF THE TRANSCRIPTIONAL ACTIVITY OF THE SOYBEAN BASIC HELIX LOOP TRANSCRIPTION FACTOR GMSAT1 WHEN EXPRESSED IN THE YEAST AMMONIUM TRANSPORT MUTANT 26972C

Mazurkiewicz D.1, Chiasson D.1, Loughlin P.2, Okamoto M.3 and Kaiser B.N.1 1School of Agriculture, Food and Wine. The University of Adelaide, Urrbrae, SA, 5064, Australia. 2School of Biological Sciences. The University of Sydney, Sydney, NSW, 2006, Australia. 3Australian Centre For Plant Functional Genomics. The University of Adelaide, Urrbrae, SA, 5064, Australia.

GmSAT1 is a soybean basic helix loop helix (bHLH) transcription factor localised to the peribacteroid membrane and nucleus of infected nodule cells. GmSAT1 was first identified by its ability to functionally complement the growth of a yeast ammonium transport mutant 26972c on low ammonium concentrations (1 mM). GmSAT1 also increases the uptake of the toxic ammonium analogue methylammonium (MA). Like in soybean, GmSAT1 is membrane bound in yeast and upon activation cleaved from its C-terminal membrane anchor and enters the nucleus. We have evaluated the transcriptional activity of GmSAT1 in 26972c cells grown for 12 hours in media containing 1mM ammonium, using microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-PCR. We have characterised one of the genes, DMO1, upregulated approximately 55-fold by GmSAT1. Analysis of the promoter region of DMO1 identifies multiple bHLH E-box DNA binding motifs. DMO1, previously uncharacterised, shows strong similarity to the predicted drug:H+ antiporters of the DHA2 family of major facilitator proteins. Overexpression of DMO1 in 26972c enhanced 14C-MA uptake in a SAT1 like manner, while loss of DMO1 activity in a 26972c:Δdmo1 mutant eliminated the GmSAT1 induced enhancement of 14C-MA uptake and related toxicity phenotype when grown at elevated MA concentrations. We are currently exploring the binding affinities of GmSAT1 to the promoter region of DMO1 and investigating the functional similarity of DMO1 homologues identified in soybean.

CHARACTERIZATION OF THE INTERACTION BETWEEN THE SNARE PROTEIN SYNTAXIN AND THE SEC/MUNC PROTEINS

Christie M.P.1, Hu S.1, Jarrott R.J.1, Latham C.F.1, 4, Lua L.H.L.2, Collins B.M.1, James D.E.3 and Martin J.M.1 1Institute for Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia. 2Protein Expression Facility, University of Queensland, St Lucia QLD 4072, Australia. 3Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia. 4Department of Biological Science, Columbia University, New York, NY 100227, USA.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are essential components of the vesicle fusion machinery. SNAREs bring about fusion by the formation of the SNARE ternary complex. The Sec/Munc (SM) family of proteins are essential regulators of fusion. However, their exact role in fusion is controversial. This is partly due to the different binding modes of SM proteins with the SNARE protein Syntaxin. Munc18a, the SM protein involved in neurotransmitter release acts as a negative regulator by interacting with its cognate syntaxin, Syntaxin1 in a closed conformation (1,2). The SM proteins Sly1p and Munc18c on the other hand interact with their cognate syntaxins, Sed5p and Sx4 via the first ten residues at the N-terminus (N-peptide) of Syntaxin (3,4). This is compatible with SNARE complex assembly (5). The interaction between Munc18a and Munc18c with the Sx N-peptide was thermodynamically characterized using isothermal titration calorimetry. These experiments showed that the interaction did not contribute to the specificity of SM-Sx interactions. We were also able to show that the N-peptide interaction plays a role in vesicle fusion regulation by SM proteins. References: 1. Misura et al., Nature 2000 404, 355 2. Burkhardt et al., EMBO J 2008 27, 923 3. Bracher and Weissenhorn EMBO J 2002 21, 6114 4. Hu et al., PNAS 2007 104, 8773 5. Latham et al., Traffic 2006 7, 1048.

PURIFICATION AND CHARACTERIZATION OF A NOVEL ALKALINE SERINE PROTEASE SECRETED BY VIBRIO METSCHNIKOVII

Park J.Y. and Lee J.S. Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University, Gwangju 501-759, Republic of Korea

A pathogenic marine bacterium Vibrio metschnikovii (V. metschnikovii) evokes serious symptoms, including pneumonia, leg ulcer, and diarrheal disease. In this study, a novel extracellular alkaline serine protease was purified and characterized. The purified Vm-AP (stands for V. metschnikovii alkaline protease) seemed to be degraded by heating; however, it could be inhibited by the addition of divalent cations such as CuCl2, ZnCl2, and NiCl2. Vm-AP was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl2. The optimal temperature and the pH for Vm-AP enzyme activity were 37°C and pH 9.5, respectively. In addition, the maximal protease activity could be found under alkalic condition ranging from pH 8.0 to 12.0, and the enzyme activity was inhibited by protease inhibitors, including PMSF and aprotinin. These results suggest that Vm-AP is an alkaline serine protease. Vm-AP could cleave various blood coagulation-associated proteins, including plasminogen, plasmin, prothrombin, and thrombin. In addition, the enzyme showed a powerful fibrin(ogen)olytic activity, as it could cleave all fibrinogen chains, fibrin polymer, and cross-linked fibrin. Taken together, the results suggest that the pathogenic bacterium V. metschnikovii may disturb the blood coagulation system by secreting an alkaline serine protease during the course of its infection.

MULTIMODAL NANOPARTICLES FOR THE STABILISATION OF ENZYMES AGAINST THERMAL INACTIVATION

Clemons T.1, 3, Evans C.1, 3, Stubbs K.2 and Swaminathan I.1 1Centre for Strategic Nano-fabrication, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia. 2School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia. 3Regenerative Neurosciences, School of Animal Biology, University of Western Australia.

Enzyme modification and immobilisation have been extensively studied and utilised to generate biocatalysts with improved stability and selectivity as well as applications in sensing and enzyme-related biotechnology. The major hindrance in developing enzyme related therapy is its inherent thermal instability and drop in activity with sometimes minute thermal fluctuations. This necessitates the use of repeated injections or local infusions for a period of days to weeks which are invasive, infection-prone, and clinically problematic. A novel strategy for enhancing thermal stability is through non-covalent surface interaction with the large surface area at the nanometer-scale. Our study investigates the thermal stabilisation of a range of enzymes through physisorption to a multimodal polymeric nanoparticle system. The nanoparticle system contains both a fluorescent probe commonly used in fluorescent microscopy as well as magnetite nanoparticles, which will allow for both magnetic imaging and enzyme recovery post treatment. The surfaces of the nanoparticles have been modified with either polyethyleneimine (PEI) or polyethylene glycol (PEG) to ultimately enhance not only the attachment of different enzymes to the nanoparticle but also increase their tolerance to thermal denaturing. Preliminary studies with a β-glucosidase and β-galactosidase, both enzymes of significant interest to industry, have shown a strong physisorption of the enzyme to the PEI modified nanoparticles.




POS-MON-335 POS-TUE-336AN EXTRACELLULAR METALLOPROTEASE FROM VIBRIO VUNIFICUS ACTIVATES TOLL-LIKE RECEPTORS 2 AND 4 TO INDUCE AN INFLAMMATORY RESPONSEPark J.E. and Lee J.S. Department of Biotechnology and BK21 Research Team for Protein Activity Control,Chosun University, Gwangju 501-759, Republic of Korea

Extracellular proteases produced by pathogenic bacteria are considered to be important enzymes because of their implication in the pathogenesis of these organisms. Vibrio vulnificus (V. vulnificus), a gram-negative halophilic marine bacterium, is an opportunistic human pathogen that causes wound infection and septicemia. We have reported that vEP metalloprotease secreted by V. vulnificus exerts many biological activities, such as prothrombin activation. To investigate that this enzyme plays an additional role in pathogenesis by modulating inflammatory-associated cytokine production, the effect of vEP on proinflammatory cytokine induction was examined in a macrophage cell line Raw264.7. vEP stimulated to induce typical inflammatory cytokines including TNF-alpha and IL-1beta, and regulators such as cyclooxygenase-2 (COX-2) and iNOS. Interestedly, the upregulation of cytokines and regulators by vEP protease occurred only when the cells were treated with wild type vEP having C-terminal domain (named C-ter100), while vEP lacking this domain showed no effect. In addition, the protease could activate the NF-kappaB signaling pathway for producing those cytokines, in which IkappaB was phosphorylated and eventually degraded after treatment with vEP. Toll-like receptors (TLRs)-2 and -4 seemed be involved in the vEP-mediated NF-kappaB signaling pathway, as judged by RT-PCR and immunoprecipitation. The C-ter100 only was also sufficient to activate the NF-kappaB signaling for the production of those kinds of cytokines and regulators, suggesting that the C-domain of vEP can act as a ligand for the activation of TLRs. Moreover, the macrophage cell lines transiently expressing RNAi molecules for TLR-2 or -4 showed significantly decreased TNF-alpha production when they were treated with vEP or C-ter100. These data suggest that the C-terminal domain of vEP may play a critical role in the induction of inflammatory response.

A SERINE PROTEASE SECRETED BY STAPHYLOCOCCUS AUREUS CAN INDUCE PRO-INFLAMMATORY CYTOKINES AND EVOKES VASCULAR PERMEABILITY

Park J.W. and Lee J.S. Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University, Gwangju 501-759, Republic of Korea.

It is been well known that Staphylococcus aureus (S. aureus) is a ubiquitous Gram-positive bacterium responsible for a majority of skin infections as well as causing toxic shock syndrome. In this study, a glutamate-specific serine endopeptidase (named VSPase) secreted by a clinical isolate S. aureus sp. strain C-66 was purified and characterized in terms of its involvement in the inductions of inflammatory response and vascular permeability. VSPase clearly increased the transcription levels of the genes for pro-inflammatory cytokines such as TNF-alpha and IL-1beta, and also for an inflammatory regulator cyclooxygenase-2. However, VSPase-derived mutant enzymes (H119L, D161A, and S237L) that are totally deficient in proteolytic activities showed no effects. VSPase could induce the degradation of IkappaB, resulting in translocation of NF-kappaB proteins into nucleus, as judged by Western blot analysis and supershift assay with anti-p65 antibody, respectively. These results suggest that VSPase can activate NF-kappaB signaling pathway through the degradation of IkappaB proteins, leading the production of pro-inflammatory cytokines and an inflammatory regulator. Interestingly, Miles assay using Guinea pig showed that wild type VSPase could cause an increased vascular permeability in a dose-dependent manner, whereas a mutant enzyme S237L could not, suggesting that the permeability is also related to the proteolytic activity of the enzyme. The data to be presented show that VSPase plays an important role in the expression of pro-inflammatory cytokines and inflammatory regulators through the activation of NF-kappaB, and also can enhance a vascular permeability during the Staphylococcal infection.

HSA-MIR-200B HAS TWO FUNCTIONAL PROMOTERS, DIFFERENTIAL METHYLATION OF WHICH IS ASSOCIATED WITH CLINICAL CHARACTERISTICS IN BREAST CANCER

Wee E.J.H.1, Peters K.1, Hulf T.2, Stein S.3, Wagner S.3, Lee S.Y.1, French J.1, Francis G.3, Clark S.2 and Brown M.A.1, 4 1School of Chemistry and Molecular Biosciences, The University of Queensland, Australia. 2Department of Pathology, Princess Alexandra Hospital, Queensland, Australia. 3Epigenetics Group, Garvan Institute of Medical Research, Sydney, Australia. 4UQ Diamantina Institute, The University of Queensland, Australia.

MicroRNAs are small non-coding RNAs of ~20nt in length that are capable of modulating gene expression post-transcriptionally. Many miRNAs have been implicated in cancer, including breast cancer, however, miRNA transcriptional regulation and the role of defects in this process in cancer is not well understood. In this study, we describe an alternate promoter of Hsa-mir-200b, P2, which is located in a CpG island approximately 2kb upstream of its 5’ stemloop. P2 has comparable promoter activity to the previously reported promoter, P1, and was able to drive the expression of miR-200b in its native genomic context. DNA methylation of both P1 and P2 is inversely associated with miR-200b expression in 8/9 breast cancer cell lines, and in vitro methylation of both promoters represses promoter activity in reporter assays. In clinical samples, decreased methylation of P1 in primary breast tumours is associated with better survival and increased DNA methylation of P2 in primary breast tumours is associated with loss of either ER, PR or HER2. In a triple negative cohort of breast cancer cases, lymph node metastases have lower methylation of P2 compared to primary tumours. These data suggest the potential use of DNA methylation status of miR-200b promoters as a prognostic marker of breast cancer progression and survival.

THERMO SCIENTIFIC SOLARIS QPCR GENE EXPRESSION ASSAYS - PERFORMANCE AND APPLICATION

Haas A.1, Jayne B.1, Haimes J.1, Covino J.1, Leake D.1, Kelley M.1 and Gillespie J.2 1Thermo Fisher Scientific, Dharmacon RNAi Technologies, 2650 Crescent Drive, Suite 100, Lafayette, CO 80026, USA. 2Thermo Fisher Scientific, Abgene House, Blenheim Road, Epsom, Surrey, KT19 9AP, UK.

Thermo Scienti-fic SolarisTM qPCR Gene Expression Assays are gene-speci-fic probe and primer pairs that utilize minor groove binder (MGB) and Superbase technologies. To test the performance of Solaris Assays, common qPCR parameters (sensitivity, dynamic range, ampli-fication ef-ficiency and r2 values) were measured. Additionally, we assessed the stability of the Solaris reagents by comparing performance and Cq values using reagents exposed to ambient laboratory conditions relative to fresh reagents. We also used the Solaris Assays to measure the consequences of RNAi-mediated knockdown where accurate determinations of changes in target gene expression are critical for appropriately interpreting the data. When employing the Solaris Assays to detect gene expression and, using a Cq method for calculating relative gene expression, we normalized to multiple reference genes to provide further confidence in the accurate measure of target gene levels. The results show that the Solaris qPCR Assays deliver repeatable, sensitive and gene-specifi-c quanti-fication.





ACTIVATION OF CAMP-DEPENDENT SIGNAL TRANSDUCTION PATHWAY IN BONE MARROW HEMATOPOIETIC STEM CELLS OF ALPHA-FETOPROTEIN-TREATED ANIMALS

Bogdanov A.Y. Scientific Center for Anti-infectious Drugs, Auezov Str. 84, Almaty, 050008, Republic of Kazakhstan.

In our earlier studies it was established that the biological activity of murine bone marrow hematopoietic stem cells (HSCs) in vitro exposed to alpha-fetoprotein (AFP) considerably depended on activation of two types of cAMP-dependent signal transduction pathways: cAMP/Epac1/2 and cAMP/PKA cascades. However the involvement of this pathways in general mechanism of AFP action in bone marrow HSCs in vivo has not been studied. In the present study we endeavored to investigate the functioning of the discovered cAMP-dependent signal transduction pathways in murine bone marrow HSCs in vivo under exogenous AFP introduction. As a result it was shown that intravenous introduction of AFP increased cAMP synthesis in every studied subpopulation of bone marrow HSCs ex vivo as compared with control animals. Analysis of the main components of revealed cAMP-dependent signal transduction pathways in vitro in cytoplasm of each investigated HSCs subpopulation demonstrated rise activity of both PKA and Rap1 in AFP-treated animals as against the control group. The content of phosphorylated Epac1/2 in each studied HSCs subpopulation prevailed over the active Epac1/2 content in analogous HSCs subpopulations of control animals. Thereby, the findings enable to state the activation of the revealed cAMP/Epac1/2 and cAMP/PKA pathways in the studied HSCs subpopulations in animal bone marrow under AFP exogenous introduction. Therefore, AFP interaction with HSCs in animal bone marrow initiates activation of the same cAMP-dependent mechanisms of signal transduction in vitro conditions, which, most probably, are responsible for generation of the biological activity modifications.

SUMO-1 MARKS LYSOSOMES IN NEURODEGENERATIVE DISEASES AND IN ANIMAL AND CELLULAR DISEASE MODELS

Wong M.B.1, Noe N.2, Meedeniya A.1, Richter-Landsberg C.2 and Pountney D.L.1 1Griffith Health Institute & ESKITIS, Griffith University, Australia. 2Molecular Neurobiology, Carl Von Ossietzky University, Germany.

Many neurodegenerative diseases are characterised by microscopically-visible protein aggregates, or inclusion bodies, within neural cells. The ubiquitin homologue, SUMO-1, has been identified in sub-domains of pathological inclusion bodies in several neurodegenerative diseases. Inclusion bodies are believed to be formed actively in a defensive response to soluble cytotoxic protein aggregates. We hypothesised that SUMO-1 may become associated with lysosomes in this response. Protein aggregation was induced in 1321N1 glioma cells by proteasome inhibition with MG132 or by transient transfection with Q74-EGFP. Fluorescence immunohistochemistry identified co-localisation of SUMO-1 and the lysosomal marker, cathepsin D, in both the transfected cells and MG132-treated cells, increasing over time at 24-96 hrs post-transfection/treatment. SUMO-1-positive lysosomes were also detected following MG132-treatment of 1321N1 cells expressing SUMO-1-GFP and stained with Lysotracker dye. SUMO-1 did not mark lysosomes in untransfected or sham treated cells. To determine if SUMO-1 also marks lysosomes in disease, we examined 5 cases of progressive supranuclear palsy (PSP), 5 cases of multiple system atrophy (MSA) and a rat Parkinson’s disease model. Punctate co-localisation of cathepsin D and SUMO-1 was consistently associated with both the tau-positive PSP inclusions and the α-synuclein-positive MSA and rat PD model inclusions. A similar pattern was also found with the OLN-t40 oligodendrocyte model. These findings suggest a role for SUMO-1 in the autophagy-lysosome pathway linked to the response to protein aggregates.

THE GENERATION OF A DOXYCYCLINE-INDUCIBLE, TISSUE-SPECIFIC AROMATASE TRANSGENIC MOUSE

Chow J.D.Y.1, 2, Price J.3, Bills M.3, Simpson E.R.1, 3 and Boon W.C.1, 2, 4 1Prince Henry’s Institute of Medical Research, Clayton, VIC, Australia. 2Anatomy & Developmental Biology, Monash University, Clayton, VIC, Australia. 3Biochemistry & Molecular Biology, Monash University, Clayton, VIC, Australia. 4Howard Florey Institute, Melbourne, VIC, Australia.

Aromatase catalyses the biosynthesis of estrogen and is widely expressed in the body. The aromatase knockout mouse (ArKO) becomes estrogen-deficient and develops unexpected phenotypes such as obesity and male-specific fatty liver and apoptosis at the hypothalamus. As circulating levels of estrogen in males are low, we hypothesize that local estrogen production in the brain may be important in regulating metabolic functions (e.g. liver lipid homeostasis) by acting in a paracrine or intracrine manner. To test this hypothesis, we are generating a brain-specific doxycycline-inducible, aromatase transgenic mouse. The transgene (pTetOAROM) consists of the human aromatase cDNA (hAROM) and a luciferase marker placed under a bi-directional tetracycline-responsive promoter (pTetO), which is regulated by transactivators (rtTA or tTA) and doxycycline. This transgene increased hAROM transcription (16-fold, p=0.01), aromatase activity (3.4-folds, p=0.0008) and luciferase activity (16-fold, p=0.0006) in transfected MBA-MB-231tet cells that stably expresses rtTA, with doxycycline induction. Pronuclear microinjection of the transgene produced four pTetOAROM founder mice (L1 to 4), which were bred with C57B6 WT mice to produce transgenic F1, with the following proportions of positive transgenics to total live births (ratios are of female to male positive pups): founder female L1 7/12 (4:3); male L2 - 7/26 (3:4); female L3 3/8 (2:1); male L4 - 9/31 (2:1). A male pTetOAROM founder mouse was bred with a female mammary gland specific-rtTA mouse (MTB) to produce MTB-pTetOAROM double transgenic. Upon doxycycline treatment via drinking water, human aromatase expression was detected (by RT-PCR) specifically in mammary glands, salivary glands and seminal vesicles of double transgenic mice. Luciferase expression was also detected in these tissues by in vivo luciferase scan and in vitro luciferase assay. In summary, we are generating a transgenic mouse model that expresses the human aromatase in a temporal- and spatial-specific manner.

LEARNING BIOCHEMISTRY BY USING THE PEERWISE SYSTEM OF STUDENT CONTRIBUTED MULTIPLE CHOICE QUESTIONS AND PEER EVALUATION

Bottomley S.1 and Denny P.2 1School of Biomedical Sciences. Faculty of Health Sciences. Curtin University. Western Australia. 2Computer Science Department. University of Auckland. New Zealand.

Biochemistry students in the second year of their degree were asked to research, design, and write their own multiple choice questions (MCQs). In addition they were also required to answer, evaluate and critique the MCQs written by their peers. The technology used to support this activity was PeerWise - a freely available, innovative web-based system that supports students in the creation of an annotated question repository. In this study we report the implementation of, and student responses to, the PeerWise system for a cohort of 120 second year biomedical science students from three degree streams studying a core biochemistry subject. The study suggests that the students are eager participants, take an active part in their learning, develop their higher order thinking skills, produce a large repository of MCQ’s that they use for revision, and rate the PeerWise system highly.





THE CATALYTICALLY-COMPETENT COENZYME ALLOCATION INTO THE ACTIVE SITE OF ANABAENA FERREDOXIN NADP+-REDUCTASE

Lans I.1, Peregrina J.R.1, Gonzalez-Lafont A.2, Lluch J.M.2, Garcia-Viloca M.2 and Medina M.11Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Institute of Biocomputation and Physics of Complex Systems. Universidad de Zaragoza. Spain. 2Departament de Química and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona. Spain.

Ferredoxin-NADP+ reductase (FNR) catalyses the electron transfer from Ferredoxin to NADP+ via its flavin FAD cofactor. Experimental kinetics and QM/MM theoretical approaches are here applied to visualise the catalytically competent interactions produced in WT Anabaena FNR with its coenzyme, NADP+, as well as in its variant Y303S. Our data indicate that the architecture of the WT active site precisely contributes to the orientation of the N5 of the FAD isoalloxazine ring and the C4 of the coenzyme nicotinamide ring in the conformation of the catalytically competent HT complex and, therefore, to the efficiency of the process. In the reactant complex of the Y303S variant the formation of a close contact ionic pair FADH-:NADP+ surrounded by the polar environment of the enzyme might be the cause of its low catalytic efficiency. However, in WT FNR the side-chain of the C-terminal Y303 appears key to provide the optimal geometry by reducing the stacking probability between the isoalloxazine and nicotinamide and by providing the required co-linearity and distance among the N5 of the flavin cofactor, the C4 of the coenzyme nicotinamide and the hydride that has to be transferred between them, factors highly related to the reaction efficiency, mechanism and reversibility of the process.

SO YOU HAVE MADE A DISCOVERY? WHAT NOW?

Tzanidis A. and YinFoo D. Phillips Ormonde Fitzpatrick.

Life as a medical research scientist is dotted with moments of discovery. Few will be “eureka!” moments, while others will merely provide a satisfying moment of vindication for the efforts that were laid down before. Whatever the response, what will you do with your discovery? Traditionally, the pressures of building one’s track record and securing grant funding will often lead to the publication of work in a peer-review journal. There is certainly merit in this approach, as it aims to ensure that only quality research reaches the public domain. However, evidence would suggest that peer-reviewed publications alone will not attract the commercial investment that is necessary to take your discovery from the bench to the bedside where it is needed. Commercial investment is likely to come only where there is valuable intellectual property (IP) inherent in your discovery. Such value will typically take the form of a registered patent. But how will you know whether your discovery is necessarily patentable? What steps can you take to obtain IP protection to attract commercial investment that will help to bring your discovery to those that need it most? Contrary to popular belief, patentability is not necessarily dependent on showing evidence that your discovery has immediate application in the clinical setting. In fact, the mere discovery of a unique signalling pathway may have practical applications that may be patentable. Practical applications may include diagnostics, screening assays, methods of medical treatment, pharmaceutical compositions and so on. This paper will guide you through your next steps and provide examples of areas of basic medical research that are inherently patentable and common criteria for patentability.

THE ROLE OF L-PROLINE AND L-PROLINE TRANSPORTERS IN THE REGULATION OF ES CELL DIFFERENTIATION IN CULTURE

Tan B.S.N.1, Lonic A.2, Morris M.2, Rathjen P.D.1, 2 and Rathjen J.1, 2 1Department of Zoology, University of Melbourne, Parkville, VIC, 3010. 2School of Molecular and Biomedical, University of Adelaide, Adelaide, S.A. 5005.

The initial step in formation of the somatic lineages from ES cells, differentiation to primitive ectoderm, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response the conditioned medium MEDII. L -proline, an amino acid found in abundance in MEDII, has been identified as a bioactive component required for EPL cell formation. Addition of L-proline to ES cells results in changes in colony morphology, gene expression and differentiation kinetics consistent with differentiation towards EPL cells. This activity is restricted to L-proline as addition of other amino acids or synthetic analogues of L-proline does not result in EPL cell formation. Here we characterise the role of L-proline uptake and amino acid transporters in ES cell differentiation. Characterisation of L-proline transport using radioactive uptake assays has shown that the amino acid System A Transporter SNAT2 (SLC38a2) is the major transporter for L -proline transport into ES cells. Uptake is sodium and pH dependent and can be inhibited by the addition of a molar excess of other amino acid substrates of SNAT2. Amino acids that block L -proline uptake also block the ability of L -proline to induce EPL cells from ES cells. This study implicates amino acids and amino acid transporters in ES cell differentiation and defines inexpensive culture supplements with potential applications in improving the maintenance, and/or differentiation, of mouse and human pluripotent cells in culture. Furthermore, this work describes a novel and amenable model to investigate amino acid regulation of intracellular signaling, transcriptional activity and cellular function.

ROLE OF IFNγ SIGNALING IN THE PATHOGENESIS OF HIV

Achuthan A.1, 2, Zhou J.1, Crowe S.1, Scholz G.2 and Jaworowski A.1 1Centre for Virology, Burnet Institute, Melbourne. 2Dept of Medicine (RMH/WH), The University of Melbourne.

Tuberculosis (TB) is the leading cause of death among HIV-positive individuals. Indeed, HIV and M. tuberculosis (Mtb) co-infected individuals have a 10% annual risk of reactivating latent Mtb infection. Macrophages are both the host cell for Mtb infection and also the cell directly responsible for Mtb killing. Hence, evasion of macrophage intracellular destruction mechanisms is pivotal to Mtb virulence. IFNγ promotes macrophage immune functions (e.g. cytokine secretion and phagocytosis) relevant to Mtb killing and immunity, in part by modulating expression and/or function of effector proteins (e.g. SNARE, autophagy and Rab proteins), controlling membrane vesicle fusion events. We report here that IFNγ-induced STAT1 phosphorylation is impaired in HIV-1-infected primary human monocyte-derived macrophages. We found IFNγ up-regulates the expression of SNARE proteins Stx11 and VAMP8 in macrophages. Significantly, CD14+ monocytes from HIV-1-infected individuals displayed decreased protein levels of VAMP8 and Stx11 as compared to controls. The effects of IFNγ-inducible Stx11 and VAMP8 on secreting cytokines and killing Mtb in macrophages will be presented and discussed.





EPIGALLOCATECHIN-3-GALLATE INHIBITS FIBRIL FORMATION BY RECOMBINANT PLASMODIUM FALCIPARUM MEROZOITE SURFACE PROTEIN 2

Adda C.G.1, Chandrashekaran I.R.2, MacRaild C.A.2, Anders R.F.1 and Norton R.S.2 1Department of Biochemistry, La Trobe University, Victoria 3086, Australia. 2The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3052, Australia.

The merozoite surface protein 2 (MSP2) is an abundant ~28 kDa GPI-anchored protein expressed on the surface of the maturing asexual blood stages of the malaria parasite, Plasmodium falciparum. MSP2 is currently being assessed as a potential component of a human malaria vaccine having shown efficacy in a Phase I/IIb clinical trial in Papua New Guinean children. Recombinant MSP2 is an intrinsically unstructured protein that has been shown to form fibrils that, under physiological conditions, have many of the characteristics of amyloid. The propensity of MSP2 to form fibrils is a potential problem for vaccine development and small molecule inhibitors of fibril formation have been investigated. The flavonoids, epigallocatechin-3-gallate (EGCG), baicalein and resveratrol, were examined for their ability to inhibit fibril formation (1). The effects on fibril formation were studied using thioflavin T binding assays, electron microscopy, NMR spectroscopy, circular dichroism and other biophysical methods. Unlike baicalein and resveratrol, EGCG demonstrated the capacity to form SDS-stable oligomeric MSP2 complexes, and inhibit fibril formation. The molecular mechanism of fibril inhibition by EGCG involves noncovalent binding followed by covalent modification of the protein. Although the addition of EGCG appears to be an effective means of stabilizing MSP2 in solution, from a regulatory perspective, EGCG-stabilized MSP2 oligomers are unlikely to be acceptable in a vaccine formulation. However, these small molecule inhibitors of MSP2 fibril formation may be useful as mechanistic probes in studying oligomerization of MSP2. (1) Chandrashekaran IR, Adda CG, MacRaild CA, Anders RF, Norton RS. (2010) Biochemistry (in press).

MOLECULAR BASIS FOR MEMBRANE RECRUITMENT OF THE PX DOMAIN-CONTAINING PROTEIN SNX17 AND ITS STRUCTURAL RELATIONSHIP TO SNX31 AND SNX27

Ghai R., Mehdi M., Teasdale R., King G. and Collins B. Institute of Molecular Bioscience, University of Queensland, ST. Lucia Campus, Brisbane, Queensland 4067.

Transmembrane protein sorting between different membrane-bound compartments inside the cell is critical for a number of physiological and pathological processes. Sorting nexin 17 (SNX17) is a member of the phox-homology (PX) domain containing family of proteins, and has been shown to regulate the endosomal trafficking of amyloid precursor protein (APP) and LDL receptor (LDLR) family proteins critical in Alzheimer’s disease. In this study we show that SNX17 and other PX domain proteins SNX27 and SNX31 define a novel sub-family of PX proteins containing an unusual FERM-like domain. SNX17 and SNX27 have been demonstrated to regulate the trafficking of cargo receptors from phosphatidylinositol-3-phosphate (PtdIns(3)P)-enriched endosomal membranes. Using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy we show that both SNX17 and SNX27 have absolute specificity for PtdIns(3)P interaction via their PX domains. The structure of the SNX17 PX domain with bound SO42-, and comparison with other PX domains reveals important insights into the mode of PtdIns(3)P binding by the PX-FERM-like proteins within a conserved basic cleft. This binding site was further confirmed by NMR spectroscopy titration experiments, molecular docking and mutagenesis. Analysis of the SNX17 PX domain structure also reveals the presence of a novel conserved surface that may be important for additional inter- or intra-molecular interactions. Overall we have defined a unique sub-family of PX-FERM-like proteins, and using a combined structural and biochemical approach have confirmed their ability to associate with the endosomal targeting lipid PtdIns(3)P via a conserved molecular mechanism.

STRUCTURAL ANALYSIS OF ALPHA HAEMOGLOBIN IN COMPLEX WITH ALPHA HAEMOGLOBIN STABILIZING PROTEIN

Dickson C.F.1, Rich A.M.3, Krishna Kumar K.2, Lay P.A.3, Mackay J.P.2 and Gell D.A.1, 2 1Menzies Research Institute, University of Tasmania. 2School of Molecular Biosciences, University of Sydney. 3School of Chemistry, University of Sydney.

Alpha haemoglobin (αHb) stabilizing protein (AHSP) is an erythroid specific protein known to be important for the development and survival of normal red blood cells. One role of this small protein is to sequester and detoxify excess αHb chains that remain in the cell after haemoglobin assembly. To fulfill this role, AHSP binds to free αHb and converts it to an inert form in which the six available coordination sites surrounding the haem-iron are occupied by αHb histidine sidechain or porphyrin ligands. The structure of this final stable state is well characterized, but the mechanism by which AHSP induces these structural changes in the haem pocket of αHb is poorly understood. To address this, we have performed nuclear magnetic resonance (NMR) studies of free αHb and the αHb:AHSP complex. These data represent the first high-resolution structure of a free Hb chain. Chemical shift analysis suggests that upon binding AHSP, conformational change occurs in the F-G helices of αHb. Helix G forms part of the αHb:AHSP interface, whereas the adjacent F-helix bears the haem-coordinating H87. Thus, these data provide a plausible mechanism by which AHSP binding effects might be propagated to the haem pocket. Extended X-ray absorption fine structure (EXAFS) indicate that a small but significant lengthening of the Fe-O2 bond of oxy-αHb upon binding to AHSP, indicating that O2 dissociation is promoted. These findings fit with our hypothesis that AHSP alters the conformation of αHb to promote autoxidation and allow haem to simultaneously bind two axial histidine sidechain ligands.

A FLEXIBLE DEVELOPMENT ENVIRONMENT FOR THE ANALYSIS OF LARGE SCALE BIOLOGICAL DATA

Milla L. and O’Connor L. La Trobe University.

Biologists today are faced with the challenge of analysing increasingly large amounts of data, sourced from a variety of instruments whose technologies are rapidly evolving. To keep up with this constantly changing environment, we are building a flexible development framework that can be used to quickly create and modify data pipelines that automate the processing and analysis of data. Using workflow tools we can read disparate sources of information, process the data by integrating existing algorithms or third party software, and produce results that are further analysed and visualised with dedicated tools. This flexible development environment allows scientists to concentrate on the analysis and interpretation of experiments rather than the development and maintenance of custom code to process and view the data.





THE X-RAY CRYSTAL STRUCTURE OF MURINE α2-ANTIPLASMIN

Law R.H.P.1, Encarnacao J.1, Zhang Q.1, Horvath A.2, Buckle A.M.1, Whisstock J.C.1 and Coughlin P.B.2 1Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800 Australia. 2Australian Centre for Blood Diseases, Monash University, Alfred Medical Research Precinct, Prahran, Victoria, 3181 Australia.

α2-Antiplasmin is a member of the serpin superfamily. It plays an important role in the regulation of hemostasis through inhibition of plasmin. Accordingly, deficiency in humans results in severe bleeding episodes. Uniquely amongst human serpins, α2-antiplasmin contains an unusual extensive N-terminal and C-terminal sequences flanking the serpin domain. The 55-residue C-terminal extension is highly conserved between species and contains important determinants for exosite interactions with the target protease. Hence, α2-antiplasmin with truncated C-terminal region reacts much more slowly with plasmin. We study the structure and function of N-terminal truncated α2-antiplasmins. Here we determined the 2.65Å structure of murine α2-antiplasmin in the native and inhibitory conformation. In contrast to coagulation factors such as antithrombin and heparin co-factor II, this structure reveals that the reactive centre loop (RCL) is not pre-inserted into the A β-sheet. Thus it suggests that α2-antiplasmin does not undergo co-factor mediated conformational change. Importantly, the structure reveals that the first 9 residues of the C-terminal sequence are tightly associated with the serpin body and form an extra strand with the C β-sheet. Such interaction would position the flexible plasmin-binding portion of the C-terminus in close proximity to the RCL. In conclusion, we suggest that the C-terminal region of α2-antiplasmin may function as a “hook” that accelerates the interaction with the target plasmin, and disruption of this interaction disrupt the rapid assembly of the initial plasmin / α2-antiplasmin complex.

RECEPTOR LOCALISATION TO CAVEOLAE MODULATES G PROTEIN COUPLING OF THE β3-ADRENOCEPTOR

Evans B.A.1, Sato M.1, Hutchinson D.S.1, Furness S.1, Bengtsson T.2 and Summers R.J.1 1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria. 2Department of Physiology, Wenner-Gren Institute, Stockholm University, Sweden.

Caveolae are plasma membrane invaginations, formed by recruitment of caveolins to membrane rafts, that are known to modulate G protein-coupled receptor signalling. For example, caveolin-3 interacts with the β2-adrenoceptor (AR) and Gαs, Gαi, adenylate cyclase, PKA and protein phosphatase 2A [1]. β3-AR signalling is also dependent on caveolae, however it is not known whether caveolin-1 is associated with the β3-AR or downstream signalling proteins [2]. We have addressed this question by taking advantage of the distinct signalling properties of mouse β3-AR isoforms [3]. The β3a- and β3b-AR differ only in their C-terminus, yet the β3b-AR couples to Gs and Gi whereas the β3a-AR couples only to Gs, suggesting β3a-AR interaction with protein(s) interfering with Gi coupling. When CHO-β3a-AR cells are treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, β3a-AR signalling becomes PTX sensitive, indicating Gi coupling. The β3a-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, contains a caveolin interaction motif. Unlike the wild type receptor, β3a-ARs with F389A/Y392A/F398A or P384S/F389A mutations display Gi coupling. A Duolink in situ proximity ligation assay demonstrates direct interaction between caveolin-1 and the wild type β3a-AR, but not the F389A/Y392A/F398A or P384S/F389A mutants. In addition, endogenous β3a-ARs acquire Gi coupling in brown adipocytes from caveolin-1 knockout mice, or in wild type adipocytes treated with filipin III. Our data demonstrate a functionally significant interaction between caveolin-1 and the β3a-AR C-terminus. [1] Balijepalli et al (2006) PNAS (USA) 103, 7500-7505. [2] Cohen et al (2005) Diabetes 54, 679-686. [3] Sato et al (2005) J Pharmacol Exp Ther 315, 1354-1361.

OXI: A NOVEL BRANCHING MUTANT IN ARABIDOPSIS THALIANA

Meyers E.L., Brewer P.B., Mason M., Dun E.A. and Beveridge C.A. School of Biological Sciences, University of Queensland, St Lucia, Queensland, 4072.

Shoot branching in plants is carefully regulated by several interacting pathways. Strigolactones are a new family of plant hormones and are involved in controlling shoot branching. Very little is known about strigolactone signalling pathways and only a handful of genes controlling their biosynthesis have been characterised. Recent work has identified several candidate genes that may be involved in shoot architecture regulation. A knockout mutant of one of these genes, OXI, displays a moderate branching phenotype. This phenotype is rescued by exogenous supply of strigolactone, consistent with other strigolactone biosynthesis mutants. Real time and GUS expression analysis has found that OXI is co-expressed with other strigolactone pathway genes. Grafting experiments have shown that oxi rootstocks restore branching inhibition to other strigolactone biosynthesis branching mutant shoots. However, unlike other branching mutants, oxi branching cannot be rescued when grafted to a wild type rootstock. These findings suggest that OXI is a novel player in the biosynthesis of strigolactones and may act downstream of several previously characterised genes in the strigolactone biosynthesis pathway. Current work to test this hypothesis involves identifying and quantifying endogenous strigolactones in the oxi mutant. The characterisation of OXI is important for elucidation of the strigolactone biosynthetic pathway and to further the understanding of shoot branching regulation.

EXPLORATION OF β(1,4)-GALACTAN BIOSYNTHESIS IN PINUS RADIATA

Weinberg C.S.1, West M.1, Phillips L.1, Flint H.1, Mast S.2 and Wagner A.1 1Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua, New Zealand. 2Prozyme, 3832 Bay Center Place, Hayward, CA, 94545, USA.

Gravitropism significantly stimulates the production of the hemicellulose β(1,4)-galactan during secondary cell wall formation in pine. However, the molecular and physiological function of this cell wall polymer is essentially unknown. The goal of the project is to uncover the biological function of β(1,4)-galactan in wood through the identification and functional testing of genes involved in the biosynthesis of this polysaccharide in Pinus radiata. However, genes involved in the biosynthesis of this cell wall polymer have not been identified to date. Therefore this study tries to explore galactan biosynthesis by using a proteomic approach and focuses initially on the galactosyl transferase (GalT), which is responsible for the biosynthesis of the polymer backbone. Initially, an HPLC-based enzyme assay was established that uses fluorescently labelled galactan oligomers as substrates. Assaying different wood types demonstrated that GalT activity levels were virtually non-existent in opposite wood, but high in compression wood. Our enzymatic data indicated that production of β(1,4)-galactan in different wood types is at least partially, if not predominantly, based on differential GalT activity levels. Preliminary work on the purification of GalT showed that this enzyme is likely to be associated with membrane bodies, potentially Golgi vesicles.





GENETIC CONTROL OF C-GLYCOSYLATION IN THE BIOSYNTHESIS PATHWAY OF FLAVONE-C-DIGLYCOSIDES IN BREAD WHEAT

Wijaya G.Y.1, 2, Asenstorfer R.1 and Mares D.J.1 1School of Agriculture Food and Wine, University of adelaide, Glen Osmond SA 5064, Australia. 2Biotechnology Faculty, Atma Jaya Catholic University, Jl Jenderal Sudirman no 51 Jakarta 12930, Indonesia.

Falvone-C-diglycosides (FCGs) have diverse and important roles in plants, ranging from pigments and bioactive compounds in food and beverages, to allelopathic compounds against parasitism, pest and microbial infection. The are produced from the intermediate naringenin, via branch of the flavonoid biosynthitic pathway, and involve C-glycosylation of the 6th and 8th carbon of A flavone ring. To gain a better understanding of the genetic control of FCG C-glycosylation, two varieties of hexaploid bread wheat (Triticum aestivum vars Tasman and Sunco, AABBDD genome), a complete set of nullisomic-tertrasomic (NT) lines of Chinese Spring, 5 lines of the diploid species Triticum monococcum (AmAm genome), and 5 lines of Aegilops tauschii (DD genome) were analysed by HPLC. The results showed that C-glycosylation in bread wheat is genetically controlled by loci on 7A, 7B, and 7D and 1B. A possible explanation for the observed variation in FCG1/FCG2 ratio is that C-glycosyltransferase coded by genes on these chromosome vary in their specificity for UDP-glucose or UDP-galactose.

HOW TO KILL TUMOUR CELLS THAT HAVE LOST P53 ACTIVITY: A NOVEL APOPTOTIC PATHWAY INDUCED BY DNA DAMAGING CHEMOTHERAPEUTICS

Delbridge A.R.D.1, 2, Scott C.1, Bouillet P.1 and Strasser A.1 1Molecular Genetics of Cancer Division, Walter & Eliza Hall Institute. 2Department of Medical Biology, University of Melbourne.

While a large body of knowledge has accumulated in relation to the p53-dependent DNA damage-induced apoptotic pathway, comparatively little is known about the p53-independent apoptotic pathways that can be activated by DNA damage, although they are known to contribute significantly to anti-cancer therapy induced tumour cell killing. In order to address this we have investigated the role of the pro-apoptotic BH3-only proteins in the response of p53-deficient thymic lymphoma derived cell lines to DNA damage induced by γ-irradiation or chemotherapeutic drugs. Our findings identify Bim as a critical initiator of apoptosis induced by DNA damage inducing drugs in the absence of p53, while Puma and Bmf may also contribute, at least in response to treatment with etoposide. Our data also implicates several mechanisms for Bim induction in the response of p53-deficient cells to DNA damage. This novel apoptotic pathway also appears to play a role in the suppression of lymphoma development. We suspect that Bim may play a crucial surveillance role in pre-leukaemic cells, acting to eliminate cells that have sustained p53 mutations particularly when they have become genomically unstable. Consistent with this hypothesis, our data show that the additional loss of Bim markedly accelerates lymphoma development in p53-/- and p53+/- mice.

COMPARATIVE ANALYSIS OF FICIN STABILITY IN DIFFERENT CHEMICAL DENATURANTS

Sidek N.A.A., Kadir H.A. and Tayyab S. Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Ficin (E.C. 3.4.22.3) is an endopeptidase which belongs to the class, cysteine proteases. We studied the effect of different chemical denaturants, i.e. guanidine hydrochloride (GdnHCl), urea and ethanol on ficin stability using far-UV CD, near-UV CD, intrinsic fluorescence, tryptophan fluorescence, acrylamide quenching and biological activity. Far-UV CD spectra of native ficin showed the presence of α-helical structure which was completely lost in 6M GdnHCl but retained in 9M urea. Loss of tertiary structure in 6M GdnHCl was also evident from near-UV CD spectra. There was total loss of biological activity at 6M GdnHCl. A red shift of about 10 nm was also observed in the emission maxima in presence of 6M GdnHCl upon excitation at 280/295 nm, suggesting major conformational change in the protein. Titration of native ficin with increasing GdnHCl concentrations using far-UV CD signal followed a two-state, single-step transition which yielded a value of ∆GD

H2O as 5186.6 cal/mol. Fluorescence intensity data showed lesser changes in presence of different concentrations of urea as well as ethanol compared to those observed with GdnHCl. Taken together, all these results suggested a strong denaturing action of GdnHCl on ficin among all the denaturants used.

BINDING OF THE LECTIN ARTINM TO MAST CELLS RESULTS IN CELL ACTIVATION

Lorenzi V.C.B.1, Roque-Barreira M.C.1, Pereira-Da-Silva G.2, Jamur M.C.1 and Oliver C.1 1Department of Cell and Molecular Biology and Pathogens Bioagents, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil. 2School of Nursing of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil.

Mast cells (MC) are essential cells in IgE-associated immune responses.FcεRI is the major receptor for MC activation. FcεRI cross linking induces MC degranulation and release of proinflammatory mediators. We have previously shown that the lectin ArtinM induces MC degranulation but the mechanism of ArtinM induced activation remains unknown. The present study was undertaken to characterize the ability of ArtinM (10μg/ml) to activate the rat MC line RBL-2H3. By confocal microscopy, it was observed that ArtinM binds to the MC surface and this binding is dependent on ArtinM carbohydrate recognition domains (CRDs). Microplate assays showed that ArtinM binds to IgE and this binding is also dependent on ArtinM CRDs. By Western blotting of total cell lysates, ArtinM also recognizes the β subunit of FcεRI. MC incubation with ArtinM results in the release of β- hexosaminidase activity in the presence or absence of IgE. The tyrosine phosphorylation of intracellular proteins was investigated. Western blotting of IgE sensitized cells stimulated with ArtinM showed that tyrosine phosphorylation of intracellular proteins was similar to that of to antigen stimulated cells. These results suggest that ArtinM may bind to glycans of FcεRI receptor and/or of the IgE (bound to FcεRI) and that such interactions could be implicated in its ability to activate and degranulate mast cells. In view of the well-established significance of mast cells in allergy and inflammation, the participation of sugars as receptors on the mast cell surface opens new possibilities to control allergic disorders.


POSTERS WEDNESDAY & THURSDAY

POS-WED-01 POS-THU-02


RHOU IS REQUIRED FOR NORMAL ACTIN DISTRIBUTION AND DIFFERENTIATION IN THE DEFINITIVE ENDODERM

Loebel D.A.F.1, 2, Studdert J.B.1, Jones V.1, Power M.1, Radziewic T.1 and Tam P.P.L.1, 2 1Children’s Medical Research Institute, Westmead NSW 2145. 2Sydney Medical School, University of Sydney, NSW 2006.

The definitive or gut endoderm forms at gastrulation and contributes to the epithelial linings of the lungs, stomach, oesophagus and trachea, as well as organs including the liver, pancreas, thyroid and thymus. To gain insights into the molecular basis of development, we have performed microarray analysis to identify endoderm-enriched transcripts in foregut of early-somite-stage mouse embryos. We identified Rhou, encoding an atypical Rho GTPase amongst the genes that were preferentially expressed in the endoderm. Overexpression or knockdown of Rhou has been shown to have dramatic effects on cell morphology and the actin cytoskeleton in cell line models. Rhou knockdown embryonic stem (ES) cell lines were generated and tested by in vitro differentiation as embryoid bodies (EBs) in the presence of Activin A. Rhou-deficient cells in the EBs display abnormal F-actin localization and impaired differentiation of endoderm derivatives. In embryos generated from the knockdown ES cell lines by tetraploid complementation, the foregut collapses and the cells lining the gut lose their normal epithelial architecture. The endodermal cells show a reduced apical concentration of F-actin but appear to form normal adherens and tight junctions. Overexpression of Rhou in endoderm cells by embryo electroporation also resulted in changes in cell shape and F-actin distribution. Our data suggest that tight regulation of levels of Rhou is required for proper F-actin distribution in the endoderm and that disruption of the epithelial organization of the endoderm significantly impairs endoderm differentiation.

HIGHLY SIGNIFICANT CORRELATION BETWEEN THE PROTAMINE CONTENT, DNA INTEGRITY AND QUALITY OF EJACULATED SPERMATOZOA FROM INFERTILE MEN

Manochantr S. 1, Chiamchanya C. 2 and Sobhon P. 3 1Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Patumthani, 12120, Thailand. 2Department of Obstetrics and Gynecology, Faculty of Medicine, Thammasat University, Patumthani, Bangkok 12120, Thailand. 3Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.

Histone-to-protamine exchange is a late spermiogenesis event, along with acrosome formation, membrane remodeling and other significant morphological and biochemical events that are necessary for normal sperm function. Therefore, protamines are considered a good marker of sperm nuclear maturity. The aims of the present study were to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduced the relationship with sperm quality as measured by conventional semen parameters. Routine semen analysis was carried out to ascertain sperm concentration, motility, and morphology. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomysin A3 (CMA3) assay and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The ultrastructural analysis of spermatozoa from infertile men indicated nuclear maturation defect in the spermatozoa of infertile men. Spearman’s correlation analysis indicated the positive correlation between the percentages of CMA3- and TUNEL- positive spermatozoa. In addition, these 2 parameters were negatively correlated with concentration, motility and normal morphology. This study demonstrated that the men with abnormal semen parameters carrying higher loads of protamine deficiency and DNA-damaged spermatozoa. Therefore, the assessment of chromatin condensation and DNA integrity appears to be a useful tool for assessing male fertility potential.

THE ROLE OF HEDGEHOG SIGNALING IN GUT COLONISATION BY VAGAL ENTERIC NEURAL CREST CELLS

McConnell S., Simkin J.E., Zhang D. and Newgreen D. Murdoch Children’s Research Institute, Flemington Rd, Parkville 3052.

The enteric nervous system is derived from a transient sub-population of cells called the neural crest (NC), specifically from the vagal (caudal hindbrain) level. These colonise the gut in a proximo-distal wave. Enteric (ie. vagal) NC cells from already colonised embryonic quail gut, rapidly colonised an abutted chick aneural gut in organ culture. But when vagal neural tube/NC and the aneural gut were abutted in culture, there was little NC cell migration into the gut although the NC cells were migratory elsewhere. By altering duration of culture and duration of abutment, we found vagal NC cells require time plus some other factor(s) to become competent to migrate into the gut. When the entire vagal tissues (neural tube/NC, plus somites, foregut endoderm and mesoderm) were cultured together, subsequent colonisation of aneural gut by NC cells was improved, relative to neural tube/NC alone. This suggested that factor(s) in the vagal tissue, plus time to respond, enhanced the migratory ability of the NC cells. Sonic hedgehog (Shh) expression in the foregut endoderm at the vagal level was found exactly when and where the vagal NC cells approach and enter the gut. Moreover, NC cells from the entire vagal tissues failed to colonise the gut when exposed to the hedgehog-antagonist cyclopamine. In contrast, older NC cells from gut already colonised, could migrate into aneural gut when cyclopamine was present. This suggests the drug is neither cytotoxic nor inhibitory to motility, and hence is consistent with hedgehog being a competency factor for gut colonisation, but that hedgehog activity is only critically required during initial approach/entry of NC cells into the gut.

MICRORNA SIGNALLING IN EARLY MALE GERM CELLS

McIver S.C.1, 2, 3, Roman S.D.1, 2, 3, Nixon B.1, 3 and McLaughlin E.A.1, 2, 3 1Reproductive Science Group. 2ARC Centre of Excellence in Biotechnology & Development. 3School of Environmental and Life Sciences, University of Newcastle, Callaghan, Australia.

MicroRNAs are short noncoding RNA sequences which bind to the 3’ untranslated region of mRNA to control translation and aberrant miRNA expression is linked to many disease states and developmental abnormalities. Of particular interest to our group is the. We are focusing on the early development of the male germ cell particularly the period in which gonocytes differentiate to become spermatogonia. This is of interest because in the human Carcinoma in Situ cells, the precursors to testicular germ cell tumours have been identified as arising from dysfunctional gonocytes which fail to differentiate into spermatogonia. Gonocytes from post natal day 1 mice and spermatogonia from day 7-9 mice were enriched by 2-4% BSA gradient sedimentation. Total RNA including microRNA was extracted and analysed in by Illumina miRNA microarray. Total RNA was also reverse transcribed using specific primers and analysed by qPCR. Three biological replicates were performed in both the microarray and qPCR experiments. Bioinformatic analysis with SAM (Significance analysis of Microarrays) identified seven significantly different miRNA molecules expressed in spermatogonia and gonocytes. qPCR analysis determined two miRNAs that were significantly upregulated in spermatogonia (743a, 463*) and three miRNAs which were significantly down regulated in spermatogonia (293, 290-5p, 291a-5p). Several miRNA molecules were selected for further study (293, 290-5p, 136, and 146a) and overexpression assays in P19 cells will help determine their function. In the future the role of these miRNA molecules in seminoma will be analysed using overexpression within a seminoma cell line. It is hypothesised that these miRNA molecules control genes involved in male development and differentiation such as stella, nanog and oct3/4 and that these molecules may also play a role in tumour development In conclusion miRNA expression is significantly different between gonocytes and spermatogonia and influence their differentiation and developmental course.





REGULATION OF FOXD3 IN THE NEURAL CREST

Mckeown S.J.1, 2 and Bronner M.2 1University of Melbourne, VIC, Australia. 2California Institute of Technology, Pasadena, CA, USA.

Neural crest cells are a uniquely vertebrate cell type vital for development. They form at the border of the neural plate and epidermal ectoderm, migrate to numerous locations and differentiate into many cell types. Many transcription factors are involved in the generation of the neural crest, including FoxD3. To better understand the regulatory interactions involved in neural crest formation, we analyzed the regulatory region of FoxD3 for the presence of enhancer elements. Elements conserved across chick, mouse and human in the FoxD3 genomic region were cloned into a reporter construct and electroporated into Stage 4 chick embryos. We identified three elements upstream of FoxD3 that direct expression in the neural crest in spatially and temporally distinct patterns, and in combination recapitulate the entire expression of FoxD3 in the neural crest. The first element directs expression only in premigratory and migratory cranial neural crest. The second element directs expression in migratory cranial neural crest and premigratory and migratory vagal and trunk neural crest. The third element directs expression in some premigratory vagal and trunk neural crest and a population of spinal cord interneurons. We reduced the size of these enhancers to identify minimal core regions and used bioinformatics analysis to identify putative transcription factor binding sites. Site directed mutagenesis of the premigratory cranial neural crest enhancer revealed two binding sites critical for activity of the enhancer: a homeodomain site and an Ets/Gata binding site. Antisense morpholinos and chromatin immunoprecipitation are being used to identify the upstream transcription factors that regulate these FoxD3 neural crest enhancers. The results provide new insight into the gene regulatory mechanisms underlying neural crest formation.

OLFACTORY GLIA ENHANCE NEONATAL AXON REGENERATION

Chrehrehasa F., Windus L., Ekberg J., Scott D., Amaya D., Mackay-Sim A. and St John J. National Centre for Adult Stem Cell Research, Griffith University.

Olfactory ensheathing cells (OECs) migrate with olfactory axons that extend from the nasal epithelium into the olfactory bulb. Unlike other glia, OECs are thought to migrate ahead of growing axons instead of following defined axonal paths. However it remains unknown how the presence of axons and OECs influences the growth and migration of each other during regeneration. We have developed a regeneration model in neonatal mice to examine whether (i) the presence of OECs ahead of olfactory axons affects axonal growth and (ii) the presence of olfactory axons alters the distribution of OECs. We performed unilateral bulbectomy to ablate olfactory axons followed by methimazole administration to further delay neuronal growth. In this model OECs filled the cavity left by the bulbectomy before new axons extended into the cavity. We found that delaying axon growth increased the rate at which OECs filled the cavity. The axons subsequently grew over a significantly larger region and formed more distinct fascicles and glomeruli in comparison with growth in animals that had undergone only bulbectomy. In vitro, we confirmed (i) that olfactory axon growth was more rapid when OECs were more widely distributed than the axons and (ii) that OECs migrated faster in the absence of axons. These results demonstrate that the distribution of OECs can be increased by repressing by growth of olfactory axons and that olfactory axon growth is significantly enhanced if a permissive OEC environment is present prior to axon growth.

CHARACTERISATION OF ENU-INDUCED IFT140 MUTANT MICE EXHIBITING EXENCEPHALY AND POLYDACTYLY

McMorran T.A.1, 2 and Farlie P.1 1Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville. 2Department of Paediatrics, University of Melbourne.

Congenital defects are abnormalities in body structure, function or metabolism present at birth resulting from genetic or environmental causes and are the leading cause of infant morbidity and mortality. Approximately one third of congenital defects involve the craniofacial structures. Affected individuals often have other abnormalities, such as defects in limb or internal organ development. The genetic basis of many craniofacial disorders and the developmental processes involved are poorly understood. An ENU mutagenic screen of mice has been undertaken to address this deficiency. A strain has been identified where affected embryos exhibit severe neural tube and craniofacial defects and have an abnormal number of digits on both the fore- and hindlimbs. The features of this strain are similar to those seen a family of human conditions including short-rib polydactyly and Jeune asphyxiating thoracic dystrophy. The gene mutated has been identified as Ift140, which encodes a component of the primary cilium, a cellular structure that functions as a signalling center within the cell and coordinates the activity of some important growth factor signalling pathways. A similar set of features has been observed in mutant mice with defective sonic hedgehog (Shh) signalling, one of the key signalling pathways that functions during development. The IFT140 protein is involved in the transport of proteins within the primary cilium and is thought to interact with components of the Shh signalling pathway, which would explain the similarity between the Ift140 and Shh mutants. Characterisation of the developmental defects observed in the IFT140 mouse strain will facilitate an investigation into the interaction between Shh and IFT140 and improve our understanding of the processes associated with mammalian development.

TRANSGENIC APPROACHES TO ANALYSE PLANAR CELL POLARITY/NON-CANONICAL WNT SIGNALLING IN ZEBRAFISH EARLY ENDODERM MORPHOGENESIS

Miles L.B.1, Mizoguchi T.2, Kikuchi Y.2 and Verkade H.1 1School of Biological Sciences, Monash University, Clayton, 3800 Victoria, Australia. 2Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526 Japan.

The zebrafish intestine, liver, and pancreas develop from the endodermal cells, which start as a broad monolayer that migrates to the dorsal midline during early development. Once at the midline the cells undergo midline aggregation, where the monolayer of individual cells coalesces to form a narrow endodermal rod at 18hpf. This endodermal rod will later undergo morphogenesis and differentiation to give rise to their final organs. The process of midline aggregation is essential for correct development of the gut but the mechanisms used are unknown. I am investigating the role of changes in cellular shape, behaviour, and polarity during midline aggregation to determine the mechanisms involved. Mutants of the non-canonical wnt / planar cell polarity (PCP) signalling pathway, vangl2/trilobite (tri) and knypek (kny), display a distinctive phenotype of shortened body axis and wider body plan resulting from a reduction in convergence and extension of the mesodermal tissues that overlie the endoderm. I will present data showing that these mutants also have reduced convergence and extension of the endodermal cells, producing a wider endodermal stripe during early-mid somitogenesis stages. In addition my preliminary studies indicate that these mutants show disrupted midline aggregation and incorrect endodermal cell shape changes during mid-somitogenesis stages. To access the timing specific role of PCP signalling during early endodermal migration and midline aggregation, I have created a Tg(hsp70l-xdvlΔDEP-mCherry) transgenic line. This line facilitates a temporal specific disruption of the PCP signalling pathway by inducing a dominant negative form of Dishevelled (xdvlΔDEP), the PCP signalling intracellular mediator.





NEOGENIN 1: A NEW PLAYER IN THE SONIC HEDGEHOG (SHH)/GLI NETWORK IN THE DEVELOPING VERTEBRATE CENTRAL NERVOUS SYSTEM

Milla L.A.1, CortéS C.1, Wainwright B.J.2 and Palma V.1 1Center for Genomics of the Cell (CGC), Faculty of Sciences, University of Chile. 2Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia.

Objective Due the multiple roles played by the Shh/Gli pathway during embryonic development and in adulthood, our work has been focused in uncovering new direct downstream targets for this signaling network. Neo1 has been recently involved in many processes during CNS development, has emerged as an interesting candidate. Here, we characterized the nature of the interaction between Shh components and Neo1 in the rodent CNS. Methods Bioinformatic analysis revealed several putative consensus Gli binding sites (GBS) in the 5´ proximal regulatory region of neo1. To verify in vivo and in vitro direct binding of Gli transcription factors to the neo1 promoter, we used Chromatin Immunoprecipitation (ChIP) in mouse embryonic neocortex (E18.5) and luciferase reporter assays. Expression of Neo1 revealed a similar profile to Gli1 in the developing dorsal CNS. To ascertain the relationship between Shh/Gli and Neo1 expression, we took advantage of different experimental approaches: pharmacological gain and loss of function experiments using mouse cerebellar primary cultures and analysis of Shh pathway gain of function transgenic mouse models. Results ChIP and reporter assays shows direct binding of Gli transcription factors to 3 diffferents neo1 consensus Gli binding sites (GBS) in its promoter, verifying our in silico analysis. Using genetic or pharmacological Shh/Gli gain or loss of function approaches, we demonstrate a regulation of Neo1 levels. The Shh pathway activation seems to be relevant to drive and regulate Neo1 expression and, more importantly, to contribute thereby to progenitor proliferation. Since Neo1 is strongly expressed in the developing cerebellum we are currently addressing the functional consequences of the Shh/Gli-Neo1 relationship in the Shh responsive granular precursor cell population. Conclusion Neogenin1 is a new critical target of the Gli Transcription Factors in vertebrate Hh signaling in the developing CNS.

TEMPORAL REGULATION OF THE SALVADOR-WARTS-HIPPO PATHWAY IN DROSOPHILA MELANOGASTER EYE DEVELOPMENT

Milton C.C.1, 2, Zhang X.1, 2, Albanese N.O.1, 2 and Harvey K.F.1, 2 1Cell Growth and Prolferation Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia. 2Department of Pathology, University of Melbourne, Parkville, VIC 3010, Australia.

The Salvador-Warts-Hippo (SWH) signalling pathway restricts tissue growth and organ size during development. Loss of SWH signalling results in tissue overgrowth as well as tumour development. The SWH pathway is made up of multiple growth inhibitors which act to limit activity of the Yorkie (Yki) oncoprotein. The complex fashion by which these inhibitors act upstream of Yki is demonstrated by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue mutant for the core SWH complex components salvador, warts or hippo is highly overgrown, while tissue lacking fat or expanded is not. We analysed the relative contributions of the SWH pathway proteins Fat, Expanded and Discs overgrown to organ size control by investigating their temporal activity throughout Drosophila eye development. We found that eye tissue which lacks fat, expanded or discs overgrown displays elevated Yki activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. In the pupal eye, Fat and Expanded do possess Yki-repressive activity, but loss of fat or expanded at this stage of development can be compensated for by Merlin. We showed that this compensation is not due to an upregulation of Merlin protein levels in pupal eye tissue lacking fat or expanded. It is likely that Fat represses Yki in the pupal eye together with Expanded as pupal eye tissue lacking both of these genes resembles single mutant tissue. This study highlights the complexity and multiple ways by which different SWH pathway proteins can control tissue growth and organ size during different stages of development.

THE GENETIC AND MORPHOLOGICAL CHARACTERISATION OF A NOVEL ZEBRAFISH DEVELOPMENT MUTANT, HL-7.2

Moore W.B.1, Sherson S.1, Ober E.2, Field H.2, Dong P.D.2, Stainier D.Y.R.2 and Verkade H.1 1School of Biological Sciences, Monash University, Clayton, VIC, Australia. 2Department of Biochemistry and Biophysics, University of California, San Francisco, USA.

The zebrafish is a useful model to examine the specification, differentiation and proliferation of various cell types over the course of development. HL-7.2 is a novel zebrafish mutant identified in an ENU mutagenesis screen that aimed to identify mutants with morphological defects in endoderm derived organs. At 36hpf, analysis using Tg(gut:GFP) transgenic, which has GFP specifically expressed in endodermal organs revealed that HL-7.2 mutants display dysmorphic budding of the liver and pancreas from the endodermal rod and no intestinal looping. Other abnormalities observed in HL-7.2 mutants include a cardiac defect, lack of pigment, malformed brain and craniofacial defects. In situ hybridization analysis of early stages of development up to 36hpf has demonstrated a wide loss of expression of differentiation markers across all germ layers, suggesting a broad developmental defect. Acridine Orange staining revealed that HL-7.2 mutants have a significant increase in the number of dying cells after 30hpf. I have mapped the HL-7.2 mutant locus to a 262kb genomic interval containing 6 genes. These 6 candidate genes are currently being sequenced to identify the mutation responsible for the HL-7.2 mutant phenotype. We hypothesize that the HL-7.2 mutant phenotype results from a cellular arrest phenotype such as cell cycle arrest, as HL-7.2 mutant embryos appear to developmentally arrest at 24hpf. I am currently exploring the mechanisms responsible for the severe abnormalities through the use of immunohistochemistry to examine cell proliferation and to examine for mitotic abnormalities in these mutants.

STEM CELL THERAPY FOR HIRSCHSPRUNG DISEASE: IN VIVO STUDIES USING A POST-NATAL MOUSE MODEL

Hotta R.1, Stamp L.1, 2, Foong J.P.1, Thacker M.1, Pontell L.1, Bergner A.J.1, Anderson R.G.1, Furness J.B.1, Newgreen D.F.2 and Young H.M.1 1Department of Anatomy & Cell Biology, University of Melbourne, Australia. 2Murdoch Childrens Research Institute, Royal Childrens Hospital, Melbourne, Australia.

Gut motility is controlled by the enteric nervous system (ENS), which is derived from the neural crest (NC). In Hirschsprung disease the ENS is absent from the distal colon. Current treatment requires surgery to remove the affected colon, but bowel dysmotility often persists. Cell therapy has the potential to treat Hirschsprung disease by replacing missing neurons. NC stem/progenitor cells can form ENS in aneural embryonic colon in vitro. However, the clinically appropriate context is post-natal colon in vivo; it is unknown whether such cells can colonize and differentiate in such circumstances. To test this, ENS-derived neurospheres were generated by FACS selection of Kikume+ or GFP+ NC cells from gut of E14.5 transgenic mice. Neurospheres were transplanted into the distal colon of P14-P21 wild-type and Ednrb-/- Hirschsprung model mice. After 1-12 weeks, the neurosphere-derived cells had migrated extensively in neural and aneural colon, formed ganglion-like groups and differentiated as neurons (Hu and PGP9.5) and glia (S100β). Neurons expressed nitrergic and cholinergic sub-type markers, and projected axons into other ENS ganglia of the recipient, with synapse-like (synaptophysin) specialisations. Electrophysiological recording established that these neurosphere-derived neurons could generate action potentials and display EPSPs indicative of neural connectivity. These results show that stem/progenitor cells can migrate and undergo neuronal and glial differentiation in the post-natal bowel in vivo. Future studies will determine whether the transplanted cells can restore gut motility patterns.





THE EFFECT OF ACRYLAMIDE EXPOSURE ON EARLY MALE GERM CELLS

Nixon B.J., Nixon B. and Roman S.D. ARC Centre of Excellence in Biotechnology & Development, School of Environmental & Life Sciences, Faculty of Science & IT, University of Newcastle, Callaghan.

Acrylamide is a common industrial compound that has been found to elicit male infertility and transgenerational toxicity in rodents through the male germline. Acrylamide has been identified in carbohydrate-rich foods cooked at high temperatures, thus humans may be at risk to acrylamide exposure in the diet. The aim of this project was to elucidate the mechanisms of acrylamide toxicity in male germ cells of mice. Freshly isolated early male germ cells were assessed for cell viability and aberrant morphology following exposure to acrylamide. DNA damage in these cells was investigated using a modified version of the Comet Assay, which allowed for adduct specificity. The regulation of cytochrome P450 gene expression was measured using real time PCR. Significant increases in cell death or aberrant morphology was not found following acrylamide exposure (1μM, 18 hours). However, a significant increase in DNA damage (125% increase in mean tail DNA assessed by Comet) was identified; which may originate from either the metabolic conversion of acrylamide to glycidamide, leading to glycidamide adducts, or from oxidative stress. Additionally, early male germ cells were found to upregulate gene expression of cytochrome P450 enzymes in response to acrylamide treatment. The results of this study support a genotoxic mode of action of acrylamide toxicity, in addition to potential oxidative damage in male germ cells. However, the mechanism by which acrylamide elicits toxicity in germ cells requires further investigation. Future outcomes of this research may provide insight into factors necessary for the healthy development of offspring and aid in the risk assessment of dietary acrylamide exposure in humans.

THE IDENTIFICATION OF A NOVEL GENE INVOLVED IN DROSOPHILA MIDGUT MIGRATION

Pert M.C., Saint R.B. and Murray M.J. Department of Genetics, The University of Melbourne, Australia.

Cell migration is an important process in animal development and disease. In Drosophila embryogenesis, cell migration is necessary for the formation of the midgut, in which epithelial patches of cells at the anterior and posterior poles of the embryo undergo an epithelial to mesenchymal transition and migrate towards one another using the visceral mesoderm as a substrate. Once the two patches of cells have met in the middle of embryo, they undergo a mesenchymal to epithelial transition to form the midgut. Currently, there is only one group of proteins known to be required for midgut migration; the extracellular matrix adhesion proteins, the integrins. It is therefore of great interest to discover new genes involved in this process. We have identified a deficiency stock that has an embryonic phenotype in which the midgut does not form due to a failure in cell migration. As this deficiency stock results in the loss of a large number of genes, a deficiency screen has been carried out to narrow down the number of possible candidates. None of these candidates have previously been associated with midgut migration. Currently, we are utilizing molecularly defined deletions, genomic rescue constructs, and injection of dsRNA to determine which of the candidate genes is responsible for the midgut phenotype. We are also using a combination of immunostaining and live imaging to better characterise the cellular basis for the migration defect. The findings of these experiments will be presented.

XENOBIOTICS; INFLUENCE ON LONG TERM OOCYTE VIABILITY

Pye V.1, Sobinoff A.P.1, Nixon B.1, 2, Roman S.D.1, 2 and McLaughlin E.A.1, 2 1School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia. 2The ARC Centre of Excellence in Biotechnology and Development, The University of Newcastle, Callaghan, NSW, Australia.

Mammalian females are born with a finite number of non-renewing primordial follicles, the majority of which can remain in a quiescent state for many years. Due to their non-renewing nature, these “resting” oocytes are particularly vulnerable to environmental and toxic insults, especially those which are capable of inducing oxidative stress. Recent evidence suggests that certain synthetic chemical compounds, known as xenobiotics, have the potential to generate oxidative stress through the production of free oxygen radicals as a byproduct of the cell’s detoxification process. Given the redox sensitive nature of the mammalian oocyte, xenobiotic exposure has been hypothesized to have long term adverse effects on oocyte viability. In this study, we attempted to identify the effects of short term xenobiotic exposure on long term oocyte viability. Female Swiss neonatal mice (day 4) were administered 7 daily consecutive doses of 4-Vinylcyclohexene diepoxide (40mg/kg/daily; 80mg/kg/daily) Methoxychlor (50mg/kg/daily; 100mg/kg/daily) or Menadione (7.5mg/kg/daily; 15mg/kg/daily). Mice were superovulated at 6wks and their oocytes collected for sperm-egg fusion assays. Sperm-egg fusion assays revealed a significant decrease in sperm egg binding/fusion (p<0.05) in a dose dependent manner for all three xenobiotic treatments in vivo, signifying a decrease in oocyte membrane fluidity. Follow-up lipid peroxidation analysis on xenobiotic cultured oocytes also showed a dose dependent increase in membrane lipid peroxidation in response to xenobiotic exposure. These results suggest that short term xenobiotic exposure can cause long term oocyte dysfunction, possibly interfering with the fluidity and/or elasticity of the oocyte plasma membrane through lipid peroxidation.

NOVEL PARACRINE SIGNALING FROM CARDIAC PROGENITOR CELLS TO EARLY NEURAL CREST IN MAMMALIAN EMBRYOS

Prall O.1, Menon M.K.2, Biben C.1, Hartley L.1, Martin J.3, Imanaka-Yoshida K.4 and Harvey R.P.2 1Walter and Eliza Hall Institute, Melbourne, Australia. 2Victor Chang Cardiac Research Institute, Sydney, Australia. 3Institute of Biosciences and Technology, Texas, USA. 4Mie University, Japan.

Congenital abnormalities of the heart and face frequently occur simultaneously due to shared aspects of their formation. In early vertebrate embryos “neural crest” (NC) detaches from dorsal neural tissue and migrates over substantial distances to supply cells to many structures including facial bones and the heart. Prior to entering the heart, NC cells exchange molecular signals with cardiac progenitors in the pharynx. This reciprocal signaling is essential for both NC and cardiac development. We have found evidence of two new molecular signals that cardiac progenitors send to NC, a morphogen bone morphogenetic protein 2 (BMP2) and an extracellular matrix protein tenascin C (TNC). In contrast to known interactions between cardiac progenitors and NC, this paracrine signaling occurs in cranial regions prior to pharyngeal development. Mutation of the cardiac transcription factor gene Nkx2-5 results in advanced migration of cranial and cardiac NC. In Nkx2-5 mutants, Tnc and Bmp2 mRNA expression are upregulated in cardiac progenitor cells. TNC protein is deposited in sub-NC mesenchyme in both control and Nkx2-5 mutants, and elevated in transgenic mice over-expressing Tnc specifically in cardiac progenitor cells. Nkx2-5 mutants lacking the BMP signaling intermediate Smad1, show upregulation of BMP2 in cardiac progenitors but not advanced NC migration. This suggests that cardiac-derived BMP2 signals to neural crest. It is possible that genetic and environmental factors perturbing early cardiac progenitors may cause congenital abnormalities through early indirect influences on NC behaviour.





IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

Redgrove K.A.1, McLaughlin E.A.1, O’Bryan M.K.2, Aitken R.J.1 and Nixon B.1 1School of Environmental and Life Science, University of Newcastle, Callaghan, NSW, Australia. 2School of Biological Sciences, The Department of Anatomy and Development Biology, Monash University, Melbourne, VIC, Australia.

Cell-cell adhesion is a highly diverse and intricate interaction that relies on the expression, and often, coordinated action of several specialised cell adhesion molecules. One of the most intriguing cell-cell interactions is the binding of spermatozoa to an oocyte. Upon ejaculation, mammalian spermatozoa are unable to recognise and bind to the female egg. They must first undergo a maturation phase termed ‘capacitation’ during which they are transformed into functionally competent cells. Emerging evidence suggests that during capacitation, a multimeric protein complex is assembled or unmasked on the sperm surface to facilitate sperm recognition and adhesion to the egg. Through the novel use of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. In this study, we aimed to characterise one such complex shown to comprise a number of putative recognition molecules, including arylsulfatase a (ASA). Using immunolocalisation techniques we demonstrated that ASA localises to the apical region of the human sperm head, a domain that is known to be responsible for mediating sperm-egg interactions. Furthermore, flow cytometry analysis revealed that ASA translocates to the surface of spermatozoa during capacitation, and that this movement may be blocked by the addition of exogenous cholesterol to the cell. Our current research is focused on determining the role of ASA in sperm-egg adhesion and the cellular mechanisms that underpin it’s translocation to the cell surface.

INVESTIGATION OF THE ROLE OF DYNAMIN IN SPERM SURFACE REMODELLING

Reid A.T., Roman S.D., Aitken R.J. and Nixon B. School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia.

Recent research from our laboratory has provided evidence that the construction of a multimeric sperm receptor complex mediates sperm/egg interaction. In addition, we have convincing evidence that this complex is assembled on the sperm surface during the final phase of maturation, a process known as capacitation. The mechanisms underpinning these capacitation-associated surface remodelling events remain poorly understood and are the subject of our current investigation. Specifically we have focused on whether this process is driven by vesicle mediated, intracellular protein trafficking. For this purpose we have investigated the presence and physiological significance of an integral part of the molecular machinery necessary for this form of trafficking, namely the GTPase dynamin. Our studies revealed that mouse spermatozoa are endowed with at least two isoforms of dynamin (1 and 2) both of which reside within the peri-acrosomal region of the sperm head, a location compatible with a role in sperm membrane remodelling associated with the acquisition of the ability to engage in oocyte interaction. Consistent with this notion, we have demonstrated that pharmacological inhibition of dynamin activity leads to a concomitant reduction in the ability of mouse spermatozoa to bind to the outer vestments of homologous oocytes. Collectively these data support the novel hypothesis that dynamin does participate indirectly in sperm membrane remodelling events by virtue of its ability to mediate intracellular trafficking.

FUNCTIONAL ANALYSIS OF KLF2 DURING EMBRYONIC SKELETAL DEVELOPMENT

Rodda F.A.1, 2, Cameron T.L.1, Gordon C.T.1, Bateman J.F.1, 2 and Farlie P.G.1, 2 1Murdoch Childrens Research Institute, Parkville, Victoria, Australia. 2The University of Melbourne, Parkville, Victoria, Australia.

Endochondral ossification is the process by which the majority of the bones of the body are formed. It occurs via the differentiation of mesenchymal cells into chondrocytes to produce a cartilage template of the skeleton (chondrogenesis), followed by replacement of this template with bone (osteogenesis). This complex process is incompletely understood. Kruppel-like factor 2 (Klf2) is a zinc finger transcription factor upregulated 30-fold during chondrogenesis. With known roles in regulating blood vessel tone, T-cell and smooth muscle cell migration, it as-yet has no known role in skeletal development. Here we provide evidence for the functional significance of Klf2 in limb development. Retroviral-driven misexpression of Klf2 in chick embryos results in reduction of overall bone length, transformations of digit identity, and an alteration of bone morphology coined the ‘web of bone’. We are currently using a viral construct containing a tissue-specific promoter to assess if the web of bone is due to a disruption of signalling from the cartilage itself, or from the surrounding perichondrial cell layer. In addition, in situ hybridisation analysis is being used to examine gene expression alterations in response to Klf2 misexpression, to identify the network in which Klf2 functions and elucidate its mode of action in chondrogenesis and osteogenesis.

EXPRESSION AND FUNCTIONAL ANALYSIS OF SOX3 IN MURINE NEUROGENESIS

Rogers N.A., Banerjee K. and Thomas P.Q. School of Molecular and Biomedical Science, The University of Adelaide.

Sry-related HMG box transcription factor 3 (Sox3) has been associated with human CNS defects, whereby duplication and mutations of SOX3 have been identified in patients with X-linked hypopituitarism (XH). Patients with XH suffer from mild intellectual disability and hypothalamic-pituitary axis dysfunction, the developmental origin and molecular pathology of which is poorly understood. In mice, we have shown SOX3 is highly expressed in the progenitor/stem cells throughout embryonic neurogenesis (12.5-18.5 dpc) and is maintained in the adult neurogenic niches. To investigate the functional role of SOX3, we compared the expression of cell type-specific neurogenesis markers in Sox3 null (EGFP knock-in) mice, which phenocopy XH patients, and wild type mice. No overt changes in dorsal telencephalic neurogenic marker gene expression were detected indicating that the lack of SOX3 has a minimal impact on neurogenesis. Similarly, in adult mice, no obvious defects were observed within the sub-ventricular zone or the sub-granular zone of SOX3 null animals. Interestingly EGFP reporter gene expression in Sox3 null mice was lost at approximately 18.5 dpc, suggesting that SOX3 expression may be autoregulatory in neural stem/progenitor cells. To further investigate the role of SOX3 in vitro, we generated neurospheres from the embryonic telencephalon and the adult neurogenic zones. No obvious difference in the proliferation and differentiation properties of wild type and null neurospheres were detected, although subtle differences in neurosphere growth rates were apparent and are currently under investigation. Ultimately we hope that these studies will provide greater understanding of the role of SOX3 within the developing CNS and the molecular pathology of XH.





CONSISTENT NUCLEOSOME RETENTION DURING CHROMATIN PACKAGING IN HUMAN SPERMATOZOA

Reid A.T.1, 2, McEwan K.2, Campbell D.M.1, 2, Jans D.A.1, 3 and Roman S.D.1, 2 1ARC Centre of Excellence in Biotechnology & Development. 2Biology, School of Environmental & Life Sciences, Faculty of Science & IT, University of Newcastle, Callaghan NSW 2308, Australia. 3Dept of Biochemistry & Molecular Biology, Monash University, Clayton Vic 3800, Australia.

In contrast to the histone packaging of somatic cells, spermatozoa are predominantly packaged by the protamine proteins. However, human spermatozoa retain ~15% histone packaging. Regions that are left nucleosome bound could either be genes that are active shortly after fertilisation or genes that are transcribed late during spermatogenesis. DNA damage at histone bound loci would be of consequence to spermatogenesis and/or to a resulting embyro post-fertilisation. Western blot analysis confirmed the presence of acetylated H3 and H4 in sperm. Interestingly, we identified the presence of these modified histones in isolated good quality sperm considered to have complete packaging. This indicates that histone retention is not a consequence of aberrant chromatin remodelling. Using a combination of ChIP (chromatin immunoprecipitation) and tiling arrays we have obtained evidence of regions bound by acetylated histone 3 (H3). ChIP-PCR confirmed histone retention at several loci identified by tiling array. For the previously identified loci in exon 1 of the TNP-2 gene we were able to narrow the region bound to the size of one nucleosome only. Using a modified form of ChIP known as carrier ChIP we were able to examine histone retention in individual ejaculates. We demonstrated that humans consistently retain acetylated H3 at the same loci. For four out of five males the TNP-2 loci was bound in 100% of the multiple ejaculates examined. In conclusion, retention of acetylated H3 is consistently maintained during packaging of the genome in spermatogenesis.

MIGRATION OF DEFINED SUBPOPULATIONS OF VAGAL ENTERIC NEURAL CREST CELLS

Simkin J.E., Zhang D. and Newgreen D.F. Murdoch Childrens Research Institute, Flemington rd, Parkville, Vic 3052.

Neural crest (NC) cells arise in the embryonic brain and spinal cord. NC from the hindbrain or vagal level (adjacent to somites s1-s7) migrate laterally in streams from each somite level, enter the foregut, and colonize the entire gut in a rostro-caudal wave. They then form the enteric nervous system. Previous studies demonstrated broad regional differences in developmental potential, depending on the NC level of origin. This study utilizes a new focal labeling method to investigate fine-scaled (single-somite length) differences within the vagal level. We ask: are particular vagal subpopulations favoured in initial entry to the gut, and is there some form of matching between subpopulation origin and region of gut ultimately colonized? We electroporated a genome-integrating GFP construct into single-somite lengths of E1.5 quail embryo premigratory NC. Using fluorescence time-lapse and still imaging, we viewed the embryos at a) E2.5-E3, when the NCCs approach/enter the foregut, and b) E6.5, when NC cells have colonized most of the gut. Initially the subpopulations of vagal NC were unmixed, entering a 0.5 mm long region of foregut subjacent their somite level of origin. Later, subpopulations become intermingled, so any sub-population could contribute stochastically to any region of the gut. However, the s3 NC had a numerical advantage, and were more likely to contribute large numbers of NC cells in any gut region, compared to all other sub-populations. These results provide insight into factors advantageous to gut colonization, which may be useful in the design of future stem cell therapies for neurocristopathies, such as Hirschprungs Disease (incomplete enteric nervous system).

THE MAMMALIAN EXPANDED HOMOLOGUE, WILLIN, CAN NOT FUNCTIONALLY SUBSTITUTE FOR EXPANDED IN DROSOPHILA

Sinha A.1, 2, Gunn-Moore F.J.3 and Harvey K.F.1, 2 1Cell Growth and Proliferation Laboratory, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia, 3002. 2Department of Pathology, University of Melbourne, Parkville, Victoria, Australia, 3010. 3Bute Building, School of Biology, University of St Andrews, St Andrews, UK. KY16 9TS.

Studies in Drosophila melanogaster have defined a novel growth inhibitory pathway known as the Salvador-Warts-Hippo (SWH) pathway. This pathway is evolutionarily conserved and most of the component members have mammalian homologues. It has been shown for a few of these members that they can functionally substitute for their Drosophila counterparts. Recently a sequence homologue of an upstream member of the pathway, Expanded (Ex), was identified and is known as Willin or FRMD6. In this study we investigated whether Willin can act in a similar fashion to Ex in Drosophila epithelial tissues called imaginal discs. When Willin was misexpressed using wing-specific drivers it showed the same sub-cellular localization as Ex, but could not rescue growth defects associated with ex deficiency. The later result was recapitulated in a clonal assay in the eye-antennal disc as well, where pathway targets such as ex and diap1 were high and unaffected by overexpressing Willin in these clones. These results suggest that Willin can not functionally substitute for the ability of ex to control SWH pathway-dependent tissue growth in D. melanogaster. These results call into question whether Willin executes the same function in mammals as Ex does in D. melanogaster.

CONSISTENT MECHANISM OF PRIMORDIAL FOLLICLE ACTIVATION IN NEONATAL MOUSE OVOTOXICITY

Sobinoff A.P.1, Pye V.1, Nixon B.1, 2, Roman S.D.1, 2 and McLaughlin E.A.1, 2 1School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia. 2The ARC Centre of Excellence in Biotechnology and Development, The University of Newcastle, Callaghan, NSW, Australia.

The mammalian female reproductive lifespan is largely defined by a finite pool of primordial follicles established around the time of birth. Overall, <1% of these follicles are destined for ovulation, with the vast majority being lost during development in a process called atresia. While atresia is a normal physiological process, it is now well documented that it can be triggered through exposure to certain synthetic chemical compounds, known as xenobiotics, causing premature ovarian senescence. In addition to follicular atresia, new evidence suggests that aberrant follicular activation may play a role in the xenobiotic ovotoxicity. In this study we attempted to identify similarities between the mechanisms of ovotoxicity for three ovotoxic agents, 4-Vinylcyclohexene Diepoxide (VCD), Methoxychlor (MXC), and Menadione (MEN), which target immature follicles. Microarray analysis of neonatal mouse ovaries exposed to these xenobiotics in vitro revealed a more than two-fold significant difference in gene expression (p<0.05) for a number of genes associated with apoptotic cell death and primordial follicle activation. Follow-up qPCR analysis on VCD and MXC cultured ovaries confirmed an increase in expression for Akt1 (5.8, 6 fold), Akt2 (2.9, 1.5 fold), and Ccnd2 (5.3, 6 fold), all three of which are involved in follicular development. Histomorphological and immunohistological analysis supported the microarray data, showing signs of primordial follicle activation and pre-antral follicle atresia in vitro and in vivo. These results indicate a consistent mechanism of primordial follicle activation in pre-antral ovotoxicity for all three xenobiotics.





PRIMARY MAMMARY EPITHELIAL CELLS (PMEC) DERIVED FROM LACTATING HUMAN TISSUE HAVE UNIQUE DIFFERENTIATION PROPERTIES IN THREE-DIMENSIONAL CULTURE

Thomas E.C.1, 2, Zeps N.2, Rigby P.J.1 and Hartmann P.E.1 1School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Perth, Australia. 2School of Surgery, QEII Medical Centre, The University of Western Australia, Perth, Australia.

A diverse body of evidence indicates that the human mammary gland contains a multipotent population that generates the lactating gland during gestation. It is also believed that loss of regulation of this population may lead to breast cancer. We have used cells expressing markers for multipotent phenotype (CD49f, CD29, CD24, p63, CK6 and Nestin) isolated from lactating human tissue via an expressed breastmilk sample to derive a primary cell population (PMEC) that can be propagated through multiple passages of monolayer culture. PMEC generate ductal-alveolus-like structures in extracellular matrix (ECM), and display unique structural and functional features compared to non-tumorigenic mammary epithelial cell lines MCF10A and HBL-100. Using structurally interrupted and growth factor reduced ECM we show that complete differentiation and organized assembly of PMEC into a basal (CD49f-positive) and luminal (CK18-positive) ductal-alveolar structures is dependant on intact and complete ECM. Complete differentiation is required for luminal cell expression of milk proteins alpha-lactalbumin and beta-casein in response to Prolactin stimulation. We also show that treatment with developmental and lactational regulators influence different stages of PMEC proliferation, differentiation and function. Using quantitative mass spectrometery (iTraq) we have profiled the proteome of PMEC-derived ductal-alveolar structures at daily intervals for seven days to identify changes associated with proliferation and differentiation. We suggest this will provide a model for normal differentiation of the mammary epithelium and provides a platform to identify pathways that may lead to aberrant growth when regulation is lost.

TWIST GENES REGULATE OUTGROWTH AND PATTERNING DURING LIMB MORPHOGENESIS

Wade C., Brinas I. and Farlie P. Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia 3052.

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of sites. We have used RCAS-mediated overexpression to investigate the function of Twist2 in limb development. Expression of Twist2 within the developing limbs begins prior to formation of the limb bud and persists within the peripheral mesenchyme until digital rays condense when Twist2 expression becomes restricted to the interdigital mesenchyme. Viral misexpression following injection into the lateral plate mesoderm results in a spectrum of hypoplastic limb phenotypes. These include generalised shortening of the entire limb, fusion of the autopod skeletal elements, loss of individual digits or distal truncation resulting in complete loss of the autopod. These phenotypes appear to result from a premature termination of limb outgrowth. In situ hybridisation analysis demonstrates that many components of the Shh/Fgf/Gremlin regulatory loop that controls early limb outgrowth are downregulated by Twist2 overexpression. This suggests that Twist2 controls limb outgrowth through the regulation of this central growth regulatory network. Given the downregulation of Gremlin and its complementary expression pattern to Twist2, Gremlin is a good candidate for a transcriptional target of Twist2, and preliminary ChIP data supports this hypothesis. However, despite loss of AER Fgf8 and other regulatory loop factors, Shh expression is sustained at normal levels. This raises the possibility of Shh being a transcriptional target of Twist2. Similar data were obtained for Twist1. These data illustrate the molecular mechanisms through which the Twist genes control morphogenesis of the limb.

THE ROLES OF FOXL2 & ZDHHC17 IN SKELETAL DEVELOPMENT

Welfare M.F. and Farlie P.G. Murdoch Children’s Reseach Institute, Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville.

Very little is currently known about which genes control skeletal development. In order to increase our knowledge of the genes involved, we are performing a large scale screen of genes that are active during skeletal development. Two of the identified genes will be examined further to determine what role they play in skeletal development. The first gene, Foxl2, will be overexpressed in the limbs. Initial studies suggest that this may cause significant shortening of the limb bones and identifying the mechanisms underpinning this phenotype (eg apoptosis or a growth deficiency) will form a major part of the project. The Foxl2 gene will be cloned into a viral vector (RCAS) and then injected into chicken embryos where the viral construct will cause the inserted gene to be expressed. The second gene, Zdhhc17, will be knocked down in the craniofacial region. This will show the effect of removing the function of Zdhhc17 during the development of the facial and skull bones. No previous studies have examined the role of Zdhhc17 in skeletal development. In this case, a knockdown construct will be cloned into RCAS. This construct encodes a RNA which is complementary to the hosts Zdhhc17 mRNA, therefore the introduced RNA will bind to the host mRNA and trigger its degradation. This prevents the Zdhhc17 protein from being made, effectively removing the functionality of the Zdhhc17 gene. The understanding gained from this analysis will enable us to further elucidate the complex mechanisms underlying skeletal development. This in turn will then lead to an increased understanding of many birth defects and syndromes with underlying skeletal defects.

INSIGHTS INTO C-TERMINAL BINDING PROTEIN (CTBP) VIA A MUTAGENESIS SCREEN IN C. ELEGANS

Yucel D.1, Crossley M.1, 2 and Nicholas H.1 1University of Sydney. 2University of New South Wales.

CtBP is a transcriptional co-repressor which plays roles in development and apoptosis. There are two highly related CtBP genes in vertebrates. Knock-out of both CtBP1 and CtBP2 results in lethality early in embryogenesis. Recently, we cloned a single CtBP in C. elegans. Like its mammalian counterparts, the C. elegans CtBP, called CTBP-1, functions as a transcriptional co-repressor and docks onto transcription factors containing an amino acid motif of the form PXDLS. We have analysed the expression pattern of CTBP-1 using a translational reporter construct (CTBP-1::GFP) which has shown that CTBP-1 is expressed in numerous cells, some of which have been identified as neurons.We are using various reporter markers to assess whether CTBP-1 is involved in the development or specification of these neurons.One such marker showed that ctbp-1 loss of function mutant worms have fewer of one particular motor neuron cell type. In order to identify novel mutations that interfere with correct CTBP-1 activity, we carried out a mutagenesis screen.We treated the CTBP-1::GFP transgenic line with EMS and isolated worms which showed changes in the expression of the fusion protein.We have isolated six mutant lines, two of which show particularly interesting phenotypes. In one of the mutants named aus5, the expression of the CTBP-1::GFP fusion protein is dramatically decreased. In the other mutant line, aus3, the fusion protein is ectopically expressed.The aus5 mutant line may have a mutation in an activator of CTBP-1 such that CTBP-1 is switched off. In the aus3 mutant line, the mutation could be in a repressor of CTBP-1 allowing CTBP-1 to be switched on ectopically.Whole genome sequencing is in progress to identify the genes affected by these mutations which alter CTBP-1 activity. Taken together, our results hold the potential to unravel the roles of CTBP-1 in neuronal regulation and to reveal novel components of the gene regulatory networks in which CTBP-1 is involved.





NEURAL CREST CELL INVASION OF DEVELOPING GUT IS PREVENTED BY EXISTING NEURAL CREST CELLS BUT NOT BY ENTERIC NEURONS

Zhang D. and Newgreen D.F. Murdoch Childrens Research Institute, Parkville, Victoria 3052.

The development of the enteric never system (ENS) depends on vagal neural crest cell (NCC) invasion. We showed previously using chick-quail gut kebab grafts that NCC invasion ability depends on cell proliferation especially in the invasion wavefront. Using various spatiotemporal combination gut grafts, we showed that the invasion ability of NCCs is high when placed at the wavefront and restricted at behind-wavefront positions. The latter position differs from the former in that 1. the gut tissue is older, 2. NC-derived neurons are present, and 3. NCC are present. To test condition 1, we grafted quail NCC donor gut into older gut from vagal neural tube-ablated chick embryos. The NCCs invaded older NCC-free and neuron-free gut avidly, therefore gut age is not primarily responsible for invasion restriction. To test condition 2, we briefly treated older chick gut (with NCCs and neurons) with the proliferation inhibitor mitomycin C. After 2 days the NCCs had disappeared but abundant neurons with neurites remained. We then grafted quail NCCs donor into the NCC-free but neuron-containing gut. The NCCs invaded this, suggesting that enteric neurons do not restrict NCC invasion. To test condition 3, we grafted quail NCC into chick host gut such that chick NCC migrate caudally and quail NCC rostrally, at a stage when neurons are almost absent. These two NCC populations mutually impeded each other, showing that presence of NCCs in the gut contributes to the restriction of invasion by new NCCs. In conclusion, the restriction of invasion ability of NCCs into developing gut depends on pre-existing NCCs but not on neurons, and this restriction is reversible by removal of NCCs.

THE REGULATORY NETWORK OF TTP, MKP-1 AND MAPK IN RESPONSE TO PREADIPOCYTE DIFFERENTIATION SIGNALS

Lin N.Y.1, Lin T.Y.2, Wang S.C.1 and Chang C.J.1, 2 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 2Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan.

The activation of mitogen activated protein kinases (MAPK) signaling pathways is necessary to initiate the differentiation process of 3T3-L1 preadipocytes, and the activation needs to be turned-off to proceed to terminal maturation. The expression of MAPK phosphatas-1 (MKP-1) can control the timing window of MAPK activity. This study demonstrated that the transient expression of MKP-1 was transcriptionally induced by ERK signaling; at the same time, ERK signaling also mediated the activation of RNA binding protein tristetraprolin (TTP) to decrease the expression level of MKP-1 posttranscriptionally. In this study, U0126 was used to block the activation of ERK signaling and it resulted in the down-regulation of TTP expression and enhanced stability of MKP-1 mRNAs at 1h after induction to differentiation. siRNAs against TTP prolonged the half-life of MKP-1 mRNA. RNA-protein interaction and functional reporter assays showed that TTP differentially bound to three AU-rich elements (AREs) of MKP-1 mRNA and negatively regulated the MKP-1 ARE-containing luciferase reporter activity. Interestingly, we found that MKP-1 can associate with TTP and possibly down-regulate TTP phosphorylation and modulate the ARE-binding activity or protein stability of TTP. Moreover, the knockdown of MKP-1 and TTP showed a slight but different effect on adipogenesis. The results suggested that TTP can regulate the MKP-1 mRNA stability to subsequently control the activation of MAPK signaling pathways in adipocyte differentiation.

PHOSPHORYLATION-DEPENDENT REGULATION OF INTRACELLULAR LOCALIZATION AND POTENTIAL TUMOR SUPPRESSOR FUNCTION OF LKB1 BY AKT AND 14-3-3

Liu L.1, Wang Y.2, Lam K.S.L.1 and Xu A.1, 2 1Department of Medicine, The university of Hong Kong. 2Department of Pharmacology, The university of Hong Kong.

Introduction: LKB1, a tumor suppressor protein kinase, is causally linked to Peutz-Jeghers syndrome, with a predisposition to cancer. Several studies indicated that the intracellular localization of LKB1 is very important for its function. Scansite predicted that serine334 of LKB1 locates in both the recognition motif of 14-3-3 and the phosphorylation site of Akt. The objectives of this study are to investigate whether LKB1 is a downstream target of Akt and a novel binding partner of 14-3-3, and to elucidate how the intracellular localization and functions of LKB1 can be regulated. Methods: Seven GST-tagged 14-3-3 isoforms were used for pull-down experiment to check whether 14-3-3s interact with LKB1. In vitro phosphorylation assay, mass spectrometry (MS) analysis and site-directed mutagenesis were employed for determination of whether serine334 is both the phosphorylation site of Akt and 14-3-3 binding site. Results: Five of the seven 14-3-3 isoforms interact with LKB1. The inhibition of Akt activity can cause both the decreased interaction between 14-3-3 zeta and LKB1 and the increased cytosolic/nuclear ratio of LKB1. In vitro phosphorylation assay result confirmed that LKB1 is a direct target of Akt and the subsequent MS analysis determined the precise site is serine334. Site-directed mutagenesis revealed that mutation of LKB1 on serine334 to alanine can lead to both the decreased interaction between LKB1 and 14-3-3zeta and increased cytosolic localization. Conclusions: LKB1 is a downstream target of Akt kinase and a novel interaction partner of 14-3-3. Serine334 is the phosphorylation site of Akt and interaction site of 14-3-3. Akt regulates the phosphorylation-dependent interaction between LKB1 and 14-3-3 and subsequently regulates the intracellular localization of LKB, which might be very significant for its tumor suppressor functions. These findings may provide a novel mechanism whereby the tumor suppressor functions of LKB1 can be regulated.

INFLAMMATION IN SEPSIS: ROLE OF ENDOTHELIAL CELLS

Low W.X.1, Muniandy S.1, Qzist R.2 and GracieOng S.Y.3 1Department of Molecfular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 3Department of Anesthesiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Severe sepsis and septic shock are the primary causes of the multiple organ dysfunctions and one of the common causes of death in intensive care units worldwide. The infecting microorganisms trigger widespread activation of multiple pathophysiological processes resulting in the clinical syndrome of multiple organ dysfunctions. Immune cells and endothelial cells play crucial roles in severe sepsis. We investigated the expression of target genes associated with triggering of endothelial cells by inflammatory molecules present in plasma of subjects with severe sepsis. Cultured human umbilical vein endothelial cells were triggered with plasma from patients with gram negative bacteraemia. Inoculation was performed at 5 different time intervals (0 hour, 4 hours, 7 hours, 12 hours and 24 hours). Results revealed that the expression of NFκB was significantly up regulated at the 24th hours after treatment of the endothelial cells. The expression of 84 key genes were screened using real time reverse transcription profiler PCR array and significantly expressed pro- and anti-inflammatory genes were identified. Prominent pro-inflammatory cytokines namely IL8, IL6 and IFNγ were also determined. These observations demonstrated that endothelial cells serve as an equally important inflammatory role as other immune cells in triggering sepsis and NFκB, inflammatory genes and inflammatory cytokines were expressed in parallel as early as 24 hours after treatment. Further studies into the inflammatory roles of the endothelial cells may help in assessing the efficacy of therapeutic interventions in near future.





PITUITARY AND OVARIAN FUNCTION IN PRIP KNOCKOUT MICE

Matsuda M. and Hirata M. Laboratory of Molecular and Cellular Biochemistry, Graduate School of Dental Science, Kyushu University.

Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and -2 double knockout (DKO) mice to gain insight into the biological function. DKO mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments indicated that the cause appeared to be on the female side. The observation of the estrous cycle in mice by histological analysis of vaginal smears showed that the estrous days were apparently increased in DKO mice. Levels of serum LH and FSH were measured for 5-6 consecutive days, and were significantly higher in the mutant, which was also confirmed by examining the secretion of LH from the explant culture of anterior pituitary glands of wild-type and DKO mice. We also examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were significantly decreased in mutant female mice, compared to that seen with wild-type, which was also confirmed by histological analysis of the ovaries after ovulation. The analysis also showed that the numbers of follicles originally were not significantly different between WT and DKO ovaries. These results suggest that PRIP plays an important role in female reproductive system, especially in gonadotropin secretion and ovarian follicle maturation.

STRESS-INDUCED SYNTHESIS OF PHOSPHATIDYLINOSITOL 3-PHOSPHATE IN MYCOBACTERIA

Morita Y.S.4, Yamaryo-Botte Y.2, 3, 1, Miyanagi K.4, Crellin P.K.2, 3, Coppel R.L.2, 3, Kinoshita T.4 and McConville M.J.1 1Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. 2Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia. 3Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia. 4Research Institute for Microbial Diseases, and WPI Immunology Frontier Research Center, Osaka University, Osaka, 565-0871, Japan.

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. In this study, we show that Mycobacterium smegmatis can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P), making this organism the first example of a prokaryote that can synthesize PI3P de novo. This lipid was transiently labeled with [3H]inositol, and its synthesis was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Sensitivity of the purified lipid to alkaline phosphatase, head group analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure; 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. The transient synthesis in response to environmental stimuli suggests that mycobacterial PI3P may be a second messenger, and we are currently attempting to identify effector molecules that may be involved in the signaling cascade.

ANALYSIS OF THE REGULATION AND FUNCTION OF KIWIFRUIT FLOWERING LOCUS T

Moss S.M.A.1, 2, Putterill J.1 and Varkonyi-Gasic E.2 1School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand. 2Plant and Food Research, Mt Albert, Private Bag 92169, Auckland Mail Centre, Auckland 1142, New Zealand.

Kiwifruit (Actinidia sp) requires synchronised, concentrated flowering to obtain good fruit quality and yield. The molecular mechanisms controlling flower initiation and development in kiwifruit are largely unknown. The genes and pathways controlling flowering have been elucidated in model plants, such as Arabidopsis thaliana. In Arabidopsis, the photoperiodic, autonomous and vernalization pathways all converge on floral integrator genes that promote the transition from vegetative to floral development. One of the key floral integrator genes is FLOWERING LOCUS T (FT). FT-like genes have been found to be major flowering regulators in other model species, including rice, tomato and poplar. In kiwifruit, a candidate FT gene (AcFT) has been identified as highly homologous to Arabidopsis FT. Overexpression of AcFT complements the Arabidopsis ft mutant illustrating gene functionality across species. The aim of this research is to analyse the AcFT promoter sequence using three techniques. The characterisation of the AcFT promoter will provide information as to spatial and temporal AcFT expression at a tissue-specific and cell-type specific level. Firstly, bioinformatics will be used to identify the potential regulatory motifs of the AcFT promoter. Secondly, the reporter genes GUS and GFP will be used in the model plants Arabidopsis and Nicotiana to locate the sites of AcFT promoter activation. Thirdly, a transcriptional network will be developed using dual transient luciferase assays. The characterisation of the AcFT promoter should help to provide insight into the initiation and maintenance of flowering in kiwifruit, and potentially other perennial species.

A TAIL OF STAT3 SPLICEFORM DIFFERENTIAL REGULATION

Ng I.H.W.1, 2, Ng D.C.H.1, Jans D.A.2 and Bogoyevitch M.A.1 1Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, VIC, Australia. 2Department of Biochemistry and Molecular Biology, Monash University, VIC, Australia.

STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor activated by a range of extracellular signals and regulates important biological processes. Two STAT3 spliceforms, STAT3α and STAT3β, have been identified with spliceform-specific functions reported. Interestingly, the only structural difference noted in the spliceforms is a unique 7 amino acid C-terminal tail in STAT3β that replaces the 55 amino acid tail of STAT3α; this tail is a unique feature of STAT3β when compared with other truncated STAT isoforms. We have now demonstrated that increased expression of STAT3β leads to a prolonged STAT3α Y705 phosphorylation in both Cos1 and HEK293 cells. This correlated with the prolonged nuclear retention of STAT3α and suggested that STAT3β levels could influence STAT3α phosphorylation. Since the heterodimerisation of STAT3α and STAT3β could complicate the analysis of spliceform-specific regulations and functions, we have developed a STAT3-inducible expression system in a STAT3-/- background. We have further studied the differential regulation of the individual STAT3 spliceforms in this system and revealed the differential phosphorylation kinetics of STAT3α and STAT3β. Thus, in the absence of STAT3β, STAT3α Y705 phosphorylation levels appeared lower and more transient whereas STAT3β Y705 phosphorylation levels were strong and sustained. Furthermore, these changes in phosphorylation correlated with the differential nuclear translocation for the individual STAT3 spliceforms. Ultimately, this STAT3-inducible expression system will allow the differential regulation of STAT3α and STAT3β to be studied in greater detail with future studies such as gene regulation and protein partner interaction studies allowing greater understanding of the regulation and actions of these STAT3 spliceforms.





IDENTIFICATION OF A NOVEL C-JUN N-TERMINAL KINASE INHIBITORY PEPTIDE

Ngoei K.R.1, Catimel B.2, Lio S.1, Cheng H.C.1, Ng D.C.H.1 and Bogoyevitch M.A.1 1Dept. of Biochemistry and Molceular Biology, Bio21 Institute, University of Melbourne. 2Ludwig Institute for Cancer Research, University of Melbourne.

An improved understanding of the roles of protein kinases in cellular signaling and disease progression has driven significant developments in the area of protein kinase inhibitor discovery. Whilst the issues of selectivity and specificity have become increasingly recognized as major hurdles to overcome undesirable off-target effects of inhibitors targeting the kinase ATP-binding site, peptide inhibitors that target the kinase protein substrate-binding site may offer greater specificity. Here, we report a novel c-Jun N-terminal Kinase (JNK) inhibitory peptide, PYC71N, that inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2, Elk1 and the microtubule regulatory protein DCX. Moreover, alanine scanning replacement studies revealed the importance of two residues within PYC71N (F9 and F11) for JNK inhibition. Intriguingly, the kinetics data suggested that inhibition was mediated by a substrate-inhibitor complex. Using biosensor analysis, PYC71N was shown to interact with both non-phosphorylated JNK1 (inactive) and the substrate, cJun but did not recognize active JNK1. In contrast, a previously characterized JNK inhibitory peptide, TIJIP, showed stronger interaction with active JNK1. Our results define novel properties of the PYC71N peptide, highlight its value as a research tool to study JNK signaling, and provide an alternative for targeting JNK for possible therapeutic benefit.

THE OXYGEN-SENSING ASPARAGINYL HYDROXYLASE FACTOR INHIBITING HIF-1A (FIH) IS AN ESSENTIAL REGULATOR OF METABOLISM

Peet D.J.1, Linke S.1, Zhang N.2, Chicher J.3, Gorman J.J.3, Poelinger L.4 and Johnson R.S.2 1School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia. 2Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. 3Protein Discovery Centre, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, QLD 4029, Australia. 4Karolinska Institute, Stockholm S-17177, Sweden.

Oxygen sensing is essential in all metazoans to enable the control of oxygen homeostasis. The major genomic response to hypoxia is mediated by the hypoxic inducible transcription factors (HIFs), and is regulated by the oxygen-dependent HIF prolyl and asparaginyl hydroxylases. These hydroxylases are members of the 2-oxoglutarate dependent dioxygenase family and they directly regulate activity of the HIFs by oxygen-dependent protein hydroxylation, thus acting as cellular oxygen sensors. The ubiquitously expressed HIF asparaginyl hydroxylase, known as Factor Inhibiting HIF (FIH), is able to regulate the transcriptional activity of the HIF-α proteins through oxygen-dependent hydroxylation of the C-terminal transactivation domain. This modification directly influences the recruitment of the CBP/p300 coactivators, thus modulating the activy of this transactivation domain. Subsequently FIH has also been shown to hydroxylate numerous proteins containing ankyrin repeat domains, including IkBα and Notch. These oxygen-dependent modifications are confined to specific, conserved asparaginyl residues, although their function in these ankyrin repeat proteins remains unclear. The recent generation and analysis of FIH-deficient mice has shed light on the importance of FIH, specifically its role in regulating key aspects of metabolism. FIH deficient mice have elevated metabolism, including respiration, cardiovascular output, and energy metabolism; they consume more food, exercise less and yet are leaner than wild type mice with improved glucose and lipid homeostasis. The mutant mice are also resistant to weight gain on a high fat diet and hepatic steatosis, and remain insulin hypersensitive, underlying the key role and therapeutic potential of FIH in regulating metabolism.

CYTOKINE RECEPTORS TURNED ON: A GENERAL MECHANISM OF ACTIVATION

Poger D.1 and Mark A.E.1, 2 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4067 Australia. 2Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4067 Australia.

Despite the structures of various cytokine receptors being known, the mechanism of activation, in particular how the binding of ligand mechanically transmits a signal through the plasma membrane, is uncertain. Atomistic molecular dynamics simulations have been used to investigate the conformational changes within the extracellular domain (ECD) of the growth hormone receptor (GHR), the prolactin receptor (PRLR) and the epidermal growth factor receptor (EGFR/Her1/ErbB1) triggered by the binding of ligand (growth hormone (GH), prolactin (PRL) and epidermal growth factor (EGF) and transforming growth factor α (TGFα), respectively), thereby shedding light on the molecular details on the mechanism of activation in these cases. For all three receptors, it is shown that the removal of the cytokine from the ligand-bound homodimeric receptor complexes results in a rotation of the two subunits relative to each other. A relative rigid-body rotation of 45° and 25° was found in the GHR and PRLR dimers, respectively. A relative twist by about 30° also occurred within the EGFR dimer between the two domains I, associated with the closure of the ligand-binding pocket between domains I and III in each ECD. In each case, control simulations showed that the GHR, PRL and EGFR apo dimers and the relative orientation of their monomers after the rotation were stable. The magnitude and direction of the motion observed in the simulations of GHR are in close agreement with experiment. The similarity of the motions in all three cases and in multiple simulations suggests that the relative rotation of the ECDs could represent a general mechanism of activation of cytokine receptors.

RNAi SCREENING FOR NOVEL KINASES AND PHOSPHATASES THAT REGULATE THE SALVADOR-WARTS-HIPPO PATHWAY IN GROWTH CONTROL

Poon C., Cuddy C. and Harvey K. Cell Growth & Proliferation, Peter MacCallum Cancer Centre.

The Salvador-Warts-Hippo (SWH) pathway controls tissue growth and organ size by limiting the function of the transcriptional coactivator Yorkie (Yki). Phosphorylation is critical to regulation of SWH activity: for example, both the assembly and activity of the core complex, comprising protein kinases Hippo and Warts (Wts) and their respective binding partners Salvador and Mats, is promoted by Hpo phosphorylation. Critically, Wts-mediated phosphorylation restricts Yki to the cytosol, thus limiting the transcriptional output of SWH signalling. Recently, phosphoproteome analysis of Drosophila embryos1 indicates previously uncharacterised phosphorylation sites on Hpo pathway proteins, suggesting that additional phosphorylation events may influence SWH signalling. Furthermore, we have a limited understanding of which kinases and/or phosphatases target the novel and, indeed, established phosphorylation events described above. To identify kinases and phosphatases that regulate the SWH pathway, we have carried out both gain- and loss- of SWH function RNAi screens in Drosophila. In our pilot screens, we have identified candidates that are known growth regulators, such as Akt, as well as candidates that are yet to be described in the context of growth control. We will validate and characterise these candidates to gain insight into the role of phosphorylation in fine tuning SWH signalling. 1 Zhai et al, Journal of Proteome Research, 2008.





NUCLING, A NOVEL APOPTOSIS-ASSOCIATING PROTEIN, REGULATES NF-κB PATHWAY

Sakai T., Liu L., Tran N.H., Kim S.M. and Fukui K. The Institute for Enzyme Research, The University of Tokushima.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an upregulated expression during cardiac muscle differentiation. Nucling is induced by proapoptotic stimuli and important for the following induction of apoptosis. We further demonstrated that Nucling regulated apoptosome complex composed of apoptotic protease activating factor 1 (Apaf-1), caspase-9 and cytochrome c following cytotoxic stimuli. We generated Nucling-KO mice to reveal physiological functions of Nucling. The KO mice were resistant to the damaging effects of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Upregulation of apoptosome was attenuated in Nucling-KO mice treated with MPTP, indicating that mitochondrial apoptosis pathway plays an important role in the neurotoxicity of MPTP. We also reported an alternative pathway for Nucling to regulate apoptosis, in which Nucling acts as an inhibitor of the anti-apoptotic molecule, galectin-3. On the other hand, Nucling is characterized as a regulator of nuclear factor-kappa B (NF-κB) operated by its cytoplasmic retention through the physical interaction with Nucling. We recently revealed that Nucling is important for the regulation of NF-κB signals in liver. Defect of NF-κB signal led several liver dysfunctions including hepatitis and cancer in Nucling-KO mice. Our latest data concerning the regulatory machineries of Nucling in NF-κB signal pathway will be presented.

THE A KINASE ANCHORING PROTEIN AKAP200 CAN SUPPRESS THE ONCOGENIC PHENOTYPES OF DV-CBL AND RASV12 IN THE DROSOPHILA EYE

Sannang R., Robertson H. and Hime G. Department of Anantomy and Cell Biology, The University of Melbourne, Parkville VIC 3010, Australia.

The fruit fly Drosophila melanogaster has been an important model organism for many years. Drosophila and humans share many genes, and like us, they are capable of developing cancer. Two oncogenes that act similarly in Drosophila and humans are Dv-cbl and rasV12, and we have previously performed an enhancer/suppressor screen in the Drosophila eye against them using the Gene Search collection (1). In this way, a number of genes capable of suppressing Dv-cbl or rasV12 were identified. One line was able to suppress both Dv-cbl and rasV12. This line mapped to Akap200, an A kinase anchor protein that was previously described as having a role in formation of ring canals in the Drosophila ovary (2). As little is known about Akap200 function in the eye, we went on to characterise Akap200 and its interactions with Dv-cbl and rasV12 in eye development. Here we show detailed analysis of Akap200 in the eye. In adults the rough eye (differentiation defects) phenotypes, caused by over expression of Dv-cbl or rasV12, are suppressed by Akap200 expression. We then looked in larval tissue at the expression of proteins known to be affected by over expression of Dv-cbl. Defects are suppressed and appear closer to the wild type configuration when Akap200 is over expressed. This helps us to determine which biochemical pathways or processes Akap200 is affecting in order to suppress the Dv-cbl over expression phenotype. We also found that Akap200 was able to suppress the cooperation of Dv-cbl and rasV12, and plan to test its ability to suppress other oncogene combinations. (1) Toba, G., et al., 1999. (2) Jackson, S.M. and C.A. Berg, 2002.

CELL-CYCLE SENSING OF OXIDATIVE STRESS IN SACCHAROMYCES CEREVISIAE BY OXIDATION OF A SPECIFIC CYSTEINE RESIDUE IN THE TRANSCRIPTION FACTOR SWI6P

Chiu J.1, Tactacan C.M.1, Lin R.C.Y.2, Wouters M.A.3 and Dawes I.W.1, 2 1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052. 2The Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, NSW 2052. 3Structural and Computational Biological Program, Victor Chang Cardiac Research Institute, Sydney, NSW 2010.

Yeast cells begin to bud and enter S phase when growth conditions are favourable during G1 phase. When subjected to oxidative stress, cells arrest at G1 delaying entry into the cell cycle allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell-cycle regulator of Saccharomyces cerevisiae , Swi6p, as a redox sensor through its intrinsic cysteine residue at position 404. Mutation of Cys404 to Ala abolished the ability of the cells to arrest at G1 upon treatment by lipid hydroperoxide. At the protein level, Cys404 residue of Swi6p became oxidised when cells were subjected to the oxidant. Furthermore, microarray analysis revealed that mutation of Cys404 to Ala alleviated the wild-type suppression of the G1 cyclins Cln1p and Pcl1p hence promoting morphogenesis and bud emergence in S-phase when cells were exposed to lipid hydroperoxide. However, genes involved in DNA replication (ALG14, DUN1, CDC45, POL1) were down-regulated in a similar manner as observed in wild-type cells indicating that the mutation did not affect the regulation of DNA replication. In conclusion, oxidative stress signalling for cell-cycle regulation in yeast occurs through the oxidation of the G1/S-specific transcription factor Swi6p and consequently leads to the suppression of G1-cyclins inhibiting entry into the cell cycle.

CLUSTERIN INDUCES MATRIX METALLOPROTEINASE-9 EXPRESSION VIA ERK, JNK, AND NUCLEAR FACTOR-κB IN RAW 264.7 CELLSShim Y.J.1, Jeon H.S.1, Jiang L.H.1, Park I.S.2 and Min B.H.1 1Department of Pharmacology and BK21 program for Medical Sciences, College of Medicine, Korea University. 2Department of Anatomy and BK21 Center for Advanced Medical Education, College of Medicine, Inha University.

Clusterin (CLU) is a multifunctional protein that has been implicated in tissue remodeling, metastasis, and inflammation. Since these processes may be linked to the degradation of extracellular matrix (ECM) and cell migration, we investigated whether CLU regulates expression of matrix metalloproteinase-9 (MMP-9). Normally, MMP-9 expression is tightly controlled and low or absent in most tissues. Here, we show that the production of MMP-9 protein and mRNA is induced by CLU in murine macrophage-like Raw 264.7 cells. CLU enhanced MMP-9 expression in a time- and dose- dependent manner, revealed by RT-PCR, Western blot and Gelatin zymogram. To explore the intracellular signaling routes involved in MMP-9 gene transcription, cells were treated with different inhibitors of major mitogen-activated protein kinase (MAPK) pathways. CLU-stimulated MMP-9 induction was significantly attenuated through inhibition of extracellular signal-regulated kinase (ERK1/2) and c-jun-N-terminal kinase (JNK) by PD98059 and SP600125, respectively, but not by SB203580 as a p38 kinase inhibitor. Indeed, CLU stimulated a time-dependent phosphorylation of both ERK1/2 and JNK with a maximal response within 60 min. Moreover, the CLU-induced MMP-9 gene expression was also mediated through the translocation of nuclear factor-κB (NF-κB) p65 into the nucleus and the degradation of IkB-alpha. The regulation of MMP-9 induction by ERK1/2, JNK, and NF-κB was further confirmed by luciferase reporter gene assay. MMP-9 promoter activity was increased by CLU in cells transfected with wild-type mouse MMP-9-Luc, which was inhibited by PD98059, SP600125. Taken together, these results suggest that phosphorylation of ERK1/2, JNK, and NF-κB transactivation is essential for CLU-induced MMP-9 production in Raw 264.7 cells. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009-009-1418)].




Cancelled


GENOME WIDE FUNCTIONAL GENOMICS

Thomas D., Gould K. and Simpson K.J. Peter MacCallum Cancer Centre.

The Functional Genomics Group at the Peter MacCallum Cancer Centre is open to the Australian medical research community for the purpose of providing access to both small interfering (siRNA) and lentiviral-based short hairpin microRNAi (shRNAmir) gene knockdown screens for functional genomics studies. The Victorian Centre for Functional Genomics (VCFG) has purchased a library of ~100,000 lentiviral based shRNAmir constructs, representing up to 5 constructs per gene from Open BioSystems. Researchers can access the whole human genome, or boutique library collections that focus on apoptosis, cell cycle, growth and proliferation, polarity and cell invasion and the kinome. We have produced lentiviral pools of 4,608 constructs per pool that permit rapid and successful screening at the whole genome level. The Australian Cancer Research Foundation (ACRF) siRNA high throughput screening facility offers a parallel screening platform using Dharmacon siRNA technology to systematically knockdown individual genes for the entire human (~18,500) or mouse (~17,000) genome. Such whole genome screens are performed in high throughput (96 or 384 well format) using our fully integrated robotics platform which includes an automated liquid handling robot, an automated cell dispenser/plate washer, a high throughput plate reader and a high throughput, high content image analyser (Cellomics VTi) for cell based microscopy assays. We are a member of the RNAi Global Initiative, a consortium of International Institutes that have invested in the whole genome screening technology. Efficient and cost-effective gene knockdown screens are offered along with Standard Operating Protocols and assay development advice.

VASCULAR TONE REGULATION BY EXTRACELLULAR UTP THROUGH P2X RECEPTOR

Sugihara M.1, Morita H.2, Matsuda M.1, Kajioka S.3, Ito Y.4, Abe K.2 and Hirata M.1 1Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science,. 2Special Patient Oral Care Unit of Kyushu University Hospital,. 3Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka,. 4Department of Health Science, Kumamoto Health Science University, Kumamoto Japan.

P2X receptors are non-selective cation channels gated by extracellular ATP, and play important roles in various physiological processes. UTP is released from sympathetic nerve terminals and various cells surrounding smooth muscle cells in vasculature. It has been generally considered that UTP regulates vascular tone through selective activation of G-protein coupled P2Y receptors. However, we here confirmed that UTP mediates contraction of vascular smooth muscle through P2X receptor activation. We performed a tension recording with a rat aortic ring preparation denuded of endothelium. UTP elicited a biphasic contraction consisting of phasic and tonic components. The phasic contraction disappeared when removal of extracellular Ca2+ or by addition of nifedipine or TNP-ATP, whereas the tonic contraction remained. Next, we examined native P2X receptor function using patch-clamp method. Application of UTP (≥10μM) induced transient inward current in arterial smooth muscle cells. The current showed inward-rectification, which was similar to those evoked by activation of P2X channel. The UTP-induced current was inhibited by pretreatment with TNP-ATP, suramin and PPADS. UTP and α,β-methylene ATP (10μM), a potent P2X receptor agonist were mutually competitive. Using RT-PCR and Western blot analysis, we have detected high expression of P2X1 subtype in cerebral and mesenteric arteries and aorta. P2Y2, 4, 6 receptors were also detected by RT-PCR. Taken together, our results suggest that UTP regulate the arterial tone through dual pathways, including P2X1-like and P2Y receptors activation.

A NOVEL NON-RAD HOMOGENEOUS ASSAY TO MONITOR POST-TRANSLATIONAL HISTONE MODIFICATIONS

Sutija M.1, Kreps A.2, Lipari F.2, Arias M.2, Lévesque-Sergerie J.P.2, Rodriguez-Suarez R.2, Cosentino G.2 and Rouleau N.2 1PerkinElmer Inc., Melbourne, Australia. 2PerkinElmer Inc., Montreal, Quebec, Canada, H3J 1R4.

Post-translational histone modifications play a part in a wide array of cellular processes including regulation of gene transcription, DNA repair, and mitosis. The enzyme classes that mediate these histone modifications include histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltrasferases (HMTs), and histone demethylases. Here we report development of Alpha technology-based assays (luminescent oxygen channelling immunoassay) to monitor histone modifications that are mediated by the activity of HATs and HMTs. To measure acetylation and methylation, full length histone H3 modification at lysine 9 was used as a model. The assays involved two steps: 1) Acetylation or methylation of the histone H3K9 with P/CAF or G9a, and 2) detection using the Alpha immunoassay. Using the optimized buffer conditions, we were able to measure histone acetylation at K9 using as little as 10 nM of enzyme and 300 nM of substrate. The acetylation detection assay was also successfully used to look at the general state of acetylated H3K9 from nuclear extract of HeLa cells treated with a known HDAC inhibitor. G9a activity was monitored by measuring the production of H3K9me2. A signal-to-background ratio over 10 was obtained using as little as 10 nM enzyme. Moreover, the evaluation of different known HMT inhibitors confirmed the specificity of the reaction, with rank order of potency in line with those found in the literature. Since the use of a full length H3 protein substrate may represent certain advantages over the use an H3-derived peptide, these novel Alpha assays will represent a powerful tool in vitro screens of novel modulators of this emerging target class.





GENOME-WIDE CHIP-SEQ ANALYSIS OF HISTONE 3 LYSINE 27 TRIMETHYLATION DURING SKELETAL MUSCLE DEVELOPMENT HIGHLIGHTS CHROMOSOME ORGANISATION AND REGULATION OF GENE EXPRESSIONTellam R.L.1, Byrne K.1, McWilliam S.1, Vuocolo T.1, Cockett N.2 and Gondro C.3 1CSIRO Livestock Industries. 2University of Utah. 3University of New England.

Skeletal muscle undergoes a major switch in its gene expression program and metabolism during the fetal to post-natal developmental transition. We hypothesized that epigenetic modifications of the genome underpin coordinated changes in gene expression which prepare skeletal muscle for movement, support against gravity and altered metabolism in the post-natal environment. Using Illumina GAII sequencing we performed genome-wide mapping of Histone H3 lysine 27 trimethylation modifications (H3K27me3 CHiP-SEQ) for ovine skeletal muscle samples taken at late fetal development (100 days; n=3) and 12 weeks postpartum (n=3). The sequences were mapped onto the bovine genome and processed using CisGenome. The H3K27me3 peaks fell into two major categories, local and regional peaks, with the latter being predominant. H3K27me3 peaks were associated with genes, transcriptional start sites and CpG islands. Gene-associated H3K27me3 was negatively correlated with gene expression. There were strong associations between genes with promoters enriched for H3K27me3 and several GO terms common to both biological states. Gene-associated H3K27me3 peaks present in the lamb samples but absent in the fetal samples were associated with the TGF-βbeta and WNT signaling pathways. Homeobox genes showed strong regional enrichments for H3K27me3 in both biological states. H3K27me3 was substantially enriched on the X chromosome of females but not males, thereby implicating this epigenetic mark in X chromosome inactivation. These analyses revealed remarkable modifications of the epigenome that comment on many aspects of chromosomal organisation and gene activity in the context of development.

DEPLETION OF THE NF-κB TRANSCRIPTION FACTORS IS REQUIRED FOR CHROMATIN REASSEMBLY AT THE GM-CSF PROMOTER FOLLOWING GENE ACTIVATION

Poke F.S., Upcher W.C. and Holloway A.F. Menzies Research Institute, University of Tasmania, 17 Liverpool St, Hobart, Tasmania 7000.

Aberrant gene expression is a contributing factor to the development and progression of disease including cancer. The chromatin environment of a gene plays an important role in regulating gene expression, whereby it governs access of the protein complexes, transcription factors and transcriptional machinery which facilitate transcription, to their binding sites. While the changes in the chromatin environment that are associated with gene activation are well characterised, little is known about the factors driving chromatin reassembly following gene transcription. We have examined chromatin reassembly at the promoter of a cytokine gene, granulocyte/macrophage colony-stimulating factor (GM-CSF) following gene activation. GM-CSF is an important gene involved in hematopoiesis, which becomes dysregulated in acute myeloid leukaemias. Following activation of the GM-CSF gene in EL-4 T cells, transcript levels decline rapidly and this occurs prior to the progressive reassembly of the promoter nucleosome. Nucleosome reassembly occurs independently of DNA replication and cell division, and requires protein synthesis. Furthermore we show that nucleosome reassembly is highly dependent on the depletion of the activating NF-κB transcription factors. These transcription factors are often constitutively expressed in leukaemias, and these results suggest they may incorrectly maintain gene transcription by preventing restoration of the chromatin environment following gene activation. This highlights the importance of knowing how genes become “switched off” for understanding aberrant, constitutive gene expression in cancer.

VARIOUS FORMS OF THE HUMAN EXOSOME COMPLEX CONTAIN DIFFERENTIALLY LOCALIZED CATALYTIC SUBUNITS: HDIS3 AND HDIS3L

Tomecki R.1, Kristiansen M.S.2, Lykke-Andersen S.2, Chlebowski A.1, Dziembowski A.1 and Jensen T.H.2 1Institute of Biochemistry and Biophysics, Polish Academy of Sciences; Pawinskiego 5A, 02-106 Warsaw, Poland. 2Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology, Aarhus University; C. F. Mollers Alle, Bldg. 1130, DK-8000 Aarhus, Denmark.

The eukaryotic RNA exosome is a multisubunit ribonucleolytic machinery involved in virtually all aspects of RNA turnover, processing and quality control. It is composed of a nine-subunit catalytically inert core that serves a structural role and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic protein, which possesses both processive exo- and endonuclease activities, whereas the latter is a distributive exonuclease restricted to the nuclear form of the yeast complex. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here we demonstrate the association of two different homologs of Dis3p: hDIS3 and hDIS3L, with the human exosome core. Only hDIS3 is able to complement reduced yeast DIS3 expression. Interestingly, the two proteins display markedly different intracellular localization in that hDIS3 is mainly nuclear, while hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases, however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.

CHARACTERIZATION OF NOVEL PKD INHIBITORS USING BIOCHEMICAL ASSAYS AND CELLULAR STUDIES

Vantus T.1, Varga A.1, Szokol B.2, Nemeth G.2, Orfi L.2, Szantai-Kis C.2, Horvath Z.2, Varga Z.2 and Keri G.1, 2 1Pathobiochemistry Research Group, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary. 2Vichem Chemie Research Ltd., Budapest, Hungary.

Protein kinase D (PKD) family is a novel group of serine/threonine kinases. PKD1 was the firstly identified member of this group and also the best characterized so far. PKD1 is critical in certain growth factor signaling pathways and has an important role in inflammation and angiogenesis. To further explore the signal transduction of PKD, there is a need for a specific inhibitor. In this study, our aim was to discover and characterize novel, small molecular PKD1 inhibitors applying biochemical and cellular assays. Using Vichem’s Nested Chemical Library, firstly we performed a high throughput screening (HTS) to test molecules using in vitro kinase assay. The optimized IMAP assay for PKD was an efficient tool to find hit molecules with different core structures those were selected for further enzyme kinetic studies. After enzyme kinetic measurements, we further characterized the signaling of our, mostly ATP binding site inhibitor hit molecules in various cellular systems including cancer cell lines and other cell types. We investigated the effect of these inhibitors on cellular phosphorylation, cell proliferation and on apoptosis induction, besides we determined certain early ADME parameters as well. In summary, we optimized a cost efficient, recombinant enzyme based HTS kinase assay platform and selected efficient drug-like lead compounds inhibiting PKD1 with nanomolar IC50 values. We further characterized the signaling of these small inhibitory molecules in several cellular assays. Acknowledgements: This work has been financially supported by the NKFP-A1-0069/2006 (Inflamin) and OM-00080/2008 (Nanodrug) grants.





GUANINE NUCLEOTIDE EXCHANGE FACTORS FOR RHO GTPASES IN ENDOTHELIAL CELLS: CRITICAL TRANSDUCERS OF ANGIOGENIC SIGNALS

Carretero-Ortega J.1, Guzman-Hernandez M.L.1, Hernandez-Garcia R.1, Vazquez-Macias A.1, Hernandez-Negrete I.1, HellerBrown J.3, Gutkind J.S.4, Reyes-Cruz G.2 and Vazquez-Prado J.1 1Department of Pharmacology. CINVESTAV-IPN. Mexico City. MEXICO. 2Department of Cell Biology. CINVESTAV-IPN. Mexico City. MEXICO. 3Department of Pharmacology, UCSD. San Diego,USA. 4Oral & Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, USA.

G protein coupled receptors activate signaling pathways related to endothelial cell migration and angiogenesis. We studied the role of Gbetagamma and P-Rex1, a Rac guanine exchange factor, as mediators of angiogenic signals. We characterized a differential inhibitor of Gbetagamma, derived from Phosducin-like protein (PhLP-M1-G149), able to interfere with the interaction between Gbetagamma and PI3Kgamma, which inhibits AKT signaling, endothelial cell migration and tubulogenesis. In addition, we tested the hypothesis that P-Rex1 is required for the angiogenic response stimulated by SDF1 and characterized the role of P-Rex1-interacting proteins in cell migration. We found that P-Rex1 is an effector of the mammalian target of Rapamycin, linking this kinase to Rac activation and cell migration, and demonstrated a critical participation of P-Rex1 in the migration and in vitro angiogenic response of endothelial cells stimulated with SDF-1/CXCL12. Together, our data indicate an important role of the Gbetagamma/PI3Kgamma/P-Rex1 signaling pathway in angiogenic responses.

THE ROLE OF KRüPPEL-LIKE FACTOR 3 IN B-LYMPHOCYTE DEVELOPMENT AND TUMORIGENESIS

Vu T.1, Funnell A.1, Gatto D.2, Brink R.2, Pearson R.1, 3 and Crossley M.1, 3 1School of Molecular Biosciences, University of Sydney, DARLINGTON, SYDNEY NSW 2008, AUSTRALIA. 2B Cell Immunology Group, Immunology Program, Garvan Institute of Medical Research, DARLINGHURST, SYDNEY NSW 2010, AUSTRALIA. 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, SYDNEY, NSW, 2052, AUSTRALIA.

Krüppel-like factor 3 (Klf3) is a member of the Krüppel-like factor family of transcription factors. Klf3 is a transcriptional repressor that is highly expressed in haematopoietic tissue. In addition, the Klf3 locus is a common site of retroviral integration in murine B cell cancers [1] and Klf3 knockout mice appears to be immuno-compromised and show a reduced vitality. The knockout mice show a significant reduction in percentage and absolute numbers of marginal zone and B-1a peritoneal B cell populations. Furthermore, staining of knockout spleen sections shows sparse distributions of follicles and thin marginal zones. Nevertheless, the Klf3 null bone marrow has an approximate 3-fold increase in the percentage of mature cells, accompanied by a significant reduction in the percentage of late pre-B and immature B-cells. Given that deregulation of Klf3 expression can lead to B-cell cancer and that B-cell development is impaired in the knockout mice, we suggest that Klf3 plays an important role in normal B-cell development. Microarray analysis comparing gene expression in splenic B cells purified from wild type and Klf3 knockout mice has revealed potential Klf3 target genes with roles in B cell proliferation, differentiation and apoptosis. We are currently validating the biological significance of these putative Klf3 target genes in B cell development and tumorigenesis.

NON-CANONICAL GONADOTROPIN SIGNALLING IN OVARIAN CANCER CELL MIGRATION

Zhang H.1, 2, Bolitho C.1, Marsh D.J.1 and Baxter R.C.1 1Hormones and Cancer Group, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney NSW 2065 Australia. 2School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

The gonadotropin hypothesis proposes that elevated levels of follicle stimulating hormone (FSH) and luteinising hormone (LH) seen in postmenopausal women increase the risk of epithelial ovarian cancer (EOC). We have reported that in EOC, gonadotropins signal through a non-canonical, cyclic AMP-independent pathway that is Ca2+-dependent but involves the Ca2+-independent protein kinase C isoform PKCδ (Mertens-Walker et al. Endocr-Relat Cancer 17:335-49, 2010). We now show that the sphingolipid sphingosine 1-phosphate (S1P) is a key intermediate in this pathway, with a role in stimulating EOC cell migration via activation of extracellular signal-regulated kinase ERK1/2. Monolayer scratch-wounding and migration across transwell membranes were employed to assess S1P involvement in gonadotropin-induced migration of the human EOC cell lines OV207 (clear-cell) and OVCAR-3 (serous). Cells were treated for 1 h with sphingosine kinase (SphK) inhibitor to inhibit S1P generation (10 µM 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole or 5 µM N,N-dimethylsphingosine), or 1 µg/ml S1P blocking antibody (Dr R. Sabbadini, LPath, Inc.), then migration was stimulated by adding 10 nM FSH or LH. Cell lysates from OV207 and OVCAR-3 were immunoblotted for ERK1/2 activation following 10 min treatment with either 10 nM FSH or LH, with or without inhibitors. In both cell lines, LH and FSH stimulated ERK1/2 phosphorylation, this effect being blocked by SphK inhibition. Gonadotropin-induced migration in both cell lines was also inhibited by SphK inhibition or S1P blockade, with little or no effect on basal migration. These data further delineate the pathway of gonadotropin action in ovarian cancer, and implicate S1P signalling in gonadotropin-dependent migration of EOC cells.

GTF2IRD1, A WILLIAMS SYNDROME-RELATED PROTEIN, IS A NOVEL SUMO SUBSTRATE

Widagdo J.1, Palmer S.J.1, Taylor K.1, Bontempo S.2 and Hardeman E.C.1 1School of Medical Sciences, Department of Anatomy, University of New South Wales, NSW, Australia. 2Department of Pathology, University of Sydney, NSW, Australia.

Williams-Beuren syndrome (WBS) is a neurological disorder that results from a hemizygous microdeletion within chromosome 7q11.23 involving 28 genes. Its features typically involve characteristic physical abnormalities and a set of cognitive and behavioural features, known as the Williams syndrome cognitive profile (WSCP). Studies of patients with smaller deletions have implicated GTF2IRD1 and its evolutionary-related homolog GTF2I in the main aspects of the WSCP. We generated a Gtf2ird1 knockout/LacZ knock-in mouse line to map its expression in the brain and to examine the consequences of gene inactivation. These mice show developmental abnormalities and cognitive impairment, reminiscent of the WSCP. However, the cellular function of GTF2IRD1 still remains elusive. Here we present the evidence that GTF2IRD1 is subjected to the post-translational modification, sumoylation. Sumoylation involves covalent conjugation of small ubiquitin-like modifier (SUMO) protein to a lysine residue in the target protein, via the sequential action of SUMO-specific E1, E2, and E3 enzymes. Overexpression of GTF2IRD1 and HA-SUMO in COS-7 cells leads to a covalent attachment of SUMO to GTF2IRD1, shown through western blotting and co-immunoprecipitation analyses. Through mutagenesis experiments, we have identified the lysine residue, contained within an evolutionary conserved motif in GTF2IRD1 that is targeted by sumoylation. We have also confirmed the interaction of GTF2IRD1 with the SUMO E2 conjugating enzyme UBC9 and E3 ligase PIASx through a yeast-two-hybrid system. We are in the process of investigating the function of GTF2IRD1 sumoylation which might affect its: subcellular localisation, protein stability, transcription factor activity and/or its interaction with other proteins. Whilst our investigation at the transcriptional level has already shown that GTF2IRD1 negatively autoregulates itself, sumoylation presents an additional mechanism. The regulations at different levels suggest that the activity of GTF2IRD1 is finely controlled and therefore, the high likelihood of dosage sensitivity would play a major contribution to the features of WBS.


INVESTIGATING MECHANISMS OF POST-TRANSCRIPTIONAL GENE REGULATION IN ZEBRAFISH GERM CELLS

Wiszniak S.E. and Jensen K.B. University of Adelaide, North Terrace, Adelaide, SA 5005.

Post-transcriptional gene regulation is essential in development, especially during development of the primordial germ cells (PGCs). In zebrafish, PGCs are specified by the inheritance of cytoplasmic determinants, termed the germ plasm, which contains maternal mRNAs, such as nanos and vasa. The restricted localisation of these mRNAs to the germ plasm and subsequent PGCs is due to cis-acting elements present in the 3’UTR. Nanos mRNA is specifically degraded by miR-430 in the soma, while in the PGCs the RNA-binding protein Dnd protects nanos from miR-430 mediated degradation. The Hu family of RNA-binding proteins consists of five members in zebrafish. HuA and HuG are ubiquitously expressed, whereas HuB, HuC and HuD show neuron-specific expression in the adult. Interestingly, HuB also has an independent role during early development. HuB mRNA is maternally deposited in the embryo, and shows PGC-specific expression. A synthetic transcript consisting of the mCherry open reading frame fused to the HuB 3’UTR shows mCherry protein expression restricted to the PGCs at 24 hpf. This result suggests sequence elements in the HuB 3’UTR can restrict protein expression to the PGCs. Deletion analysis has narrowed down the PGC-specific element to a 150nt segment of the 3’UTR, and further mutational analysis has identified distinct regions responsible for somatic degradation of the message, and for PGC-specific stabilisation. There is no miR-430 site present in the HuB 3’UTR, implying a novel mechanism is responsible for the post-transcriptional regulation of HuB mRNA in the zebrafish embryo. Several approaches are being taken to identify candidate RNA-binding proteins that are able to stabilise HuB mRNA in the PGCs, including development of an RNA-tethering assay and also RNA pull-downs combined with mass spectrometry.




DIFFERENTIAL PATTERNS OF DNA METHYLATION AND MICRORNAS IN ADPKD KIDNEY TISSUES BY GENOME-WIDE ANALYSIS

Woo Y.M.1, Chung H.Y.2, Lee M.J.1, Bae J.B.2, Shin S.A.2, Lyu J.M.2, Yoo K.H.1, Seo M.J.1, Kim Y.J.2 and Park J.H.1 1Dept. of Biological Science, Sookmyung Women’s University, Seoul, 140-742, Korea. 2Dept. of Biochemistry, Yonsei University, Seoul, 120-749, Korea.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common human genetic disease and characterized by the formation of multiple fluid-filled cysts, which result in end-stage renal failure. Increased cell proliferation is one of the key features of the disease. We already examined the gene expression levels and methylation patterns of genomic DNA from kidney of normal and ADPKD patients via genome-wide analysis. As a result, overall genome of ADPKD patients was hypermethylated compared with normal samples. While the most of genes hypermethylated in promoter region of ADPKD patients were correlated to proliferation, apoptosis and calcium ion channel, those in genebody region were associated with notch signaling pathway. Especially, genebody of PKD1 which is major gene causing ADPKD was hypermethylated in ADPKD. To investigate how chromosome block is prone to hypermethylated in ADPKD patients, we confirmed the gene expression levels of the factors, which affect DNA methylation, such as DNMTs (DNA methyltransferases), DNA demethylases, HDACs (histone deacetylases). In this study, altered expression levels of these genes may induce an increase of DNA methylation in ADPKD. Interestingly, upon treatment of DNMT inhibitor, such as 5-aza-2’-dexoycytidine and zebularine, into the ADPKD primary cells, DNA methylation and mRNA levels were restored. Furthermore, microRNA (miRNA) DNA methylation and its expression levels were also differential between normal and ADPKD kidney tissues. It is suggested that epigenetic silencing of miRNAs by CpG island hypermethylation might affect on miRNA expression levels and result in cyst formation in ADPKD.

CHARACTERISATION OF THE ARABIDOPSIS DEHYDRIN, XERO2

Wright E.J. and Parish R.W. Department of Botany, La Trobe University, Bundoora, Vic. 3086, Australia.

Crop damage caused by frost events early in the growing season is a major issue for Australian agriculture. One genetic approach towards improving the frost tolerance of agronomic crops involves identifying proteins that protect plant tissues during periods of low temperature. The expression of these proteins can then be altered in genetically modified varieties so that protective proteins are more active or concentrated in plant tissues that require greater cold protection, such as sensitive floral tissues. The Arabidopsis gene, Xero2, is a candidate gene for this genetic approach towards improved frost tolerance. The Xero2 gene is expressed in floral and vascular tissues and is strongly upregulated in leaf tissues in response to cold and abscisic acid exposure. The XERO2 protein also has elements similar to other known dehydrative response proteins. The strong expression of Xero2 in Arabidopsis florets, even at control temperatures, suggests that XERO2 could play a role in the protection of floral tissues from low temperature damage. Transgenic plants have been generated to investigate the role of the XERO2 protein within the plant, however research is also being undertaken to understand how cold responsive genes, such as Xero2, are induced and regulated in the early stages of cold treatment. Cold treated Arabidopsis suspension cells are being used to decipher the transcriptional changes that take place within the plant cell after the onset of low temperature and immediately prior to the induction of Xero2. Transcriptional information from this study will add to our knowledge of how the strong expression of this cold responsive gene is regulated in the early stages of cold exposure.

DISCOVERY AND CHARACTERISATION OF C-JUN N-TERMINAL KINASE BINDING PROTEINS

Yeap Y.Y.C., Ng D.C.H. and Bogoyevitch M.A. Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, VIC, Australia.

Protein-protein interactions dictate protein actions and so these interactions have been increasingly studied to probe protein functions. We are interested in the formation of intracellular signalling protein complexes, and have employed the Yeast two-hybrid (Y2H) approach to discover and characterise proteins that interact with c-Jun N-terminal Kinase (JNK). JNK, a member of the Mitogen-activated Protein Kinase (MAPK) proteins, can phosphorylate a variety of nuclear or non-nuclear substrates and so mediates diverse cellular events including cell death, differentiation or proliferation. To confirm the fidelity of our Y2H interaction system, we included JNK1 bait with known interacting partners (c-Jun and JIP1 prey constructs) as positive controls. Approximately 30 million clones from a human fetal heart library were screened with JNK1 as bait. From the positive colonies identified, we confirmed JNK-specific interaction with 5 prey proteins: SYPL, COXIII, NEBL, EIF2AK3 and WDR62. Retransformation and quantitative liquid (CPRG) assay measurements confirmed interaction with JNK1 as well as showing interaction with JNK2. Based on structural information of the JNK1-JIP1 interaction, we also engineered and tested JNK single mutant (R127 or E329A) and JNK double mutants (R127A/E329A) for their interaction with the prey proteins. Quantitative liquid (CPRG) assay measurements revealed differing effects of the R127A, E329A and R127A/E329A changes in both JNK1 and JNK2 on their interactions with the tested prey proteins. These results suggest different modes of recognition of the partners by the JNKs and lay the foundation for further structural and biochemical studies addressing detailed aspects of these interactions.





AN ANALYSIS OF STRESS-STIMULATED STATHMIN (STMN) PHOSPHORYLATION BY 2D-PAGE

Yip Y.Y., Bogoyevitch M.A. and Ng D.C.H. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia.

Stathmin (STMN) is a cytoplasmic phospho-protein that has critical functions in regulating microtubule (MT) dynamics during cell migration and mitosis. STMN triggers MT disassembly by binding and sequestering α/β tubulin dimers. STMN activity is negatively regulated by multi-site phosphorylation on four conserved serine residues (Ser 16, 25, 38 and 63) which renders it unable to bind or sequester free tubulin subunits. STMN is phosphorylated by a number of kinases in response to extracellular stimuli such as mitogenic growth factors. In addition to growth factors, STMN is also phosphorylated in response to cellular stress (eg. osmotic stress and heat stress). While growth factor-stimulated STMN phosphorylation is well characterised, STMN phosphorylation in response to cellular stress and the signaling mechanisms involved have not been as well studied. Using a combination of 2-dimensional gel electrophoresis (2D-PAGE) and western blotting with site-specific phospho-STMN antibodies, we revealed differences in STMN multi-site phosphorylation in response to hyperosmotic stress compared to nerve growth factor (NGF) stimulation in PC12 cells. These altered phosphorylation states allude to separate mechanisms mediating STMN responses to environmental stressors compared to growth factors. Our findings also highlight that STMN function in regulating microtubules in stressed cells may differ when compared to normal growth conditions.

MARSUPIALS, MYTHS AND MASTER REGULATORS

Young L.J.1, Flenady S.1 and Belov K.2 1Centre for Environmental Management, CQUniversity Australia. 2Faculty of Veterinary Sciences, University of Sydney.

Members of the four-helical type I cytokine family are essential for the regulation of adaptive immunity in mammals. Interleukin-2 (IL-2) supports the growth, survival and differentiation of T cells. Interleukin-4 upregulates expression of antigen recognition and receptor molecules such as Major Histocompatibility Class II and IgM and is essential for the immunoglobulin class switch from IgG to IgE. Both ligands bind to a common receptor, the IL-2 gamma chain (or common gamma chain receptor), which is essential for effective downstream signalling. Despite repeated efforts over more than a decade, the expression of neither IL-2 nor IL-4 has been identified in any marsupial species. Indeed, literature reports have documented these difficulties and speculated about possible implications for the complexity of immune responses in marsupials as a consequence of the apparent lack of expression of these cytokines. Our view, after working with cell culture systems for the tammar wallaby to optimise in vitro T cell responses, was that the complexity of the adaptive immune response was most likely similar to other mammals and that reports of aberrant cell-mediated and humoral immune responses in marsupials were attributable to other, unidentified causalities. Renewing efforts to detect the expression of these genes, we combined information about the conserved regions in the structure of 4-alpha helical cytokines with nucleotide sequence data for phylogenetically distant species and the model marsupial, Monodelphis domestica and used RNA extracted from T cells in active blastogenesis (from our previous studies) as the template for homologous cloning techniques. We report the identification and expression of two master immune regulators in marsupials (IL-2 and IL-4), along with their common receptor (IL-2Rgamma) and discuss the implications for marsupial immunity.

EVOLUTIONARY CONSERVATION AND FUNCTION OF ESCRT-II: A KEY ENDOSOMAL SORTING COMPLEX

Ilievska J., Singh J., Dortenzio R., Annesley S., Fisher P. and Bishop N. Department of Microbiology, La Trobe University, Bundoora VIC.

The ESCRT (endosomal complex required for transport) machinery is essential for multivesicular body (MVB) biogenesis and degradation of endocytosed proteins. ESCRT components also play a crucial role in down-regulation of receptor signalling, retroviral budding and cell division. The ESCRT machinery is comprised of four complexes 0, I, II and III in metazoan and fungal cells. An ATPase, Vps4, plays a role in dissociation of the ESCRT complexes from membranes, and in fission of intraluminal vesicles within MVBs. The ESCRT-III interaction with Vps4 predates the divergence of Archaea and Eukarya and functions in archaeal cell division. While ESCRT complexes I-III are present in all major eukaryotic supergroups, some clades have lost genes encoding components of ESCRT-I and/or ESCRT-II. We present our findings on the evolutionary history of ESCRT-II and highlight the presence of orthologs in the amoeba, Dictyostelium discoideum. Dysfunctional ESCRTs are associated with a number of neurodegenerative and lysosomal diseases, and functional studies on the ESCRT-II in model organism D. discoideum are being used to determine the contribution of ESCRT-II to these diseases.

MEMBRANE MICRO-DOMAIN ORGANIZATION OF CAVEOLIN-1 MODULATES LIPID RAFT PROTEINS

Inder K.L.1, Loo D.1, Zheng Y.Z.1, 2, Foster L.J.2, Parton R.G.3 and Hill M.M.1 1Diamantina Institute, University of Queensland and Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia. 2Centre for High-Throughput Biology and Department of Biochemistry and Molecular Biology, 2125 East Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4. 3Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

Caveolin-1 is a cholesterol-binding membrane protein that forms specialized domains on the plasma membrane termed caveolae. Until recently, caveolins were thought to be the only proteins needed for the formation of plasma membrane pits called caveolae. We recently discovered that polymerase I and transcript release factor (PTRF) is a necessary coat protein required for caveolae formation. Caveolin-1 and PTRF are proposed to mediate signal transduction through the formation of caveolae. Several lines of evidence implicate over-expression of caveolin-1 in progression of advanced prostate cancer, with the mechanism still unknown. In the metastatic prostate cancer cell line PC3, caveolin-1 is present on flat plasma membrane due to the lack of PTRF expression. We hypothesized that non-caveolar caveolin-1 mediates metastatic ability. In agreement, ectopic expression of PTRF in PC3 cells resulted in caveolae formation, reduced cell migration and anchorage-independent growth. To further elucidate the caveolar and non-caveolar functions of caveolin-1 and PTRF we performed quantitative proteomics analysis using SILAC (stable-isotope labeling with amino acids in culture) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of subcellular fractionated proteins. Quantitative proteomic analysis revealed a number of signaling proteins differentially recruited to lipid rafts in PTRF expressing PC3 cells. Thus the organization of caveolin-1 on the membrane modulates recruitment of signaling proteins to membrane micro-domains. Altered caveolin function may contribute to diseases such as prostate cancer.





PROTEIN KINASE C CONTROLS APOLIPOPROTEIN E SECRETION FROM HUMAN MACROPHAGES

Karunakaran D.1, Kockx M.1, Owen D.2, Gaus K.2, Jessup W.1 and Kritharides L.1, 3 1Macrophage Biology Group, Centre for Vascular Research, UNSW, Sydney, Australia. 2Membrane Biology Group, Centre for Vascular Research, UNSW, Sydney, Australia. 3Dept. of Cardiology, Concord Repatriation General Hospital, USyd, Sydney, Australia.

Apolipoprotein E (apoE) is a multifunctional protein secreted by macrophages. It plays an important role in reducing atherosclerosis. However, precise signalling mechanisms regulating apoE secretion remain unclear. Recently, we demonstrated protein kinase A (PKA), protein phosphatase 2B (PP2B) and calcium regulate apoE secretion. Research suggests PKA interacts with protein kinase C (PKC), and we therefore investigated the potential role of PKC in apoE secretion from macrophages. Human monocyte derived macrophages (HMDMs) treated with pan-PKC inhibitors, Calphostin C (CalpC), Ro-31-8220 and Bisindolylmaleimide-I resulted in a significant dose-dependent decrease in apoE secretion. The PKC inhibitory peptide also significantly reduced apoE secretion, albeit less effectively. Further, Go6976 (inhibitor of classical PKC isoforms) resulted in ~40% decrease in apoE secretion from HMDMs. Treatment of HMDMs with Apolipoprotein A-I (stimulator of apoE secretion) or PMA (PKC activator) increased apoE secretion, which was reduced with CalpC. Preliminary metabolic labelling studies, where HMDMs were 35S-L-Met/Cys labelled and chased with or without CalpC, suggest that CalpC has no effect on apoE synthesis or degradation but directly inhibits the secretion of apoE. Further, apoE-vesicular movement observed by live cell imaging of HMDMs transiently transfected with apoE-GFP, was arrested by CalpC and Ro-31-8220. Quantification of vesicular speed demonstrated a marked decrease in average speed of vesicles from 0.42μm/s in non-treated HMDMs to 0.14μm/s or 0.15μm/s in CalpC- or Ro-31-8220-treated HMDMs respectively. This is the first report of a role for PKC in mediating basal or stimulated cellular apoE secretion, and implies current clinical applications of PKC inhibitors in humans may need to consider unexpected effects on atherosclerosis and inflammatory processes.

DISRUPTION OF EARLY ENDOSOME INTERACTION WITH HELICOBACTER PYLORI PHAGOSOMES

Keep S.J.1, Borlace G.N.1, 2, Butler R.N.1 and Brooks D.A.1, 2 1Cell Biology of Diseases Research Group, Sansom Institute for Health Research, University of South Australia. 2Department of Paediatrics and Reproductive Medicine, University of Adelaide.

Helicobacter pylori has successfully infected over 50% of the global human population causing gastritis, ulcers and gastric cancer. Despite evoking a strong immune response in the host, H. pylori persists, avoiding being killed by phagocytic cells. H. pylori may avoid degradation and clearance by these immune effector cells by altering the process of phagosome maturation. Primary human macrophages infected with H. pylori for 0, 5, 15, 30 minutes, and 2 hours were investigated by confocal immune fluorescence microscopy, using markers specific for H. pylori and early endosomes: Rabex5, Rab5, Rabaptin5, PI(3)P and EEA1. H. pylori phagosomes acquired Rabex5 immediately after infection and maintained this association for 15 minutes whereupon Rab5 became associated with the phagosomes. This recruitment of Rabex5 and Rab5 to H. pylori phagosomes was consistent with other models of phagosome maturation. In contrast, H. pylori phagosomes did not acquire PI(3)P until 30 minutes after infection. Moreover, Rabaptin5 and EEA1, which are downstream effectors of Rabex5 and Rab5, were recruited to H. pylori phagosomes immediately after infection and were retained for at least 2 hours, which represented a significant departure from normal phagosome maturation. These observations indicate that H. pylori phagosomes may sequester Rabaptin5 and EEA1 independently, interfering with early endosome-phagosome interaction. Perturbation of the molecular mechanism that results in this disrupted phagosome maturation could reinstate the efficacy of phagocytic killing. This has obvious significance for minimising H. pylori pathogenicity by enhancing the killing and clearance of H. pylori following a host immune response.

EXPRESSION OF CALCIUM-SENSING RECEPTOR HETERODIMERS AT THE CELL SURFACE USING THE GABAB SORTING SYSTEMKhan M.A., Chan R. and Conigrave A.D. School of Molecular Bioscience, University of Sydney, NSW 2006, Australia. The extracellular calcium-sensing receptor (CaR) is a class C GPCR that is expressed at the cell surface as homodimers where it selectively activates G-proteins in response to a variety of activators including extracellular Ca2+(Ca2+

o). Mutations in the CaR may modify its trafficking and/or signalling properties. While the effects of mutations in CaR homodimers have been well characterised, the effects of mutations in heterodimers have not been studied due to the presence of multiple dimeric species. The GABAB sorting sytem has been used to deliver class C GPCR dimers to the cell surface based on the shielding of an ER retention signal in the GABAB1 C-terminal by the GABAB2 C-terminal. We therefore decided to investigate whether the GABAB sorting system could be used to selectively target CaR heterodimers to the cell surface. The following constructs were generated in pcDNA3.1: (i) CaR-B1 in which the CaR C-tail (residues 876-1078) was replaced by the mGABAB1 tail (residues 856-961); (ii) R185Q-B2 in which residues 901-1078 of a dysfunctional CaR mutant (R185Q) were replaced by residues 760-961 of the human GABAB2 tail. HEK 293 cells were then transfected with CaR-B1 or R185Q-B2, individually or together, to characterise receptor responses to elevated Ca2+

o using Fura 2-AM to monitor intracellular Ca2+ mobilisation. Cells expressing CaR-B1 or R185Q-B2 individually, had reduced maximal responses (Vmax= 9±1.4, 8±0.6, respectively) when compared with WtCaR expressing cells (Vmax= 13±0.4) and EC50 values for Ca2+

o were markedly increased for CaR-B1 (18±5 mM) and R185Q-B2 (21±3 mM) when compared with WtCaR (5±0.3 mM). The reduction in maximal responses and decreased Ca2+

o sensitivity for CaR-B1 indicate receptor internalisation and for R185Q-B2 indicate impaired function. However, cells co-expressing the CaR-B1 and R185Q-B2 constructs exhibited a significant recovery comparable to WtCaR in the maximal response (12±0.7) as well as a recovery in Ca2+

o sensitivity (EC50=14±1.4). The results are consistent with successful trafficking of CaR-B1 and R185Q-B2 heterodimers to the cell surface. We conclude that the GABAB sorting system can be used to successfully traffick CaR heterodimers to the cell surface. This system will permit the study of specific mutations in heterodimers.

THE C-TERMINAL PROPEPTIDE OF A PLANT DEFENSIN CONFERS CYTOPROTECTIVE AND SUBCELLULAR TARGETING FUNCTIONS

Lay F.T.1, Heath R.L.2, Barbeta B.L.1, Poon S.2, McGinness B.2, Connelly A.A.2 and Anderson M.A.1 1Department of Biochemistry, La Trobe University, Melbourne VIC 3086 Australia. 2School of Botany, The University of Melbourne, Melbourne VIC 3010 Australia.

Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that are found in most plant species and tissues. They can be divided into two classes based on the presence or absence of a C-terminal propeptide (CTPP). The antifungal floral defensin from Nicotiana alata, NaD1, is a member of Class II. Its CTPP is removed during maturation of the precursor and the defensin is deposited in the vacuole. The function of the CTPP is not well understood but roles in vacuolar targeting and/or detoxification of the mature defensin domain have been suggested (Lay et al., 2003, Plant Physiol; Lay and Anderson, 2005, Curr Prot Pept Sci). Here we report that NaD1 accumulates in the vacuoles of transgenic cotton plants transformed with the full-length coding region and that removal of the CTPP prevents vacuolar deposition of NaD1. Transgenic cotton plants produced from constructs encoding NaD1 without the CTPP have an abnormal morphology, suggesting that NaD1 is phytotoxic if it is not deposited in the vacuole. Furthermore, we show that the CTPP is sufficient to direct green fluorescent protein to the plant vacuole in transient plant expression assays.





MITOTIC FUNCTION AND LOCALISATION OF IQGAP1 DURING GLIOBLASTOMA CELL DIVISION

Lian A.T.Y., Robinson P.J. and Chircop M. Children’s Medical Research Institute, The University of Sydney, Locked Bag 23, Wentworthville, Sydney, NSW 2145, Australia.

Cytokinesis is the final stage of cell division generating two independent daughter cells. Failure of this process results in enlarged and multinucleated cells, contributing to the initiation and/or progression of tumourigenesis. These cells are histopathological hallmarks for many tumours including glioblastoma multiforme (GBM). Cytokinesis in animal cells requires membrane ingression followed by abscission. Our recent unpublished discovery reveals a molecular pathway associated with the completion of abscission. During cytokinesis, Ca2+ influx activates the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin (CaN), dephosphorylating a suite of proteins such as dynamin II (dynII), triggering abscission. This occurs at a new sub-compartment, the Flanking Midbody Rings (FMRs), that reside on either side of the midbody ring at the intracellular bridge between two daughter cells. IQGAP1 binds actin and calmodulin and is essential for cytokinesis in fission yeast. We demonstrate that IQGAP1 is localised to the cleavage furrow and the intracellular bridge of SMA-560 murine glioblastoma cells that are undergoing cytokinesis. This is consistent with its actinomyosin ring localisation in fission yeast cytokinesis. Western blot analysis shows that IQGAP1 is upregulated upon mitotic entry and decreased during cytokinesis. Overexpression of GFP-IQGAP1 leads to an increased number of multinucleated cells. Our findings suggest that IQGAP1 participates in cytokinesis and its expression must be tightly regulated to ensure successful completion of this process. IQGAP1 is highly expressed in glioblastoma tumours and cytokinesis failure contributes to genomic instability. Thus, our results provide a possible mechanism for how IQGAP1 overexpression may increase oncogenic potential of glioblastoma.

CHARACTERIZING THE ROLE OF ADAPTOR PROTEIN 1A IN THE BASOLATERAL TARGETING OF KAE1

Netsawang J.1, 2, 3, Ngaojanlar P.1, Sawasdee N.1, Malasit P. 1, 2, Limjindaporn T.1, 4, Yenchitsomanus P.1 and Cordat E.3 1Medical Molecular Biology Unit, Office for Research and Development, 12th Floor Adulyadejvigrom Bld., Faculty of Medicine Siriraj Hospital, Prannok, Bangkok Noi, Bangkok, Thailand 10700. 2Department of Immunology,11th Floor Adulyadejvigrom Bld., Faculty of Medicine Siriraj Hospital, Prannok, Bangkok Noi, Bangkok, Thailand 10700. 3Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. 4Department of Anatomy, Faculty of Medicine Siriraj Hospital, Prannok, Bangkok Noi, Bangkok, Thailand 10700.

Distal renal tubular acidosis (dRTA) is characterized by defective acid secretion by the α-intercalated cells located in distal nephron. Most of the reported dominantly inherited forms of the disease revealed the importance of mutations in the kidney anion exchanger 1 (kAE1) gene. Previous studies demonstrated the crucial role of C-terminal residues in basolateral targeting of kAE1. Our preliminary data from yeast-two hybrid screening displayed the adaptor protein 1A (AP-1A) as one interacting partner for the C-terminus of kAE1. AP-1A is located in the trans-golgi network and endosomes and is involved in the cation-independent mannose-6-phosphate receptor (CI-MPR) trafficking. We hypothesized that AP-1A is important for kAE1 targeting to the cell surface in kidney cells. We have generated stably transfected MDCK expressing WT kAE1 and kAE1 mutated in the 11 C-terminal residues (R901X, Y904A, and Y904A/Y907A) in order to characterize the interaction. Endogenous AP-1A was co-immunoprecipitated only with WT kAE1, not with the C-terminal mutants. Far western blots confirmed the interaction, which predominantly occurred with AP-1A, not with 1B. In addition to kAE1, erythrocyte AE1 (eAE1) also interacts with AP-1A. Immunofluorescence experiments demonstrated that only intracellular WT kAE1 predominantly co-localized in a perinuclear region with AP-1A. Moreover, WT kAE1 co-localized with CI-MPR, another TGN marker that interacts with AP-1A. Pulse-chase experiments were performed to determine when this interaction occurs. We finally found that AP-1A was specifically immunoprecipitated with WT kAE1 after 2 hours chase time. Therefore, we conclude that kAE1 protein transiently interacts with AP-1A during its biosynthesis.

CHARACTERISATION OF THE ARCHITECTURE IN MUCOPOLYSACCHARIDOSIS IIIA NEURONS

Parkinson-Lawrence E.J.1, 2, Hemsley K.2, Khalessi-Rad M.1, Winter M.1, Hopwood J.J.2 and Brooks D.A.1, 2 1Sansom Institute for Health Research, University of South Australia, Adelaide, South Australia. 2Lysosomal Diseases Research Unit, SA pathology (at the Women’s and Children’s Hospital), North Adelaide, South Australia.

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder which is caused by a deficiency in the activity of sulphamidase; an enzyme involved in the degradation of heparan sulphate. MPS IIIA is characterised by progressive neurological dysfunction, but the effect of storage on neurons is largely unknown. Adult dorsal root ganglia neurons cultured from MPS IIIA mice demonstrated significant heparan sulphate storage together with fibrillogranular and zebra body inclusions. Neurite outgrowth was observed for both MPS IIIA and control neurons, but there were differences in both neurite length and network formation between these two groups. In control cells the cytoskeleton protein F-actin was diffusely organised with focal aggregates in the axoplasm. In MPS IIIA cells there were was an increase in small F-actin foci indicating cytoskeletal alterations. In control dorsal root ganglia neurons LAMP-1 staining was mainly observed in the cell body with negligible staining in the neurites. In contrast, MPS IIIA dorsal root ganglia neurons had a large number of punctate LAMP-1 positive vesicles in the neurites, which correlated with the distribution of the F-actin foci. There was also a significant increase in a high molecular weight form of LAMP-1 in MPS IIIA compared to control mouse brain tissue. The alterations in the F-actin cytoskeleton and differential distribution of LAMP-1-positive vesicles, together with altered LAMP-1 processing supported the concept that intracellular architecture and intracellular transport is altered in MPS IIIA neurons. This could have a direct impact on neuronal function and help explain the development of neuropathology in MPS IIIA.

REGULATING SECRETION IN INNATE IMMUNITY: DROSOPHILA 14-3-3ε’S ROLE IN ANTI-MICROBIAL PEPTIDE SECRETION

Shandala T.1, 2, 3, Woodcock J.M.2, Ng Y.1, 2, Lopez A.F.2 and Brooks D.A.1 1Sansom Institute for Health Research, University of South Australia, Adelaide, SA5001, Australia. 2Division of Human Immunology, Centre for Cancer Biology, Adelaide, SA5000, Australia. 3School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5000, Australia.

The secretion of anti-microbial peptides is recognized as an essential step in innate immunity, but little is known about the molecular mechanism controlling the release of these effectors from immune response cells. Here we report that Drosophila 14-3-3ε mutants exhibited reduced survival when infected with either Gram+ or Gram- bacteria, indicating a functional role for 14-3-3ε in innate immunity. In Drosophila 14-3-3ε mutants, there was an accumulation of the anti-microbial peptide Drosomycin and Rab11-positive vesicles at the plasma membrane, of cells from immune response tissues. This phenotype correlated with a reduced release of Drosomycin to the hemolymph. The accumulation of Drosomycin and Rab11-positive vesicles in immune cells from Drosophila 14-3-3ε mutants was similar to that observed in response to the depletion of the vesicular protein Syx1A. In contrast, RNAi silencing of endogenous Rab11 resulted in the accumulation of Drosomycin in the peri-nuclear region, preventing delivery to the cell surface. The depletion of another vesicular protein Lyst caused a less pronounced accumulation of vesicles at the plasma membrane, but enlarged Rab11-positive vesicles within immune cells. The Lyst phenotype was partially suppressed by the loss of 14-3-3ε, suggesting that 14-3-3ε may act as a negative regulator of Lyst. In cells from wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, Hrs and Syntaxin positive vesicles. We conclude that 14-3-3ε is required for antimicrobial peptide secretion during an innate immune response and for the functional interaction of Rab11 positive vesicles with the plasma membrane.





THE TROPOELASTIN R515 RESIDUE: ROLES IN TROPOELASTIN ASSEMBLY AND STRUCTURE

Yeo G.C.1, Dyksterhuis L.B.2, Mithieux S.M.1 and Weiss A.S.1 1School of Molecular Bioscience, University of Sydney, NSW 2006, Australia. 2CSIRO Molecular and Health Technologies, Clayton, VIC 3168, Australia.

Elastic fibres provide structural integrity and confer elasticity and resilience in tissues such as skin, lungs and blood vessels. The major component of elastic fibres is elastin, which, in turn, is formed from its soluble precursor, tropoelastin. The process of assembling tropoelastin into elastic fibres consists of distinct stages of coacervation, deposition on microfibrils, and cross-linking into mature elastin. It has been proposed that tropoelastin may be cleaved after its arginine 515 (R515) residue at some stage during elastin assembly. Interestingly, this R515 site resides within a section of domains crucial for coacervation and cross-linking. It is also proximal to a hinge region thought to contribute significantly to the tropoelastin structure. To explore the likelihood of tropoelastin cleavage during elastogenesis and the potential significance of the tropoelastin R515 site, we produced a protease-resistant R515A tropoelastin mutant and compared its assembly, cell interaction, and structural properties with the wild-type and the cleaved species. Both the R515A and the cleaved tropoelastin isoforms exhibited greater temperature and time requirements for coacervation, abnormal cross-linking, and decreased cell attachment relative to the wild-type species. Our results suggest that tropoelastin is unlikely to be cleaved until after cross-linking has been initiated, and that a percentage of monomers may remain uncleaved to allow necessary elastin-cell interactions. Structural analysis of the R515A species indicates that apart from its proposed role as a cleavage site in tropoelastin, R515 most likely contributes to the protein structure as part of a flexible bridge region. Mutation of this residue leads to a significant distortion of the bridge region and consequent displacement of the C-terminal region. Clinically, disruptions to the tropoelastin C-terminus have been associated with elastin disease phenotypes such as cutis laxa and supravalvular aortic stenosis. Our findings provide direct evidence of the contributions of the R515 residue to tropoelastin structure and function.

LIGHTING UP THE INNER MEMBRANE COMPLEX OF PLASMODIUM FALCIPARUM

Yeoman J.A.1, 2, Dearnley M.1, Dixon M.W.A.1, Maier A.G.1, Hanssen E.3, Baum J.4 and Tilley L.M.1, 2 1Department of Biochemistry, La Trobe University, VIC, Australia. 2Centre of Excellence for Coherent X-ray Science, La Trobe University, VIC, Australia. 3Bio21 Institute, University of Melbourne, VIC, Australia. 4Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, VIC, Australia.

The most deadly malaria parasite, Plasmodium falciparum, has a complex life cycle consisting of asexual and sexual stages within its host cell, the human erythrocyte. Host cell invasion is powered by an actin/myosin motor complex (glideosome) that is linked to an inner membrane complex (IMC) via a membrane anchor, glideosome-associated protein-50 (GAP50). We have generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 to study the formation of the IMC. Using long term live cell imaging, we show that PfGAP50-GFP is initially located in the endoplasmic reticulum and redistributed to a ring-like structure at the apical end of the nascent merozoite once the parasite commences division. Structured illumination microscopy reveals the early stage of the IMC as a double-holed flat ellipsoid that divides to form claw-shaped apposed structures. We have also found that PfGAP50-GFP interacts with glideosome proteins in the previously uncharacterised gametocyte IMC during the sexual part of the life cycle. P. falciparum gametocytes develop a characteristic elongated banana-like shape as they mature and our results suggest that the glideosome proteins may have a role in this cellular modification.

BINDING OF PHOSPHOLIPASE C-RELATED BUT CATALYTICALLY INACTIVE PROTEIN TO SNARE PROTEINS

Zhang Z., Takeuchi H., Gao J. and Hirata M. Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.

PRIP (Phospholipase C-related but catalytically inactive protein) was first identified as a novel Ins(1,4,5)P3 binding protein. We have recently found that the secretion of various peptide-hormones from multiple organs were upregulated in PRIP knock-out mice, indicating that PRIP negatively regulates the steps of exocytic event common to dense-core vesicles. To explore the molecular mechanism underlying the inhibitory role of PRIP in exocytosis, we examined the role of C2 domain of PRIP (PRIP-C2). Although many of the functional C2 domains reported have been shown to bind to acidic phospholipids in a Ca2+ dependent manner, we could detect no trace of lipid binding of PRIP-C2 in a lipid overlay assay. On the other hand, in PC12 cells, ectopically expressed EGFP-fused PRIP-C2 was partially co-localized with endogenous SNARE (soluble N-ethylmaleimid-sensitive factor attachment protein receptors) complex proteins which serve as the minimum machinery of membrane fusion. In addition, using pull-down assay and proteo-liposome floatation assay, we found that the both isolated PRIP-C2 and full-length PRIP interact with the components of SNARE complex including SNAP-25 (25-kD synaptosome associated protein) and syntaxin1. The results suggest that PRIP-C2 is involved in PRIP-mediated inhibition of regulated exocytosis through the protein-protein interactions: SNARE binding of PRIP might mediate recruiting PRIP which has an inhibitory role via other domains to the site of exocytosis or competes with other protein(s) which has a stimulatory role in exocytosis.

PROTEIN ENGINEERING AND AMYLOID β MUTATIONS

Nisbet R., Nuttall S., Caine J., Varghese J., Sankovich S., Bartone N. and Streltsov V. CSIRO Molecular and Health Technologies, and Preventative Health Flagship, 343 Royal Pde, Parkville 3052, AUSTRALIA.

Alzheimer’s disease (AD) is a highly prevalent, irreversible, neurodegenerative disease characterised by large amyloid plaques in the brains of affected individuals. The major constituent of amyloid plaques in AD is the Amyloid β (Aβ) peptide which is generated by sequential cleavage of the amyloid precursor protein (APP). Emerging evidence suggests that it is not the insoluble amyloid plaque that is neurotoxic in AD, but rather the soluble Aβ oligomers. We have utilised advanced protein engineering to trap regions of Aβ within protein scaffold domains allowing protein crystallisation. The predominant oligomeric species is a tightly associated Aβ dimer which we believe to be the smallest toxic species of Aβ. Several mutations within Aβ have been shown to reduce the tendency of Aβ to aggregate in vitro. Conversly, the Japanese familial mutation within Aβ, ΔGlu22, increases the aggregation of Aβ. We have engineered these specific mutations within our model system and investigated Aβ oligomerisation. Here we show that the effect of Aβ mutation on the oligomerisation of Aβ within the scaffolds are consistent with those previously observed providing support for our model system.





A TALE OF TWO SCAFFOLDS - PROTEIN ENGINEERING OF THE AMYLOID-β PEPTIDE

Nuttall S.D., Caine J., Varghese J., Nisbet R., Sankovich S., Bartone N. and Streltsov V. CSIRO Molecular and Health Technologies, and Preventative Health Flagship, 343 Royal Pde, Parkville 3052, AUSTRALIA.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the presence of misfolded protein depositions or amyloid plaques. Plaques consist predominantly of amyloid β-peptide (Aβ), which is produced by cleavage from the membrane-bound amyloid precursor protein (APP) via the α/β/γ secretase pathway. However, current views suggest that soluble Aβ oligomer intermediates, and not the plaque burden, may be the major drivers of Aβ-mediated neuronal dysfunction. Here, we utilise protein engineering using protein scaffold domains to trap and analyse several forms of the Aβ peptide. We describe a 2.2 Å resolution crystal structure of the P3 fragment, which represents an unusually folded dimeric form.

CALCIUM INDUCES ALPHA-SYNUCLEIN AGGREGATES IN SOLUTION, ON SURFACES AND IN CULTURED CELLS

Pountney D.L.1, Nath S.2, Goodwin J.1 and Engelborghs Y.2 1School of Medical Science, Griffith University, Gold Coast, Australia. 2Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven, Belgium.

Parkinson’s and Parkinson’s-plus diseases are associated with abnormal, aggregated forms of the protein, α-synuclein. We have investigated the effects of calcium on α-synuclein aggregation in vitro and in vivo. We treated monomeric α-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Our in vitro data suggests two distinct modes of aggregation: surface-dependent aggregation and aggregation in solution, both of which are accelerated by calcium, but at different concentrations. Incubation of monomeric α-synuclein (24 hrs) at low concentration (10 μM) with calcium resulted in surface aggregates (1.5±0.7 μm) saturating at a half-maximum calcium concentration of 80 μM, whilst incubations without calcium showed few protein aggregates. In the presence of calcium, plaques (0.5-1 μm) of α-synuclein aggregates comprising 10-20 nm globular particles were observed by scanning electron microscopy. Incubation of α-synuclein at high concentration (75 μM; 6 hrs) resulted in soluble oligomeric aggregates detected by fluorescence correlation in a calcium dependent process, saturating at a half maximum calcium concentration of 180 μM. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-synuclein-GFP construct and observed calcium flux and α-synuclein aggregation by fluorescence microscopy. The in vivo data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic α-synuclein aggregates 12 hrs post-treatment (P, 0.01). Our data indicates that calcium accelerates α-synuclein aggregation in vitro and in vivo and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.

TRIAZINE DERIVATIVES DECREASE LPS-INDUCED CELL DEATH BY INHIBITING NF-κB AND COX-2 IN NEURON-LIKE PC12 CELLSRamin M.R. and Khodagholi F. Neuroscience Res Ctr., Shahid Beheshti University of Medical Sciences, Iran

Introduction: Triazine derivatives are one group of compounds possessing a wide array of biological activities. Some derivatives have been introduced as anti inflammatory, radical scavenger, and β-sheet breaker agents. Lipopolysaccharide (LPS) is a pro-inflammatory substance present in the cell wall of Gram-negative bacteria. When activated by LPS, macrophages produce inflammatory cytokines, which in turn activate unprimed macrophages and other nearby cells. PC12 cell is an established cell line that is derived from rat pheochromocytoma. It has been used extensively as an in vitro model system to study neuronal cell fate, including survival, proliferation, differentiation, and apoptosis. Investigations have proved that LPS potentiates the effects of Nerve Growth Factor (NGF) in inducing neural differentiation of PC12 cells. In this study, we investigated the effect of Triazine derivatives on LPS induced neuron-like PC12 cell death via inhibiting caspase-3 activation. We further studied their effect on NF-κB and COX-2. Methods: Cell viability was determined, using conventional MTT assay, in the presence of triazine derivatives synthesized at Tehran University of Medical Sciences. Inflammation was induced by LPS. Then western blot analysis of caspase-3, NF-κB and COX-2 was done to determine if these drugs inhibit cell death or not. Results: The present study indicates that neuroinflammation resulting from LPS can be inhibited in the presence of triazine derivatives. This protection was associated with a marked reduction of caspase-3 activation. It shows they can inhibit cell death. Furthermore, it was proved that NF-κB and COX-2 were also decreased remarkably that shows the anti-inflammatory effect of triazine derivatives. Conclusion: As inflammatory stress is a critical event in the pathogenesis of neurodegenerative diseases, having neuroprotective effects along with anti-inflammatory properties implies the possibility of using triazine derivatives as a candidate for treating neurodegenerative diseases like Alzheimer’s Disease (AD).

AN INVESTIGATION OF THE FACTORS THAT REGULATE MUSCARINIC RECEPTOR EXPRESSION IN SCHIZOPHRENIA

Seo M.S.1, 2, Scarr E.2, 3 and Dean B.1, 2 1Department of Psychiatry, The University of Melbourne Parkville, Victoria, Australia. 2The Rebecca L. Cooper Research Laboratories, The Mental Health Research Institute, Parkville, Victoria, Australia. 3Centre for Neuroscience, The University of Melbourne Parkville, Victoria, Australia.

Muscarinic receptors (CHRM) belong to the superfamily of G-protein coupled receptors and are crucial for normal functioning of the central nervous system. We previously reported decreased levels of CHRM1 but not Sp1 in BA6 from subjects with schizophrenia. We have since identified a subgroup of subjects with schizophrenia, who have a 75% decrease in their cortical CHRM1 levels. This subgroup of subjects is termed the muscarinic receptor deficiency schizophrenia (MRDS). We have extended our previous study by measuring the density of CHRMs 1 and 3 using radioligand binding with autoradiography in BA6 in 20 MRDS subjects, 18 Non-MRDS subjects and 20 control subjects. Levels of Sp1 and Sp3 were also measured in these subjects using Western blotting. The density of [3H]pirenzepine binding was decreased in both non-MRDS (p<0.05) and MRDS (p<0.001) compared to controls. [3H]4-DAMP binding density was also decreased in MRDS (p<0.001) compared to control subjects. However, Sp1 and Sp3 levels were not altered in either group of subjects with schizophrenia. This study suggests the decreased levels of CHRMs in MRDS extend across the CHRM family, identifying further biochemical differences between MRDS and non-MRDS subjects. However this decrease in CHRMs does not appear to simply be regulated by local levels of Sp1 and Sp3, suggesting that other factors are involved in mediating low cortical CHRM expression. Identifying these factors could be seminal in the development of therapeutic agents to treat the cognitive deficits associated with schizophrenia.





THREE SITES PHOSPHORYLATED BY PROTEIN KINASE A IN HUMAN TAU PROTEIN AFFECT ITS INTERACTION WITH 14-3-3ZETA

Sluchanko N.N., Seit-Nebi A.S. and Gusev N.B. Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russian Federation.

Neuronal specific tau protein belongs to the group of microtubule-associated proteins and regulates formation, dynamics and functioning of microtubules. Point mutations, limited proteolysis, oxidation of SH groups and hyperphosphorylation promote aggregation of tau leading to accumulation of paired-helical filaments, a hallmark of many neuronal diseases, the so-called tauopathies and among them Alzheimer disease. Since universal adapter protein 14-3-3zeta co-localizes with aggregated tau, it is supposed that 14-3-3 might somehow affect association and accumulation of insoluble aggregates. We tried to analyze effect of tau phosphorylation on its interaction with 14-3-3. Using native gel electrophoresis, size-exclusion chromatography and chemical crosslinking we found that catalyzed by cAMP-dependent protein kinase (PKA) phosphorylation of the shortest isoform of human tau strongly increases its interaction with recombinant human 14-3-3zeta. We supposed that phosphorylated Ser156 of the shortest tau isoform (or homologous Ser214 of the largest isoform) located in the consensus sequence recognized by 14-3-3 are predominantly responsible for 14-3-3 binding. However, mutation S156A preventing phosphorylation of this site by PKA, decreased, but did not completely prevent tight interaction of phosphorylated tau with 14-3-3 thus indicating the presence of other phosphorylation sites playing important role in tau-14-3-3 interaction. By using step-by-step mutagenesis we found that phosphorylation of at least three sites (Ser156, Ser235 and Ser267) of the shortest tau isoform is important for 14-3-3 binding. Thus, phosphorylation-dependent sites of 14-3-3 binding are located both in the Pro-rich region (Ser156) and in tubulin-binding motifs (Ser235 and Ser267) of tau protein. This work was supported by Russian Foundation for Basic Research.

THE BH3-ONLY PROTEIN BIM: POSSIBLE LINK BETWEEN ER STRESS AND APOPTOSIS IN NEURAL CELLS EXPRESSING MUTANT SOD1

Soo K.Y.1, 2, 3, Atkin J.D.1, 3, 4, Farg M.1, Walker A.K.1, 3, 4, Horne M.K.3, 4 and Nagley P.2 1Department of Biochemistry, La Trobe University, Vic 3086, Australia. 2Department of Biochemistry and Molecular Biology, Monash University, Vic 3800, Australia. 3Brain Injury and Repair Group, Howard Florey Institute, University of Melbourne, Parkville, Vic 3010, Australia. 4Centre for Neurosciences, University of Melbourne, Parkville, Vic 3010, Australia.

In cellular models of motor neuron disease (ALS), cells bearing mutant Cu,Zn-superoxide dismutase 1 (SOD1) inclusions undergo mitochondrial apoptotic signaling. Dispersed SOD1 proteins, either wildtype (WT) or mutant, were found to partially protect cells against apoptosis; such protection is upstream of mitochondria. We previously showed ER stress to be linked to neurotoxicity associated with mutant SOD1 inclusions. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cells, Neuro2a, Bim knockdown by siRNA significantly reduced nuclear apoptotic features in cells bearing mutant SOD1 inclusions. After Bim knockdown, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased in such inclusion-bearing cells. However, CHOP translocation to nucleus, a marker of ER stress, was not reduced by Bim knockdown. Significantly, the neuroprotection afforded by dispersed WT SOD1 was substantially enhanced by Bim-depletion, observed in Bim-depleted cells exposed to various apoptotic insults. In cells not subjected to Bim knockdown, kinetic studies indicated CHOP translocation to nucleus to occur prior to formation of mutant SOD1 inclusions. Interestingly, Bax recruitment to mitochondria (but not apoptotic nuclei) was also observed before formation of mutant SOD1 inclusions. These findings suggest that neurotoxicity is induced by a toxic structure derived from mutant SOD1 that activates stress responses in cells much earlier than the appearance of grossly aggregated SOD1 in inclusions.

CHANGING THE SOLVENT ACCESSIBILITY OF THE PRION PROTEIN DISULFIDE BOND MARKEDLY INFLUENCES ITS TRAFFICKING AND EFFECT ON CELL FUNCTION

Tabrett C.A.1, Harrison C.F.2, Schmidt B.1, Bellingham S.A.2, 3, Hardy T.1, Sanejouands Y.4, Hill A.F.2, 3 and Hogg P.J.1 1Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, NSW 2052 Australia. 2Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010 Australia. 3Mental Health Research Institute of Victoria, University of Melbourne, Victoria 3010 Australia. 4Laboratoire de Physique, Ecole Normale Superieure, 46 allees d’Italie, 69364 Lyon Cedex 07, France.

Prion diseases are fatal, transmissible, neurodegenerative diseases that result from structural conversion of the prion protein into a disease-associated isoform. The prion protein contains a single disulfide bond. Our analysis of all NMR structures of the prion protein containing an explicit disulfide bond reveals that the bond exists predominantly in a stable, low-energy state, but can also adopt a high-energy configuration. Side chains (tyrosine and phenylalanine residues) control access of solvent to the disulfide bond by rotating away in the high-energy state. The importance of these aromatic residues for protein function was analysed by mutagenesis, biophysical and cell biological approaches. While the mutant protein behaved similarly to wild-type prion protein in recombinant systems, the mutants were retained in the endoplasmic reticulum of mammalian cells and degraded by the proteasomal system. The cellular behaviour of the aromatic residue mutants was similar to the cellular behaviour of a disulfide bond mutant prion protein, a result which is consistent with an unstable disulfide bond in the aromatic residue mutants. These observations suggest that the conformation of the prion protein disulfide bond may have implications for correct maturation and function of this protein.

EXPRESSION OF NF-κB REGULATED CYTOKINES TO EVALUATE EARLY DIAGNOSIS OF NEURODEGENERATIVE DISORDERS MAINLY HUNTINGTON’S DISEASE AND SPINOCEREBELLAR ATAXIA

Potdar P.D. and Tatwani S. Department of Molecular Medicine & Biology, Jaslok Hospital & Research Centre, 15, Dr. G. Deshmukh Marg, Mumbai 400026, India.

In1999, Celera Diagnostics sequenced the first human chromosome. Thereafter, scientists started developing molecular diagnostic tests to define involvement of any specific gene mutation in various genetic disorders. Huntington’s disease (HD), and Spinocerebellar ataxia (SCA1,2,3,6,7&17) are progressive neurodegenerative disorders caused by polyglutamine protein aggregates, which are known as “CAG triplet repeat disorders.” However, it was observed that many times diagnosis of these diseases, only by detecting CAG repeats, is not sufficient. Tabrizi et al (2005) have recently shown that IL-6 is consistently elevated in HD. Several NF-κB dependent genes are either up or down regulated in the expanded polyglutamine expressing cells depending on disease conditions. Recent reports have also indicated that TNF-α is up regulated in SCAs. Though the basic works suggest the role of NF-κB activation in the causation of these diseases, there is a need for using these biomarkers as molecular diagnostic tests for early diagnosis of these diseases. Our lab has already set up PCR based assays for studying CAG repeats in HD and SCAs but it is found that addition of these markers will help us to confirm their diagnosis more precisely. We therefore decided to study the expression of NF-κB regulated cytokines in HD and SCAs patients. We found that HD patients showed significant increase in IL-6 expression whereas, SCAs patients showed significant high expression of TNF-α with down regulation of IκB-α. Thus, this study indicates that these biomarkers will be important for evaluating HD & SCAs patients whose diagnosis by currently employed CAG repeat methods is negative or uninformative.





CHANGES IN SIGNALLING PROTEINS IN MUSCARINIC RECEPTOR DEFICIT SCHIZOPHRENIA

Udawela M.1, 2, Scarr E.1, 5, Hannan A.J.3, Thomas E.A.4 and Dean B.1, 5, 6 1Rebecca L Cooper Research Laboratories, Mental Health Research Institute of Victoria, VIC, Australia. 2Centre for Neuroscience, University of Melbourne, VIC, Australia. 3Howard Florey Institute, Florey Neuroscience Institutes, University of Melbourne, VIC, Australia. 4Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA. 5Department of Psychiatry, University of Melbourne, VIC, Australia. 6Department of Psychiatry, University of Melbourne, VIC, Australia.

Schizophrenia is a debilitating mental illness, affecting approximately 1% of the population [1]. Cholinergic muscarinic receptors (CHRMs) have been shown to be decreased in the CNS of subjects with schizophrenia [2] and are thought to be implicated in cognitive deficits associated with schizophrenia. Our laboratory recently showed in post-mortem tissue that around 25% of subjects with schizophrenia have a 74% reduction in binding to cortical CHRM1 in Brodmann’s Area (BA) 9 [3]. Our recent microarray data identified decreased expression in the CHRM1 downstream signalling protein phospholipase C beta 1 (PLCβ1) mRNA in schizophrenia in BA46, therefore to determine whether patients with CHRM1 deficit also had disrupted downstream signalling pathways we investigated changes in PLCβ1 in our subset of patients with low CHRM1 binding. Using qPCR and Western blot analysis we found PLCβ1 mRNA and protein expression is reduced in BA 9 from patients with schizophrenia with normal levels of CHRM1 binding, but not in those with low levels of binding, compared to age/sex matched controls. In BA 46, mRNA was decreased in both groups of patients compared to controls with no change in protein levels. Understanding changes in downstream effector pathways may reveal other possible therapeutic approaches to treating symptoms in schizophrenia. [1] Black and Andraeson. (1994) Textbook of Psychiatry, The American Psychiatric Press, Washington, DC [2] Raedler, T. J., et al. (2007) Mol Psychiatry 12, 232-46 [3] Scarr, E., et al. (2009) Mol Psychiatry 14, 1017-23.

SOCS2 REGULATES NEURITE OUTGROWTH AND NEURONAL SURVIVAL THROUGH TRK RECEPTOR INTERACTIONS

Uren R.T., Faux C.H., Turbić A., Wong A.W., Murray S.S. and Turnley A.M. Centre for Neuroscience, University of Melbourne, Parkville, Victoria, 3010, Australia.

Overexpression of Suppressor of Cytokine Signalling-2 (SOCS2) promotes increases in neurite length and neurite number in PC12 cells and cortical neurons. The mechanisms by which SOCS2 regulates the signals that control neurite outgrowth and neuronal differentiation are unresolved but appear to involve Trk neurotrophin receptors. Wildtype or mutant SOCS2 proteins were expressed in PC12 cells and neurite outgrowth examined under basal proliferative conditions and with Nerve Growth Factor (NGF) which promotes neuronal differentiation. Expression of full length SOCS2 promoted neurite outgrowth in both the absence and presence of NGF and the amount of endogenously expressed TrkA was increased. Expression of SOCS2 lacking the SOCS box blocked NGF-induced neurite outgrowth, thus behaving as a potent dominant negative mutation. To examine SOCS2 and Trk interactions in primary neurons, the morphology of TrkA expressing Dorsal Root Ganglion (DRG) neurons from SOCS2 overexpressing (SOCS2-Tg) mice and mice lacking SOCS2 (SOCS2 -/-) was compared to wildtype neurons. DRG neurons were obtained from 1 day post-natal mice, dissociated neurons were cultured and neurite morphology scored. DRG neurons from SOCS2-Tg mice demonstrated an increased proportion of neurons with complex neurite morphology. In contrast, SOCS2 -/- derived DRG neurons displayed reduced neurite length and number. Furthermore, the survival profile of SOCS2-Tg and SOCS2 -/- DRG neurons cultured with 50 ng/mL NGF was examined and the number of viable DRG neurons (large, phase bright cell bodies) was recorded at 24 hour intervals. At 48 and 72 hours post-plating, the number of viable SOCS2-Tg DRG neurons was greater than controls, whereas the number of viable SOCS2 -/- DRG neurons was reduced.

IDENTIFICATION OF OLFACTORY SIGNALLING GENES IN DROSOPHILA MELANOGASTER

Liu Y.-C., Honda T., Beale M., De Bruyne M. and Warr C.G. School of Biological Sciences, Monash University, VIC 3800, Australia.

Drosophila melanogaster has emerged as a powerful model organism for the study of the peripheral olfactory system due to the availability of sophisticated molecular genetic techniques for studying gene function, coupled with sophisticated electrophysiological tools for measuring single olfactory receptor neuron (ORN) responses. In Drosophila odour signals are detected by a large family of 62 seven-transmembrane receptor proteins, the odorant receptor (Or) family. Unlike mammalian Ors which are G protein-coupled receptors, the insect Ors appear to encode directly ligand-gated ion channels. To identify genes involved in peripheral olfactory function in Drosophila we have performed genetic screens using electrophysiological recording techniques. This approach has identified two EMS-generated mutant strains which have greatly reduced electro-antennogram responses to all tested odours. The two mutations show interesting differences in their electrophysiological phenotypes, indicating one (called ll2) may affect signal transduction in ORNs, whereas the other (called O88) may affect the function of the accessory cells that support the ORNs. We have deficiency mapped each mutation, the ll2 mutation maps to a region in 87A containing 12 annotated genes, and the O88 mutation to a region in 33E containing 6 annotated genes. We are using multiple approaches to identify the gene affected in each mutant: candidate gene sequencing, expression analysis, RNA interference experiments, and rescue experiments. Recent in vivo RNAi experiments indicate that the gene affected in the O88 mutation is bru-2, an essentially uncharacterised member of the bruno family of RNA-binding proteins, and that its function is required in the accessory cells. Rescue experiments are currently in progress to confirm this.

GSTP ISOLEUCINE 105 TO VALINE POLYMORPHISM MAY PLAY A ROLE IN DETERMINING SUSCEPTIBILITY TO PEPTIC ULCER

Kangsadalampai S., Gamnarai P., Rojpibulstit P. and Vilaichone R.K. Faculty of Medicine, Thammasat University (Rangsit Campus), Klong-Luang, Pathumthani, 12121. Thailand.

ABSTRACT A case-control study on a relationship between glutathione S-transferase class pi (GSTP) Ile105Val polymorphism and a susceptibility to gastric ulceration in Thai dyspeptic patients was carried out. The GSTP Ile105Val genotype was determined by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The genotype frequency in the patients with ulcer (case; n = 105) was significantly different from that in the non-ulcer dyspeptic patients (control; n = 194). The patients who were homozygous Val105 seemed to have higher risk to peptic ulceration [OR = 4.290 (95% CI, 1.100-16.722), p = 0.030]. In contrast, the Ile105Val genotype appeared to act as a protective genotype [OR = 0.563 (95% CI, 0.333-0.952), p = 0.031]. However, other confounding factors such as sex, age, and H. pylori infection should not be overlooked. Therefore, stratified analysis was performed. The result of the analysis revealed that sex and age were not the confounders but the H. pylori infection exhibited the influence on the association. Thus, besides the genetic factors, the environmental factors could be taken into an account when genotype susceptibility to disease was studied.





STUDIES ON THE ROLE OF P73 PROTEINS IN INTESTINAL STEM CELLS AND TUMOURS

Kass L.1, Horvay K.1, Stiewe T.2, Hime G.R.3, 4 and Abud H.E.1 1Monash University, Department of Anatomy and Developmental Biology, Clayton VIC 3800. 2Philipps-University Marburg, Institute of Molecular Biology and Tumor Research Marburg, Germany. 3University of Melbourne, Department of Anatomy and Cell Biology, Parkville VIC 3010. 4ARC Centre of Excellence in Biotechnology and Development.

This research is focussed on understanding the role of a dominant-negative form of the p73 protein (DNp73) in regulating the differentiation of intestinal epithelial cells and the initiation of tumours. p73 is a homologue of the p53 tumour suppressor gene and produces two major protein isoforms via use of alternative promoters. Several studies have suggested that the longer TAp73 protein has tumour suppressor activity and the N-terminally truncated DNp73 protein can act as an oncogene. Our studies show that p73 is expressed in epithelial cells at the base of intestinal crypts which suggests that p73 may have a role in regulating normal homeostasis within the intestinal stem cell niche. Several studies including ours have found that the overall level of p73 is overexpressed in human colorectal cancer (CRC) cell lines and that the DNp73/TAp73 ratio is increased. We are investigating the consequences of disrupting the DNp73/TAp73 ratio in the murine intestine using a transgenic mouse model that permits inducible conditional expression of DNp73 in the intestinal epithelium. Initial analysis of the effects of overexpressing DNp73 has revealed an interesting phenotype where there appears to be an expansion of the progenitor pool at the expense of differentiated cell types. We are currently analysing this phenotype in more detail.

ANTI-CANCER EFFECT BY KNOCK-DOWN OF THE PROTEINS OVER-EXPRESSED IN LUNG CANCER

Kim J.E.1, 2, 3, Koo K.H.1, 2, 3, Kim Y.H.1, 2, 3, Sohn J.1, 2, 3 and Park Y.G.1, 2, 3 1Dept. of Biochemistry. 2Division of Brain Korea 21 Program for Biomedical Science. 33Cancer Research Center for Lung and Breast/Ovarian Cancers, Korea University College of Medicine, 136-701, Seoul.

Lung cancer is a malignant tumor with a very high incidence and mortality. The majority of lung cancer patients have been diagnosed at late stage and surgical resection cannot be the best choice for successful treatment. Therefore, the therapeutic molecular targets are necessary for improving clinical outcome of lung cancer patients. In this study, we performed comparative proteomic studies to find the potential therapeutic targets of lung cancer. We used the different histopathologic types of lung cancer including squamous cell carcinoma and adenocarcinoma. 62 proteins were up-regulated and 13 proteins were down-regulated in lung cancer tissues. 16 of 75 identified proteins were verified by performing western-blot analysis in the same lung tissues used for proteomic analysis, the cell lines representing multi stage human lung carcinogenesis and lung carcinoma cell lines. Three proteins were selected to evaluate the roles in lung cancer cell growth, because the three proteins were up-regulated in both lung cancer tissues and cell lines. It has been observed down-regulation of the proteins significantly suppressed cell proliferation and clonogenicity in the three NSCLC cell lines, A549, H1299 and H1573. It is likely that the three proteins contribute maintenance of lung cancer. Our results indicate that the three proteins play a significant role in cell proliferation and are potential therapeutic targets for lung cancer.

APOPTOSOME APPARATUS AND EXPRESSION OF ITS REGULATORS, XIAP, APIP AND NUCLING, IN NON-SMALL CELL LUNG CANCER CELLS AND TISSUES

Moravcikova E., Krepela E., Prochazka J., Cermak J. and Benkova K. University Hospital Bulovka, Budinova 2, 18081 Prague, Czech Republic.

Dysfunction of apoptosome apparatus (AA) contributes to tumour growth and progression and therapy resistance of tumours. To evaluate AA functionality in non-small cell lung carcinoma (NSCLC) we studied AA activation in cell-free cytosols from NSCLC cells and tissues. Although NSCLCs expressed Apaf-1, procaspase-9, -3 and -7 proteins, AA was activated by cytochrome-c (cyt-c) and dATP only in 2 of 6 examined NSCLC cell lines and in 18 of 59 NSCLC tissues obtained from surgically treated patients. Although XIAP is a direct inhibitor of some caspases, its level in NSCLC tissue cytosols did not correlate with the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Moreover, XIAP neutralizing peptides AVPIAQK or ATPFQEG derepressed only slightly the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity in tumour cytosols. NSCLC cell lines expressed both APIP2 mRNA and protein and both nucling (UACA) mRNA and protein. NSCLC tissues showed significantly decreased expression of both APIP and nucling mRNAs as compared to matched lungs. In the tumours, there was no correlation between the expression of APIP mRNA or nucling mRNA and the endogenous or the (cyt-c + dATP)-induced caspase-3-like activity in cytosols. In conclusion, these results indicate that AA activation is suppressed in a high proportion of NSCLCs, that XIAP is not the major factor responsible for apoptosome dysfunction in NSCLCs, and that downregulation of APIP and nucling expression in NSCLCs has no obvious impact on AA activation. Acknowledgements. Supported by research projects NS/9715-4 and MZO00064211 from the Ministry of Health, Czech Republic.

DNA ANALYSIS IN FEMALES WITH 46,XY KARYOTYPE-ASSOCIATED SEX DEVELOPMENT DISORDERS

Krepelova A.1, Malikova M.1, Simandlova M.1, Plevova P.2 and Gaillyova R.3 1Department of Biology and Medical Genetics, University Hospital Motol and 2nd Medical School, Prague, Czech Republic. 2Department of Medical Genetics, University Hospital Ostrava, Czech Republic. 3Department of Medical Genetics, University Children Hospital Brno, Czech Republic.

Disorders of sex development (DSD) in females with karyotype 46,XY represent a very heterogeneous group of disruptions of normal gender ontogeny. Underlying genetic defects include disorders of genes determining development of testes, e.g. SRY gene, disorders of androgens synthesis or impaired function of the androgen receptor. In many cases of 46,XY DSD the molecular basis has not been identified yet. We studied 21 female patients with 46,XY DSD. We used PCR amplification and direct DNA sequencing to detect mutations in several genes including SRY, SF1, AR and 17βHSD3. We used MLPA analysis to detect DAX2 gene duplications. We detected a novel mutation c.146_153del8ins15 (p.Gly49fsX10) in the SRY gene in a girl with gonadal dysgenesis. In six cases with clinical diagnosis of complete androgen insensitivity (CAIS) we found five different mutations in the AR gene. Three mutations are novel: c.33_37dupCCCTC (p.Arg13ProfsX23), c.122_136del15ins5 (p.His41ProfsX130) and c.827_828dupGC (p.Val277LeufsX17), and two were observed previously by others: c.2194G>A (p.Asp732Asn), and c.2543dupA (p.Asn848LysfsX32). In one CAIS patient we identified a homozygous mutation in the 17βHSD3 gene: c.[325+4A>T]+[325+4A>T]. This mutation was reported previously to cause an abnormal mRNA splicing. In thirteen remaining patients the molecular cause of DSD was not yet determined. In conclusion, the heterogeneity of DSD requires to search for defects in multiple candidate genes in order to idenfity the exact molecular basis of DSD. This is essential for the genetic prognosis, therapeutic decision, and psychological and social care in these patients. Supported by Grant MZO 00064203.





FUNCTIONAL ANALYSIS OF ZINC TRANSPORTERS OF ZNT/SLC30A FAMILY MEMBERS AMONG A MAMMARY GLAND DISORDER OF REDUCED ZINC SECRETION INTO MILK

Kumar L., Michalczyk A., Freestone D. and Ackland L. Centre for Cellular and Molecular Biology, School of life and environmental sciences, Deakin University, VIC, 3125, Australia.

Zinc is an essential trace element required for normal growth, development and reproduction. Zinc deficiency can cause dermatitis, diarrhoea, alopecia, loss of appetite, hypogonadism, impaired growth and diminished immune response. An adequate supply of zinc is required particularly during the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. Hereditary disorders of impaired zinc secretion from the mammary gland into milk during lactation can cause acquired zinc deficiency in infants exclusively fed on breast milk. It usually occurs in pre term babies 27-33 week gestation due to their greater bodily demand and lower bodily zinc stores. Mammalian zinc transporters, which may play an important role in secretion of zinc into milk, are broadly classified into SLC30A and SLC39A families. Transcriptional levels were measured for identified members of mammalian SLC30 family to test the hypothesis that defects in one or more of them may be responsible for impaired zinc secretion into the breast milk. Significantly reduced levels of ZnT5 and ZnT6 mRNA transcript were found in patients lymphoblast and fibroblast compared to control cells. These finding suggested that mutation in ZnT5 and/or ZnT6 may underline the disorder of reduced zinc secretion into the milk. Sequence analysis of ORF was carried out but no differences were found between the patients and control samples. Analysis of 5/3 UTR (untranslated region), promoter region, protein expression, localization, alkaline phosphatase activity and mi-RNAs of both genes was carried out. In summary ZnT5 and ZnT6 may indirectly contribute to this zinc deficiency disorder.

ISOMALYNGAMIDE A, A-1 AND THEIR ANALOGS SUPPRESS CANCER CELL MIGRATION IN VITRO

Li W.-S.1, Chang T.1, More S. 1, Lu I.-H. 1, Hsu J.-C.1, Chen T.-J.2, Jen Y.1, Jao S.-C.3 and Lu C.-K.2 1Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan. 2Division of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine, Taipei 112, Taiwan. 3Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

Isomalyngamide A, A-1 and their analogs suppress cancer cell migration in vitro Tzu Ting Chang,a Shivaji V. More,a I-Hsuan Lu,a Jui-Ching Hsu,a Ting-Ju Chen,b Ya Ching Jen,a Shu-Chuan Jao,c Chung-Kuang Lu,*b and Wen-Shan Li*a aInstitute of Chemistry, Academia Sinica, Taipei 115, Taiwan, bDivision of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine, Taipei 112, Taiwan, and cInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Abstract: There are considerable pharmacological interests associated with marine metabolites because of their demonstrated value as antimalarial, antitubercular, anti-HIV, and antitumor agents. However, recent advances in search of antimetastatic agents are mainly from either natural products or small synthetic libraries, lacking the participation of marine metabolites and their man-made analogues. Metastasis is mutiple biological events resulting in migration of tumor cells from their initial site to secondary organs. Inhibition of any of these events could hamper the whole metastatic process. Therefore, antimetastatic agents, an alternative of anticancer drugs, have attracted much attention lately. We have identified highly potent inhibitors of tumor progression from marine metabolites, isomalyngamide A (1) and A-1 (2), which show therapeutic potential in breast cancer not only by bolcking cell proliferation with low micromolar IC50 values but also by inhibiting metastasis with low nanomolar IC50 values. Notably, since analog 9 generated in this study can present a bioactive mimic of isomalyngamide A and A-1, it provides a channel of devotedly study integrin-mediated antimetastatic pathway without the interference of cytotoxic effect.

IDENTIFICATION AND FUNCTIONAL CHARACTERISATION OF WDR35, A GENE MUTATED IN SHORT RIB POLYDACTYLY SYNDROME

Amor D.J.1, Fitzpatrick E.2, Mountford H.2, Bahlo M.3, Mill P.4, Hall E.4, Bromhead C.3, Pope K.1, Aftimos S.5 Jackson I.4, Delatycki M. 2, Savarirayan R1 and Lockhart P.J.2 1Victorian Clinical Genetics Service, Murdoch Childrens Research Institute, Australia. 2The Bruce Lefroy Centre, Murdoch Childrens Research Institute, Australia. 3Walter and Eliza Hall Institute, Australia. 4MRC Human Genetics Unit, UK. 5Northern Regional Genetics Service, New Zealand.

The short rib-polydactyly (SRP) disorders are a heterogenous group of autosomal recessive lethal skeletal dysplasias characterised by short ribs and limbs. The presence of additional clinical features such as abnormal viscera, cranium and palate can be used to classify the disorders into specific subtypes. We previously identified a family with an unclassifiable SRP that displayed laterality defects suggestive of ciliary dysfunction (1). We performed SNP analysis on five family members and identified three regions with a LOD>1. Analysis of inferred haplotypes suggested that a single region at chromosome 2p24.1 was homozygous by descent in affected members. CNV analysis identified a homozygous deletion within the linkage peak that disrupted the WDR35 gene. WDR35 encodes a novel highly conserved protein with homology to known cilial proteins. Immunocytochemical analysis localised WDR35 to the cilial axoneme and basal body of control fibroblasts, however cilia were completely absent in fibroblasts derived from our SRP patients. Targeted disruption of wdr35 in the mouse resulted in abnormal cilia, randomised laterality, polydactyly and neural tube defects. These features were consistent with the human phenotype and suggested deficits in hedgehog (Hh) signalling contributed to disease pathogenesis. Quantitative real-time PCR analysis demonstrated that downstream effector genes, such as Ptch1 and Gli1, were not responsive to Hh signalling in MEFs derived from mutant mice. In conclusion, our studies have identified a novel ciliopathy gene and extended our understanding of the pathogenesis of SRP. (1) Kannu P et al (2007) AJMG Part A 143:26073.

IDENTIFICATION OF ACTIVATED KINASE PROTEINS IN CHOLANGIOCARCINOMA

Loilome W.1, 2, Dokduang H.1, 2, Juntana S.1, 2, Namwat N.1, 2 and Yongvanit P.1, 2 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Dysregulation of protein kinases activity due to overexpression and/or mutation has been found to be implicated in various tumor types and also in cholangiocarcinoma (CCA), the malignant tumor arising from bile duct epithelia which is the common cancer and a major public health problem in northeast Thailand. This study aims to identify the activated kinases which are involved in CCA development in order to use as the drug target in particular cancer. Using human phospho-RTKs and human phospho-kinases array analysis demonstrated that multiple RTK and protein kinase signaling pathways are activated in both CCA cell lines and CCA tissues including MAPK kinase, PI-3K/Akt, Wnt/b-catenin, JAKs/STAT, PKA and PKC signaling pathways whereas, activated RTKs which related to tumor angiogenesis such as VEGFR, FGFR and Tie found only in CCA tissues. We also have tested a panel of small molecule protein kinase inhibitors including those FDA approved, clinical and pre-clinical drugs. The multi-targeted protein kinases inhibitor, sorafenib and sunitinib seems to be the most effective drug for inhibiting CCA cells growth due to the lowest IC50 observed. The present work indicating that targeting multiple kinases which are activated in CCA would be benefit for particular tumor therapy. Although, the molecular mechanism by which those drugs inhibiting cell growth as well as efficiency of drugs in vivo will be further investigated.





THE FUNCTIONAL CHARACTERISATION OF HUMAN RMND5 PROTEINS IN PROSTATE CANCER

Louw A.1, 2, Dawson L.1, Harvey J.2 and Bentel J.1, 2 1Anatomical Pathology, Royal Perth Hospital. 2University of Western Australia, Perth, Western Australia.

Human RMND5A and RMND5B are hypothetical proteins named after their yeast orthologue, RMD5 (required for meiotic nuclear division), an E3 ubiquitin ligase and component of the yeast Gid/Vid30c complex. RMND5A and RMND5B contain a RING finger-like domain and LisH, CTLH and CT-11 RanBPM (CRA) domains, the functions of which are uncharacterised. In particular, E3 ubiquitin ligase activity of RMND5A or RMND5B has not been demonstrated. RMND5A and RMND5B are expressed in prostate cancer cell lines and are localised in the nucleus and cytoplasm following transfection into these cells. In addition, each of the human orthologues of Gid/Vid30c components, RanBPM, muskelin, Twa1, EMLP, ARMC8α and RMND5A, are expressed in prostate cancer cell lines, providing evidence that the human Gid/Vid30c complex orthologue, the CTLH complex, is able to form. In LNCaP prostate cancer cells, both RMND5A and RMND5B interact with RanBPM, whose yeast orthologue is a proposed core component of yeast Gid/Vid30c, further supporting formation of a human CTLH (Gid/Vid30c) complex and potentially indicating that CTLH complexes can contain either (or both) RMND5A, as previously reported, and the highly homologous family member, RMND5B. RMND5A, RMND5B and RanBPM are each localised in the nucleus and cytoplasm of prostate cancer cells with extensive co-localisation in both cell compartments. While the biological activity of RMND5 proteins is currently unknown, RMND5B interacts with the prostatic tumour suppressor, NKX3.1, enhancing its transcriptional repressor activity. Furthermore, the RMND5B gene is located within a prostate cancer susceptibility locus and disruption of RMND5A and RMND5B expression is detected in a variety of cancers, indicative of important cellular functions of these highly conserved and ubiquitously expressed proteins.

CHARACTERISATION OF HUMAN ACETYL COA CARBOXYLASE -A TARGET FOR OBESITY THERAPEUTICS

Macaulay S.L.1, Castelli L.A.1, Jarvis K.1, Liepa A.J.1, Fernley R.T.1, Pilling P.1, Epa V.C.1 and Kemp B.E.1, 2 1CSIRO Preventative Health Flagship, Parkville, Vic. 2St Vincents Institute, Fitzroy, Vic.

Acetyl CoA carboxylase (ACC) 1 and 2 are required for fatty acid synthesis and regulation of fatty acid oxidation respectively. Both are potential therapeutic targets for the treatment of obesity and type 2 diabetes. Difficulties in expression of full length proteins have hampered investigation of the enzymes as targets. Two recent reports described the expression of ACC1 and truncated ACC2 (Kim et al 2007, Cheng et al 2007). Here we describe the expression of full length human ACC2 and ACC1 with a C-terminal FLAG tag in insect cells and inhibitors of enzyme activity. Both proteins were active and had a Km of 12-15 μM for acetyl CoA. The yield of protein was modest, 0.2mg/L. The proteins were inhibited by low molecular weight compounds previously studied as potential herbicides, with the best of the inhibitors having IC50 of 200nM. To further investigate inhibition by related compounds, FLAG tagged carboxyltransferase (CT) domain was expressed and purified from insect cells. This protein expressed at higher levels than the full length enzyme (25mg/L). Activity of this domain was inhibited by the same compounds that inhibited the full length enzyme. Crystallisation trials of this domain are in progress together with medicinal chemistry around the inhibitors. Structure of a similar construct of yeast CT domain was obtained at 3.0Å with inhibitor and is being refined. The inhibitors stimulated fatty acid oxidation in L6 muscle cells, and, in high fat fed mice, decreased respiratory exchange ratio and improved glucose tolerance confirming them as potential leads for therapeutic development.

OSTEOPONTIN IS REQUIRED FOR NORMAL SKELETAL MUSCLE DEGENERATION AND REGENERATION

Uaesoontrachoonm K., Tudor E.M., Pagel C.N. and Mackie E.J. School of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia.

Osteopontin is an extracellular matrix glycoprotein which is expressed in muscle and regulates myoblast behaviour. To investigate the role of osteopontin in muscle regeneration, we utilized the method of whole muscle grafting in osteopontin-null and wildtype mice; the extensor digitorum longus (EDL) muscle was removed from and then returned to its bed, causing vascular disruption and muscle degeneration, followed by muscle regeneration. Osteopontin expression as detected by immunohistochemistry was strongly up-regulated in wildtype EDL 3 and 5 days after grafting but not at later time points. Three days after grafting, in wildtype mice the diameter (determined as minimum Ferret’s diameter) of the whole EDL and of individual muscle fibres was significantly smaller in grafted than in sham-operated muscles; in osteopontin-null mice, there was no significant difference in either parameter between graft and sham. There were significantly fewer muscle fibres 3 days after grafting in wildtype compared to osteopontin-null muscles; the fibre number increased from day 3 in wildtype grafts, whereas a decrease in fibre number was observed up to day 7 in grafted osteopontin-null muscles. There were significantly fewer centrally nucleated muscle fibres, a sign of regeneration, in osteopontin-null than in wildtype grafts up to 20 days after grafting. There were fewer neutrophils at day 3 and fewer macrophages at days 3 and 5 in osteopontin-null grafts than in WT grafts. These results suggest that degeneration of muscle fibres is delayed in the absence of osteopontin, which can be attributed in part to a lack of phagocytosis resulting from reduced infiltration of osteopontin-null grafts by macrophages and neutrophils. Furthermore, these observations indicate that osteopontin is important in the regulation of the early stages of muscle regeneration.

FINDING NEW SIGNALING PATHWAYS THAT CONTRIBUTE TO BREAST CANCER PATHOGENESIS

Majewski I.J., Bosma A., Severson T., Kerkhoven R., Velds A., Linn S.C. and Bernards R. The Netherlands Cancer Institute (NKI-AVL).

Breast cancer is a heterogeneous disease. Our work focuses on two very different types of breast cancer, invasive lobular carcinoma (ILC) and triple negative breast cancer (TN), which together represent 25% of all breast cancers. TN tumors lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and Her2 (Erbb2), greatly restricting treatment options. Lobular breast cancers are generally ER+ and can be treated with hormonal therapies; however, these tumors often reoccur and have a poor outcome. New therapies are desperately required for the treatment of these cancers. Kinases are important mediators of cell signaling and are generally considered to be good therapeutic targets. We have developed an exon capture system for high-throughput mutation analysis that will allow us to identify kinases that are mutated in ILC and TN breast cancer. The kinome sequencing technology will be validated in cell line models, but we aim to expand this work to survey several hundred cancers from patients with ILC and TN breast cancer.





CLUSTERIN (APO J) AND COMMD1: CHAPERONES THAT FACILITATE THE DEGRADATION OF THE MAMMALIAN COPPER TRANSPORTERS, ATP7A AND ATP7B

Materia S.1, Cater M.2, Klomp L.3, Mercer J.1 and La Fontaine S.1 1School of Life and Environmental Sciences, Deakin University, Burwood, Victoria, 3125, Australia. 2The Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, 3002, Australia. 3Laboratory of Metabolic and Endocrine Diseases, University Medical Center, Utrecht, The Netherlands.

Copper is essential for human health and is important in the aetiology and pathology of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and prion diseases, in addition to its well-established role in the genetically inherited disorders, Menkes and Wilson diseases. The two mammalian copper-transporting P-type ATPases, ATP7A and ATP7B regulate the copper status of the body. They have dual functions that consist of copper transport into the secretory pathway for incorporation into essential enzymes, and relocation to the cell periphery at high copper levels to rid the cell of excess copper. Clusterin (Apo J) and COMMD1 are two proteins that interact with the Cu-ATPases and their misfolded variants to facilitate their degradation. Clusterin has properties similar to the small heat shock proteins with chaperone-like activities, binding to stressed, partially unfolded proteins. ATP7B is predominantly degraded by the lysosomal pathway and Clusterin overexpression facilitated its degradation. Partial degradation of ATP7B by the proteasomal pathway involves COMMD1. Clusterin and COMMD1 overexpression and knockdown affected Cu-ATPase expression levels and cellular copper levels as determined by a copper-sensing luciferase reporter assay. It is emerging that both Clusterin and COMMD1 are key components required for Cu-ATPase quality control, which in turn is necessary for the maintenance of normal copper homeostasis.

THE AGILENT TECHNOLOGIES’ SURESELECT TARGET ENRICHMENT SYSTEM FOR NEXT-GENERATION SEQUENCING DEMONSTRATES HIGH PERFORMANCE AND ENABLES DIVERSE APPLICATIONS

Roberts D.1, Happe S.2, McInnes R.3, Pabon C.1, Giuffre A.2, Novak B.1, Visitacion M.1, Joshi S.2, Ong J.2 and Leproust E.1 1Agilent Technologies, Santa Clara, California. 2Agilent Technologies, Cedar Creek, Texas. 3Agilent Technologies, Melbourne, Victoria.

The discovery of rare polymorphisms, structural variants, and novel transcripts has been accelerated dramatically by next-generation sequencing technologies. However, it remains cost-prohibitive to sequence entire genomes in large cohort studies. To enable larger sample size, Agilent Technologies has developed the SureSelect platform, allowing focused analyses on specific genomic loci at considerable cost savings. Agilent is continuing to expand its portfolio to increase the number of applications available to users. First, we demonstrate high performance across Illumina, SOLiD, and 454 platforms, as measured by capture efficiency, uniformity, reproducibility, and SNP detection. Custom design across all platforms and multiple model organisms is simplified using a desktop version of the eArray software. Next, we create multiple unique SureSelect panels such as sequences encoding the human kinome and whole exome to highlight flexibility across a wide range of target size and complexity. These catalog designs enable standardized studies across multiple research sites. Third, for smaller targeted regions, we demonstrate the ability to index samples in a single lane, thereby further decreasing the cost of large studies. For more comprehensive genome analysis, we reveal expanded utility and scope of the All-Exon catalog product by increasing read depth uniformity and target coverage to include regulatory regions, small RNAs, and content from multiple databases including CCDS and RefSeq. SureSelect applications will continue to expand as users harness the power of next-generation sequencing.

CANINE MAMMARY TUMOURS: A MODEL FOR HUMAN BREAST CANCER?

Milley K.M.1, Toribio R.2, Slavin J.3, Scott-Dowell K.4, Papadopoulos R.1, Richardson S.J.1 and Danks J.A.1 1School of Medical Sciences, RMIT University, Bundoora 3083, Australia. 2School of Veterinary Medicine, Ohio State University, Columbus, USA. 3St. Vincent’s Hospital, Fitzroy 3065, Australia. 4Gribbles Pathology, Heidleberg 3084, Australia.

Canine mammary tumours (CMTs) have been suggested as a model for human breast cancer (HMC) since the 1970s. This assertion was based on the histopathology of CMTs compared with HMC. In addition, CMTs mimic HMC in regards to hormone dependence, age of onset, tumour size and cell kinetics. Unlike other animal models, dogs live in a natural environment and are exposed to the same carcinogens as humans. To date utilization of CMTs for research has lagged behind other animal models such as mice and rats. Sequencing of the canine genome in 2005 has provided renewed impetus for comparative research between humans and dogs. Our research focused on demonstrating the application of the HMC molecular sub-typing classification system in 30 benign and malignant CMTs using immunohistochemistry. In addition, we have localized other commonly used tumour markers such as oestrogen receptor (ER), Ki-67, E-cadherin and β-catenin. The variation in staining of these markers between different subtypes and histological classifications was then compared. Both benign and malignant CMTs demonstrated similar staining patterns to the tumour types found in humans. In particular the reduction of staining both E-cadherin and β-catenin in malignant tumours compared to benign tumours. The increased proliferation of malignant tumours was demonstrated by the higher Ki-67 indices. The results of this research further strengthen the hypothesis that CMTs are a good model for HMC. In particular, that the progression of both the human and dog disease follow a similar course potentially advocating CMTs as a model for HMC.

INTERACTION BETWEEN THE ATAXIA TELANGIECTASIA MUTATED (ATM) AND TUBEROUS SCLEROSIS COMPLEX (TSC) PATHWAYS

Newman J.V.1, Crampton E.M.1, Kozlov S.2 and Watters D.1 1ESKITIS Institute for Cell and Molecular Therapies and School of Biomolecular and Physical Sciences, Griffith University, Nathan Campus, Kessels Rd. Brisbane, Australia, 4111. 2Queensland Institute of Medical Research, Herston Rd. Brisbane, Australia, 4029.

Ataxia Telangiectasia is a progressive neurodegenerative disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. Nuclear ATM has a well established role in response to DNA damage, however ATM has also been localized outside the nucleus where it has been demonstrated to participate in the insulin signalling pathway by phosphorylating eIF-4E binding protein(4EBP1). 4EBP1 is a target of Mammalian target of Rapamycin (mTOR) and its phosphorylation releases it from eIF-4E enabling translation of mRNA and protein synthesis. The Tuberous Sclerosis Complex (TSC) proteins, hamartin (TSC1) and tuberin (TSC2) act as a heterodimer to regulate mTOR activity. mTOR exists as two complexes, mTORC1 (rapamycin sensitive) and mTORC2 (insensitive to short term rapamycin). These complexes control many cellular functions including protein synthesis, autophagy, lipid metabolism, mitochondrial biogenesis and cytoskeletal organisation. Mutations in either of the TSC1 or TSC2 genes lead to Tuberous Sclerosis, an autosomal dominant, multisystem disorder of benign tumour growth and neurological abnormalities. Studies in our laboratory have demonstrated for the first time that ATM interacts with both tuberin and hamartin. The effects of ATM deficiency on the mTOR pathway under different growth conditions and stresses will be described.





THE VITAMIN E ANALOGUE γ-TOCOTRIENOL BLOCKS OSTEOCALSTOGENESIS AND PROMOTES OSTEOBLAST MATURATIONOdysseos A.D.1, 2, Liapis V.3, Hay S.3, Christou Y.A.1, 2, Maltezou E.2, Keramidas A.2 and Evdokiou A.3 1EPOS-IASIS, R&D, Nicosia, Cyprus. 2University of Cyprus, Nicosia, Cyprus. 3University of Adelaide, Royal Adelaide Hospital, Adelaide, Australia.

In this study we investigated the effects of the vitamin E analogue, γ-Tocotrienol on (a) apoptosis induction in bone metastatic cells and (b) osteoclast differentiation and bone resorptive activity using three independent in vitro-model systems of osteoclastogenesis. When human peripheral blood mononuclear cells (PBMCs) and the RAW264.7 murine monocytic cell line were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), both formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and bone resorption were increased. In combination with RANKL, γ-Tocotrienol dose-dependently inhibited RANKL-induced osteoclastic differentiation and bone resorption in both cell models. Similarly, it inhibited the bone resorptive activity of mature osteoclasts that were isolated from human Giant Cell Tumours (GCT) of bone when cultured on dentine slices. RANKL activated osteoclastogenesis essential pathways NFKB, ERK1/2 and p38/MAPK, whereas γ-Tocotrienol profoundly inhibited RAKNL-induced activation of NFKB and P38/MAPK but not ERK1/ERK2. The effect of γ-tocotrienol on osteoblast function was investigated in mineralized bone nodule-forming primary human osteoblast cultures. γ-Tocotrienol progressively increased matrix-containing mineralized nodules in osteoblast cultures when compared to untreated cells with a concomitant increase in alkaline phosphatase activity. Comparable concentrations of γ-Tocotrienol induced profound caspase-independent apoptosis in highly metastatic osteolytic and osteoblastic breast and prostate cancer cell lines, respectively. Taken together these results demonstrate for the first time that γ-Tocotrienol has a combined inhibitory effect on osteoclast activity and on survival of bone metastatic cancer cells whereas it significantly promotes osteoblast activity serving as promising treatment for osteolytic bone disease.

MUTATIONS IN THE BHLH/PAS TRANSCRIPTION FACTOR SIM1 MAY UNDERPIN A NOVEL MONOGENIC OBESITY DISORDER

Raimondo A.1, Ramachandrappa S.2, Farooqi S.2 and Whitelaw M.L.1 1School of Molecular and Biomedical Science, University of Adelaide, Adelaide, AUSTRALIA. 2Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK.

Single-Minded 1 (SIM1) is a member of the basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) family of transcriptional regulators that possess diverse roles in development and homeostasis. Members of this family heterodimerise with a shared partner protein, such as ARNT1 or ARNT2, to regulate expression of their direct target genes. Sim1-/- mice die perinatally due to hypodevelopment of hypothalamic neuroendocrine populations critical for appropriate feeding control. Sim1+/- mice, however, survive to adulthood and display a severe early onset obesity phenotype characterised by hyperphagia, hyperinsulinemia and hyperleptinemia, without any decrease in energy expenditure. Similar characteristics have been reported in a number of human clinical cases involving translocations or deletions within the SIM1 locus. We have begun functional analysis of sixteen SIM1 point mutations recently identified in the UK Genetics of Obesity Study (GOOS). These mutations are over-represented within the GOOS cohort relative to control individuals, and are associated with severe early onset obesity (<10 years old) with a BMI standard deviation score >3. Several of these mutations reproducibly result in decreased SIM1 functionality in dual luciferase reporter assay experiments, and do not appear to do so by altering protein stability. We are therefore pursuing other experimental lines of evidence to determine whether these mutants are compromised in their ability to dimerise with ARNT1 and ARNT2, and/or regulate expression of endogenous SIM1 target genes. In this way, we hope to highlight the molecular basis of a novel, heritable form of severe monogenic obesity, which may facilitate the development of effective treatments in patients associated with SIM1 mutations.

A RANDOM shRNA SCREENING STRATEGY TO IDENTIFY NOVEL DRUG TARGETS

Ramadas R.D.1, Dawes I.W.1 and Arndt G.M.2 1School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney 2052, Australia. 2Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Sydney 2052, Australia.

The main goal of the project is to devise a strategy to use a random small hairpin RNA (shRNA) library to identify novel targets to study disease mechanisms. This strategy can be used to identify modulators of chemotherapeutic resistance, as well as to pinpoint key factors involved in a variety of diseases such as viral infection. A random shRNA library was constructed using an optimized enzymatic method, which has as its starting point an oligonucleotide incorporating 19 random nucleotides. The shRNA library was cloned into a doxycycline-dependent retroviral vector, and transduced into HEK293 cells by calcium phosphate transfection to establish a pooled population of cells containing individual members of the library. The random shRNA library was successfully constructed after several modifications to increase the amount of shRNA-encoding DNA inserts and consequently more clones with greater representation. The doxycycline–dependent system was characterized using a luciferase gene target, and associated shRNA, and the most effective transduction conditions optimized. The selected population of cells were then treated independently with three common chemotherapeutic agents: cisplatin, carboplatin and paclitaxel. Cells displaying resistance to these agents (surviving cells after shRNA induction by doxycycline addition) were enriched and studies to characterize the resident constructs are currently underway. It is hoped that using a random shRNA library will aid in the identification of novel factors that may be important in overcoming cancer drug resistance. This strategy will also be expanded to identify previously unknown factors contributing to viral infection.

CRITICAL ROLE OF THE HEPATIC GLUCOCORTICOID RECEPTOR IN SYSTEMIC BILE SALT HOMEOSTASIS WHICH IMPACTS UPON CHOLESTEROL-GALLSTONE DISEASE

Rose A.J., Berriel Díaz M., Reimann A. and Herzig S. Molecular Metabolic Control Group, German Cancer Research Center, Heidelberg, Germany.

Normal physiological bile salt handling in the fast-feeding cycle is essential for proper digestion of ingested fat and impacts upon systemic energy homeostasis. On the other hand, aberrant regulation of hepatic bile salt flux can lead to cholesterol-gallstone disease. Our preliminary data suggested that the glucocorticoid receptor (GR) can regulate genes involved in hepatic cholesterol and bile salt metabolism. However, it remained unclear whether hepatic GR action exerts a regulatory function for systemic bile salt handling in vivo. Short-term liver-specific GR knockdown resulted in higher serum bile salt levels as well as lower gallbladder bile volume and cholesterol solubility, especially when mice were fed a lithogenic diet. In addition, when long-term hepatocyte-specific GR knockdown was imposed on gallstone susceptible mice and these mice were fed a lithogenic diet, there was a higher incidence of cholesterol gallstones. Furthermore, the aberrant bile salt homeostasis was recapitulated in mice with deficient GR DNA binding activity suggesting that it is the direct genomic actions of the GR that modulate transcriptional events of key genes in the liver that lead to this phenotype. Overall, using several different models of liver GR deficiency, we show that reduction of hepatic GR action results in aberrant bile salt handling and lower gallbladder bile cholesterol solubility. Together, our studies establish the GR signalling axis as a critical regulatory checkpoint for bile salt homeostasis and the susceptibility for cholesterol-gallstone disease. We are currently investigating the transcriptional mechanisms by which the GR affects liver bile salt handling.





INDUCTION OF METALLOTHIONEIN IN CULTURED HUMAN KERATINOCYTES BY ZINC: DETECTION WITH A COMMERCIALLY AVAILABLE MONOCLONAL ANTIBODY

Ross S. and Gray P.J. Human Protection & Performance Division, DSTO Australia.

Metallothionein is a low molecular weight protein of around 6,000 Daltons, which has a high capacity for binding metal cations. It is expressed in bacteria, eukaryotes and plants. It exists as several isoforms, each being 60 - 63 amino acids long; 20 of these are cysteines. It is through the thiol groups of these cysteines that the protein can bind multiple cations (up to seven) of various metals, including zinc, copper, cadmium and mercury. Metallothionein is thought to have an antioxidant function. There is also evidence for it as a factor contributing to resistance of some tumour types to alkylating chemotherapy agents. This protective function appears to be more complex than simply acting as a sequesterer of nucleophilic species. Increased levels of intracellular metallothionein in a normal cell type was proposed as a potential pre-exposure countermeasure to the vesicant agent sulphur mustard. This chemical agent is a potent alkylating agent, similar in action to alkylating chemotherapy drugs - therefore, increased intracellular metallothionein in a target cell population might improve resistance to mustard toxicity. A direct ELISA assay for metallothionein in cell extracts was developed to monitor intracellular expression. Protein pre-loading with free metallothionein proved difficult - however, incubation with zinc (as chloride) induced an increase in intracellular metallothionein in cultures of normal human keratinocytes. Zinc chloride has a cytotoxic effect on keratinocytes at concentrations >50μuM; however, at 20 - 40μuM, increased expression of metallothionein of to 14-fold increase compared to control levels over seven days of incubation was achieved. Pulse-chase experiments showed that intracellular levels of metallothionein returned to baseline after zinc incubation was stopped.

PHARMACOLOGICAL SCREENING FOR THE THERAPY OF FRIEDREICH ATAXIA

Li L.1, Van Den Hengel S.2, Cook J.1, Sandi C.4, Pook M.4, Ioannou P.A.2, 3, Delatycki M.B.1, 3 and Sarsero J.P.1, 2, 3 1Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. 2Cell and Gene Therapy Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. 3Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. 4Hereditary Ataxia Research Group, Centre for Cell and Chromosome Biology, School of Health Sciences and Social Care, Brunel University, Uxbridge UB8 3PH, United Kingdom.

Friedreich ataxia (FRDA) is characterised by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein, frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological upregulation of FXN expression may restore frataxin to therapeutic levels in patients. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we have established a genomic reporter assay consisting of stable HeLa cells containing an FXN-EGFP fusion construct (in-frame fusion of the EGFP gene with the entire normal human genomic FXN locus on a BAC clone). The cell line was used to established a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library of FDA-approved compounds and natural extracts has been screened and analysed. Compound hits identified by HTS have been further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels have been measured in cell lines derived from individuals with FRDA and in a GAA expansion mouse model of FRDA. Any compound that specifically increases frataxin levels by several-fold in FRDA patients could serve as a potential pharmacological therapy for Friedreich ataxia.

AN ONCOGENOMICS-BASED IN VIVO CDNA SCREEN PINPOINTS CO-AMPLIFIED CCND1 AND FGF19 AS THERAPEUTIC TARGETS FOR LIVER CANCER

Sawey E.T.1, Cai C.1, Bakleh A.1, Liu Q.1, French D.M.2, Finn R.S.3, Lowe S.W.1 and Powers S.1 1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY USA 11724. 2Department of Pathology, Genentech Incorporated, South San Francisco, CA USA 94080. 3Department of Medicine, Geffen School of Medicine, University of California, Los Angeles, CA USA 90095.

Liver cancer is a leading cause of cancer death worldwide. Although several risk factors for liver cancer are known, the exact way in which these factors cause normal liver cells to become cancerous is only partially understood, making this one of the most difficult-to-treat cancers. We aimed to establish a screen to identify genes that promote liver tumorigenesis in order to better understand the molecular basis of liver cancer. Using array CGH analysis of hepatocellular carcinomas (HCCs), we identified genes within focal amplicons and introduced corresponding cDNA clones into sensitized liver progenitor cells. We discovered 18 genes that promote tumor formation, including 10 novel oncogenes as well as CCND1 and its neighbor FGF19. These latter two genes, which are co-amplified in 15% of HCCs, cooperated in malignant transformation and formed a feed-forward network whereby Fgf19 signaling increases CyclinD1 expression. RNAi-mediated inhibition of CCND1 or FGF19 inhibited clonogenicity and tumorigenicity of amplified HCC cells, while having no phenotypic effect in non-amplified cells. Amplification of CCND1/FGF19 also predicted response to a neutralizing monoclonal antibody directed against Fgf19. The result of this work has been the identification of novel oncogenes in HCC. In addition, these results show that gene amplification predicts response to Fgf19 inhibition, and that oncogenomic cDNA screens are an effective new tool to identify therapeutic strategies that are directly linked to cancer genotypes.

ROLE OF EXOGENOUS ADENOSINE IN CARDIAC REPERFUSION INJURY UNDER HIGH GLUCOSE STRESS

Sermsuvitayawong K.1 and Wilairat P.2 1Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University. 2Department of Biochemistry, Faculty of Science, Mahidol University.

The role of exogenous adenosine (0.5-1.0 mM) in regulating the protective pathway in ischemic reperfusion injury remains unclear. Rat cardiac myoblast H9C2 cells following 21 hr of hypoxia (1% oxygen) and 3hr of normoxic condition in the presence of 25 mM glucose and 1 mM adenosine in DMEM demonstrated elevated apoptosis through up-regulation of pro-apoptotic Bnip3 but not Bax and Nix compared to normoxic condition (with high glucose but without the addition of adenosine), a phenomenon not observed with 0.1 mM adenosine. These results indicate that using high concentration of exogenous adenosine in protecting against ischemic reperfusion injury may be contra-indicated. Keywords: Reperfusion injury, high glucose, adenosine, Bnip3.





PENTACYCLIC TRITERPENOID LANTADENE A: ANTITUMOR ACTIVITY AND EXPRESSION OF TRANSCRIPTION FACTORS

Sharma M.1 and Sharma N.2 1Jaypee University of Information Technology. 2Maharaja Singh College.

Lantadene A (LA, 22β-angeloyloxy-3-oxoolean-12-en-28-oic acid) a pentacyclic triterpenoid isolated from leaves of obnoxious weed Lantana camara L. was evaluated for its antitumor activity. LA showed apoptosis in human leukemia HL-60 cell line. Typical morphological changes including cell shrinkage, chromatin condensation and characteristic DNA ladder formation in agarose gel electrophoresis were observed. LA induced marked concentration and time dependant inhibition of cancer cell proliferation with IC50 value of 19.8 ± 0.10 &956;g/ml following 48h incubation. Flow cytometric analysis showed suppressed cell proliferation associated with cell cycle arrest in the G0/G1 phase. LA significantly inhibited cell proliferation of HL-60 cells and induced cell apoptosis through down regulating Bcl-2 and up regulating Bax expression. The peptidic caspase-3 inhibitors DEVD-CHO (NH2-Asp-Glu-Val-Asp-CHO, 2µM), increased the viability of HL-60 cells, previously treated with LA. The LA was further evaluated for its tumor suppression potential in DMBA/TPA induced squamous cell carcinogenesis in Swiss albino mice. A significant decrease in the incidence of number of lesions in mice was observed in LA treated groups as compared to DMBA/TPA alone. The RNA of tumors were isolated, after 20 weeks study and evaluated for various signaling factors like p53, p65 and c-jun. Significant increase in the protein levels of c-jun, p65, and p53 by ELISA were observed in DMBA/TPA treated mice tumors whereas less expression was observed in LA treated tumors. Immunohistochemical localization of transcription factors was studied which also showed less localization of c-jun, p65, and p53 in LA treated tumors as compared to localization in DMBA/TPA treated tumors.

CHARACTERISATION OF BCHE POLYMORPHISMS IN SMALL AUSTRALIAN DEFENCE FORCE COHORT

Shields K.A. and Lewis J. Defence Science and Technology Organisation, 506 Lorimer St. Fishermans Bend, Vic 3207, Australia.

There is a wide range of chemicals and drugs that interact with the cholinergic nervous system including: nerve agents, nerve agent pre-treatments, insecticides and therapeutic drugs. Sensitivity to cholinesterase inhibitors and effective nerve agent pre-treatment rely on the activity of ACHE, BCHE and PON1, and these genes are subject to genetic variation. Genetic variations in these genes have been shown to alter the level and activity of their enzyme products and therefore effective treatment may therefore be influenced by the genotype of the individual. BCHE is a high affinity stoichiometeric scavenger of toxic organophosphate (OP) compounds that has a role in protection against OP exposure. Individuals carrying heterozygous or homozygous inactivating alleles have been shown to have a lower capacity to interact with and detoxify drugs. Our initial work has focused on characterisation of a small cohort (n=52) of Australian Defence Force (ADF) personnel. Using the LightCycler 480 system, a protocol was established using High-Resolution Melting Curve Analysis to screen the BCHE gene. Any PCR amplicons displaying altered melting profiles were subjected to direct sequencing and variants were identified. Initial screening has identified both known and novel polymorphisms and future work will involve the functional characterisation of novel variants. In summary this work provides important information in the characterisation of genetic variation in an ADF population. It is likely that functional variants unique to Australian populations exist and that these will need to be identified in order to accurately predict chemical and drug response.

CARDIOPROTECTIVE EFFECT OF VASOENDOTHELIAL GROWTH FACTOR AT MYOCARDIAL INFARCTION

Shurygin M.G., Shurygina I.A., Dremina N.N. and Kanya O.V. Scientific Center of Reconstructive and Restorative Surgery SB RAMS.

The aim is to investigate regulatory effects of vasoendothelial (VEGF) in a reparation process by an example of experimental myocardial infarction. Female Wistar rats (N=85, weight 220-250 g) at the age of 9 month were selected in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 85-23, revised 1985). The experiment was carried out in accordance with the principles of humane treatment of animals set out in the directives of the European Community. We used myocardial infarction model with diathermocoagulation of coronary artery. The study comprised two groups of animals: “VEGF” (N=40), “Control” (N=45). Animals from “VEGF” group had 100 ng VEGF164 (Sigma V3638-10UG Lot 064K1239) injection into left ventricle at a 1.5 h period after operation. We used methods of light microscopy, immunohistochemistry, morphometry. We found out that the high VEGF level extended infiltrative phase of inflammation. VEGF have increased the synthesis of collagen. The collagen volume on 30 day in “VEGF” group was higher than in “Control” group (58.56 vs 40.51%, p<0.05). At the same time, excessively high concentrations of VEGF led to the development of focal cardiosclerosis in the intact myocardium, which is a by-effect of this growth factor. VEGF provided a significant effect on angiogenesis. We discovered that the injection of intracardiac VEGF experimental animals had increased survivability of cardiomyocytes in the infarction zone. VEGF has cytoprotective effect on cardiomyocytes. Thus, VEGF is important regulators of the reparation process in a myocardial infarction. This study was supported by a grant P1250 from the Russian Federal Purposive Program “Scientific and scientific-pedagogical staff of innovation Russia”.

RECOMBINANT EXPRESSION AND CHARACTERISATION OF PROTEASE INHIBITOR-RESISTANT TRYPSINS FROM THE MIDGUT OF H. PUNCTIGERA

Stevens J.A., Dunse K.M. and Anderson M.A. Department of Biochemistry, La Trobe University, Melbourne VIC 3086.

Protease inhibitors (PIs) from the flowers of Nicotiana alata (NaPI) show promise for the control of Helicoverpa insect pests. This has been demonstrated using artificial diet bioassays and by expressing NaPI in transgenic cotton plants. PIs function by blocking the digestive proteases in the larval gut, limiting the availability of amino acids from dietary protein. As a consequence the larvae are arrested in development and eventually die. In addition some larvae adapt to the presence of serine PIs in the diet by over-production of PI-insensitive trypsins and chymotrypsins. This induction of protease expression is a common mechanism by which herbivorous insects overcome plant PI defenses. Even though trypsins are the major digestive proteases of Helicoverpa species and have been a prime target for pest control, little is known about their production, regulation and structure. We have identified a family of PI-resistant trypsins that are produced in the gut of H. punctigera after ingestion of serine PIs. Recombinant expression of these trypsins is underway to provide insights into the structural features that prevent PI inhibition. A detailed understanding of the interaction between insect trypsin and plant PIs will assist in the rational design of new inhibitors to protect crops from insect damage.





CHEMOKINE BINDING TO SULFOTYROSINE RESIDUES IN CHEMOKINE RECEPTORS

Stone M.J.1, Millard C.J.1, Clayton D.1, Zhu J.Z.2, Simpson L.S.2, Widlanski T.S.2 and Payne R.J.3 1Monash University. 2Indiana University. 3University of Sydney.

The trafficking of leukocytes to sites of inflammation is orchestrated by the interactions of chemokines with chemokine receptors. Chemokine receptors commonly contain sulfated tyrosine residues in their amino-terminal regions, which are critical for recognition of their chemokine ligands. Eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 are chemokines involved in recruitment of eosinophils, basophils and Th2 cells through activation of the receptor CCR3. The amino-terminal region of CCR3 contains two adjacent putatively sulfated tyrosine residues. In this study, we have investigated the role of tyrosine sulfation of N-terminal CCR3 peptides in controlling binding affinity and selectivity for its cognate chemokines. Peptides containing sulfotyrosine were generated by applying a novel sulfate protection strategy in Fmoc-based solid-phase peptide synthesis. We prepared four peptides containing sulfation on Tyr-16, Tyr-17, both Tyr residues, or neither Tyr residue. NMR spectroscopy was then used to determine the affinity of each peptide for each chemokine and to identify the chemokine surface interacting with each peptide. Although all three chemokines utilise the same surface for binding to CCR3-derived sulfopeptides, the energetic effect of sulfation of the two adjacent tyrosine residues was either additive or cooperative for different chemokines. The structure of a chemokine-sulfopeptide complex obtained using isotope-filtered/edited NMR experiments will be presented.

ANDROGEN REGULATION OF P27 IN BREAST CANCER CELLS

Tay J.W.1, 2, Azzam D.G.1, 2, Harvey J.2 and Bentel J.M.1 1Department of Anatomical Pathology, Royal Perth Hospital. 2School of Pathology and Laboratory Medicine, University of Western Australia.

p27, an important cell cycle inhibitor that regulates the G1/S checkpoint is frequently deregulated in breast cancers where its expression is downregulated or the protein is mislocalised to the cytoplasm. Treatment of MCF-7 breast cancer cells with the androgen, 5α-dihydrotestosterone (DHT) inhibits cell proliferation in association with increased p27 levels. Approximately 30% of breast cancers exhibit increased activity of kinase signalling pathways and are resistant to conventional therapies, however DHT treatment of stably transfected MCF-7 cells (Δ6B) with elevated extracellular signal-regulated kinase (ERK1/2) activity inhibits proliferation and increases p27 levels. The function of p27 as a cell cycle inhibitor is dependent on its localisation in the nucleus, and the intracellular localisation of p27 is regulated via its phosphorylation by kinases such as AKT and ERK1/2. DHT treatment of the androgen receptor (AR) expressing breast cancer cell lines, MCF-7, MT3, Δ6B, T47-D, BT-474 and MDA-MB-453 results in the nuclear accumulation of both endogenous p27 and transiently transfected GFP-p27. Increased cytoplasmic GFP-p27 is observed in Δ6B and BT474 cells with high ERK1/2 activity, potentially as a direct or indirect consequence of ERK1/2 phosphorylation of p27, however DHT treatment also enhances p27 nuclear localisation in these cells. These findings indicate that in addition to increasing p27 protein levels, AR activation promotes the nuclear accumulation of p27 to initiate the inhibition of cell proliferation. Subcellular fractionation and MALDI-TOF analysis of p27 in breast cancer cells with normal or hyperactivated ERK1/2 signalling and following DHT treatment will identify novel phosphoresidues that direct p27 nuclear localisation and its cell cycle inhibitory function, thereby identifying potential therapeutic targets for human breast tumours.

MARCKS EXPRESSION DURING INFLAMMATORY PROCESS IN LIVER FLUKE-ASSOCIATED CHOLANGIOCARCINOMA

Techasen A.1, 2, Loilome W.1, 2, Namwat N.1, 2, Thanan R.1, 2 and Yongvanit P.1, 2 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated in PKC-mediated membrane-cytoskeletal alterations which underlines such LPS-induced macrophages responses. Our previous study found that MARCKS was significantly overexpressed in CCA and inflammatory cells. It is therefore postulated that MARCKS may involve in inflammation-associated CCA. The aims of this study are to investigate localization pattern of MARCKS in hamster tissue during cholangiocarcinogenesis and examine the involvement of MARCKS in inflammation by determining the expression and localization in Raw 264.7 macrophage cell line. The semi-quantitative real time RT-PCR and immunofluorescence analysis were performed to determine MARCKS mRNA level and protein localization in hamster tissues at different time points after administrationwith either liver fluke (Opisthorchis viverrini, Ov) or N-nitrosodimethyamine (NDMA) or both. The expression of MARCKS induced by inflammation was also confirmed by incubating Raw 264.7 macrophage cell line with LPS or crude Ov antigens. Moreover, co-expresssion between MARCKS and MAC387 which is a marker for macrophage was also investigated in human CCA tissues. Our study showed the moderate to strong expression of MARCKS in inflammatory cells at early stage and also in bile duct cancer at the end stage of cholangiocarcinogenesis in hamster. In addition, MARCKS mRNA and protein levels were also induced by stimulation of LPS or Ov crude extract as a dose- and time- dependent manner in Raw 264.7 macrophage cell line. Moreover, the co-expression between MARCKS and MAC387 was observed in human CCA tissues. This finding can be concluded that MARCKS was associated with inflammation-related CCA induced by Ov. Further study on functional analysis of MARCKS is under investigation.

CONSTRAINTS WITHIN MAJOR-HISTOCOMPATIBILITY COMPLEX CLASS I –RESTRICTED PEPTIDES: PRESENTATION AND CONSEQUENCES FOR T-CELL RECOGNITION

Theodossis A.1, Guillonneau C.2, Welland A.1, Ely L.K., Clements C.S.1, Williamson N.A., Webb A.I.3, Doherty P.C.2, McCluskey J.2, Purcell A.W.3, Turner S.J.2 and Rossjohn J.1 1The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria 3800, Australia. 2Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia. 3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.

Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of “independent pegs” that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of αβ-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many self- and viral-peptides show extensive and direct inter-residue interactions, an unexpected finding that has led us to the idea of “constrained” peptides. Mutational analysis of two constrained peptides (the HLA B44-restricted self-peptide (B44DPα - EEFGRAFSF) and an H2-Db-restricted influenza peptide (DbPA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLENFRAYV → SSLENARAYV) within the constrained PA peptide resulted in a diminished cytotoxic T-lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between individual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, while the perturbation of such constraints can lead to previously unappreciated mechanism of viral escape.





EVALUATION OF PROSTATE CANCER ANTIGEN-1 AS A MOLECULAR TARGET FOR PROSTATE CANCER AND PANCREATIC CANCER

Tsujikawa K.1, Koike K.1, Hase T.1, Kitae K.1, Ueda Y.1, Shimada K.2, Syo M.2, Konishi N.2, Nakajima Y.2 and Yamamoto H.1 1Grad. Sch. Pharm. Sci., Osaka University, Osaka 565-0871, Japan. 2Nara Medical University, Nara 634-8521, Japan.

We found a novel gene prostate cancer antigen (PCA)-1 that was highly expressed in prostate carcinoma cells but not in the adjacent normal tissues. Immunohistochemical analysis of prostate carcinoma specimens showed that PCA-1 was strongly expressed in prostate cancer lesions, including preneoplastic lesions, but there was little or no expression in normal epithelium or benign prostatic hyperplasia. Recently, high expression of PCA-1 was also detected in pancreatic adenocarcinoma specimens. To evaluate PCA-1 as a molecular target for these cancers, therefore, we knocked down the PCA-1 gene in both prostate cancer cell lines (DU145 and PC3) and pancreatic cancer cell lines (Punc-1 and MIA-Paca-2) by using the RNA interference technique. Transfection of PCA-1 small interfering RNAs suppressed PCA-1 mRNA expression and then inhibited proliferation of these cancer cells by apoptosis. Colony formation in soft agar was significantly inhibited in DU145 and PC3 cells in which PCA-1 was stably knocked down. Real-time PCR analysis indicated that PCA-1 knockdown suppressed the expression of XIAP and urokinase-type plasminogen activator in these cell lines. Moreover, immunoblot analysis indicated that PCA-1 knockdown induced dephosphorylation of Akt in the prostate cancer cell lines, suggesting that PCA-1 is an important regulatory molecule in the Akt signaling pathway. Furthermore, the administration of PCA-1 siRNA markedly attenuated tumor formed by DU145 cells and Panc-1 cells in nude mouse xenograft in vivo. These results indicate that PCA-1 may be a promising molecular target for prostate cancer as well as pancreatic cancer.

BIOMARKERS DISCOVERY IN THE NEOADJUVANT BREAST CANCER TREATMENT

Tvrdik D.1, Povysil C.1, Dundr P.1, Velenska Z.1, Melcakova S.1, Skalova H.1 and Petruzelka L.2 1Institute of Pathology, 1st Faculty of Medicine, Charles University and General University Hospital in Prague. 2Department of Oncology, 1st Faculty of Medicine, Charles University and General University Hospital in Prague.

Neoadjuvant chemotherapy, also called preoperative chemotherapy, is now widely used in the treatment of breast carcinoma. It has several potential advantages compared to the traditional strategy of surgery followed by adjuvant chemotherapy. Neoadjuvant chemotherapy substantially reduces the size of the primary tumor and lymph node metastasis in more then 80% of cases, increasing the probability that breast-conserving surgery can be performed instead of mastectomy. The study is aimed at investigation of the biomarkers in predicting pathologic response to the therapy and include 10 patients with histologically proven invasive breast cancer. Transcriptional profile of the biomarkers were determined by DNA microarray technology with biochips including 168 human genes associated with apoptosis and multidrug resistance. The quantification of the changes in expression of individual genes due to the treatment was performed by qRT-PCR. In each case, analysis of transcription profile was performed before and after the treatment, respectively. In good prognosis group, based on pathological features and clinical outcome, the analysis showed that the expression of FAS, caspase 14 and DFFA were significantly up-regulated at the RNA level after the treatment. In contrast, the expression of BCL2-related gene MCL1 was down-regulated due to the treatment. On the other hand, in poor prognosis group, we found over-expression of ABCC gene. As we have shown, gene expression profiling using microarray or Real-Time PCR assay is a valuable research tool for investigation of molecular markers and prognostic factors which may better reflect tumor biology and treatment response. This work was supported by Grant IGA MZ CR No. NS10575-3/2009.

LUNG METASTATIC CHOLANGIOCARCINOMA CELL LINE ACQUIRES HIGHER LEVEL OF CD133 EXPRESSION AND EXHIBITS HIGHER PROLIFERATION IN VIVO

Vaeteewoottacharn K.1, 2, Seubwai W.1, 2, Wongkham S.1, 2 and Okada S.3 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. 3Division III, Center for AIDS Research, Kumamoto University, Kumamoto, Japan.

While cancer stem cell (CSC) is rapidly progressed, the information regarding CSC in cholangiocarcinoma (CCA), cancer of bile duct, is limited. CCA is a rare cancer worldwide but the incidence is highest in Thailand particularly in the Northeastern part. With a major risk factor of liver fluke (Opisthorchis viverini) infection, CCA in Thai patients exhibit unique genetic signatures when compared to CCA from other causes. Even though CCA is a slow growing, early diagnosis is difficult due to lacking of cancer associated symptoms and specific markers. In this study, we aimed to identify the expressions of cancer stem cell markers in CCA cell line, KKU-M214 and its lung metastatic cell line, M214L5, and characterize cells with particular marker in vivo. Six markers (CD29, CD34, CD44, CD90, CD117 and CD133) that have been successfully detected in several CSC including hepatocellular carcinoma were detected in this study. Among 6 markers, only CD44 and CD133 were differentially expressed in both cell lines. We found increased expression of CD133 in KKU-M214L5 when compared to KKU-M214. When CD133 expressed M214L5 cells (CD133+ M214L5) and CD133 non-expressed M214L5 (CD133- M214L5) were separated and were subsequently injected subcutaneously into NOD/SCID Jak3 deficient mice, tumors were detected from both CD133+ M214L5 and CD133- M214L5 injected groups. However, tumors derived from mice injected with CD133+ M214L5 were larger than tumor derived from CD133- counterpart. These results indicated that CD133 in liver fluke associated CCA might be a potential prognostic marker. Further validation is being investigated.

CHARACTERISATION OF PROTEIN DISULPHIDE ISOMERASE FAMILY MEMBERS IN NEURODEGENERATIVE DISEASES

Walker A.K.1, 2, 3, Farg M.A.1, Turner B.J.2, Soo K.Y.1, Horne M.K.2, 3, 4 and Atkin J.D.1, 2, 3 1Department of Biochemistry, La Trobe University, Victoria. 2Howard Florey Institute, University of Melbourne, Victoria. 3Centre for Neuroscience, University of Melbourne, Victoria. 4Department of Neurology, St Vincent’s Hospital, Victoria.

Protein disulphide isomerase (PDIA1) is the prototypical member of a 19-member family of human disulphide bond-modulating chaperone proteins. Recently, differences in substrate specificity and expression patterns of PDI family members have been identified, suggesting different responsibilities for each protein. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily affecting motor neurons. PDIA1 is up-regulated and shows sub-cellular redistribution in spinal cords of ALS mouse models and human patients, and PDIA1 function is compromised in Alzheimer’s and Parkinson’s disease brains. In neuronal cell culture, PDIA1 over-expression decreases aggregation and toxicity associated with mutant superoxide dismutase 1 (SOD1), which causes a subset of familial ALS, and a small molecule mimic of the PDI active site protects motor neurons against mutant SOD1 misfolding. The largely uncharacterised family member PDIp/PDIA2 is up-regulated in Parkinson’s disease models, while ERp57/PDIA3 is protective against misfolded prion protein and is also up-regulated in ALS. Here we report further investigations of the protein interactions and functions of PDI family members relevant to neurodegenerative disease. In ALS patient spinal cord tissue, PDIA1 was found to be modified by S-nitrosylation, which causes inactivation of the protein by inhibition of critical active site cysteine residues. Furthermore, PDIA2 expression was identified in spinal cord in contrast to earlier reports of pancreas-specific expression, indicating possible involvement of this PDI family member in ALS. These results suggest that PDIA1 and other PDI family members are important for modulation of protein aggregation and cell death pathways in ALS and other neurodegenerative diseases.





EXPRESSION AND SPECIFIC ACTIVITY OF MANGANESE SUPEROXIDE DISMUTASE (MnSOD) IN RAT BREAST AND BLOOD DURING CHEMICAL CARCINOGESIS INDUCED BY DIMETHYLBENZ(A)ANTHRACENEWanandi S.I.1, Nida K.2, Wuyung P.E.3 and Dewi S.1 1Department of Biochemistry and Molecular Biology, Faculty of Medicine University of Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA. 2Master Program in Biomedical Sciences, Faculty of Medicine, University of Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA. 3Department of Pathology Anatomy, Faculty of Medicine University of Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA. Manganese superoxide dismutase (MnSOD) is a major antioxidant in matrix mitochondria which plays a pivotal role on the prevention of oxidative stress. Until now, data about the expression and activity of MnSOD in various cancer cells are still controversial. Several studies have suggested that MnSOD is a tumor suppressor, whereas others have reported that the increase of MnSOD expression in cancer cells indicates a bad prognosis. The aim of this study is to unravel the role of MnSOD during early stages of chemical carcinogenesis. In this study, 4 mg of DMBA (7,12-dimethylbenz(a)anthracene), a chemical carcinogen, was given twice a week for 1, 2, 3, 4, or 5 weeks to induced breast cancer in 30 rats. Breast tissues and blood were taken as samples and analyzed for MnSOD specific activity using xantin oxidase inhibition assay and mRNA expression using Real Time RT-PCR, as well as for MDA (malone dialdehyde) level using TBA assay. Histopathology of breast tissue and determination of sialic acid level in breast and blood resulted in gradual breast carcinogenesis. MDA level in breast was significantly increased (5.1 – 9.3 times) during carcinogenesis compared to that in plasma, indicating that DMBA specifically induced lipid peroxidation in breast cancer. The mRNA expression of MnSOD in breast was significantly enhanced (2.8 – 9.7 times) during the DMBA induction. However, its specific activity was slightly enhanced (1.4 times) only during the first week and then gradually decreased. Therefore, we could conclude that the gene expression of MnSOD is up regulated during early stages of chemical carcinogenesis in order to increase the antioxidant activity which is suppressed by the oxidative stress. Further studies are required to analyze the signal transduction pathway of MnSOD gene expression during chemical carcinogenesis.

MEASUREMENT OF LIVER ALKALINE PHOSPHATASE IN HUMAN SERUM BY IMMUNOASSAY

Wang H.T., Kim H.J., Kim T.K. and Choi E.Y. Dept. of Biomedical Science, Hallym University, Chuncheon, Korea.

Alkaline Phosphatase(ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotide, protein, and alkaloid. ALP levels in plasma will rise when people get liver or bone disease, so ALP can be a marker for liver disease or bone disease. Now, almost all kinds of ALP assay are used to check total alkaline phosphatase in blood. To measure liver ALP isoenzyme mass directly, we want to develop an immunoassay to get our goal. Firstly, gene cloning technique was used to get positive recombinant which can express liver ALP in E.coli; Secondly, purified liver ALP was injected into mice to make monoclone Ab against ALP; Thirdly, ALP Ab was labelled; Currently, we are going to check liver ALP level in serum. Key words: liver alkaline phosphatase monoclone immunoassay.

HEPCIDIN-INDEPENDENT IRON RECYCLING IN A MOUSE MODEL OF HAEMOLYTIC ANAEMIA

Wilkins S.J., Frazer D.M., Darshan D., Whitelaw N.C., Whitelaw E. and Anderson G.J. The Queensland Institute of Medical Research.

The liver-derived peptide hepcidin plays a central role in body iron homeostasis by inhibiting cellular iron export. Stimulated erythropoiesis reduces hepcidin expression which in turn increases macrophage iron release and intestinal iron absorption. However, in certain chronic haemolytic anaemias, iron absorption is not elevated despite a high erythropoietic rate. To investigate this in more detail, we studied the MommeD7 mouse, a novel model of chronic haemolytic anaemia, and compared it to mice with acute haemolytic anaemia induced by phenylhydrazine (PHZ). MommeD7 carries a dominant ENU-induced mutation, but the gene affected is unknown. Liver, spleen, duodenum and blood were collected from six week old MommeD7 mice, from littermate controls and from age-matched wild-type mice three days after PHZ treatment. In both the chronic and acute anaemias, raised reticulocytes and an enlarged spleen suggested stimulated erythropoiesis, and we confirmed enhanced plasma iron turnover by demonstrating increased plasma iron clearance in PHZ-treated animals. Despite increased iron turnover, the expression of hepcidin and intestinal iron transporter mRNAs was unaltered in MommeD7 mice, and hepatic iron levels were normal. In contrast, PHZ treatment, a stronger erythropoietic stimulus, led to a reduction in hepcidin expression, increased duodenal iron transporter expression, increased iron absorption and liver iron accumulation. These data suggest that in low-grade chronic haemolytic anaemia, such as that in MommeD7 mice, the increased erythroid iron requirements can be met through enhanced macrophage iron release without the need to increase iron absorption. Since this increased iron recycling can occur without changes in hepcidin, the data imply that hepcidin is not the sole regulator of macrophage iron release.

SECRETED ADIPOCYTE FATTY ACID BINDING PROTEIN (FABP4) FORMS THE MOLECULAR BASIS OF THE ADIPO-INSULIN AXIS

Wu L.E.1, 2, James D.E.1 and Cantley J.1 1Diabetes and Obesity Program, Garvan Institute of Medical Research. 2Bioanalytical Mass Spectrometry Facility, University of New South Wales.

Adiposity is closely linked with fasting insulin levels and β-cell mass. We hypothesised this to be a result of an inter-organ communication between adipose tissue and the β cell, which may involve a secretory component. Using in vitro models, we showed that treatment of pancreatic islets with conditioned media from hypoxic, but not normoxic control 3T3-L1 adipocytes led to an increase in glucose stimulated insulin secretion of 70%. A quantitative mass spectrometry screen was performed to identify hypoxia regulated adipokines. The adipocyte fatty acid binding protein FABP4 was one of the few secretory proteins that increased in conditioned media during hypoxia. Western blotting confirmed increased secretion of FABP4 from adipocytes during hypoxia. Serum concentrations of FABP4 were raised in high fat fed mice, along with circulating insulin concentrations. In vitro treatment of islets with physiologically relevant concentrations of recombinant FABP4 enhanced glucose stimulated insulin secretion to degree similar to hypoxic adipocyte conditioned media. Hypoxia inhibits mitochondrial function, and the mitochondrial poisons DNP and oligomycin also stimulated FABP4 secretion. Treatment of adipocytes with insulin during hypoxia reduces FABP4 secretion. Removal of glucose from media and substitution with pyruvate reverses inhibition of secretion during hypoxia by insulin, indicating that the primary role of FABP4 secretion is to alleviate energy crisis by increasing glucose uptake into fat through increased insulin secretion. Taken together, these data are indicative of an adipo-insulin axis mediated by FABP4, with the aim of coordinating insulin secretion and glucose uptake in response to the prevailing needs of adipose tissue.





THE GASTRIN GENE IN HUMAN GASTRIC CANCER CELLS IS TRANSCRIPTIONALLY ACTIVATED BY HYPOXIA

Xiao L., Kovac S., Baldwin G. and Patel O. The Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia 3084.

Background and Aim: Hypoxia affects the transcription of approximately 1.5% of genomic genes in solid tumour cells via the activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal cancers. However the effect of hypoxia on gastrin gene regulation is largely unknown. Here we have investigated the effect of hypoxia on transcription of the gastrin gene in human gastric cancer (AGS) cells. Methods: Gastrin promoter activity under hypoxia was examined using the dual-luciferase reporter assay, and gastrin mRNA was measured by real-time PCR. Results: Hypoxia induced by either low oxygen tension (0.5% oxygen) or 300µM cobalt chloride (CoCl2) increased the gastrin promoter activity in AGS cells overexpressing the gastrin receptor (AGS-CCK2R) by 2.4 ± 0.2 and 3.3 ± 1.3 fold (P<0.05), respectively. The gastrin promoter activity induced by CoCl2 in AGS-CCK2R cells was higher than in wild-type AGS cells. Hypoxia increased Gastrin mRNA concentrations in AGS and AGS-CCK2R cells by 1.6 ± 0.2 and 6.1 ± 2.2 fold (P<0.05), respectively. Deletion of the putative HIF-binding sites in the gastrin promoter did not affect gastrin promoter inducibility under hypoxia, indicating that the hypoxic activation of the gastrin gene may be HIF-1 independent. Mutational analysis of previously identified regulatory elements in the gastrin promoter failed to abrogate the induction of promoter activity by hypoxia. Conclusion: Our study is the first to demonstrate that hypoxia upregulates the gastrin gene in AGS cells. The observation that this effect is possibly enhanced by the presence of gastrin receptors provides potential targets for GI cancer therapy.

WNT5A ACTS AS AN ONCOGENE IN EBV-ASSOCIATED NASOPHARYNGEAL CARCINOMAYap L.F.1, Munirah A.2, Zabidi M.M.1, Chai S.J.1, Chu T.L.2, Tan S.K.2, Lim P.3, Wei W.4, Teo S.H.1, 5 and Khoo A.S.B.2 1Cancer Research Initiatives Foundation, Sime Darby Medical Centre, 47500 Subang Jaya. Selangor. Malaysia. 2Institute for Medical Research, Molecular Pathology Unit, 50588 Kuala Lumpur. Malaysia. 3Tung Shin Hospital, 55100 Kuala Lumpur. Malaysia. 4Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT. United Kingdom. 5University of Malaya, 50603 Kuala Lumpur. Malaysia. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr Virus (EBV)-associated cancer which is particularly prevalent in Southern China and Southeast Asia. In Malaysia, NPC is the fifth most common cancer overall and third common in men. Over 70% of cases present with late stage disease and the 5-year survival rates are less than 50%. Novel therapeutic approaches to manage this disease are urgently required. Using expression microarrays, we identified the Wnt5a gene as being overexpressed in primary NPC tissue samples relative to cancer-free controls. Further, comparison with a published microarray study using 36 normal human organs revealed that the level of Wnt5a mRNA in NPC is significantly higher than in a wide range of normal organs. Wnt5a is one of the most highly studied Wnts which acts primarily through the non-canonical pathway. With respect to cancer biology, there is conflicting evidence whether Wnt5a has a tumour-promoting or -suppressing role, and its role in NPC has never been investigated. The upregulation of Wnt5a was validated in 12 NPC tissue samples by quantitative PCR, and its low expression level was confirmed in 16 normal human organs by RT-PCR. In NPC cell lines, however, the expression of Wnt5a was heterogenous. Nonetheless, a dramatic increase in the Wnt5a expression was shown in the only EBV-positive line, C666.1, suggesting a potential role of EBV in regulating the expression of Wnt5a. In addition, we assessed the functional role of elevated Wnt5A on tumour cell behaviour in vitro. Ectopic expression of Wnt5a in NPC cell lines significantly promotes cell proliferation, migration and invasion. Taken together, Wnt5a appears to function as an oncogene in NPC, and its overexpression might be regulated by EBV. These data suggest that Wnt5a could be a useful therapeutic molecular target for NPC.

INDUCTION OF ETHENO-DNA ADDUCT BY CARCINOGENIC OXYSTEROLS IN IMMORTALIZED CHOLANGIOCYTEYongvanit P.1, 2, Jusakul A.1, 2, Loilome W.1, 2, Namwat N.1, 2, Dechakhamphu S.1, 2 and Kobayashi N.3 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 3Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.

Oxysterols are oxygenated derivatives of cholesterol which are produced by non-enzymatic reaction through reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). Oxysterols, which may be produced upon the liver fluke (Opithorchis viverrini; Ov) infection, leading to inflammatory process and consequently overproduction of ROS, are also suspected to play roles in genesis of cholangiocarcinoma (CCA). Our previous data showed that the level of particular oxysterols including cholestan-3β, 5α, 6β-triol (Triol) and cholest-4-ene-3-one (3K4), which are categorized as carcinogenic forms, were significantly higher in liver tissues of Ov-induced hamster CCA. We proposed to examine the effect of oxysterols including Triol and 3K4 on the induction of DNA damage and the alteration of cellular phenotype in human immortalized cholangiocyte (MMNK-1). The immunocytochemical detection of etheno-DNA adduct in oxysterols-induced MMNK-1 cell was performed to determine the level of DNA adduct that caused by oxysterols. This study showed the increased level of carcinogenic etheno-DNA adducts (1-N6-ethenodeoxyadenosine or εdA) that is formed by ROS via the reaction of lipid peroxidation. The effect of oxysterols on cell apoptosis induction was also analyzed by using flow cytometer. It was shown that these oxysterols induced cells death via apoptotic pathway as a dose-dependent manner. These evidences suggest that cell death induced by Triol and 3K4 which is generated during carcinogenesis may have the procarcinogenic impact, since oxysterol could also cause DNA damage via oxidative stress and if mutated clonal expansion occurs, it may play role in the changing of cellular phenotype from normal to malignancy. The proposed mechanistic involvement is under investigation.

INCIDENCE OF INOSINE TRIPHOSPHATE PYROPHOSPHOHYDROLASE (ITPA) DEFICIENCY IN PATIENTS WITH HAEMATOLOGICAL MALIGNANCIES

Zamzami M.1, 2, Catley L.1, Marinaki A.3, Bowling F.1 and Duley J.1 1The University of Queensland, Brisbane, Australia. 2King Abdulaziz University, Jeddah, Saudi Arabia. 3St Thomas Hospital, London.

Mutations affecting genes which produce nucleotide imbalances may represent mutational risk factors for the development of haematological malignancies e.g., myelodysplasias (MDS), where chromosome rearrangement or gene instability is implicated. We tested the novel idea that low ITPase activity in ITPA carriers may be associated with onset of haematologic malignancies common in older age-groups. We hypothesised that accumulation of rogue nucleotides such as deoxy-ITP may cause nuclear DNA mutations. Aim: The study assessed the incidence of the ITPA 94C>A and IVS2+21A>C mutant polymorphisms associated with ITPase deficiency among stable untreated patients with e.g., chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML) and MDS. Methods: 22 MDS, 28 CLL and 18 AML patients were screened for the ITPA 94C>A and IVS2+21A>C SNPs. The international prognostic scoring system (IPSS) values were not available for this patient base. Standard PCR primers were used to amplify exons 2,3 and 4 (Sumi et al, 2002). Thermocycler conditions were 95°Cx5min, 35 cycles (94°Cx20s, 56°Cx20s, 72°Cx30s), ending 72°Cx10min, followed by sequencing. Results and discussion: For Caucasian populations, the expected frequencies of 94C>A, and IVS2+21A>C are 6% and 13% respectively. In this study, the MDS frequency of 94C>A was 11.4% (p=0.134) and IVS2+21A>C 20.5% (p=0.141). For CLL, these frequencies were 7.1% (p=0.719) and 12.5% (p=0.911), and for AML 2.8% (p=0.416) and 8.3% (p=0.405) respectively. The ITPA frequencies were not statistically different from normal for MDS, CLL and AML, but 94C>A and IVS2+21A>C frequencies were trending to significantly higher for MDS, perhaps reflecting rogue nucleotide effects on mutation rates. Further studies of ITPA effects on prognosis (IPSS) are needed.





NEDD9 IS REQUIRED FOR MESENCHYMAL INVASION OF GLIOBLASTOMA CELLS

Zhong J.1, 2 and O’Neill G.M.1, 2 1Kids Research Institute, The Children’s Hospital at Westmead, NSW, Australia. 2Discipline of Paediatrics and Child Health, University of Sydney, NSW, Australia.

Glioblastoma is the most common primary brain cancer with average patient survival of 12-15 months. Despite advances in technology and therapies over the last two decades, there has been little improvement in survival and the final mortality rate remains at almost 100%. Therefore, there is an urgent need to develop new drugs to target specific molecules in signalling pathways that have been implicated in glioblastoma. Given that individual glioblastoma cells can diffusely infiltrate into surrounding healthy brain tissue and thus lead to recurrence of the disease and ultimately death, targeting migration signalling molecules could prove promising. An exciting candidate molecule is NEDD9. The docking molecule NEDD9/ HEF1/Cas-L is an in vivo regulator of cell migration and has been linked to metastasis of melanoma and lung cancer and has recently been demonstrated to promote migration in glioblastoma cells. Thus, the goal of this study is to investigate the role of NEDD9 in glioblastoma cell migration using three-dimensional (3D) culture systems to better recapitulate the in vivo micro-environment. We screened 7 human glioblastoma cell lines and observed high level NEDD9 expression in half the cell lines, correlating with mRNA levels as determined by quantitative real-time PCR. We also established a 3D-collagen gel model which revealed distinct morphological differences when compared with cells on 2D substrates. Subsequently, siRNA targeting of NEDD9 was shown to cause ~80% decrease in NEDD9 expression in 3 high-expressing cell lines and siRNA-mediated down-regulation of NEDD9 significantly inhibited glioblastoma cell migration and invasion in 3D collagen gels. Significantly, our data is the first demonstration that NEDD9 plays a role in 3D invasion of glioblastoma cells.

CHARACTERISATION OF SH-SY5Y NEUROBLASTOMA CELLS AS A MODEL FOR NERVE AGENT NEUROTOXICOLOGY

Thompson G., Thirkettle-Watts D. and Mawdsley D. Defence Science and Technology Organisation, 506 Lorimer St, Fishermans Bend, VIC 3207, AUSTRALIA.

The molecular mechanisms that underlie long-term central nervous system damage caused by organophosphorus (OP) nerve agents, as well as the native mechanisms employed by neurons to combat OP toxicity, are poorly understood. We are evaluating the SH-SY5Y human neuroblastoma cell line as a model for nerve agent neurotoxicity that is amenable to genetic, biochemical and cell biology analyses and manipulation. SH-SY5Y cells have been examined to determine their ability to model typical neuronal features and to determine quantifiable signs of OP toxicity. Following exposure to OP chemicals, innate cellular detoxification pathways have been examined to determine pathways that may provide protection from toxic effects and insights into possible novel therapeutic targets.

IMPROVED PROCESSING OF SHORT HAIRPIN RNA MOLECULES TARGETING H5N1 INFLUENZA

McLachlan K.P.1, 2, Muralitharan M.2, Hinton T.M.1, Tyack S.G.1 and Doran T.J.1 1Australian Animal Health Laboratory, CSIRO, Geelong, VIC, Australia. 2School of Life and Environmental Sciences, Deakin University, Geelong, VIC, Australia.

RNA interference (RNAi) is a natural cellular process designed to degrade foreign RNA. Exploitation of this pathway allows sequence specific silencing of targeted viral genes. Vector based expression of short hairpin RNA (shRNA) molecules is one of the strategies used to induce RNAi. The shRNAs expressed from these vectors are processed by the RNAi pathway into small interfering RNA duplexes (siRNAs), and the antisense strand goes onto trigger silencing of the target gene. Although shRNAs may be highly expressed from a vector, they are often inefficiently processed into siRNAs- resulting in lower silencing efficiency. As native microRNA (miRNA) hairpins are highly processed we decided to investigate optimising the silencing and processing efficiency of our shRNAs by modelling them on native miRNAs, starting with the loop sequences. We selected two different miRNAs native to the chicken based on their high expression from microarray and Solexa sequencing data, and a third miRNA based on knowledge of it being efficiently processed from the precursor miRNA hairpin through to the mature miRNA duplex. We identified the loop regions of these miRNAs and used these sequences in place of a standard nine nucleotide sequence used in many shRNA molecules. The shRNAs selected for optimisation are targeted to conserved regions of Influenza A viruses, including H5N1. The silencing efficiency of the optimised molecules (termed miRloop shRNAs) against their target genes has been assayed using enhanced Green Fluorescent Protein fusion constructs containing portions of the viral genes, and haemagglutination assays. The processing of the miRloop shRNAs has been analysed using northern blots. Results indicate that use of miRloops does have an affect on the processing and silencing ability of shRNAs.

UTILISING A PHOTOACTIVATABLE FLUORESCENT PROTEIN (DENDRA) TO INVESTIGATE ESSENTIAL PROCESSES IN THE MALARIA PARASITE, PLASMODIUM FALCIPARUM

McMillan P.J., Rozano L., Mwesigye E.E., Hanssen E. and Tilley L. Deptartment of Biochemistry and Centre of Excellence for Coherent X-ray Science, La Trobe University, VIC, Australia.

Photoactivatable fluorescent proteins (PAFPs) are a new class of fluorescent proteins which are capable of undergoing an irreversible switch in fluorescence. In this study a PAFP called DENDRA, which can undergo a switch from a green form to a red form, is used to monitor protein & organelle trafficking events in two vital processes in the human malaria parasite, Plasmodium falciparum. During the symptomatic stages of infection the parasite drastically modifies its host cell (red blood cells; RBC) in order to survive and replicate, which it does in two ways. Firstly, the parasite exports virulence factors to the RBC membrane that mediate adherence of the infected cell to capillaries walls, allowing the infected cell to avoid clearance by the spleen. As the RBC lacks any protein trafficking machinery, the parasite generates an exomembrane system in the host cell which is responsible for the trafficking of virulence factors to the RBC membrane. DENDRA fusion proteins will be used to investigate the trafficking of these virulence factors and the generation of this exomembrane system. Secondly, the parasite ingests the host cell cytoplasm, which primarily consists of haemoglobin, to provide amino acids and nutrients. The haemoglobin is taken up via a process of micropinocytosis and is degraded by a number of proteases to produce small peptides. The haem released from this digestion is crystallised into an inert compound called Haemozoin, which is stored in an acidic compartment called the digestive vacuole. DENDRA will be used as a marker for haemoglobin uptake to allow us to follow the uptake process in vitro using confocal microscopy.





VACUOLAR TURNOVER OF THE NUCLEUS IN SACCHAROMYCES CEREVISIAE BY LATE NUCLEOPHAGY

Mijaljica D., Prescott M. and Devenish R.J. Department of Biochemistry and Molecular Biology, Monash University, Clayton campus, Victoria 3800, Australia.

Autophagic degradation of superfluous intracellular components and reuse of the breakdown products enables cells to survive stress from the external environment (e.g. nutrient deprivation) as well as internal stresses (e.g. accumulation of damaged organelles). The yeast nucleus is subject to a form of autophagic degradation by a process called Piecemeal Microautophagy of the Nucleus (PMN). Thus, following short periods of nitrogen starvation (up to 3 hours), interactions between the outer nuclear envelope membrane protein, Nvj1p, and the vacuolar membrane protein, Vac8p, contribute to the formation of nuclear-vacuole junctions and ultimately of membrane vesicles containing nuclear material that are subsequently released into the vacuole. Using a nucleoplasm-targeted fluorescent reporter, NAB35-Rosella, we observed nucleophagy (designated LN for late nucleophagy) only after longer periods of nitrogen starvation (20+ hours). Dual labeling of cells with a nuclear membrane reporter, Nvj1-EYFP, and a nucleoplasm reporter, NAB35-DsRed.T3, confirms that initiation of the delivery of the two respective nuclear reporters to the vacuole is temporally separated. Furthermore, our data suggest that LN occurs by a mechanistically different process to PMN because it can occur in nvj1 or vac8 null cells. Nevertheless, several components of the core macroautophagic machinery are required for LN as it is efficiently inhibited in null mutants of several genes belonging to this group. Moreover, the inhibition of LN in these mutants is accompanied by alterations in nuclear morphology. Collectively, these results suggest that different compartments of the nucleus are selected for delivery to the vacuole by mechanistically distinct forms of nucleophagy.

NUCLEAR-LOCALIZED IMPORTIN ALPHA REGULATES GENE EXPRESSION IN MAMMALIAN SPERMATOGENESIS

Miyamoto Y.1, Yasuda Y.2, Yamashiro T.2, Nagai M.2, Masui A.2, Yoneda Y.2, 3, Jans D.A.1 and Loveland K.1 1Department of Biochemistry and Molecular Biology, School of Biological Sciences, Monash University, Australia. 2Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Japan. 3Department of Biochemistry, Graduate School of Medicine, Osaka University, Japan.

Importin α is known classically as a nuclear localization signal receptor. Although it shuttles between the cytoplasm and nucleus under normal conditions, we previously reported that importin α accumulates in the nucleus in response to cellular stresses, such as oxidative stress, resulting in the down-regulation of the classical nuclear import pathway by removing importin α from the cytoplasm. In this study, we show that nuclear-retained importin α bound a DNaseI-sensitive nuclear component(s). Microarray analysis showed that nuclear importin α induced the up-regulation of specific genes, including the serine/threonine kinase 35 (STK35). Binding of importin α to the promoter region of STK35 was demonstrated by chromatin immunoprecipitation. Constitutive overexpression of STK35 protein was associated with cell death under oxidative stress conditions, suggesting importin α plays a novel role in gene expression in response to stress leading to cell death. Interestingly, the selective and exclusive presence of importin α2 and α4 proteins in mouse within the nucleus of male germ cells in adult testes is concordant with expression of the Stk35 transcript. In situ hybridization analysis reveals that Stk35 gene expression is remarkably reduced in the testis of importin α4 KO mice. Collectively, these results indicate that nuclear importin α may play a role in quality control during sperm production by up-regulating the Stk35 gene and thereby cell death.

CYCLIC AMP DEPENDENT PROTEIN KINASE A STABILISES BIM EL DURING APOPTOSIS

Moujalled D.1, 2, Weston R.1, Carter H.1, Goyal P.1, Ninnis R.1, Coley A.M.1, 2, Strasser A.3 and Puthalakath H.1 1Department of Biochemistry, La Trobe Unversity, Kingsbury Drive Bundoora, VIC 3086, Australia. 2The Cooperative Research Centre for Biomarker translation, Department of Biochemistry, La Trobe univesity. 3Walter & Eliza Hall institute of medical research, IG Royal Parade, Parkville, VIC 3050 Australia.

The pro apoptotic BH3-only protein Bim can induce apoptosis in a wide variety of physiological settings including calcium flux, growth factor withdrawal, endoplasmic reticulum stress, T cell receptor activation and B cell receptor activation. Bim is considered to be the convergent point for cyclic adenosine monophosphate (cAMP) and glucocorticoid induced cell death pahtways (Zhang & Insel 2004). The pro-apoptotic activity of Bim is subject to stringent control both at the transcriptional and post-translational level. Conventionally, Bim can be phosphorylated by MAPK/ERK and JNK pathways. To identify additional kinases involved in its regulation, we carried out a yeast 2-hybrid screen. Here we report that the cAMP dependant protein kinase A (PKA) is a key regulator of BimEL(the major isoform in most, if not all cells and tissues) function. Phosphorylation of BimEL by PKA stabilizes the protein and thus increases the apoptotic activity of the protein. This is in contrast to what is reported for other kinases such as ERK where the consequence of phosphorylation is increased turnover of the protein through the ubiquitine-proteasome pathway.

IN THE PRESENCE OF HIGH LEVELS OF RIPK3, TNF CAN CAUSE CELL DEATH INDEPENDENT OF CASPASE 8 AND RIPK1

Moujalled D.1, 2, Gentle I.1, Wong L.1, Evans J.1, 2, Deed S.1, Hanssen E.4, Strasser A.3 and Vaux D.L.1, 2 1Department of Biochemistry, La Trobe University, Kingsbury Drive, Bundoora, VIC 3086, Australia. 2Cooperative Research centre for Biomarker Translations (CRC), La Trobe University Bundoora, VIC, 3086 Australia. 3Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. 4Bio21 Molecular Science and Biotechnology Institute,The University of Melbourne,Flemington Rd, Australia.

TNF receptor super-family members bear death domains are known to be able to recruit death domain adaptor proteins and cause caspase-8 dependent apoptosis. There are an increasing number of reports that in some circumstances these death receptors can sometimes induce cell death in a caspase-independent manner, for example by mechanisms involving Receptor Interacting Protein Kinases (RIPKs) or by autophagy. We have used a genetic approach to determine the mechanisms by which TNF causes death of mouse embryonic fibroblasts (MEFs). In the presence of cycloheximide, or when IAPs are depleted by an IAP antagonist compound, TNF causes apoptosis of WT MEFs, but caspase 8 and RIPK1 deleted MEFs survive. In contrast, induction of RIPK3, which is usually expressed at low levels, synergises strongly with TNF to cause death of caspase 8-/- MEFs. Interestingly, induction of RIPK3 also greatly increases the ability of TNF to kill RIPK1-/- MEFs, indicating that the activation of caspase 8 independent death mechanisms by TNF is enabled by RIPK3, even when RIPK1 is not present to recruit it to the TNFR1 associated signalling complex. Furthermore, we show that death of MEFs caused by TNF in the presence of RIPK3 was not blocked by caspase inhibitors, the RIPK1 inhibitor necrostatin, autophagy inhibitors, or absence of pro-apoptotic Bcl-2 family proteins Bax and Bak. Therefore, our data suggest that RIPK3 functions as the determinant for cell death in response to TNF in a caspase 8 and RIPK1 independent manner.





ACTIVATION OF THE CALCIUM-SENSING RECEPTOR BY GLUTATHIONE ANALOGS: PHYSIOLOGICAL SIGNIFICANCE AND INSIGHTS INTO THE MECHANISM OF ACTIONMun H.C.1, Broadhead G.K.1, Avlani V.A.1, Jourdon O.1, Christopoulos A.2, Church W.B.3, Delbridge L.4 and Conigrave A.D.1 1School of Molecular Bioscience, University of Sydney, NSW 2006. 2Department of Pharmaceutical Sciences, Monash University, Victoria, Australia. 3Faculty of Pharmacy, University of Sydney, NSW 2006, Australia. 4University of Sydney Endocrine Surgical Unit, Royal North Shore Hospital, St Leonard, NSW 2065, Australia. The human calcium-sensing receptor (CaR) plays a central role in calcium homeostasis. The CaR is activated physiologically by various L-amino acids and pharmacologically by phenylalkylamines including the clinical agent cinacalcet, which bind respectively in the receptor’s Venus Flytrap and heptahelical domains. Recent molecular modeling indicates that the amino acid side-chain binding pocket also accommodates short peptides in which the free α-amino and α-carboxylate groups are preserved. We have now tested the physiological significance of γ-glu-peptides including the dipeptides γ-Glu-Cys and γ-Glu-Ala and tripeptide γ-Glu-Cys-Gly (glutathione) and its analogs, S-methylglutathione (SMG) and S-propylglutathione on intracellular Ca2+ (Ca2+

i) mobilization and cAMP levels in CaR-expressing HEK293 cells and Ca2+

i mobilization and parathyroid hormone (PTH) secretion in normal human parathyroid cells. In addition, to explore the mechanism of action, we compared the effects of SMG and L-Phenylalanine (L-Phe) on HEK293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-Phe leaving responses to elevated the extracellular Ca2+ intact. We found that γ-glutamyl peptides are potent positive allosteric modulators of CaR that stimulate Ca2+

i mobilization and suppress intracellular cAMP in CaR-expressing HEK293 cells and enhance Ca2+

i mobilization and inhibit PTH secretion in normal parathyroid cells. Furthermore, T145A/S170T exhibited markedly reduced sensitivity to both L-Phe and SMG on Ca2+

i mobilization and cAMP suppression. The results indicate that extracellular glutathione and its natural analog SMG modulate CaR function in a physiologically relevant manner and that L-amino acids and glutathione analogs share a common mechanism of action.

YEAST GENOME-WIDE ANALYSIS IDENTIFIES CELLULAR PROCESSES AFFECTING AGGREGATION OF AMYLOID BETA 42-GFP FUSION REPORTER

Nair S., Traini M., Dawes I.W. and Perrone G.G. School of Biotechnology and Biomolecular Sciences, UNSW, 2052 Australia.

Alzheimer’s disease is the leading cause of dementia in individuals over 65 years of age; and the risk of developing Alzheimer’s disease increases steadily with age. A major neuropathological feature of this disease is the presence of amyloid-beta (Aβ) plaques, which are primarily composed of aggregates of Aβ peptides generated via amyloidogenic processing of the amyloid precursor protein. It was recently hypothesised that increased presence and aggregation of intracellular Aβ peptides may play a role in the progression of Alzheimer’s disease. In our study, we have expressed Aβ42 fused to GFP (Aβ42-GFP) in each of the mutants of the Saccharomyces cerevisiae genome-wide deletion library. This approach has identified on a genome-wide level the proteins and cellular processes that affect intracellular Aβ42-GFP aggregation. In addition, sub-cellular localisation of Aβ42-GFP was also identified in these mutants. The data generated in this study may have important implications for our understanding of cellular mechanisms that affect intracellular Aβ42 aggregation and the factors affecting the disease progression of Alzheimer’s disease.

TARGETING OF THE INNATE IMMUNE REGULATORY PROTEIN MUL1 TO MITOCHONDRIA

Ng H.1, 3, Khoo J.1, 2, 3, Jenkins K.1, 2, Mansell A.1, 2, 3, Gabriel K.1 and Nagley P.1, 3 1Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia. 2Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University. 3ARC centre of Excellence in Structural and Functional Microbial Genomics, Monash University.

The protein Mul1 associates with the MAVS complex on the outer mitochondrial membrane (OMM) and is involved in regulation of the mammalian innate immune response. Since Mul1 is a deubiquitinase that negatively regulates innate immune anti-viral signalling via RIG-1 and MDA5, understanding the targeting of Mul1 to mitochondria and determination of its topology will clarify the molecular basis of its role in the MAVS complex. Mul1 (352 aa) is thought to have two transmembrane domains, TM1 close to the N-terminus, and TM2 lying towards the C-terminus. To test targeting roles of TM1 and TM2, we constructed a systematic set of deletions of Mul1, each tagged N-terminally with HA and expressed individually in HeLa cells. Immunocytochemical analysis of HA, together with MitoTracker Red labelling of mitochondria, enabled determination of localisation to mitochondria by confocal microscopy. Full length Mul1 was precisely targeted to mitochondria. Mul1ΔTM1 (aa 9-28) and Mul1ΔTM1&TM2 (aa 9-28, aa 242-259) each failed to target correctly to mitochondria. However, Mul1ΔTM2 (aa 242-259) achieved substantial localisation to mitochondria. The data indicate that TM1 is essential for mitochondrial targeting. Studies of the topology of Mul1 with respect to the OMM are currently in progress. Strategies include expression of miniature derivatives of Mul1 containing TM1 alone with appropriate N- and C- terminal tags, and incorporation of a TEV1 protease cleavage site into intact Mul1 between TM1 and TM2. A combination of immunochemical and biochemical analyses will allow determination of the precise orientation of the N- and C- termini of Mul1 across the OMM.

A PLEIOTROPIC EFFECT OF SIRT1 ON HSF1-DEPENDENT GENE ACTIVATION?

Nguyen M.T., Láng J., Bulhardt O., Csermely P. and Sőti C. SEMMELWEIS UNIVERSITY, DEPARTMENT OF MEDICAL CHEMISTRY, BUDAPEST, HUNGARY.

Resveratrol is polyphenolic phytoalexin that has been recognized to promote longevity in several organisms. Its primary target is proposed to be the Sir2 histone deacetylase. However, resveratrol has been identified in our laboratory as a chaperone inducer and SIRT1 has been shown to activate HSF1. Besides, in C. elegans we demonstrated that both the genetic activation of SIR-2.1 by overexpression as well as by a pharmacologic activation by resveratrol requires HSF1 for increased lifespan and thermotolerance. To analyze the conservation of the genetic interaction between Sir2 and HSF1 we have set up SIRT1 transfection in the COS-7 mammalian cell line. Interestingly, both the wild-type and the dominant negative mutant (deacetylase deficient) SIRT1 inhibited Hsp70 induction suggesting that it may be other, deacetylase independent functions of SIRT1 that influence the heat shock response. To further test the interaction we employed SIRT1 overexpressing and KO mouse embryonic fibroblasts. We found that both SIRT1 overexpression and KO induced Hsp70 protein expression. Moreover, the transfection of human SIRT1 in SIRT1 KO MEFs inhibited Hsp70 promoter activation. In summary, our findings suggest a conserved, but more complex connection of Sir2 and the heat shock response than previously thought.





CHARACTERIZATION OF PUTRESCINE AND SPERMIDINE UPTAKE BY THE HUMAN MALARIA PARASITE, PLASMODIUM FALCIPARUM

Niemand J.1, Birkholtz L.1, Louw A.I.1 and Kirk K.2 1Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0002 South Africa. 2Research School of Biology, The Australian National University, Canberra, ACT 0200 Australia.

Polyamines and their biosynthetic enzymes are present in high levels in proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite but does not cure infections in vivo. This is possibly due to exogenous polyamine salvage from the host which replenishes the intracellular polyamine pool. This implies that in order to disrupt parasitic polyamine metabolism as an antimalarial chemotherapy strategy, it may be necessary to target both polyamine biosynthesis and transport simultaneously. Polyamine transport into both P. falciparum infected erythrocytes and parasites isolated from the erythrocyte were investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected cells; spermidine uptake occurs via the “new permeability pathways” induced by the parasite in the erythrocyte membrane. Once inside the erythrocyte cytoplasm, polyamines cross the parasite membrane via a temperature-and glucose dependent, saturable process. The uptake of both putrescine and spermidine were competed for by the other polyamines and biosynthesis inhibition led to increased total uptake of both putrescine and spermidine. Polyamine uptake was pH dependent with increased uptake upon increasing pH, but did not appear to be coupled to the Na+, K+ or H+ gradient. Membrane potential perturbations influenced polyamine uptake, consistent with the transport being an electrogenic process.

MID49 AND MID51, NOVEL MITOCHONDRIAL OUTER MEMBRANE PROTEINS ACTING AS DRP1 EFFECTORS IN MITOCHONDRIAL DIVISION

Palmer C.S.1, Osellame L.D.1, 2, Laine D.1, Koutsopoulos O.S.1, Frazier A.E.1 and Ryan M.T.1, 2 1LaTrobe Institute for Molecular Science. 2ARC Center of Excellence for Coherent X-ray Science, La Trobe University.

Mitochondria form appropriate cellular networks through regulated fission, fusion and distribution events. Here, we identified two homologous proteins termed Mitochondrial Distribution proteins of 49 kDa and 51 kDa (MiD49 and MiD51) that are anchored in the mitochondrial outer membrane and which influence mitochondrial morphology in mammalian cells. Overexpression of either MiD49 or MiD51 led to the mitochondria being remodeled into perinuclear clusters with fused mitochondrial tubules extending outwards that associated with actin. MiD49 and MiD51 formed discrete foci and apparent rings around potential mitochondrial constriction sites like that of the mitochondrial fission mediator dynamin-related protein 1 (Drp1). Furthermore, expression of MiD49 and MiD51 led to the recruitment of the cytosolic pool of Drp1 to the mitochondrial surface. In contrast, knockdown of MiD49 and MiD51 reduced Drp1 association with mitochondria and resulted in the formation of a mitochondrial net-like phenotype. We propose that MiD49 and MiD51 are novel mediators of mitochondrial division in mammals that regulate Drp1 action at the mitochondrial surface. Further work to determine the role of Drp1 as a potential binding partner for the MiD proteins is being pursued. Additional investigations are also being conducted to determine the role and activity of the MiD proteins in mitochondrial distribution and dynamics, including the generation of truncation mutants and identification of potential protein complexes through chemical cross-linking.

CELLULAR AND EXTRACELLULAR FORMS OF PODOCALYXIN (PODXL)

Fernandez D.2, Larrucea S.3, Nowakowski A.2, Vilar-Egea M.P.2,Ayuso M.S.1, 2 and Parrilla R.1, 2 1Centre of Biological Research, CSIC, Calle Ramiro de Maeztu 9, 28040 Madrid, Spain. 2CIBER de Enfermedades Raras, ISCII. 3Hospital de Cruces, Madrid, Spain.

CHO cells were stably transfected with cDNA encoding a fusion protein of PODXL and green fluorescence protein (PODXL-GFP). Soon after seeding, PODXL-GFP was detected in vivo, by widefield microscopy, in the Golgi and later on as microvesicles, randomly distributed all over the cytoplasm and at the plasma membrane. After 10 h of incubation, the PODXL microvesicles were preferentially located at the leading edges of the cell, progressing along the filopodia, to finally be released into the extracellular medium. CHO-PODXL-GFP cells showed the presence of two distinct anti-PODXL immunoreactive bands of ~200 and ~160 KDa, respectively. The first one reacted with anti-GFP whereas the smaller one did not, suggesting the absence of the carboxyterminal domain of PODXL. Those two bands were also detected in the insoluble fraction of extracellular medium and were identified as PODXL-GFP or PODXL-Δ by mass spectrometry analysis. An additional anti-PODXL reactive form, of ~ 70 KDa was also detected in the soluble extracellular fraction. Either intact or truncated forms of immunoreactive PODXL increased in the insoluble or soluble fractions of extracellular medium upon incubation of CHO-PODXL-GFP cells with PMA, effect that was prevented by either PKC inhibitors, or matrix metalloproteinase inhibitors. It is concluded that PODXL is released from the cell mainly as a cargo of microvesicles and also as a soluble cleaved fragment of the PODXL ectodomain. Thus, in studying the cellular actions of PODXL, the combined action of membrane-bound and free (truncated) forms of this protein should be considered.

RAPID PURIFICATION OF NATIVE HISTIDINE-RICH GLYCOPROTEIN FROM HUMAN PLASMA UTILIZING A STEPWISE PURIFICATION METHOD

Patel K.K., Hoogenraad N.J., Poon I.H. and Hulett M.D. La Trobe University, Department of Biochemistry, Bundoora, Melbourne, Victoria 3086.

Histidine-rich glycoprotein (HRG), a relatively abundant (~100-150 μ/ml) 66 kDa plasma glycoprotein, has a multidomain structure that can interact with many ligands including heparan sulphate, phospholipids, plasminogen, fibrinogen, IgG and complement. The ability of HRG to interact with various ligands simultaneously has suggested that HRG can act as an adaptor molecule and regulate numerous biological processes such as immune complex/necrotic cell/pathogen clearance, cell adhesion, angiogenesis, coagulation and fibrinolysis. In this study, we have developed a rapid stepwise purification method for HRG involving metal affinity purification, followed by Anion exchange chromatography. The final purified protein was analysed by MALDI-MS. The purification of HRG was assessed at each stage by measuring the specific activity of the protein, as well as the yield and purity. We will report on fold structure of the protein. Thus this purification procedure provides a ready source of HRG that would enable structural studies and functional assays to accurately assess its biological functions.





REDOX HOMEOSTASIS AT THE CELLULAR AND SUBCELLULAR LEVEL

Perrone G.G.1, Ayer A.1, Minard A.1, Fife C.1, Fellermeier S.1, Tan S.-X.1, Meyer A.J.2 and Dawes I.W.1 1School of Biotechnology and Biomolecular Sciences, UNSW, Australia. 2Heidelberg Institute of Plant Sciences, University of Heidelberg, Germany.

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite this it is unclear how optimal redox potential is sensed and set, and, the processes that impact redox on a cellular/organellar level. The advent of genetically encoded redox probes including redox-sensitive green fluorescent protein (roGFP2) show promise in overcoming some limitations of traditional disruptive means to analyse compartmental redox since roGFP2 facilitates in vivo analysis of redox potential in a real time, dynamic and relatively non-invasive manner. Compartmentally targeted roGFP2 and the model organism Saccharomyces cerevisiae are being exploited to investigate the biological processes and environmental factors affecting redox homeostasis at a compartmental level. This approach will utilise complete sets of S. cerevisiae deletion/mutation libraries of non-essential/essential genes, and gene over-expression libraries to identify genes and cellular processes that affect the redox environment in each compartment of interest. Testing of a broad range of environmental factors including nutrient source and availability, stress conditions and/or exposure to various drugs on the compartmental redox has also been initiated. This approach is ongoing, and to date has revealed some surprising data indicating the cellular functions and biochemical processes that impact on redox potential in a given compartment. Elucidation of the underlying mechanism(s) associated with altered compartmental redox under a given condition will contribute to our understanding of what factors influence or help to sense/set the redox potential in each cellular compartment and the manner in which changes in the redox environment in one compartment influence that in others.

LOCALIZATION OF SERINE HYDROXYMETHYLTRANSFERASE ISOFORMS IN PLASMODIUM FALCIPARUM

Pornthanakasem W., Leartsakulpanich U., Uthaipibull C., Kongkasuriyachai D. and Yuthavong Y. National Center for Genetic Engineering and Biotechnology, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120 THAILAND.

Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme in the dTMP cycle, converts serine and tetrahydrofolate to glycine and methylene tetrahydrofolate. Since Plasmodium malaria parasite relies solely on de novo pyrimidine biosynthesis pathway which differs from human, SHMT has been proposed to be a potential antimalarial drug target. Based on the DNA sequence search from PlasmoDB, there are two genes encoding SHMT in P. falciparum; one of which has a putative transit peptide at N-terminus. The presence of two isozymes in P. falciparum that may implicate the drug development is yet to be established. Along with other studies on gene disruption and enzyme characterization to validate these proteins as the drug targets, the localization study of these isoforms is investigated. The putative cytosolic and mitochondria SHMT genes were cloned in frame with DsRed and green fluorescence protein respectively. These plasmids were successfully transfected into P. falciparum and SHMT-fusion proteins were expressed. Results from laser-scanning confocal microscope showed different distribution patterns of SHMT-fusion proteins in transfected parasites with specific organelle localization. Additionally, IFA is being used to confirm the results and expression levels of proteins in each blood stage will be measured by RT-PCR. This study provides evidence that these proteins are function at different compartments.

REAL-TIME MONITORING AND COMPOSITION OF THE POTASSIUM EFFLUX AT EARLY STAGES OF APOPTOSIS IN LEUCEMIC T-CELLS

Pottosin I.1, Valencia-Cruz G.1, Bonales-Alatorre E.1, Shabala L.2, Shabala S.2 and Dobrovinskaya O.1 1Universidad de Colima, Colima, Mexico. 2University of Tasmania, Hobart, Australia.

Apoptotic K+ loss contributes to the cell shrinkage and creates a low intracellular K+ environment, favorable for the activation of apoptotic enzymes, caspases and nucleases. The ways by which potassium ions leave the cells are largely elusive as respective candidate channels are assayed under artificial conditions. In this study we have adapted unique non-invasive MIFE technique to monitor in vivo K+ fluxes from a population of mammalian cells (human lymphoma Jurkat), undergoing apoptosis. Apoptosis was induced either by intrinsic (staurosporine, STS) or extrinsic (anti-CD95) stimuli. The identity of K+ channels involved was verified by the use of channels´ blockers and cross-checked by patch-clamp measurements in the whole cell mode. Caspase 3 activity and apoptotic volume decrease were monitored in parallel. Anti-CD95 caused a relatively slow (peaked at 2 hrs) K+ efflux, immediately preceding the caspase-3 activation. On the contrary, 1 μM STS caused a rapid transient (peaked at 15 min) and massive K+ influx, followed by a slower phase. Combination of patch-clamp and MIFE, and use of K+ channels blockers suggested that the early component of K+ efflux is mediated by TRESK-like double pore K+ channels, recently identified by our group in Jurkat cells. These channels transiently activated upon STS-stimulation and completely inactivated after 0.5 hr, leading to the membrane depolarization. From that moment, the apoptotic K+ efflux was dominated by margatoxin-sensitive Kv1.3 channels. Channel-mediated K+ efflux provoked a substantial cellular shrinkage and affected the caspases activation. Supported by CONACyT (Mexico) and small grants from Universities of Colima and Tasmania.

MALATE TRANSPORTERS IN THE SOYBEAN (GLYCINE MAX)- RHIZOBIA SYMBIOSIS

Qu Y.1, Clarke V.C.1, Loughlin P.C.1, Day D.A.2 and Smith P.M.C.1 1School of Biological Sciences, University of Sydney, Sydney, 2006. 2Flinders University, Adelaide, SA, 5001.

The legume-rhizobia symbiosis describes the phenomenon of bacteria infecting legume root cortex cells and forming a novel organ known as the nodule after being engulfed by the host plant. The host plant can grow better by the grace of atmospheric nitrogen being fixed into a biologically available form by the differentiated rhizobia (bacteroid) within the nodule. In return for nitrogen, the plant provides bacteroids with nutrients to support its growth. A specialised, plant derived membrane, termed the symbiosome membrane (SM), surrounds the bacteroids and plays an integral role in regulating the exchange process, therefore making the study of transporters on the SM an important topic. Amongst all these nutrients, malate is thought to be the primary carbon energy source provided by the plant to the bacteroid, though a transporter for it on the SM has yet to be identified. Using a bioinformatic analysis based on previously characterised malate transporters in Arabidopsis and Medicago we have identified several potential soybean malate transporters. The expression pattern of candidate transporters has been investigated using qRT-PCR and candidate genes with enhanced nodule expression will be studied using GFP-fusion constructs to determine their localisation. Functional analysis will be done to confirm that our candidates can transport malate.





CELLULAR ROLES OF THE PUF1/PUF2 RNA BINDING PROTEINS IN YEAST

Quenault T.L. and Traven A. Monash University.

Puf proteins are a family of RNA-binding proteins that regulate mRNA translation and stability. Highly conserved in eukaryotes, Puf proteins are characterised by the presence of repeated Pumilio-homology domains, which form an arc-like structure that binds to the 3’-untranslated regions of target mRNAs. We are investigating the roles of the Puf proteins in two distantly related yeast species, the model yeast Saccharomyces cerevisiae and the fungal pathogen Candida albicans. We are focusing on two highly homologous S. cerevisiae Pufs; Puf1 and Puf2. Puf1 and Puf2 have been shown to bind mRNA targets that primarily encode membrane-associated proteins and could therefore be involved in the biogenesis of fungal membranes and cell walls. Biosynthesis of fungal membrane and wall components is targeted by the two main classes of antifungal drugs, the azoles and the echinocandins. Our data indicates that Puf1 modulates the sensitivity of S. cerevisiae cells to the echinocandin caspofungin. Our current research aims at identifying the relevant mRNA target of Puf1 for its role in caspofungin tolerance. We are also studying the conservation of Puf1 functions between S. cerevisiae and C. albicans at the genomic level through to the transcriptome and proteome, to understand how posttranscriptional regulation evolved between these two distantly related yeasts.

A NOVEL INHIBITOR OF RIBOSOMAL RNA GENE TRANSCRIPTION IDENTIFIES COUPLING OF POLI TRANSCRIPTION TO MULTIPLE CELL CYCLE CHECKPOINTS AND SENESCENCE

Quin J.1, 2, Sanij E.1, Bywater M.1, 2, Drygin D.3, Anderes K.3, McArthur G.1, 4, Pearson R.1, 2, 5 and Hannan R.1, 2, 5 1Research, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. 2Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, VIC, Australia. 3Cylene Pharmaceuticals, San Diego, CA, USA. 4Department of Medicine, St Vincents Hospital, Melbourne, VIC, Australia. 5Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia.

The transcription of the 45S ribosomal RNA (rRNA) genes by RNA Polymerase I (Pol I) is a major rate limiting step for ribosomal biogenesis, and consequently for cell growth and proliferation. Dysregulation of Pol I transcription of the rRNA genes is an almost universal feature of cell transformation and cancer, and work from our laboratory has shown that targeting this process is a promising new approach for cancer therapy (M.J. Bywater, Unpublished Results). A small molecule inhibitor of Pol I transcription (CX-5461) has recently been developed by Cylene Pharmaceuticals, which provides an opportunity to examine the acute response of both non-malignant and transformed cells to perturbation of rDNA transcription. We have utilized CX-5461 to examine the response of BJ (hTert) immortalized primary human fibroblasts and isogenically matched BJ cell lines, at defined stages of transformation, to inhibition of Pol I transcription. Following inhibition of Pol I transcription, BJ (hTert) cells display a p53-independent proliferation defect, which is associated with both G1 and G2 cell cycle arrest, and senescence. Pre-malignant BJ (Large-T, hTert) cells display increased sensitivity to the inhibitor, and undergo apoptosis. However, transformed BJ (Large-T, Small-T, hTert, Ras) cells are resistant to CX-5461 treatment. We will present results of our ongoing studies, which include experiments to address the molecular mechanisms underlying these differing responses.

IDENTIFICATION OF LIGAND(S) FOR HUMAN LEUKOCYTE IMMUNOGLOBULIN LIKE RECEPTOR 4 (LILRA3)

Rajasekariah P., An H., Bryant K. and Tedla N. Infalmmation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052.

Leukocyte Immunoglobulin like Receptors (LILRs) are a superfamily of cell-surface immunoregulatory receptors. They are expressed in a variety of leukocytes, macrophages, dendritic cells, B lymphocytes, and mast cell progenitors. Based on their structure, these are classified into activating, inhibitory and soluble forms. LILRA3 is expressed as a soluble form which lacks transmembrane domain (1). To date the functional role and ligands for LILRA3 are not clearly known although the deletion of the functional gene (1-7 exons) is implicated in Multiple Sclerosis (MS) and Sjögren’s syndrome (2, 3). Using Alkaline Phospatase tagged LILRA3 (APLILRA3) recombinant protein, Alkaline Phosphate (AP) binding assays and cytochemistry assays, we have demonstrated that LILRA3 binds to Peripheral Blood Mononuclear Cells (PBMCs) and U937 cells. Two cDNA libraries were constructed using these cell types in pMT21 mammalian expression vector and DNA from several pools of these libraries was transfected into COS1 cells. The transient transfectants were screened with APLILRA3 recombinant protein and the positively stained pools were rescreened. After a few rounds of screening, several cDNA clones were selected. Currently these cDNA clones are being sequenced in order to establish their identity. References: 1.Arm, J.P., et.al., (1997) J. Immunol. 159, 2342-2349. 2.Koch, S., et.al., (2005) Genes Immun. 6, 445-447. 3.Kabalak, G., et.al., (2009) Arthritis and Rheumatism 60, 2923-2925.

CHARACTERIZATION OF THE α-SYN EXPRESSION PATTERN BY IMMUNOFLUORESCENCE ASSAY WITH COMMERCIALLY AVAILABLE ANTI-α-SYN ANTIBODIES THAT RECOGNIZE DISTINCT REGIONS OF α-SYN

Nam M.K., Han J.H., Park D.W., Kim S.S., Kim G.Y., Shin H.A., Moon J.M. and Rhim H. Department of Biomedical Sciences/ Research Institute of Molecular Genetics The Catholic University of Korea.

Alpha-synuclein (α-Syn) is one of the major components of Lewy bodies observed in the brains with Parkinson’s disease. To date, various antibodies (Abs) against α-Syn have been commercially available for western blotting and immunofluoresence assays (IFA) to detect α-Syn. Nevertheless, there are no reports for epitope mapping and immunostaining patterns of α-Syn using those Abs. We used various purified recombinant α-Syn fragments to identify the region specifically recognized by three different Abs and compared staining patterns of α-Syn in various cell lines by IFA. We also confirmed the specificity of three Abs by an Ab depletion assay incubating the specific Ab with the recombinant α-Syn protein. Broad expression patterns of α-Syn in the cytosol were observed by the monoclonal Ab (A mAb) which recognizes the region containing the NAC domain. Although both polyclonal Ab (B pAb) and another C mAb recognize the following same fragments containing the C-terminal region of α-Syn: aa 1-140, 61-140, 50-140, and 96-140, tubular-like structured- and punctuated-expression patterns of α-Syn were observed by staining with the B pAb and C mAb, respectively. Additionally, a nuclear pattern of α-Syn was able to detect by staining with the B pAb and C mAb, but not with the A mAb. In this study, we have demonstrated that several commercially available anti-α-Syn Abs that recognize different region of α-Syn show different subcellular localization of α-Syn. These results suggest that conformational changes of α-Syn may occur in the different cellular compartments and influence the differential functions of α-Syn in the cell.





BIOCHEMICAL ANALYSIS OF FLAVOPROTEIN SUBUNIT ISOFORMS IN HUMAN MITOCHONDRIAL COMPLEX II (SUCCINATE-UBIQUINONE REDUCTASE)

Sakai C.1, Tomitsuka E.1, Miyagishi M.2 and Kita K.1 1Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 221st Century COE Program, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

We previously reported the presence of two isoforms of flavoprotein (Fp) subunit of human mitochondrial complex II (succinate-ubiquinone reductase, SQR). Type I Fp and type II Fp differs in two amino acid residues and the difference of the biochemical features of two isoforms are almost unknown. In this study, we constructed cell strains that express predominantly type I Fp or type II Fp by RNA interference to investigate the biochemical properties of two isoforms. Although SQR activities of the complex II with each isoform are almost the same, the complex II with type I Fp (type I complex II) showed a pH optimum with the physiological pH of mitochondrial matrix (pH 8) while that for the complex II with type II Fp (type II complex II) was pH 7.5. Moreover, the type I complex II has about 3.5 times higher affinity for succinate than the type II complex II at pH 8.0. On the other hand, the type II complex II showed about 3.7 times higher affinity for succinate than the type I complex II at pH 7.5. These results indicate that the type I complex II mainly functions as SQR in mitochondria of the cells under normal condition and the type II complex II may function in the cells with relatively low pH condition of mitochondrial matrix in such cases as hypoxia or nutrition deprivation. The investigation of the physiological role of type II Fp is now in progress.

DECODING THE MECHANISM OF CELL-ELASTIN ADHESION BY MODIFYING TROPOELASTIN

Seneviratne M.G., Mithieux S.M., Bax D.V. and Weiss A.S. School of Molecular Bioscience, The University of Sydney.

Elastin is an extracellular matrix protein composed of a mesh of cross-linked tropoelastin molecules, which provides elasticity to a variety of tissues and organs including the skin, lungs and arteries (Vrhovski & Weiss, 1998). Although a range of cell types have been shown to adhere to tropoelastin (Broekelmann et al., 2005), the adhesion mechanism is not fully understood. Recent evidence points towards an interaction between αvβ3 integrins on the cell surface and a highly-conserved RKRK motif at the C-terminus of the tropoelastin molecule (Bax et al., 2009). We aim to better understand the role of this RKRK motif. Using a synthetic human tropoelastin gene developed by our lab, we generate a panel of tropoelastin constructs with deletion and insertion modifications to the RKRK sequence. Experiments are underway to evaluate these synthetic proteins on the basis of cell adhesion, spreading and integrin binding, in order to elucidate the biochemistry of tropoelastin adhesion signaling. Developing a better understanding of this adhesion mechanism, and our potential ability to regulate it, has important implications for elastin-based biomaterials used as scaffolds for tissue regeneration.

INFLUENCE OF P38 MAPK INHIBITOR ON INFLAMMATION AFTER SURGICAL WOUND

Shurygina I.A., Shurygin M.G., Zelenin N.V., Granina G.B. and Lepekhova S.A. Scientific Center of Reconstructive and Restorative Surgery SB RAMS.

To study the role of p38 MAPK mechanisms in the regulation of inflammatory response and on this basis to develop ways to control inflammation. 60 Male Wistar rats were selected in accordance with the “Guide for the Care and Use of Laboratory Animals”. The study was approved by the Local Ethics Committee. We studied the effect of an inhibitor of p38 MAP-kinase SB203580 in the development of inflammatory response in models of skin-muscle wounds. We used methods of light microscopy, immunohistochemistry, tensiometry. We revealed that the inhibition of p38 cascade significantly affects the duration and severity of the phases of inflammation, modifies the formation of connective tissue scar in the wound process. Application of p38 blockers reduced the duration and severity of neutrophil infiltration of the damaged area. The application of SB203580 to the end of observation (30 days) in the area of surgical wound formed tender scar, the degree of development of connective tissue in the scar area was significantly lower than in the controls group. Blocker p38 substantially alter the mechanical characteristics of the forming scar in the skin-muscle wounds. Thus, the use of blocker p38 allowed to reach a 30 day wound process indicators modulus, breaking load and the transition point of elastic deformation of the plastic, similar to that of the intact skin. The results show the importance of studying regulators of cell differentiation as potential drugs significantly affecting the outcome of the pathological processes. This study was supported by a grant P803 from the Russian Federal Purposive Program “Scientific and scientific-pedagogical cadres innovation Russia”.

IMMUNE-ENHANCING ANTITUMOUR RNA INTERFERENCE THERAPY FOR CERVICAL CANCERSinghania R., Gu W. and McMillan N. UQ Diamantina Institute for Cancer, Immunology and Metabolic Medicine. Cervical cancer, caused by the over-expression of human papillomavirus E6 and E7 oncogenes, contributes to almost 12% of the global cancer burden in women. Even though prophylactic vaccines have been developed, they are of limited use to women who are already infected. Also, owing to the prolonged course of cervical carcinogenesis, cancer burden will not be reduced significantly for several years even after the availability of these preventive vaccines. So, an effective therapeutic vaccination strategy is urgently required. In recent past, RNA Interference (RNAi) technology has emerged as an effective way to silence cancer-causing genes. The recent findings of our lab show that when short hairpin RNA is targeted downstream of the cytotoxic T cell epitope in E7 gene, 5’ mRNA intermediates are generated, which get translated into incomplete proteins and result in increased presentation of the target protein and generation of a tumour-protective immune response in mice. It is known that certain viral proteins are rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products (DRiPs) by the virus. Our work aims to bring the concept of RNAi and DRiP together and we hypothesize that RNAi-enhanced adaptive immune response against tumour antigens occur via increased presentation of defective proteins. Accordingly, we have investigated the fate of RNAi-cleaved mRNA and found that shRNA treatment reduced the half-life of full-length and 3’ mRNA fragment of the E6/7 and TRP2 mRNA, but the 5’ mRNA fragment appeared to be more stable. This would favour its translation into truncated protein, which we will show from polysome analysis and identify the sub-cellular locations of 5’ mRNA fragment and its truncated protein using RNA-immunoprecipitation and confocal microscopy, respectively. Also, we will investigate the levels of peptides presented on the cell surface after RNAi treatment by mass spectrometry to address the hypothesis that immune-enhancement occurs via increased presentation of defective proteins to the immune system. In summary, the results till now indicate that this therapy need not kill or be delivered to every cancer cell to be effective, thus demonstrating its potential to overcome the current efficacy and delivery constraints.





INDUCED POLYPLOIDISATION AND MUTAGENESIS FOR GENERATION OF NOVEL PASTURE GRASS FUNGAL ENDOPHYTESLudlow E.1, 2, Guthridge K.M.1, 2, Forster J.W.1, 2, 3 and Spangenberg G.C.1, 2, 3 1Department of Primary Industries, Victorian AgriBiosciences Centre, La Trobe University Research and Development Park, Bundoora, Victoria 3083, Australia. 2Molecular Plant Breeding Cooperative Research Centre, Australia. 3Department of Botany, La Trobe University, Bundoora, Victoria 3086, Australia. Fungal species of the genus Neotyphodium form endophytic symbioses with agronomically important pasture grass species such as perennial ryegrass (Lolium perenne L.) and tall fescue (Lolium arundinaceum [Schreb.] Darbysh.). The fungal grass endophyte association results in both beneficial and deleterious agronomic effects for pasture grass production. Lolitrem B and ergovaline alkaloids produced by the fungal endophyte are responsible for herbivore toxicity syndromes (ryegrass staggers and fescue toxicosis), while deterrence of invertebrate herbivore feeding is attributable to alkaloids such as loline and peramine. Novel variants of the perennial ryegrass endophyte Neotyphodium lolii are being generated through induced polyploidisation and mutagenesis. N. lolii mycelium has been exposed to the mitotic spindle inhibitor, colchicine (0 - 0.2% [w/v]), which has been shown to be capable of successfully inducing autopolyploidisation in higher plants and a limited selection of fungal species. Protoplast preparations enabled recovery of single cells and analysis of ploidy changes. Flow cytometry studies have identified N. lolii populations with altered ploidy status and permitted assessment of the stability of ploidy changes over a number of generations. Phenotypic variation attributable to ploidy changes will be evaluated by assessment of toxin biosynthesis and other traits following isogenic inoculation into specific host plant genotypes. Ionising radiation, historically the first mutagenic agent to be described, is capable of inducing a broad range of mutagenic lesions and has been found to be very effective for a wide range of species. N. lolii mycelia were exposed to a range of ionising radiation doses and assessed for recovery and regeneration from single cells. Screening for mutations, in particular deletions within the lolitrem biosynthesis gene cluster, was optimised using a modified non-radioactive colony screening method. Novel variation within the lolitrem biosynthesis gene cluster will be further assessed following isogenic inoculation into specific host plant genotypes.

REGULATION OF NA+ IN THE INTRAERYTHROCYTIC MALARIA PARASITE

Spillman N.J., Allen R.J.W. and Kirk K. Research School of Biology, The Australian National University, Canberra, ACT, 0200.

All cells maintain tight control over their ionic composition and the malaria parasite, Plasmodium falciparum, is no exception. When the parasite first invades the erythrocyte host it encounters a low extracellular [Na+]. However, approximately 12-16 hours after invasion, there appear in the red blood cell membrane ‘New Permeability Pathways’ which increase the permeability to a large variety of unrelated solutes, including Na+. The increased host permeability to Na+ leads to an increase in the intraerythrocytic [Na+] and the mature stages of the parasite are therefore exposed to a high Na+ environment. The trophozoite form of the parasite maintains a low intracellular [Na+] of ~10 mM despite a high extracellular [Na+] (~130 mM) in the erythrocyte host cytosol. The resulting electrochemical gradient is important for the survival of the parasite, energising the uptake of essential nutrients, such as inorganic phosphate, via Na+-coupled symport processes. The mechanisms by which the malaria parasite regulates its intracellular [Na+], were investigated using the Na+-sensitive, fluorescent dye sodium-binding benzofuran isophthalate (SBFI). The functional characterisation of Na+ efflux pathways involved investigation of: pH sensitivity, inhibitor sensitivity, ATP requirements and kinetics. The data are consistent with two Na+ extrusion mechanisms operating in parallel in the mature, trophozoite stage of Plasmodium falciparum, postulated to be a Na+/H+ exchanger and a P-type Na+-ATPase.

ROLE OF APP COPPER BINDING DOMAIN IN APP METABOLISM AND STRUCTURE

Spoerri L.1, 2, Vella L.J.1, 2, Pham C.L.L.1, 2, Barnham K.J.1, 2, 3 and Cappai R.1, 2 1The Department of Pathology, The University of Melbourne, Parkville, VIC, Australia. 2The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia. 3The Mental Health Research Institute of Victoria, Parkville, VIC, Australia.

Two major pathological characteristics of Alzheimer’s disease (AD) include accumulation of β-amyloid (Aβ), a toxic peptide derived from the Amyloid Precursor Protein (APP), and abnormal copper distribution in the brain. A relationship exists between copper and APP, whereby copper can modulate APP processing and APP can regulate copper homeostasis. A better understanding of the molecular mechanisms involved in these events could lead to novel therapeutics to treat the disease. The aim of this work was to investigate the potential role of the putative copper binding residues of the APP N-terminal copper binding domain (CuBD) on APP metabolism. HEK293 cell lines overexpressing APP WT and APP CuBD mutants were employed to study this process. This investigation was complemented by structural studies using a yeast expressed recombinant protein encompassing the APP CuBD. We demonstrate that His149 and His151, located on the α-helix of the APP CuBD, are important for APP metabolism and structural stability. Replacement of these residues results in modulation of APP localization, impairment of APP maturation and decreased sAPPα and sAPPβ generation in HEK293 cell line. The decreased sAPPβ formation may imply decreased Aβ generation. No contribution to this effect is observed by replacement of other residues on the same α-helix (Leu148, Thr152 and Glu160), indicating a specific role for His149 and His151. Replacement of His149 and His151 on the recombinant protein results in changes in secondary structure. Further studies will be aimed to elucidate the mechanisms underlying these modulations.

INHIBITION OF BAK ACTIVATION BY VDAC2 IS DEPENDENT ON THE BAK TRANSMEMBRANE ANCHOR

Stojanovski D.1, Lazarou M.1, Frazier A.E.1, Kotevski A.1, Dewson G.2, Kluck R.M.2, Vaux D.L.1 and Ryan M.T.1 1La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia. 2The Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia.

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol, and upon activation is targeted to the mitochondrial outer membrane, where it releases cytochrome c and other factors that cause caspase activation. While Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. Here, its activation is believed to be inhibited by the voltage dependent anion channel isoform 2 (VDAC2), but it is not known how it does so. Using blue native gel electrophoresis, we show that endogenous, inactive Bak exists in a novel 400 kDa complex that requires the presence of VDAC2. Activation of Bak correlated with its release from the 400 kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1, prevented association of Bak with the VDAC2 complex, and increased the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 sequesters Bak in an inactive state in the mitochondrial outer membrane, thereby raising the threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.





RECLINOMONAS AMERICANA: A SNAPSHOT OF MITOCHONDRIAL EVOLUTION

Tong J., Gabriel K. and Lithgow T. Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC, Australia.

Mitochondria has evolved from an engulfed bacterial symbiont and throughout evolution, the mitochondrial genome has undergone radical changes. In mammals, the mitochondrial genome encodes only 13 proteins. Curiously, a unicellular eukaryote known as Reclinomonas americana harbours a mitochondrial genome with the largest gene set, encoding a total of 67 proteins. Using a combination of yeast genetics, biochemical and genomic analyses, we have examined some of the genes in the R. americana mitochondrial genome. We have identified the existence of both bacterial and classic mitochondrial translocation machineries that are encoded in the mitochondrial genome of this unusual organism. This might reflect an evolutionary snapshot in mitochondrial evolution. Furthermore, one of these genes encodes a protein with homology to Cox11, which is encoded for by the nuclear genome in “higher” eukaryotes. In mammals and fungi, Cox11 has an amino-terminal targeting signal that directs its translocation into the mitochondria from the cytosol. Although the R. americana Cox11 protein does not possess such a clearly defined targeting signal, it is still able to enter yeast mitochondria when expressed in the cytosol. This indicates that at the time of gene transfer to the nucleus, some proteins may have had sufficient signals for targeting back to mitochondria. Over time these targeting signals may have become more elaborate and efficient.

HAMLET BINDING TO α-ACTININ DISRUPTS FOCAL ADHESIONS AND FACILITATES TUMOR CELL DETACHMENT

Trulsson M.1, Hao Y.1, Gisselsson L.2, Chao Y.3, Urbano A.3, Aits S.1, Mossberg A.K.1, Mok K.H.4 and Svanborg C.1, 3 1Institute of Laboratory Medicine, MIG, Lund Univerity, Lund, Sweden. 2Phase Holographic Imaging, Lund, Sweden. 3Singapore Immunology Network (SIgN), A*STAR, Singapore. 4Trinity College Dublin, University of Dublin, Dublin, Ireland.

HAMLET is a protein-lipid complex that triggers tumor cell death, in parallel with a change from a distended to a rounded morphology and from an adherent to a detached state. In this study, HAMLET was shown to recognize α-actinin-4, a major F-actin cross-linking protein and focal adhesion constituent. Synthetic peptide mapping revealed that HAMLET recognizes the N-terminal actin-binding domain of α-actinin-4, suggesting that HAMLET might disrupt the α-actinin-4-dependent scaffold between the actin cytoskeleton and β integrins. HAMLET was shown by co-immunoprecipitation to interact with α-actinins in tumor cell extracts and to colocalize with α-actinin-4 at the membrane of carcinoma cells. Focal adhesions containing paxillin, vinculin and α-actinin-4 were readily observed by confocal microscopy in adherent tumor cells but were rapidly lost after HAMLET treatment. In addition, FAK and ERK1/2 phosphorylation decreased and β1integrin staining was lost in detached cells, consistent with a disruption of focal adhesions. Cell detachment in response to HAMLET was previously observed in bladder cancer patients following intra-vesical HAMLET instillation and cell detachment in response to HAMLET was confirmed in vitro, using adherent carcinoma cells. α-Actinin siRNA increased detachment of tumor cells while α-actinin-4 over-expression significantly delayed morphological changes in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins may disrupt focal adhesion complexes, leading to tumor cell detachment, in parallel with their cell death response.

REGULATORY ROLE OF PRIP IN THE BONE FORMATION

Tsutsumi K.1, Matsuda M.1, Jimi E.2 and Hirata M.1 1Lab of Mol & Cell Biochem, Fac of Dent Sci, Kyushu Univ. 2Dept of Biosci, Cent for Oral Bio Research, Kyushu Dental College.

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel signaling protein isolated in this laboratory. PRIP-deficient mice (PRIP-KO) mice showed an increased gonadotropin, but a decreased sex steroid hormone. The difference was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed bone condition of PRIP-KO mice. We first performed three dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume was higher in the mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness and osteoblast number were accelerated in the mutant, indicating that the increased bone mass is caused by the enhancement of bone formation ability. We then performed the primary culture of calvaria prepared from both genotypes. In the mutant mice, osteoblast differentiation as assessed by alkaline phosphatase activity was accelerated. These results indicate that PRIP is implicated in the regulation of the bone formation.

THE ROLE OF PRIP IN THE REGULATION OF AUTOPHAGY

Umebayashi H.1, Matsuda M.1, Kanematsu T.2 and Hirata M.1 1Lab. of Mol.and Cell. Biochem. Fact. of Dent. Sci. Kyushu Univ. 2Dept. of Dent.Pharmacol. Grad. Sch. of Biomed. Sci. Hiroshima Univ.

Autophagy is an intracellular bulk degradation system, through which a portion of cytoplasm is delivered to lysosomes to be degraded under starvation and stress. Of 33 autophagy-related (ATG) proteins identified so far, ATG8 (MAP1LC3 in mammal) is used as a specific marker to monitor autophagy. We isolated a novel protein as an inositol 1,4,5-triphosphate binding protein molecule similar to phospholipase C, but catalytically inactive, then we termed it PRIP (PLC-related catalytically inactive protein). Later, we found that PRIP bound GABARAP (GABA receptor associated protein), a homologue of MAP1LC3. Therefore, we studied the possible involvement of PRIP in autophagy. We first confirmed the interaction between PRIP and MAP1LC3. PRIP had an interaction with not only GABARAP but also MAP1LC3. Then, autophagy activities were monitored by Western blotting analysis using lysates obtained from wild-type (WT) and PRIP- deficient (PRIP-KO) mouse embryonic fibroblast (MEF) starved by amino acid-deprivation. The level of LC3-II, a lipidation form of MAP1LC3, was more increased in PRIP-KO MEF. Furthermore, we generated WT and PRIP-KO mice, expressing transgene of GFP-LC3 by mating and prepared the MEF. Enhanced autophagic activity, as assessed by more GFP-LC3 puncta observed under a confocal microscopy, was observed in the PRIP-KO GFP-LC3 MEF. These results suggest that PRIP plays a nagative role in autophagy.





INHIBITION OF ATP-BINDING CASSETTE TRANSPORTER G2 INCREASED MITOCHONDRIAL LOCALIZATION OF PPIX IN ALA TREATED CELLS AND ENHANCED THEIR APOPTOTIC CELL DEATH INDUCED BY PHOTODYNAMIC TREATMENT

Utsumi K.1, Fujita H.1, Uchida M.1, Yamamoto M.1, Inoue K.2, Utsumi T.3 and Sasaki J.1 1Okayama University Graduate School, Medicine, Dentistry and Pharmaceutical Sciences. 2Kochi Medical School. 3Graduate School of Medicine, Yamaguchi University.

Since protoporphyrin IX (PpIX) preferentially accumulates in cancer cells treated with 5-aminolevulinic acid (ALA), photodynamic therapy (PDT) has been applied for patients with cancer. In this context, apoptotic but not necrotic cell death of the targeted cancer cells is required to increase the clinical availability. It has been postulated that intracellular localization of photoactive ligands, such as PpIX, plays critical role in the induction of apoptosis of cells subjected to PDT. Recent study revealed that a family of ATP binding cassette transporters (ABC) plays important roles in intracellular localization and accumulation of ALA-derived PpIX and that ABCG2 distributed in mitochondrial cristae. The present work shows that silencing of ABCG2 using siRNA and inhibition of ABCG2 by reserpin, a pan-ABC inhibiter, increased the accumulation of ALA-mediated PpIX in ABCG2 expressed cells, such as T24, HSC-4 and Cos-1 cells. Furthermore, treatment of cells with reserpin increased PpIX levels preferentially in mitochondria (but not in cytosol and plasma membranes) and enhanced PDT-induced apoptosis of cells.

EVIDENCE FOR A NOVEL, DEVELOPMENTALLY REGULATED, AMYLOID PRECURSOR PROTEIN PROCESSING PATHWAY

Vella L.J.1, Munter L.M.3, Fodero-Tavoletti M.T.1, Roberts R.2, Masters C.L.2, Barnham K.J.1, 2 and Cappai R.1, 2 1Department of Pathology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Australia. 2The Mental Health Research Institute of Victoria, Parkville, Victoria. 3Institut fur Chemie und Biochemie, Freie Universitat Berlin, Germany.

A major pathological characteristic of Alzheimer’s disease are the extracellular plaques composed of β-amyloid (Aβ) peptides, which are derived from the larger Amyloid Precursor Protein (APP). While much is known about the production of β-amyloid (Aβ), many questions remain concerning APP function and APP metabolism. In this study, we investigated the occurrence of novel N-terminal APP (NTFs) fragments in human and mouse tissue and aimed to establish cell models to investigate the cellular pathways implicated in the generation of these fragments. Our results suggest a novel, developmentally regulated, processing pathway produce the 17-28kDa APP NTFs. As APP is a known neurite outgrowth promoting molecule, we suspect that the secreted NTFs may perform this function and promote neuronal proliferation and differentiation. Our data suggests that APP transmembrane dimerisation may be important for APP N-terminal processing and consequently the function of APP. Further studies will be aimed at determining the relevance of these isoforms to the normal cellular function of APP and Alzheimer’s disease pathophysiology.

THE ROLE OF MITOCHONDRIAL PROTEIN, CISD2, IN ADIPOGENESIS

Wang C.H.1, Chen Y.F.2, Wu C.Y.2, Tsai T.F.2 and Wei Y.H.1, 3 1Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan. 2Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan. 3Department of Medicine, Mackay Medical College, Sanjhih, Taipei County 252, Taiwan.

Cisd2, the causative gene for Wolfram syndrome 2 (WFS2), is located on human chromosome 4q, where a genetic component for longevity has been mapped. In a previous study, we demonstrated that Cisd2 is related to the life-span of the animals. Cisd2 is a mitochondrial protein and the Cisd2 deficiency causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and leads to a wide spectrum of premature aging phenotypes in mice. However, the functions of Cisd2 in a specific cell type remain unclear. Here, we report that the conditional Cisd2 knockout mice displayed lower weight of epididymal white adipose tissues and interscapular brown adipocytes as compared with age-matched control mice. These findings suggest that Cisd2 deficiency leads to defect in the formation or maintenance of adipose tissues. We then investigated whether the loss of Cisd2 causes defect in adipogenesis. We observed that the mRNA and protein expression levels of Cisd2 were up-regulated during adipogenesis. In addition, we examined the adipogenesis of mouse embryonic fibroblasts (MEFs) from the mouse embryos of different Cisd2 genotypes including wild-type, heterozygous and double-knockout. The results showed that the content of triglycerides and the expression levels of some adipogenic genes were decreased in the adipocytes differentiated from the MEFs of mice with heterozygous mutation or double-knockout of Cisd2 gene, respectively. In summary, in animal and cell models we observed that Cisd2 is required for adipogenesis.

THE CHARACTERIZATION OF C10ORF65 A PUTATIVE COMPLEX I ASSEMBLY FACTOR

Wang X., Wang W., McKenzie M. and Ryan M.T. Department of Biochemistry, La Trobe University.

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) is the largest enzyme complex in the mitochondrial respiratory chain. It is the entrance point for electrons into the respiratory complexes, which couple electron transport with proton pumping to create an electrochemical gradient to drive ATP generation. In order to achieve its enzyme activity, a total number of 45 subunits need to be assembled correctly to form the mature complex. To aid this assembly, a group of proteins, termed as “complex I assembly factors”, exist. C10orf65 is a putative assembly factor recently identified by phylogenetic profiling of different species containing or lacking complex I, however, its function is poorly understood. After translation in the cytosol, C10orf65 is imported into mitochondria via the protein import machinery of mitochondria. In vitro import assays revealed that C10orf65 has a cleaved pre-sequence which is removed to form the mature protein following import. Mitochondrial localization of C10orf65 was further confirmed by generating a C-terminal GFP-tagged construct and epi-fluorescence microscopy, while, sonication and sodium carbonate treatment of isolated mitochondria showed that C10orf65 is partially associated with the inner membrane. Blue Native (BN)-PAGE analysis revealed that GFP-tagged C10orf65 was present in a ~400 and ~500 kDa complexes which may be assembly intermediates of complex I. The function of C10orf65 in complex I biogenesis requires further investigation, however, its study may provide insights into complex I biogenesis and how disruption of this process can lead to complex I deficiency and mitochondrial disease.





DECIPHERING THE MOLECULAR DETAILS OF TYPE V SECRETION SYSTEMS IN ENTEROPATHOGENIC ESCHERICHIA COLI (EPEC)

Webb C.T., Selkrig J.P., Celik N. and Lithgow T.J. Host-Pathogen Molecular Biology Unit, Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Australia.

In order to colonise human hosts and initiate productive infections, pathogenic bacteria rely on protein transport systems. The proteins secreted by these systems, ”effectors”, function as adhesins, toxins, and in other activities that contribute to virulence. Understanding how these protein transport systems work is essential to understanding bacterial pathogenesis. Consequently some of these systems constitute attractive drug targets to disarm Gram-negative bacteria. Type V secretion systems (T5SS) are elegant devices that secrete effector proteins resulting in a myriad of disease states such as meningitis, sepsis, pneumonia and diarrhoea. Using a bioinformatic approach, we are exploring the T5SS proteins from the recently sequenced enteropathogenic Escherichia coli strain, E2348/69. Several novel proteins have been identified and we are using these to dissect the inter-related processes of protein insertion in the outer membrane and effector domain assembly. Here we present our current findings for one such candidate which we hypothesise to belong to the subfamily of functionally and structurally related autotransporters, SAATs (self-associating autotransporters).

ANTIPROLIFERATIVE AND PROAPOPTOTIC EFFECTS OF CURCIN FROM JATROPHA CURCAS ON HUMAN SMALL CELL LUNG CANCER CELL LINE

Wetprasit N.1, Bhorncharoennop C.1, Uyen U.1, Abhornsuwan T.2, Promboon A.3, Roytrakul S.4 and Ratanapo S.3 1Department of Biotechnology, Faculty of science, Ramkhamhaeng University, Bangkok 10240, Thailand. 2Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand. 3Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. 4Genome Institute, National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand.

Curcin is one of type I ribosome-inactivating proteins which can inhibit growth of several tumor cells but the molecular mechanism of this effect remains elusive. The cDNA encoding curcin gene from leaves of Jatropha curcas KUBP 78, was cloned and overexpressed in Escherichia coli. The partially purified protein was obtained by filtration through membrane with molecular weight cut off 50 kDa. In order to understand a possible involvement of genes regulating apoptotic signaling pathways, transcriptional expression levels of Caspase-3, -7, -8 and -9 genes were analyzed after incubating partial purified curcin with NCI-H187 cell line (human small cell lung carcinoma) at various times using Real-time PCR. Partially purified curcin was able to inhibit the growth of NCI-H187 cell line even at a very low concentration. Incubation of target cells with curcin could enhance the caspase-7 and -9 expressions which could be observed since day 1 and reached the peak at day 3 and 5, respectively. Delayed up-regulation of Caspase-3 at day 3 was also found but the level of Caspase-8 was not changed. These results indicated that curcin may induce apoptosis in NCI-H187 via caspase dependent pathway.

THE EFFECTS OF WEAKENED MITOTIC CHECKPOINTS: MOLECULAR EVENTS THAT FOLLOW AND THE RELEVANCE TO CANCER THERAPY

Wong H.1, Murray M.2, Gregory S.1 and Saint R.1 1University of Melbourne. 2University of Adelaide.

The spindle assembly checkpoint (SAC) is the major cell cycle control mechanism that ensures correct chromosome segregation in mitosis. Its disruption causes genomic instability and is linked to neoplastic transformation in cancer. Drugs targeting mitosis are commonly used in cancer therapy and their mode of action is dependent on a functional SAC. The lost of checkpoint functions are linked to drug resistance in clinical settings, but the cellular fates following a weakened SAC remains unclear. Using Drosophila melanogaster, this project follows the fate of cells with weakened Mitotic Arrest Deficient 2 (MAD2), a key SAC component, using flies expressing a tissue specific and inducible RNAi construct targeting MAD2. The project screens a set of 450 kinase and phosphatase RNAi lines for those that enhance the phenotype of MAD2 knockdown to lethality. In this screen we have identified MAD2 interactors for genes involved in the regulation of centromeres, histones and telomeres, JNK signalling, DNA damage responses and growth, metabolic and stress signaling. With almost no knowledge of the link between a defective SAC and apoptosis, and because apoptosis is the preferred clinical outcome of cancer treatment, the components of the JNK apoptotic pathways are key candidates for further characterization. Using fixed tissue analysis and fluorescent activated cell sorting, we are now analyzing the cellular role of these MAD2 interactors in cells with a defective SAC.

OPTIMIZATION OF HETEROGENEOUS ION CHANNEL EXPRESSION IN HEK-293T CELLS

Yaakob N.S., Exintaris B. and Irving H.R. Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia.

The 5-hydroxytryptamine receptor subtype 3 (5-HT3) is the only member of the serotonin receptor family that functions as a ligand-gated ion channel (LGIC). The 5-HT3 receptor has the potential for complex heterogeneity as five HTR3-like genes have been discovered that code for 5-HT3A, 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E subunits. Functional receptors consist of pentamers of 5-HT3A subunits only or combinations of the 5-HT3A and other subunits. The goal of our research is to compare the electrical activities between different receptor subunit combinations expressed in HEK-293T cells using patch-clamping. However, to perform efficient patch-clamping, high expression levels of the receptors are required. We have manipulated a range of parameters to optimize expression of the 5-HT3 receptor subunits in HEK-293T cells using green fluorescent protein as a marker for transfection efficiency. We are currently expressing tagged heterogenous receptor combinations to determine relative expression levels using a multi-plasmid co-transfection approach. We are also preparing multi-cistronic vectors to express two or three subunit genes in a single vector. We will report on comparisons between the two methods by assessing transfection efficiency, protein expression levels via western blotting and immunohistochemistry and finally patch clamp experiments.





A SINGLE AMINO ACID POINT MUTATION IN FIBROBLAST ACTIVATION PROTEIN (FAP) IN HUMANS IS ASSOCIATED WITH LOSS OF FUNCTION AND CELL SURFACE LOCALISATION

Yao T.W.1, Osborne B.1, Wang X.M.1, Yu D.M.T.1, Topaloglu K.A.2 and Gorrell M.D.1 1Centenary Institute DO6, University of Sydney, NSW 2006, Australia. 2Cukurova University, Faculty of Medicine, Department of Pediatric Endocrinology and Metabolism, Adana, Turkey.

Fibroblast activation protein (FAP) is a type II cell surface glycoprotein of the prolyl oligopeptidase family. FAP possesses a rare catalytic activity, hydrolysis of the post-proline bond two or more residues from the N-terminus of target substrates. FAP is undetectable in normal adult tissues, but its expression is significantly induced in tumours, inflammation and at sites of the interface of tissue fibrosis1. We have previously shown in vitro that FAP overexpression in LX2 and HEK293T cell lines has an important role in cell adhesion, apoptosis, proliferation and cell migration2. The FAP gene of 26 exons spans 72.8 kb at human 2q24.3. A homozygous loss-of-function single nucleotide mutation in this FAP gene encoding a residue in the 6th blade of the beta-propeller domain of the protein was identified. We demonstrated that this mutation caused ablation of FAP enzyme activity and an accumulation of FAP in the ER rather than at the cell surface. This mutation was associated with ER stress and unfolded protein response, as indicated by upregulation of the ER chaperone protein, BiP/GRP78, in both a heterologous expression system [transfected HEK293 cells] and cultured patient fibroblasts. A monoclonal antibody that binds to a conformational epitope of FAP bound poorly to the mutant FAP protein, suggesting a conformational change in the FAP structure. This is the first loss of function single amino acid point mutation outside the catalytic site characterised in the prolyl oligopeptidase gene family. 1. Wang XM, Yao T-W, Nadvi NA, Osborne B, McCaughan GW, Gorrell MD 2008 Front Biosci 13:3168-80. 2. Wang, XM, DMT Yu, GW McCaughan, MD Gorrell 2005 Hepatology 42: 935-45.

THROMBIN-TREATED CYCLOOXYGENASE-2 EXPRESSION AND PROSTAGLANDIN E2 PRODUCTION BY PRIMARY MYOBLASTS IS MEDIATED BY PROTEASE-ACTIVATED RECEPTOR-1

Yoo H.J.1, Pagel C.N.1, Pike R.N.2 and Mackie E.J.1 1School of Veterinary Science, The University of Melbourne, Cnr of Flemington Rd and Park Dr, Parkville, Victoria, Australia. 2Department of Biochemistry and Molecular Biology, Monash University, Wellington Rd, Clayton, Victoria, Australia.

Thrombin treatment of primary skeletal muscle myoblasts stimulates proliferation and intracellular calcium mobilisation and inhibits differentiation and apoptosis induced by serum deprivation. Previous studies have demonstrated that the effect of thrombin on myoblast apoptosis and differentiation are not mediated by the main thrombin receptor, protease-activated receptor-1 (PAR-1). The aim of this project was to identify the role of thrombin and PAR-1 in myoblast function. Thrombin treatment of serum-deprived primary myoblasts increased cyclooxygenase-2 (COX-2) gene expression, and prostaglandin E2 (PGE2) and interleukin-6 (IL-6) production. In addition, PGE2 inhibited apoptosis and differentiation induced by serum deprivation in primary myoblast cultures. We hypothesised that PGE2 production may be related to the mechanism of thrombin’s inhibitory effect on myoblast apoptosis and differentiation. However stimulation of COX-2 gene expression and both PGE2 and IL-6 production by thrombin occurred in PAR-1 dependent manners. This result suggests that COX and PGE2 may be important mediators of PAR-1 dependent effects of thrombin on myoblasts.

THE ROLE OF WW AND C-TERMINAL DOMAINS IN YORKIE/YES-ASSOCIATED PROTEIN

Zhang X., Milton C.C., Humbert P.O. and Harvey K.F. Peter MacCallum Cancer Centre.

The Salvador/Warts/Hippo (SWH) pathway regulates tissue growth through inhibiting the activity of Yorkie (Yki) in D. melanogaster and Yes-associated protein (YAP) in mammals, which act as transcriptional co-activators. Yki and YAP have a conserved N-terminal domain, through which they associate and cooperate with the transcription factors, Scalloped (Sd)/TEAD, to promote tissue growth. In addition, Yki and YAP possess two conserved WW domains that mediate interaction between several proteins that either promote or inhibit YAP’s ability to drive transcription. Furthermore, the C-terminus of YAP includes three further motifs: a SH3 domain-binding motif, transcription activation domain and a PDZ domain-binding motif, none of which are conserved in Yki. To study the role of the Yki/YAP WW domains and the YAP C-terminus, several Yki/YAP mutants were constructed and their function was analyzed with assays for tissue growth in Drosophila, or with assays for colony growth, cell transformation, migration and invasion in MCF10A or NIH-3T3 cells. Results showed that: 1. WW domain played an inhibitory role in migration and invasion in MCF10A cells, but promoted NIH-3T3 cell growth and transformation and drived tissue growth in D. melanogaster. 2. Deletion of YAP’s transcription activation domain abolished its ability to activate TEAD2, as well as the ability to stimulate invasion of MCF10A cells. However, this mutant promoted growth and transformation of both MCF10A and NIH-3T3 cells. 3. Mutation of YAP’s PDZ domain-binding motif lowered its ability to promote invasive behavior of MCF10A cells, but promoted 3D growth and transformation. Taken together, these results suggest that YAP is able to mediate tissue growth, transformation and invasion through different domains and the total effect of YAP on growth and invasion is dependent on the coordination of protein interactions via its WW and transcriptional activation domains, as well as its PDZ domain-binding motif.

LARGE-CONDUCTANCE CA2+-ACTIVATED POTASSIUM AND ETHER-à-GO-GO POTASSIUM CHANNELS REGULATE PROLIFERATION OF HUMAN MESENCHYMAL STEM CELLS

Zhang Y.Y. and Li G.R. Department of Medicine, The University of Hong Kong, Hong Kong SAR China.

Bone marrow-derived mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, cellular physiology is not fully understood in human MSCs. The present study was to determine the potential role of the dominant functional ion channels, large-conductance Ca2+-activated potassium (BKCa) channel, ether-à-go-go potassium (hEAG1) channel, and sodium channel, in regulating proliferation of human MSCs using electrophysiological, cell proliferation assay, and biochemical approaches. We found that paxilline (1 μM), astemizole (1 μM), and tetrodotoxin (TTX, 30 nM) respectively reduced BKCa, hEAG1, and sodium current in human MSCs. The cell proliferation assay with MTT and 3H-thymidine incorporation methods revealed that the inhibition of BKCa with paxilline and hEAG1 with astemizole, but not sodium current with TTX, decreased cell proliferation and reduced DNA synthesis rate in a concentration-dependent manner. Flowcytometry analysis displayed that paxilline and astemizole accumulated human MSCs at G0/G1 phase, and decreased cell population of S phase. Moreover, lentivirus-based shRNAs targeted to BKCa or hEAG1 channel remarkably reduced both mRNA and protein expression of BKCa or hEAG1 channel; and proliferation of human MSCs was also reduced by BKCa-shRNAs or hEAG1-shRNAs. These results demonstrate the novel information that BKCa and hEAG1 channels, but not sodium channel, participate in the regulation of cell proliferation by promoting G0/G1 cells into cell cycling progression in human MSCs.





CHARACTERISING THE INHIBITION OF LEGUME NODULATION CAUSED BY LOW pH CONDITIONS

Lin M.H., Ferguson B.J., Indrasumunar A. and Gresshoff P.M. ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St. Lucia, Brisbane, QLD, Australia.

Lateral organ differentiation and its internal and external control are critical frontiers of developmental plant genetics. Legume nodulation offers an elegant system to study the genetic and biochemical basis of these processes where mutational analysis has coupled structure to function. Legumes form nitrogen-fixing root organs known as nodules after establishment of a symbiotic relationship with rhizobia. Nodule formation is tightly regulated by the plant and can be inhibited by a number of external factors, such as soil acidity. This is of global importance as many of the world’s legume crops, such as soybean, are grown in acidic soils. Despite this, the precise plant mechanism(s) by which acidic conditions inhibit soybean nodule development remains poorly characterised. Here, we present evidence that the inhibition of soybean nodulation by acidic conditions may be controlled by the plant and that this control may have a systemic component. In addition, a separate inoculation-independent inhibition pathway may also exist. Furthermore, we show that the inhibition of nodulation in acidic conditions likely acts during the early stages of signal perception during nodule meristem establishment. Next generation deep sequencing will be used to determine RNA expression changes and novel genes functioning in acid-stressed soybean roots inoculated with or without a compatible Bradyrhizobium strain. Transgenic soybean chimeric plants expressing a visual reporter system containing the red fluorescent protein, DsRed2, were generated, and the nodulation-deficient mutant nod49 was successfully complemented by over-expression of the soybean Nod Factor Receptor, GmNFR1α. This transformation system will be used to investigate the role of the soybean GmNFR1α, in wild type and GmNFR1α-complemented nod49 plants grown under acid stress.

CHARACTERIZING A NOVEL LEA PROTEIN FROM RESURRECTION GRASS

Ling H.Y., Blomstedt C.K., Hamill J.D., Walmsley A.M. and Neale A.D. School of Biological Sciences, Monash University, Melbourne, Australia.

The grass Sporobolus stapfianus Gandoger exhibits homochlorophyllous desiccation tolerance, with plants able to resist to cellular degradation due to severe water deficit. This resurrection plant can revive to a fully hydrated state within 24 hours after loss of more than 95% of its total water content. This rare trait is found only about 0.2% of all vascular plant species but is widespread in primitive clades for example algae, lichens and bryophytes. A novel LEA (late embryogenesis abundant) protein was identified from dehydration-stressed leaf tissue of S. stapfianus. LEA proteins were first described in orthodox seed development and have a crucial role as a protectant in vegetative plants, invertebrates and bacteria during water deficit. The expression of LEA proteins from S. stapfianus during dehydration and rehydration has been compared with the LEA mRNA expression profile in leaf tissue. In addition, subcellular localisation of the LEA protein using transient expression in Nicotiana benthamiana revealed high accumulation of this protein in the chloroplast. The LEA gene was introduced into Arabidopsis to elucidate its role in improving dehydration tolerance and in response to other abiotic stresses. The ability of the LEA protein to protect enzyme activity in vitro using a lactate dehydrogenase enzymatic assay was investigated. This study is important to provide an understanding of the LEA protein in desiccation tolerance.

INVESTIGATING THE ROLE OF SHORT RNAS IN TOMATO FRUIT DEVELOPMENT

Lopez-Gomollon S. and Dalmay T. University of East Anglia, Norwich, NR4 7TJ, UK.

The availability and consumption of fresh fruit depends upon the quality attributes and physiological integrity of these products. For all fruit products it is essential that the quality characteristics of color, flavour and especially texture meet consumer expectations.Tomato is the model system for studying development and ripening in fleshy fruits due to excellent genetic and genomic resources. Regulatory short RNAs have been recently discovered and become one of the most intensively studied fields in plant molecular biology. However, to date this field has been restricted mainly to Arabidopsis and it is now time to investigate the role of short regulatory RNAs in crop plants, like tomato. Understanding the molecular mechanisms of specific plant traits such as fleshy fruit development and ripening is key in future breeding programs. One of the most recently discovered gene regulation mechanisms is post-transcriptional and involves 21-24 nucleotide small RNA molecules (sRNA). To get a complete picture of sRNA profiles in fruit ripening and development, 10 separate libraries from different stages of fruit development were created. We found that the pattern of 10 known miRNA change across the timeserie, suggesting a role in fruit development. Moreover, 6 new miRNA identified for first time in tomato, show a expression profile that change during development. These results were confirmed by Northern blot. To study the role of the new miRNA in vivo, tomato plants overexpressing the precursor of each miRNA will be developed, as well as plant overexpressing target mimicries, to inhibit the activity of each miRNA. Our aim is to develop a platform to study regulatory networks involved in development and maturation of commercially important fruits.

CHARACTERISATION OF THE PHYTOPHTHORA ZOOSPORE SECRETOME

Ludowici V.A., Blackman L.M. and Hardham A.R. Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia.

Phytophthora species are fungus-like plant pathogens in the Class Oomycetes. They cause millions of dollars of losses to agricultural industries and threaten natural ecosystems across the globe. Because of their phylogenetic distance from true fungi, chemical controls that target fungi are not effective against Phytophthora pathogens. A greater understanding of the molecular and cellular mechanisms of host infection could lead to the development of novel control measures. During initial plant infection, motile Phytophthora zoospores encyst and the contents of three types of peripheral vesicles undergo regulated secretion. One of the proteins secreted during this stage is a complement control protein (Ccp) which is found in so-called large peripheral vesicles. The objectives of this project are to investigate the role of the large peripheral vesicles during plant infection and also to identify other proteins secreted from zoospores during encystment. Expression of PnCcp in P. nicotianae during development has been analysed by quantitative real-time PCR and showed that this gene is more highly expressed during sporulation and in zoospores than in vegetative hyphae or 3-hour germinated cysts. Immunofluorescence microscopy showed that biogenesis of large peripheral vesicles begins prior to the development of ventral surface vesicles. Immunofluorescence microscopy was also used to study the timing of secretion from these vesicles and indicated that PnCcp is secreted within 2 minutes after induction of encystment. The secretion of two other peripheral vesicle proteins, Vsv1 found in the ventral surface vesicles and Cpa found in the dorsal peripheral vesicles, also occurred within this time period. To identify other secretome components, proteins from young cysts will be solubilised and proteomic techniques employed for their analysis.





TOWARDS THE DOMESTICATION OF A WILD RICE RELATIVE, MICROLAENA STIPOIDES USING LARGE SCALE GENE SEQUENCING

Malory S.1, Shapter F.M.1, Elphinstone M.S.1, Chivers I.H.2 and Henry R.J.1 1Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW. 2Native Seeds Pty Ltd, Cheltenham, VIC.

Microlaena stipoides, commonly known as weeping grass, is a distant relative of rice. It is a drought, frost and shade tolerant perennial evergreen plant and produces seeds similar to rice. M. stipoides can be used for grain production and additionally it can be grazed as a pasture. This species responds well to nitrogen application and also regular irrigation, making commercial production possible and making it a target for domestication. Extensive sequencing and comparative mapping has established a high degree of conservation between rice and other grasses, allowing the isolation of the corresponding homologues of important rice genes in other grasses. In this study the rice genome sequence is being used for comparison with corresponding genes in M. stipoides. A high range of variability occurs within the natural populations for domestication traits and this can be harnessed for selective breeding programs. An induced mutation population has also been established to capture desired traits for crop improvement. High-throughput next generation sequencing using the Illumina Genome Analyser IIx is being used to discover single nucleotide polymorphisms (SNPs) in M. stipoides which can be used in establishing domesticated lines of M. stipoides. Once domesticated, M. stipoides will become a new cereal crop for commercial food production. This technique can also be utilised for other wild grasses to screen for desirable domestication traits and possibly to create new crops for food consumption.

A QUIESCENT MIR159C:TARGET GENE REGULATORY MODULE IMPLIES THE ANCIENT MIR159 FAMILY HAS VERY FEW TARGETS OF BIOLOGICAL RELEVANCE IN ARABIDOPSIS

Allen R.S.1, 2, Li J.1, 2, Alsonso-Peral M.M.1, White R.G.2, Gubler F.2 and Millar A.A.1 1Research School of Biology, Australian National University, Canberra, ACT. 2CSIRO, Division of Plant Industry, Canberra, ACT.

A current challenge regarding microRNA (miRNA) biology is the identification of biologically relevant target genes. Bioinformatics and molecular approaches have predicted that the Arabidopsis miR159 family may regulate more than twenty such genes. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b appears limited to only two genes, MYB33 and MYB65. Here we show that the expression of the remaining family member, MIR159c, overlaps with a subset of these predicted target genes in anthers. Despite this, a mir159c mutant does not display any anther defects and a mir159abc triple mutant is indistinguishable from mir159ab. Consistent with this, transcription of MIR159c was low and processing of pre-MIR159c poor, resulting in a low abundance of miR159c and no deregulation of potential miR159 target genes was evident in mir159c. Conversely, examination of potential miR159c targets, MYB101 and MYB120, found that despite the detection of miR159-guided cleavage products, neither their spatial nor temporal expression domain appeared miR159-regulated. Furthermore expression of miR159-resistant versions of MYB101 or MYB120 does not lead to anther defects. Thus the potential miR159c-MYB101/MYB120 regulatory relationship appears to be of little or no biological importance, but may represent the remains of a former miRNA:target module. Such genetic remnants from the death of conserved miRNA modules may be leading to the overestimation of the complexity of miRNA regulation and illustrates the dynamic nature of regulatory relationships that exists even within ancient miRNA families.

EXAMINATION OF RESIDUES THAT INFLUENCE INTERACTION BETWEEN RUBISCO AND RUBISCO ACTIVASE

Milward S.E. and Whitney S.M. Division of Plant Sciences, Research School of Biology. RN Robertson Building 46, Australian National University, Canberra, ACT.

Ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) is the key CO2 fixing-enzyme in the biosphere and frequently limits photosynthetic carbon assimilation in higher plants. In addition to a unique activation process (requiring non-substrate CO2 and Mg2+ binding) the activity of Rubisco in higher plants is regulated by a nuclear-encoded “helper” protein, Rubisco activase (RA). Using energy generated from ATP hydrolysis, RA facilitates the removal of inhibitory sugar phosphate ligands from the Rubisco active sites via a poorly understood interactive mechanism (hindered by the inability to obtain a crystal structure of RA). Prior mutagenic studies suggest Rubisco-RA interactions are defined by surface residues 89 to 94 in the catalytic Rubisco large (L-) subunit and those in the C-terminal domain between codons 311 and 314 of RA. To examine Rubisco-RA interactions in tobacco, three transplastomic lines producing Rubisco with L-subunit mutations at codons 89 and 94 have been generated. Light-transient CO2-assimilation assays suggest that while both single L-subunit mutations have little effect on RA activation capacity (at 25°C),in the double mutant activation is impaired by ~25%. Presented here is an update of gas exchange and biochemical analysis of the effects of these mutations on the interaction between tobacco Rubisco and RA.

OVER EXPRESSION OF SNAKIN-1 AND SNAKIN-2 GENES UNDER A POTATO LIGHT INDUCIBLE LHCA3 PROMOTER IN TRANSGENIC POTATOES FOR BROAD SPECTRUM DISEASE RESISTANCE

Mohan S.1, 2, Meiyalaghan S.1, Jones E.E.2, Jacobs J.M.E.1, Pringle J.M.1 and Conner A.J.1, 2 1New Zealand Institute for Plant & Food Research. 2Agriculture and Life Science Division, Lincoln University.

Abstract Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular weight antimicrobial peptides produced in potato tubers. Such proteins are thought to play important roles in the innate defence against invading microorganisms. Over-expression of StSN genes is known to provide broad spectrum activity against a wide range of bacterial and fungal pathogens in potato. Based on homology to other genes, these proteins have also been hypothesised to be involved in diverse biological processes, including: cell division, cell elongation, cell growth, transition to flowering, and signalling pathways. StSN1 and StSN2 genes were isolated from potato cv. Iwa. The coding regions of these genes were individually cloned into the intragenic expression cassette harbouring the Lhca3 (cab) promoter and the Lhca3 (cab) terminator. The resulting intragenic cab5’-StSN-cab3’ chimeric genes were cloned into transgenic binary vector pMOA33. The binary vectors pMOA33cabStSN1 and pMOA33cabStSN2 were individually used for Agrobacterium-mediated transformation of potato. Thirty-three and thirty-two independently derived SN1- and SN2-transgenic plants were regenerated respectively. Multiplex-PCR confirmed the presence of the nptII selectable marker gene and the specific gene in all regenerated lines. Based on quantitative RT-PCR of each SN gene, seven lines were selected for each of the SN1- and SN2-transgenic plant populations for disease resistance assessment. Pathogenicity bioassays showed that the over-expression of either StSN1 gene or StSN2 gene from the intragenic cassettes conferred resistance against the enterobacterial phytopathogen Pectobacterium atrosepticum (formally Erwinia carotovora ssp. atroseptica).





THE ROLE OF ATMYB5 AND ATMYB23 IN SEED COAT AND TRICHOME DEVELOPMENT

Napoli R., Parish R. and Li S. The Department of Botany, LaTrobe University, Melbourne, Victoria, Australia.

The cell wall and mucilage (Pectin) are important in protecting seeds from damage, maintaining dormancy and facilitating germination under favourable conditions. Knowledge gained on trichome development may lead to modifictaions which improve crop pest ressistance. MYB genes encode transcription factors which regulate several developmental and metabolic pathways in plants. MYB transcription factors function by activating or repressing other genes involved in secondary metabolism, cell development, signaling pathways, disease resistance and responses to environmental stresses. Arabidopsis is well suited to study the function, structure and regulation of MYB genes. This project aims to characterise the AtMYB5 and AtMYB23 regulatory pathways in seed coat and trichome development. The AtMYB5 and AtMYB23 MYB transcription factors are found in the model plant Arabidopsis. AtMYB5 and AtMYB23 are both expressed in the developing seed and leaf trichomes. An atmyb5/atmyb23 double mutant revealed a possible redundancy between the two proteins in trichome development. Both MYB transcription factors interact with bHLH and HD-ZIP proteins to form an activation complex capable of activating target genes. Characterisation of these pathways involves expression analysis of AtMYB5/AtMYB23 target genes in trichome development. Such putative target genes were identified by microarray data from immature seeds of the atmyb5 mutant. These genes include GLYCOSYL HYDROLASE 810 (GH10810) which encodes a putative xylanase enzyme and two hydrolase genes ABE1 and ABE4. EMSA analysis is being utilised to study protein-DNA interaction between AtMYB5 and the promoters of selected target genes. The AtMYB5 and AtMYB23 pathways provide a useful system for the study of cell wall synthesis.

CHARACTERISATION OF THE SERK GENE FAMILY IN LEGUMES INVOLVED IN DEVELOPMENT AND DEFENSE

Nolan K.E. and Rose R.J. ARC CILR, School of Environmental and Life Sciences, University of Newcastle. Australia.

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins to function both in developmental and defense signalling pathways, which occur in response to both peptide and steroid ligands. SERKs are generally present as small gene families in plants, with five SERK genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. This seemingly broad requirement for this small family of genes indicates their underlying importance for plant growth and defense. The SERK1 gene of the model legume, Medicago truncatula functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation [1, 2]. However, other SERK genes in M. truncatula and other legumes are largely unidentified and their functions are unknown. To aid the understanding of signalling pathways in M. truncatula, we have attempted to identify and annotate all SERK genes in this species. Using degenerate PCR and database mining, eight more SERK-like genes have been identified and these have been shown to be expressed. One of them was found to contain a number of splice variants, which has not previously been reported in this gene family. The sequence information obtained from M. truncatula was used to identify potential SERK family genes in the recently sequenced soybean genome. The phylogenetic relationship of members of the SERK gene family to development and defense function is presented. 1. Nolan et al (2003) Plant Physiology, 133 218-230. 2. Nolan et al (2009) Journal of Experimental Botany, 60 1759-1771.

ENVIRONMENTAL REGULATION OF THE CYANOGENIC GLYCOSIDE, DHURRIN, IN SORGHUM

O’Donnell N.1, Blomstedt C.1, Stuart P.2, Neale A.1, Hamill J.1 and Gleadow R.1 1School of Biological Sciences, Monash University, VIC Australia. 2Pacific Seeds, QLD Australia.

Sorghum is a C4 plant with high water use efficiency, making it drought and heat tolerant. Forage sorghum is used as fodder for cattle in the dry tropics, but is cyanogenic, releasing hydrogen cyanide (HCN) when plant tissue is disrupted. The HCN potential (HCNp) of sorghum differs greatly between varieties and within a variety sorghum is subject to spatial and temporal regulation. The HCNp of sorghum is also affected by environmental factors and varieties can differ from one season to the next, HCNp is known to be influenced by numerous factors, including maturity, drought, frost and soil fertility. Experienced farmers know that young sorghum plants, or older plants that have been subjected to drought, are often toxic and can kill cattle. However there are no known scientific articles that have shown that HCNp in sorghum is increased under drought conditions. We have used various concentrations of polyethelene glycol (PEG), an osmoticant, added to the nutrient solution of sorghum plants grown in hydroponics, and have found a positive correlation between PEG and HCNp. The increase in HCNp under higher PEG concentrations could be due to either the up-regulation of the cyanogenesis genes, or the constitutive expression of cyanogenesis genes and subsequent accumulation of dhurrin due to reduced growth, or both. Although we have found a definite correlation between HCNp and PEG concentration, we found that the HCNp only increased when the plants were grown in the PEG for a number of weeks. These findings are relevant to the efficient production and use of sorghum for farmers globally.

ONTOGENETIC VARIATION IN EUCALYPTUS DEFENSE PROFILES

O’Gorman K.L.1, Miller R.E.1, Cody N.1, Burns A.E.1, Foley W.J.2 and Gleadow R.M.1 1School of Biological Sciences, Monash University, Clayton, VIC, Australia. 22. Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, ACT, Australia.

Plants have evolved a range of physical and chemical mechanisms which have been shown to play a role as herbivore deterrents. Chemical defences are broadly categorised as carbon-based or nitrogen-based. According to the Carbon Nutrient Balance Theory, carbon-based compounds tend to be more prevalent in plants which have evolved in non-light limiting, nutrient poor, environments. Variations in defence strategies also occur within an individual during its development. Eucalyptus, a dominant Australian genus, is typically well defended with carbon-based compounds, and a few species also invest in the nitrogen-based defence cyanogenesis. Polymorphism exists in the expression of the cyanogenic glycoside, prunasin, even within closely related populations. Recently, variation in the expression of cyanogenic glycosides has been found to change with ontogeny. The objective of this study was to characterise ontogenetic variation in chemical and physical defenses in seven closely related Eucalyptus species over a two year period. As the plants developed they were found to increase the amount of foliar carbon relative to nitrogen. This was associated with an increase in the concentration of carbon-based defences, and leaf thickness. Two species were found to be cyanogenic at 6 months of age, and three were cyanogenic by 15 months of age, suggesting ontogenetic variation in the ability to ‘switch on’ cyanogenesis. These species appear to have “defence syndromes” which differ in the extent of ontogenetic control on defence traits.





THE CONSEQUENCES OF ALTERING N-TERMINAL POST-TRANSLATIONAL PROCESSING OF RUBISCO IN TOBACCO CHLOROPLASTS

Orr D.J.1, Houtz R.L.2 and Whitney S.M.1 1Plant Sciences, Research School of Biology, Australian National University, GPO Box 475, Canberra, ACT, 0200. 2Department of Horticulture, University of Kentucky, 401D Plant Science Building Lexington KY 40546-0312 USA.

The co- and post-translational modification of proteins during maturation is an almost ubiquitous process which has long been observed to be essential for protein regulation, activation, and degradation in a large range of organisms. In higher plant chloroplasts assembly of the photosynthetic CO2-fixing enzyme, Rubisco, is a highly complex process that necessitates the co-ordinated expression and assembly of its 8 large (L) and 8 small (S) subunits into a hexadecamer (L8S8). The L subunits house the catalytic sites and are subject to the post-translational aminopeptidase removal of Met-1 and Ser-2, and the acetylation of the new N-terminal Pro-3. The function of these modifications are undefined. The L-subunit gene (rbcL) is located in the chloroplast genome (plastome) and can be genetically manipulated in tobacco with surgical precision via the homologous recombinatorial method of plastome transformation. We have generated transplastomic tobacco lines containing mutations in rbcL at codons Ser-2 and Pro-3. Changes in the N-terminal processing of the mutant L-subunits have been identified by mass-spectrometry. Presented will be an update on how these alterations in N-terminal processing influence leaf L-subunit translation, Rubisco content and the growth of these transgenic tobacco lines.

PROTEIN AND DNA ARE TAKEN UP BY PLANT ROOTS

Paungfoo-Lonhienne C.1, Lonhienne T.G.A.2, Rentsch D.3, Mudge S.R.1, Webb R.4 and Schmidt S.1 11School of Biological Sciences, The University of Queensland, St. Lucia QLD, 4072, Australia. 2ARC Centre of Excellence for Integrative Legume Research, School of Biochemistry and Molecular Biosciences, The University of Queensland, QLD, 4072, Australia. 3Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland. 4Centre for Microscopy and Microanalysis, The University of Queensland, QLD, 4072.

We examined the long-held view that plants rely on inorganic forms of nutrients and depend on microbial conversion of soil organic nitrogen (N) and phosphorus (P) prior to uptake into roots. Using green fluorescence protein (GFP) and fluorescently labelled phosphorothioate oligonucleotides (S-DNA) as sources of organic N and P, we show that protein and DNA are taken up by plant roots. Comparable results were obtained using pollen tubes as analogues of root hairs as both have similar elongated and polarized growth. We further show that addition of DNA to growth medium containing inorganic P enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with inorganic P only. These findings change our view of the spectrum of the N and P sources accessible to plants and demonstrate that DNA triggers morphological responses which may enhance root proliferation in organic matter-rich soil. Our research challenges the current paradigm that plants rely on microbes and soil fauna for break-down of organic matter and provide further evidence for heterotrophy in plants.

DICHOTOMY IN THE NRT GENE FAMILIES OF ARABIDOPSIS AND CEREAL SPECIES

Plett D.1, Toubia J.1, Baumann U.1, Garnett T.1, Tester M.1 and Kaiser B.N.2 1Australian Centre for Plant Functional Genomics, The University of Adelaide, Waite Campus, Urrbrae, South Australia, 5064, Australia. 2School of Agriculture Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, South Australia, 5064, Australia.

A large proportion of the nitrate (NO3-) acquired by plants from

soil is actively transported via members of the NRT families of NO3-

transporters. In Arabidopsis, the NRT1 family has 7 functionally characterized members and is predominantly comprised of low-affinity transporters; the NRT2 family contains 7 members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. Our bioinformatic analyses of four fully sequenced cereal genomes (maize, rice, sorghum, Brachypodium) indicates fundamental differences between Arabidopsis and the cereal species in the gene number and family structure of all three families of NRT transporters. Quantitative-PCR analysis of the expression of the NRT genes in maize adds to the apparent dichotomy between Arabidopsis and the cereals and begs the question: Is Arabidopsis a suitable model for NO3

- transport in cereal crop species?.

GENETIC ARCHITECTURE OF ROOT SYSTEMS IN COTTON

Prodhan M.A., McGee P.A. and Saleeba J.A. School of Biological Sciences, The University of Sydney, NSW 2006, AUSTRALIA.

Genetic architecture refers to the underlying genetic basis of a phenotypic trait. The spatial spread of roots or root system architecture (RSA) is an agronomically important trait that has been investigated in a few plants. Here we summarize the progress in determining genetic architecture of RSA of cotton. We chose several genes influencing RSA in Arabidopsis thaliana and assayed the RSA of their knock out (KO) lines. Data analysis revealed that KO and WT lines vary in RSA indicating the effect of the corresponding gene on RSA. The gene sequences were compared with the nucleotide and protein databases of Gossypium (cotton) at The National Center for Biotechnology Information. Results suggested that at least two of the genes have homologues in cotton, which prompted us to investigate them further as candidate genes for cotton RSA. Progress will be reported on the role of the genes in root system development in cotton.





MODELLING SCENARIOS FOR BIOMASS OPPORTUNITIES IN SUGARCANE

Rae A.L.1, Lam D.1 and O’Shea M.G.2 1CSIRO Plant Industry, 306 Carmody Rd, St. Lucia, Qld. 4067, Australia. 2BSES Limited, 50 Meiers Rd, Indooroopilly, Qld. 4068, Australia.

Sugarcane has potential as a biomass feedstock to provide materials and energy for the future. New technologies for producing ethanol from cellulose may enable more profitable use of residual fibre. In this study, an economic model was used to assess the profitability of a theoretical biorefinery producing sugar, electricity and ethanol from cellulose. The model takes into account all costs and profits relating to sugarcane production, and has the capacity to predict returns if these values change. The aim of the study was to highlight areas where research into plant variety development should be targeted. Therefore the model was used to test a number of scenarios based on changes to feedstock characteristics. The outputs defined the levels of change in each parameter which would be required for profitability. The feasibility of achieving these changes was tested by comparison to data on current and experimental plant varieties. The results showed that the scale of biomass input was a critical factor. For an average sized mill area, increasing the cane yield (t/ha), the bagasse yield (% dry matter) or the ethanol yield (L/t) individually were shown to achieve a profitable system. However, the increases required for some of these factors were outside the range of feasibility. Varying two factors in conjunction was a more feasible way to achieve profitability. Plant varieties with characteristics that can address these targets could be selected from existing germplasm or developed by breeding and selection.

CLE PEPTIDE INDUCTION OF SOYBEAN NODULE REGULATION

Reid D.E., Li D., Hayashi S., Ferguson B. and Gresshoff P. ARC Centre of Excellence for Integrative Legume Research, University of Queensland.

The ability of legumes to enter into a symbiotic relationship with nitrogen fixing bacteria is one of their defining characteristics. The host plant maintains control of this symbiosis in response to external and internal stimuli, including environmental nitrate availability, and via a systemic autoregulation of nodulation (AON) mechanism. The current model for these regulatory mechanisms involves a root-derived cue (Q) that is perceived by an LRR receptor kinase called GmNARK in soybean. Nitrate-induced Q appears to be perceived locally, whereas rhizobia-induced Q is transported in the xylem to the shoot where it is perceived by GmNARK. This activity triggers the production of a shoot-derived inhibitor that is transported back to the roots where it prevents further nodule development. Due to the high degree of molecular similarity between GmNARK and the LRR receptor-like kinase CLAVATA1 in Arabidopsis, it has been proposed that GmNARK-dependent regulation may be induced by a similar ligand/s to that interacting with CLAVATA1, a small peptide called CLAVATA3. We have identified candidate CLEs that respond to inoculation and/or nitrate treatment and which inhibit nodulation in a GmNARK-dependent manner when expressed in the root. In order to confirm if these or other candidates are required for the initiation of AON, we have developed a bioassay to detect nodulation-dependent signals in xylem sap. This bioassay is reliant on differential gene expression responses in the leaves of soybean plants fed with xylem sap presumed to either contain Q, or be devoid of it. To identify the differential responses, complete transcriptome sequencing of soybean leaves using the Illumina GAIIx has been employed.

CHARACTERISATION OF THE WHEAT MULTI-PATHOGEN RESISTANCE GENE Lr34/Yr18/Pm8Risk J.1, Solomon P.2, Richardson T.1, Viccars L.1, Chandramohan S.1, Tabe L.1, Spielmeyer W.1 and Lagudah E.1 1CSIRO Plant Industry, Black Mountain Site, GPO Box 1600, Canberra ACT 2601, Australia. 2Research School of Biology, RN Robertson Building, The Australian National University, Canberra 0200 ACT, Australia.

According to the Food and Agricultural Organization of the United Nations over one billion people are reliant on wheat for food and their livelihoods. Wheat biotrophic pathogens, such as rust and powdery mildew, can have a devastating effect on crop yields resulting in up to 60% yield loss. Recently a rust resistance gene, Lr34, that also confers multi-pathogen resistance (Yr18/Pm38), was identified and shown to be integral to durable wheat resistance to fungal pathogens for over 100 years (Krattinger et al., 2009, Science. 323, 1360-1363). Analysis of this gene revealed that Lr34 encodes an adenosine triphosphate-binding cassette transporter (ABC transporter) and that it shows homology to the Arabidopsis plasma membrane-localized protein PENETRATION3 (PEN3) (Stein et al., 2006, The Plant Cell. 18, 731-746.). While both genes are involved in pathogen resistance, LR34 confers resistance against adapted pathogens whereas PEN3 is associated with non-host resistance. Several approaches are being taken to gain insight into LR34 conferred resistance. Analysis of LR34 expression is being investigated using GFP-tagged LR34 and anti-LR34 antibodies. Brachypodium and barley transformant lines are also being developed to ascertain whether resistance can be gained from addition of Lr34 in other cereals, and to provide alternate model systems for Lr34 characterisation. Finally, metabolite profiling using GC-MS is being used to analyse whole leaf extracts, ultimately leading to the identification of the metabolite being exported by LR34.

AN INVESTIGATION ON THE INFLUENCE OF DGAT1 AND POLYOLEOSIN ON TRIACYLGLYCERIDE PRODUCTION

Roldan M.B., Winichayakul S., Scott R. and Roberts N.J. AgResearch Grasslands, Forage Biotechnology, Palmerston North 4442, New Zealand.

Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyses the last and only committed step in the synthesis of triacylglycerides (TAG). We are currently using a quadruple mutant of Saccharomyces cerevisiae, which is incapable of producing storage lipids, to investigate potential differences between monocot and dicot DGATs. Differences observed in the TAG profiles will be presented in this poster. We have also co-expressed a mutated form of the dicot DGAT1 with several forms of polyoleosin (a novel protein invented at AgResearch) in the leaves of Arabidopsis. TAG accumulation in the leaves was analysed by Gas Chromatography-Mass Spectrometry. The preliminary results indicate that one form results in the accumulation of substantial amounts of TAG, compared to control plants and plants transformed with other forms of polyoleosin. These results are also presented and the potential to apply this technology in forages is discussed.





AN UNUSUAL GA AND ABA SYNERGISM PROMOTES SOMATIC EMBRYOGENESIS IN MEDICAGO TRUNCATULA 2HA: IMPLICATIONS FOR TRANSCRIPTIONAL REGULATION

Rose R.J., Kurdyukov S., Zhang X.Y. and Nolan K.E. ARC CILR School of Environmental and Life Sciences, The University of Newcastle, NSW 2308, Australia.

In higher plants somatic embryogenesis (SE) induction in the appropriate genotype and tissue commonly requires auxin (in Arabidopsis) or auxin plus cytokinin (in Medicago) and has been studied since the 1950s. A focus with SE studies in Arabidopsis has been the LEAFY COTYLEDON transcription factors LEC1 and LEC2 which when overexpressed are able to induce SE. It has been suggested that LEC transcription factors (TFs) may make cells competent to respond to auxin and undergo SE by catabolising gibberellic acid (GA). Additional studies involving the MADS box TF AGL15 has lead to the hypothesis in Arabidopsis involving GA/ABA ratios; with GA suppressing SE and abscisic acid (ABA) acting antagonistically to enhance SE. How GA and ABA influence embryonic competence is not known. However, SE induced in the pickle (pkl) mutant of Arabidopsis can be suppressed by GA. The PKL gene encodes a chromatin remodelling protein. In M. truncatula what have been surprising results are the SE dependence on ethylene and new data that GA and ABA act synergistically to enhance SE induced by auxin plus cytokinin severalfold. LEC1 and LEC1-LIKE are not expressed during the induction of SE and are only expressed in the developing embryo. LEC2 has not been demonstrated to be present in M. truncatula. We have argued that the TF MtSERF1 whose transcription is auxin, cytokinin and ethylene dependent acts as a nexus between stress-induced ethylene and auxin and cytokinin to promote SE. However, we also have evidence that the 2HA line is an epigenetic mutant that is weakly ethylene insensitive with the MtEIL1 TF down-regulated. We suggest that in Medicago ABA and GA modulate a different set of TFs compared to Arabidopsis to enable the induction and then development of embryos in vitro, with modulation of ethylene signalling being critical.

IN PLANTA PRODUCTION OF RECOMBINANT ENZYMES FOR THE PROCESSING OF BIOFUEL FEEDSTOCKS

Scott R.W., Bartho J.D. and Roberts N.J. Forage Biotechnology, AgResearch Grasslands, Palmerston North, New Zealand.

Up to 50 % of the cost of producing ethanol-based biofuels from cellulosic feedstocks (maize, wood, etc) can be attributed to the cost of the cellulase enzymes. Cellulases break down the complex cellulose molecules into simple sugars, which can be subsequently fermented into alcohols. Expression of recombinant cellulases in plants offers a means of producing the enzymes in a low maintenance organism, with little or no processing required prior to being added to the primary feedstock. Previous studies have shown that it is possible to express recombinant cellulases in plants. In order to develop a cost-effective system we are investigating methods for sequestering high concentrations of the enzymes in planta. Four subcellular regions were chosen for targeting: (1) endoplasmic reticulum, (2) chloroplast, (3) protein storage vacuole, and (4) cytoplasm via fusion to oleosins (oil body proteins). Supporting this research we are also investigating the potential of utilising recombinant cellulases originally isolated from the rumen bacterium Butyrivibrio proteoclasticus. The cellulases chosen for study were: (A) GA1 (predicted endocellulase) and (B) GA25 (predicted endocellulase). Cellulases from the industrial standard cellulolytic fungus, Trichoderma reesei, were used as positive controls: (C) Cel7a (exocellulase) and (D) Cel7b (endocellulase). The presentation discusses the preliminary results and our plans to enhance production and enzyme activities.

P-BODY PARTICIPATION IN CELLULAR REPROGRAMMING OF CULTURED MESOPHYLL PROTOPLASTSBhullar D.S., Sheahan M.B. and Rose R.J. ARC CILR, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia, 2308.

Regeneration initiated from somatic cells of plants represents an important mechanism of morphogenic adaptation to stress and entails the acquisition of a totipotent state. The acquisition of totipotency requires dedifferentiation, a process that reprograms somatic cells to a stem cell-like state. Inherent to this reprogramming are massive changes in gene expression. Here we investigated processes that influence transcriptome reprogramming during the regeneration of tobacco mesophyll protoplasts. Processing bodies (P-bodies) are cytoplasmic RNA-protein complexes that both store and degrade mRNA and play an important role in developmental transitions. Moreover, P-bodies are dynamic structures that can change in response to environmental stress. We therefore investigated P-body dynamics during dedifferentiation and cell division initiation of tobacco mesophyll protoplasts. To visualise P-bodies, we created stable tobacco transformants expressing a YFP fusion to the C-terminal domain of VARICOSE (VCSc), a P-body localised protein that facilitates mRNA decapping. YFP-VCSc labelled spherical structures that ranged in size from 7-150 μm in leaf epidermal cells. In protoplasts, P-bodies exhibited variable size but also abundance per cell. While average P-bodies size in protoplasts remained relatively constant during culture, the number of P-bodies per cell increased significantly during early (24 h) culture and then again preceding cell division. P-body size and abundance was significantly reduced by cycloheximide and actinomycin D treatments suggesting P-bodies require continual recruitment of translationally-repressed mRNA. Transiently expressing a fusion of DCP2 (a decapping enzyme) to CFP, revealed an apparent heterogeneity in the P-body population with co-expression of VCSc promoting localisation of DCP2 to P-bodies. Our results suggest P-bodies are dynamic structures that potentially participate in reprogramming the transcriptome of mesophyll cells.

ROOT GROWTH RESPONSES IN BARLEY (HORDEUM VULGARE L.) TO SALT STRESS

Shelden M.C.1, Roessner U.1, Tester M.2 and Bacic A.1 1Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, Parkville, 3010 VIC. 2Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, 5064 SA.

Soil salinity is a major agriculture problem resulting in decreased growth and yield of crops. Cereal crops, such as barley and wheat, are of major economic importance to the Australian agricultural industry. The root system is essential for plant growth and plays a critical role in determining the crop yield. In response to salt stress (osmotic stress) there is an immediate reduction in the root elongation rate (RER). Often this is followed by a partial recovery and a new steady rate of elongation is obtained. Root growth occurs within a few mm of tissue from the root apices, in the root elongation zone (REZ). How root elongation is maintained in saline soils is largely unknown. The aim of this study is to identify candidate genes involved in the maintenance of root elongation in response to salt stress. To identify the mechanisms that regulate root elongation we are utilizing both physiological and molecular techniques. Currently we are screening a variety of barley genotypes to determine the effect of salt (NaCl) on RER. Barley seedlings are grown at different NaCl concentrations on agar plates and seminal root growth measured. Cultivars able to maintain root elongation in response to salt stress will be selected for molecular profiling of the root zones (root cap, elongation zone, maturation zone). The identification of genes involved in salt tolerance is necessary for improving crop productivity.





A COMPARATIVE GENOMICS APPROACH FOR IDENTIFYING EFFECTORS IN VENTURIA INAEQUALIS

Shiller J.B.1, Mesarich C.2, Jones D.1, Bowen J.2, Templeton M.2 and Plummer K.1 1La Trobe University. 2Plant and Food Research, New Zealand. 3The University of Auckland.

Venturia inaequalis is a hemi-biotrophic fungus that causes apple scab disease. The disease is widespread, and is found in almost every country where apples are grown. Currently, control of this disease is through fungicide use and resistant cultivars. However, there are races of V. inaequalis that can infect these previously resistant cultivars and there are also emerging cases of fungicide resistance in V. inaequalis. Whether a race of V. inaequalis is able to cause disease on a given cultivar of apple is governed by the ability of the host to detect the invading pathogen and mount a successful resistance response. At a genetic level this is dependent on a gene-for-gene relationship involving effector genes in the pathogen and resistance genes in the host. Identifying the effector genes (or gene mutations) in V. inaequalis that enable the fungus to evade the host immune system and cause disease will allow for rational development of resistant cultivars in the future. Whole genome sequences, obtained by pyrosequencing will soon be available for V. inaequalis and the closely related pear pathogen V. pirina. Using bioinformatics and functional genomics we will attempt to identify, characterise and compare effectors from these fungi. This poster will outline our progress in the project so far.

MANIPULATION OF THE NON-PHOSPHORYLATING PATHWAY ALTERS IONIC HOMEOSTASIS

Smith C.A.1, Barthet M.M.2, Whelan J.3, Smith P.M.C.2, Day D.A.1 and Soole K.L.1 1School of Biological Sciences, Flinders University, SA, 5042. 2School of Biological Sciences, University of Sydney NSW 2006. 3ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, WA, 6009.

The alternative respiratory pathway of the mitochondria is an essential bypass to adenylate control of redox poise in the cell. In most animals, reduction of mitochondrial NADH occurs through the cytochrome c pathway of cellular respiration by the rotenone-sensitive NADH dehydrogenase. In the model plant Arabidopsis thaliana, type II NDs are encoded by a small gene family with a total of seven members, NDB1-4, NDA1-2, and NDC1, as well as Alternative Oxidase (AOX). Although the nature of these enzymes as a bypass to adenylate control of cellular redox regulation implies a critical role for these enzymes in plant function, little is currently known regarding the impact of these enzymes on whole plant and cellular physiology. Using A. thaliana plants with knockdown of Atndb4 and up regulation of Atndb2 and Ataox1a, we have observed an alteration in the ionic state of these plants with increased levels of zinc. Microarray analyses on these lines at both 6 days and 5 weeks, showed an increase in nicotianamine synthase at 6 days which is the enzyme responsible for synthesizing nicotianamine. Nicotianamine has been linked to improved uptake and mobilisation of metal ions around the plant, and may explain why higher levels of zinc have been found in the Atndb4 knockdown lines as compared to wild type plants. A further exacerbation of this altered ionic homeostasis was observed in these plant lines during salinity stress, where in the presence of NaCl, leaves has higher calcium and lower boron and sodium levels.

BROWNING OF CINNAMON MYRTLE (BACKHOUSIA MYRTIFOLIA) LEAVES

Sommano S.1, Joyce D.1, D’Arcy B.1 and Joyce P.2 1The University of Queensland, Centre for Native Floriculture, School of Land, Crop and Food Sciences, Gatton, Qld. 4343, Australia. 2BSES Limited, 50 Meiers Road, Indooroopilly, Qld. 4068, Australia.

Physiology and biochemistry potentially associated with unsightly browning of harvested Cinnamon Myrtle (Backhousia myrtifolia) leaves were investigated. Chlorophyll content (Chl a & b), chlorophyll fluorescence (CF), electrolyte leakage (EL), malondialdehyde (MDA) content, polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonialyase (PAL) enzyme activities, total phenolic content and antioxidant capacity (DPPH) were measured for heat-induced browning. Green versus yellowish leaves were contrasted because of observed differential susceptibility to browning. After heating at 60°C for 60 min, yellow leaf tissue browned more than did green leaf tissue. Green leaves contained more Chl. a & b than did yellow leaves. Low CF ratios (Fv/Fm) in both leaf types suggested that this sub-tropical species suffered from low temperature stress (Gemel et al. 1997) as the potted plants were grown in the open during winter. Heat treatment of harvested leaves had generally similar effects on physiological and biochemical parameters in both leaf types. However, increased browning susceptibility was associated with greater post-treatment EL, relatively high pre-treatment (initial) PPO activity, and high pre- and post-treatment PAL activity in yellow tissue. High PPO activity has been associated (Steffens et al. 1994; Thipyapong et al. 2004) with elevated levels of reactive oxygen species (ROS) potentially leading to chlorophyll bleaching and browning susceptibility. References Gemel J et al. (1997) Plants for Environmental Studies p. 209. Steffens JC et al. (1994) Recent Advances in Phytochemistry p. 275. Thipyapong P et al. (2004) Plant Science 167, 693-703.

FROM ZYGOTE TO OIL AND PROTEIN BODIES IN THE SEED OF THE MODEL LEGUME MEDICAGO TRUNCATULA

Song Y.1, Wang X.-D.1, Garg M.2 and Rose R.1 1ARC CILR School of Environmental and Life Sciences, The University of Newcastle, NSW 2308, Australia. 2School of Biomedical Sciences and Pharmacy, The University of Newcastle, NSW 2308, Australia.

Medicago truncatula is a genetic and genomic model for legumes and is used to study seed development as well as plant-microbe interactions. However, the developmental and cell biology of embryo development to produce the oil and protein rich cotyledons has received little attention in M. truncatula. We have carried out a baseline study of the developing embryo and related this to oil and protein biosynthesis. Embryo sac and zygote development is similar to the classical models. The embryo however develops an extensive multicellular suspensor and there is extensive early development of the procambium connecting the shoot and root apical meristems. What was of particular interest in the mature cotyledon was the organization of the oil and protein bodies. Electron microscopy showed that the oil bodies 0.5-2.0 um in diameter are aligned around the periphery of the protein bodies and along the plasma membrane in an apparent specific cellular arrangement. The fatty acid composition is C16:0, C18:1n-9, C18:2n-6, and C18:3n-3. The major component is C18:3n-3, accounting for 35% of the total. Fatty acid accumulation is evident as the cotyledons start to develop. An OLEOSIN gene, which encodes an oleosin protein inserted in the phospholipid monolayer enclosing the triacylglycerols of the oil bodies, prevents coalescence of the oil bodies and is expressed at a similar stage. Genes expressing the storage protein vicillin commence expression at a similar time to OLEOSIN but LEGUMIN A initiates expression earlier in development. These data will assist in linking the biochemistry and cell biology to the genetic regulation of the biogenesis of oil and protein bodies.





AN AUTOACTIVE FLAX R PROTEIN IS PURIFIED WITH ATP BOUND IN ITS NUCLEOTIDE BINDING POCKET

Williams S.J.1, Sornaraj P.1, Decourcy-Ireland E.1, Menz R.I.1, Kobe B.2, Ellis J.G.3, Dodds P.N.3 and Anderson P.A.1 1The School of Biological Sciences, Flinders University, Adelaide, Australia. 2School of Chemistry & Molecular Bioscience; Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia. 3CSIRO, Plant Industry, Canberra.

The flax rust resistance protein, M, confers immunity in flax to strains of the flax rust fungus that encode the effector molecule, AvrM. The M protein is a member of the most common class of resistance proteins, containing a central NB-ARC domain and a c-terminal leucine-rich repeat (LRR). When strains of the rust pathogen carrying the AvrM effector encounter a flax plant carrying the M rust resistance gene, a recognition event takes place that culminates in localised cell death at the infection site. This recognition event involves the direct interaction between the M and AvrM and presumably results in the activation of the M protein to instigate cell death signalling. We have previously reported the purification of flax M and L6 resistance proteins using the Pichia pastoris expression system (Schmidt et al 2007). A simplified method of protein production has been developed and with this we have found that purified recombinant M protein is associated with ADP. A mutation in the P-loop motif of M, shown to inactivate the protein in planta, abolishes this ADP association. Furthermore, purified M protein containing a mutation in the conserved MHD motif of the ARC2 subdomain, that gives rise to an autoactive M protein in planta, is associated with higher levels of ATP than ADP, and this association is also dependent on the integrity of the P-loop. Our data validates aspects of the current preferred model of NB-ARC-LRR protein activation, suggesting that ADP/ATP binding controls the inactive/active, state of the protein (Tameling et al 2006). Schmidt et al (2007) Plant J. 50:1107-1117 Tameling et al (2006) Plant Physiol. 140:1233-1245.

JASMONIC ACID AND ETHYLENE SIGNALING PATHWAYS ARE INVOLVED IN CITRUS DEFENSES AGAINST A. ALTERNATA “TANGERINE PATHOTYPE”

Stuart R.M.1, 2, Kubo K.S.1, 2, Boava L.P.2, Bastianel M.2 and Machado M.A.2 1State University of Campinas UNICAMP, Campinas, SP, Brazil. 2Centro de Citricultura Sylvio Moreira, Cordeiropolis, SP, Brazil.

Alternaria alternata “tangerine pathotype” causes the disease known as Alternaria brown spot (ABS), which affects mandarins and several of their hybrids. The symptoms include brown to black necrotic spots surrounded by a yellow halo, and affect twigs, young leaves and fruits. The fungus produces the host-selective toxin (HST) ACT, which is responsible for the pathogenesis. The molecular mechanisms involved in plant susceptibility or resistance are obscure. In order to better understand the plant defense responses to ABS, key genes involved in salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways were analyzed by RT-qPCR in response to A. alternata “tangerine pathotype” inoculation. Leaves of susceptible Murcott tangor (Citrus sinensis x C. reticulata), resistant Cleopatra mandarin (C. reshni), and resistant Clementine (C. clementine) were inoculated with fungal spores suspension. cDNA was synthesized from the total RNA extracted from leaves after 6 and 12 hours post inoculation. Resistant Clementine and Cleopatra mandarin showed up regulated genes involved in JA/ET pathways, like RlemLOX2 (lipoxigenase), RlemMPL2 (miraculin like protein 2), PR-3 (Chitinase II), and ACC oxidase after inoculation. In contrast, genes involved in JA/ET pathways, like RlemLOX2 and PR-3, and also genes involved in SA/flavonoids pathways, like MPK1 (MAP kinase phosphatase 1), PAL (Phenylalanine ammonia lyase), CHS (chalcone synthase), were down regulated in susceptible Murcott tangor. This suggests that JA/ET play important roles in plant resistance against A. alternata. Financial support: FAPESP (07/00931-3) and CNPq (INCT).

A TRANSCRIPTIONAL AND ENZYMATIC EVALUATION OF MALIC ACID METABOLISM IN DEVELOPING WINEGRAPES

Sweetman C.1, Ford C.M.2 and Soole K.L.1 1Flinders University of South Australia, Sturt Road, Bedford Park, SA 5042. 2University of Adelaide, Hartley Grove, Glen Osmond, SA 5064.

The acidity of Vitis vinifera berries is largely determined by tartaric acid and malic acid. Unlike tartaric acid, malic acid is intricately involved in cellular metabolism, and is degraded as the fruit ripens. As such, the concentration of malic acid in winegrapes is often an influential factor for harvesting and winemaking decisions, and can ultimately affect sensory qualities of the wine. The importance of malic acid in berry ripening can be demonstrated by the significant change in metabolism at veraison, whereby net malic acid accumulation gives way to net degradation. This metabolic “switch” coincides with the initiation of berry softening, sugar accumulation and pigment development. The aim of this research is to improve understanding of the control of malic acid metabolism during berry development, particularly at the metabolic switch. As malic acid has numerous metabolic roles (including photosynthesis and respiration), specific activities of several enzymes have been measured to determine which pathways of metabolism may be favoured during malate accumulation and degradation phases, and the potential functions of these pathways. Enzyme assays were tested from mitochondrial and cellular isolates of grape berries at various stages of development and expression of genes of key enzymes were determined using qRT-PCR. Data are used to evaluate known metabolic functions of malic acid in plants, and to determine which enzymes may be important in regulating the content of malic acid in developing grape berries.

INVESTIGATING THE ROLES OF BARLEY CESA GENES IN CELL WALL BIOSYNTHESIS

Tan A.H.T., Burton R.A. and Fincher G.B. Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, The University of Adelaide, Urrbrae, SA.

Barley contains at least eight cellulose synthase (CesA) genes of which have been transcript profiled in a range of tissues (Burton et al., 2004). CesA1, CesA2 and CesA6 have higher transcript levels in tissues more likely to be undergoing the synthesis of primary cell walls, while CesA4, CesA5/7 and CesA8 are more likely to be involved in the synthesis of secondary cell walls (Burton et al., 2004). Growing and expanding cells usually consist of primary walls and secondary cell walls are usually deposited upon cessation of cell growth (McNeil et al., 1984). In this study, a gain-of-function approach was used to study the roles of the CesA genes in barley cell wall biosynthesis. Five sets of transformed barley plants transgenic for constructs carrying the CesA1, CesA2, CesA4, CesA6 or CesA8 genes driven by the constitutive 35S promoter were analysed for transcript levels, phenotype and tissue morphology. Preliminary results showed that transcript levels of the CesA transgenes were lower than expected even though they were driven by a strong constitutive promoter. The transgenic lines transformed with the secondary cell wall CesA4 exhibited a phenotype where xylem cells in the vascular tissue appear to be irregular in shape and are partially collapsed. This resembles the irx mutants described in Arabidopsis (Taylor et al., 1999). A “brittle node” phenotype was also observed in barley transgenic for the 35S:HvCesA8 which also may be affecting secondary cell wall synthesis. Further studies will include stem strength measurements and cellulose content assays of the transgenic barley stems.





HIGH ENERGY FORAGE WITH ELEVATED AND PROTECTED TRIACYLGLYCERIDES

Winichayakul S., Scott R., Roldan M., Cookson R., Richardson K., Bryan G. and Roberts N. AgResearch Grasslands, Forage Biotechnology, Palmerston North, New Zealand.

To ensure benefits to farming both financially and environmentally while maintaining low input agricultural systems, our main goal is to elevate the metabolisable energy available in forages to the grazing ruminants. Raising dietary intake of lipids mostly in the form of triacylglycerides (TAGs) is standard practice in the feed lot industry and results in a significant positive influence on the feed conversion efficiency of sheep and beef as well as an increase in milk production and reproductive performance. We proposed to emulate this practice via accumulation of TAG in the leaves of forage species by over-expressing diacylglycerol acyltransferase1 (DGAT1), an enzyme that catalyses the last and only committed step in the synthesis of TAG. Our first modification in ryegrass showed approximately 30-40% of total lipids increased in the leaves over-expressing Arabidopsis thaliana DGAT1, under the control of the CaMV35S promoter. Further improvements included the optimisation of the DGAT1 nucleotide sequence for transcription stability, mutation of the peptide sequence to prevent posttranslational regulation, placing of the gene under control of a leaf specific promoter, and co-expression of DGAT1 with modified oleosin - a unique protein that encapsulates TAG forming discrete oil bodies (OBs) in the seed. Modified oleosins are engineered in our laboratory to stabilise OB integrity and protect TAG from (1) remobilisation within the leaf, (2) biohydrogenation within the rumen. The efficacy of these modifications was determined in Arabidopsis and showed that one of our modifications leads to 70% increase in total leaf lipids and a substantial portion of this was in the form of TAG. Results to date and our projection for future work are presented here.

CHARACTERIZATION OF REGULATED PROTEIN SECRETION IN PHYTOPHTHORA ZOOSPORES

Zhang W., Hardham A.R. and Blackman L.M. Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia.

Motile zoospores are key players in establishing infection in many species of Phytophthora and other pathogenic oomycetes. Three types of vesicles in the peripheral cytoplasm of zoospores may play important roles during infection. Having reached a potential host, Phytophthora zoospores encyst and become immobilised on the plant surface. During zoospore encystment, ventral and dorsal vesicles are exocytosed rapidly, delivering adhesives and a putative protective coating onto the surface of the cysts. By contrast, during encystment large peripheral vesicles move away from the plasma membrane, become randomly distributed within the cyst cytoplasm and after several hours are degraded. In recent research, we have found that a 12 kDa protein, designated PnCcp, within the large peripheral vesicles is selectively secreted during encystment. This discovery provides evidence of a highly novel mechanism for differential protein secretion in eukaryotes. Studies have shown that in sporangia and zoospores PnCcp colocalises with Lpv, a high molecular weight glycoprotein also resident in the large peripheral vesicles. However, in hyphae, in some cases the large peripheral vesicles contain only Lpv and in some cases, PnCcp appears to form a layer surrounding Lpv within the vesicles. These observations suggest that the two proteins may be transported to the large peripheral vesicles at different times during their development. Constructs encoding GFP-tagged versions of PnCcp are being made and will be transformed into Phytophthora nicotianae to facilitate studies of the synthesis and dynamic movement of the large peripheral vesicles and the timing of PnCcp secretion.

TERMINATION OF DNA REPLICATION IN E. COLI: NOT EVERY TER SITE MEDIATES TERMINATION OF REPLICATION

Moreau M.J. and Schaeffer P.M. James Cook University, School of Pharmacy and Molecular Sciences, Angus Smith Drive, Townsville, QLD, 4811.

The replication of the circular chromosome of E. coli starts at the unique origin of replication oriC where two replisomes are assembled and progress in opposite directions to replicate the chromosome. Termination of replication occurs roughly opposite to oriC and is mediated by the Tus protein bound to 21 bp sequences called Ter. Fourteen Ter sites have been identified but only 10 (TerA-J) were shown to mediate DNA replication fork arrest. The 10 primary Ter sites are arranged in two clusters of five, each cluster being on either side of the region directly opposite to oriC. These clusters act in a polar manner to constrain forks meeting in the terminus region of the chromosome and prevent fork progression in the terminus to origin direction by forming a Tus-Ter-Lock structure. We studied the affinity and kinetics of complex formation of Tus with the 10 primary Ter sites and their Tus-Ter-Lock variants using surface plasmon resonance and our new GFP-based protein stability assay (GFP-Basta). GFP-Basta measures the increased stability of a protein induced by its binding to a specific ligand. It is a powerful technique capable of detecting very small changes in binding affinity (Moreau et al. (2010) submitted to Molecular BioSystems). Both methods showed for the first time that Tus binds differently to the 10 Ter sites and the Tus-Ter-Lock variants. The results also revealed that not all Ter sites are able to form the Tus-Ter-Lock structure. The significance of these new results, along with the development of GFP-Basta will be discussed in detail.

GENOTYPE AND ENVIRONMENT EFFECTS ON PROTEIN COMPOSITION OF WHEAT GRAINS

Md Zin N.H., Copeland L. and Wilkes M. Faculty of Agriculture, Food and Natural Resources, University of Sydney, NSW 2006.

The amount and type of proteins are important in determining the quality of wheat grains for specific uses in foods. Protein composition is determined by the variety of grains and environment in which it is grown. To increase our knowledge of genotype by environment interactions on quality proteins, three wheat varieties were grown in four different locations in New South Wales, Australia. Proteins were extracted from the grains and separated by SDS-PAGE and bands were identified by peptide mass fingerprinting. Quantitative changes were observed in electrophoretic patterns of the polypeptides of molecular mass in the 32 kDa to 60 kDa range from the different wheat varieties grown in different locations. Proteins in this size range are known to be associated with quality characteristics of wheat grains. In comparison, polypeptides with molecular mass smaller than 32 kDa or greater than 60 kDa showed essentially no variability of expression in gels, indicating the influences of genotype by environment interactions on protein composition were selective.





STRATEGY FOR COMPREHENSIVE IDENTIFICATION OF FUNCTIONAL HUMAN N-MYRISTOYLATED PROTEINS USING AN INSECT CELL-FREE PROTEIN SYNTHESIS SYSTEM

Moriya K.1, Suzuki T.2, Ando E.2 and Utsumi T.1 1Graduate School of Medicine, Yamaguchi University. 2Shimadzu Corporation.

To establish a strategy for the comprehensive identification of functional human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and mass spectrometric analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from ~2,000 KOP (Kazusa ORFeome project) human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal 10 amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated. Thirteen proteins out of these 18 novel N-myristoylated proteins were functionally unknown proteins. The rest of the proteins were key players in various cellular signal transduction pathways, such as phosphatase, ubiquitin E3-ligase, cytoskeletal regulatory protein, apoptosis-related protein, and amino acid transporter. Analysis of intracellular localization of EGFP-fusion proteins of wild type and G2A-mutant of these proteins revealed that protein N-myristoylation strongly affected intracellular localization of many of these proteins. These results indicate that the strategy proposed in this study is useful for the comprehensive identification of functional human N-myristoylated proteins from human cDNA resources.

β7-STRAND IS ESSENTIAL FOR MONOMER INTERACTION IN HOMO- AND HETEROOLIGOMERS OF HUMAN SMALL HEAT SHOCK PROTEINS

Mymrikov E.V., Bukach O.V., Seit-Nebi A.S. and Gusev N.B. Department of Biochemistry, School of Biology, Moscow State University.

Ten small heat shock proteins (sHsp) form a family of chaperones widely expressed in different human tissues. sHsp prevent aggregation of denatured proteins and regulation of many intracellular processes. All sHsp contain conservative α-crystallin domain and tend to form homo- and heterooligomers. Irregular oligomer composition and high mobility prevent crystallization of full-size sHsp and make desirable development of alternative approaches for investigation of protein-protein interaction within sHsp oligomers. In order to solve this problem we obtained point mutants of human Hsp20, Hsp22 and αB-crystallin with replacement of all endogenous Cys residues by Ser and a single Cys residue inserted in position homologous to Cys137 of human Hsp27. This unique Cys residue is located in the β7-strand of α-crystallin domain, i.e. in position of putative monomer contacts within Hsp27 homooligomers. Mutations introduced do not affect secondary, tertiary or quaternary structure and chaperone-like activity of human sHsp and therefore are suitable for investigation of monomer interaction. Mild oxidation of thus obtained Cys-mutant of sHsp leads to formation of disulfide cross-linked dimers in the case of homooligomers of Hsp20, Hsp22, αB-crystallin and wild type Hsp27. Effective disulfide crosslinking was observed in heterooligomers containing Hsp20, Hsp27 and αB-crystallin. In heterooligomers containing Hsp22 and any other sHsp formation of crosslinked heterodimers was significantly reduced. Thus, the β7-strand of α-crystallin domain is essential for monomer contacts in homo- and heterooligomers of sHsp and Hsp22 is less effective in formation of heterooligomers than any other sHsp analyzed. Acknowledgement. This investigation was supported by Russian Foundation for Basic Science..

RATIONAL DRUG DESIGN OF INHIBITORS FOR THE SCHIZOPHRENIA THERAPEUTIC TARGET KYNURENINE AMINOTRANSFERASE-I

Nadvi N.A.1, 2, Akladios F.1, Salam N.K.1, Park J.H.2, Hibbs D.E.1, Hanrahan J.R.1, Kapoor V.3, Gorrell M.D.2 and Church W.B.1 1Faculty of Pharmacy, University of Sydney NSW. 2Centenary Institute, University of Sydney NSW. 3School of Medicine and Pharmacology, University of Western Australia, Perth, WA.

Kynurenic acid (KA) is an intermediate derived from the Kynurenine pathway of tryptophan metabolism. It is an endogenous antagonist at the glutamate-binding site of the NMDA receptor and high levels of KA often accompanies the occurrence of several neurological disorders including Schizophrenia. Overactivity in the enzyme human Kynurenine Aminotransferase-I (hKAT-I) which produces this metabolite, is believed to be an important link in the pathophysiology and makes the kynurenine pathway a valuable target for the treatment of Schizophrenia. We require to express large amounts of purified hKAT-I to obtain co-crystal structures of enzyme-inhibitor complexes, test the lead compounds and continue subsequent optimisation to design potent inhibitors. Recombinant hKAT-I was expressed using the baculovirus-insect cell system. Computational biochemistry and bioinformatics have been used to generate potential inhibitors for hKAT-I. In particular in silico docking calculations involving both ligand and structure-based descriptors have been used to predict lead compounds, and a number synthesized. Two sets of recombinant hKAT-I have been prepared: a semi-purified sample for high-throughput screening of lead inhibitors and a very pure, crystal-grade sample for crystallisation trials and comprehensive investigation into the inhibition study with the most promising lead inhibitors. In preliminary assays eight out of 11 candidate compounds were found to inhibit KAT-I, with the strongest possessing an apparent Ki of 2.8 μmol/l. These assays were performed with semi-purified recombinant hKAT-I and further compounds are available for testing. An in-depth analysis of inhibition kinetics with crystal-grade hKAT-I samples and corresponding co-crystal structures will aid in elucidating and designing novel KAT-I specific inhibitors for therapeutic treatment of Schizophrenia.

DESIGNER SINGLE-STRANDED RNA-BINDING PROTEINS

Nguyen C.D., Weisbrodt S., Mansfield R. and Mackay J.P. School of Molecular Bioscience, University of Sydney, NSW, Australia.

It has become clear that a large number of RNA species are widely involved in gene regulation and diseases in complex organisms. Their functions are regulated by RNA-binding proteins (RBPs). Therefore, engineered RNA-binding proteins with the capacity to target any chosen RNA sequence would be powerful tools with which to manipulate gene expression for both therapeutic and research applications. Given spectacular achievements of designer DNA-binding proteins assembled from zinc fingers (ZnFs) and preliminary data from our laboratory, we propose that classical ZnFs and RanBP2 ZnFs can be excellent scaffolds from which to create sequence-specific RBPs. Mutagenesis has been extensively used to make variants of ZRANB2 ZnFs which is a member of RanBP2 ZnFs family, then their binding affinities to relevant single-stranded RNA (ssRNA) sequences are measured by using fluorescence anisotropy. So far, a couple of mutants have been found to have specificity for different ssRNA triplets. Nuclear magnetic resonance spectroscopy and/or X-ray crystallography will be recruited to determine the structural basis for these protein-RNA recognitions. Moreover, the composition and length of inter-domain linker will be optimized in order to construct modular single-stranded RNA-binding proteins that can recognize desired six/nine/twelve-base ssRNA sequences.





NATURAL SUBSTRATES OF THE N-END RULE PATHWAY IN ESCHERICHIA COLI

Ninnis R., Spall S., Talbo G., Truscott K. and Dougan D. La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing a destabilising N-terminal amino acid (N-degron) are degraded by ClpAP in an ATP-dependent manner. The degradation of these substrates is controlled by the adaptor protein ClpS, which is directly responsible for recognition of the N-degron and its delivery to the ClpAP protease(1). We have isolated 23 ClpS interacting proteins from E. coli. Our data demonstrate that two of these proteins, DNA protection during starvation protein (Dps) and putrescine aminotransferase (PATase), are post-translationally modified to generate an N-degron. In the first example, the N-degron of Dps appears to result from an endoproteolytic cleavage by an unknown protease. In the second case, the N-degron of PATase is generated by the addition of primary destabilising residues Leu and Phe to the N-terminus of the protein. In this case generation of the N-degron is dependent on leucyl/phenylalanyl-tRNA protein transferase (LFTR) and determines the stability of the protein in vivo. Independent of the type of post translational modification, both N-end rule substrates are degraded by ClpAP in a ClpS dependent manner. We conclude that the modification of PATase, by LFTR, is directly responsible for its ClpAPS-mediated turnover in E. coli and hence the N-end rule pathway may play an important role in putrescine homeostasis. (1)Erbse A, Schmidt R, Bornemann T, Schneider-Mergener J, Mogk A, Zahn R, Dougan DA, & Bukau B 2006 ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature 439, 753-756.

ANALYZING LIM-DOMAIN MEDIATED INTERACTIONS

Nisevic I., O’Connell M., Mansfield R. and Matthews J.M. School of Molecular Bioscience, The University of Sydney, NSW 2006.

LIM-homeodomain (LIM-HD) proteins are transcription factors involved in cell specification in many different tissue types. The twelve LIM-HD proteins in mammals are expressed in different combinations in different cells at various stages of development, thereby forming a combinatorial “LIM-code”. Each LIM-HD protein has 2 N-terminal tandem LIM-domains (LIM1 and LIM2) and a central homeodomain. The essential cofactor protein LIM-domain binding protein 1 (Ldb1) contains a ~30-residue region, known as the LIM-interaction domain (LID) which allows it to bind the tandem LIM domains of all members of the LIM-HD family. We want to determine the binding affinities for all of these interactions to establish which LIM-HD proteins are most likely to bind to the cofactor Ldb1. It is not straight-forward to achieve this aim because the isolated LIM domains from these proteins tend to be insoluble and/or aggregate in solution. Fortunately, they are stabilised by the presence of Ldb1-LID or other binding partners. Thus, we have been assessing the use of several different types of competition assays, including ELISAs and Fluoresence-based assays. The implementation of these assays and different strategies for fluorescently labelling the proteins will be discussed.

STRUCTURE-FUNCTION STUDIES OF ANNEXINS IN INFECTIOUS DISEASES

Osman A. and Hofmann A. Structural Chemistry Program, Eskitis Institute for Cell & Molecular Therapies, Griffith University, Don Young Road, Nathan, Brisbane, 4111 Qld, Australia.

Many biochemical and histological properties are shared within the annexin family of proteins, because of their highly conserved three-dimensional fold. The landmark feature of annexins is the calcium-dependent binding to acidic phospholipid membranes. Recently, parasite annexins have attracted attention due to their immunogenic properties in the case of cysticercosis and giardiasis. It is hypothesized that their localization at the cyst wall (Taenia solium) and adhesive ventral disk (Giardia lamblia) provides a potential link to the parasite attachment to or invasion of the host. As such, parasite annexins may be novel targets to combat infectious diseases. In this study, the crystallization and biochemical properties (membrane and glycosaminoglycan binding) of parasite annexins are investigated. Crystals of alpha-1 giardin from G. lamblia were obtained and diffracted to 1.9 Å. The crystals belong to the orthorhombic space group P212121. The determination of a parasite annexin structure is essential to provide better insight into their biological roles and to support drug discovery efforts against infectious diseases. Only few biochemical characteristics of parasite annexins are known to date. Recent studies of parasite annexins in our lab demonstrate lectin properties for alpha-1 giardin and annexin B1 from T. solium, but not for annexin (Sm)1 from Schistosoma mansoni and alpha-3 giardin from G. lamblia. Similarly, membrane binding varies for the different parasite annexins, depending on the presence of the canonical calcium binding motif, the endonexin sequence. The difference in the binding ability of parasite annexins towards glycosaminoglycan and lipid membranes may suggest diverse biological functions.

AN INVESTIGATION IN PARTNERSHIPS: PARASPECKLE PROTEIN- RNA INTERACTIONS

Passon D.M.1, 2, Lee M.1, Bythell-Douglas R.R.1, Fox A.H.2 and Bond C.S.1 1School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, 6009 WA. 2Western Australian Institute of Medical Research, University of Western Australia, Perth 6000 WA.

Paraspeckles are dynamic sub-nuclear bodies, located next to splicing speckles, within eukaryotic cells. The formation of paraspeckles depends both on the transcription of a long non-coding RNA, NEAT1 and on the presence of core paraspeckle proteins. The best evidence thus far for paraspeckle function points to a role in the control of gene expression via nuclear retention of certain mRNA species. This is mediated by paraspeckle proteins binding to adenosine-to-inosine edited inverted repeat motifs and targeting of the RNA transcript to paraspeckles. The molecular mechanisms behind these processes are still unclear. Our hypothesis is that the characterisation of paraspeckle proteins and their RNA interaction partners will give functional and mechanistic insights into this method for controlling gene expression. The core paraspeckle proteins PSP1 and P54nrb are located in paraspeckles in an RNA-dependent manner. Both proteins have highly similar sequences, contain RNA-recognition motifs and form a heterodimer. I will present preliminary data of potential RNA binding partners of PSP1 and p54nrb and I will describe experiments aimed at establishing in vitro and in vivo RNA-protein interaction assays in order to identify potential RNA binding partners for subsequent crystallization of protein-RNA complexes.





THE ANTIFUNGAL ACTIVITY OF A PLANT DEFENSIN REQUIRES THE FUNGAL CELL WALL

Payne J.A., Hulett M., Anderson M.A. and Van der Weerden N. Department of Biochemistry, La Trobe University, Plenty Road, Bundoora 3086.

Plant defensins are small, cysteine-rich proteins involved in the innate immune system of plants. NaD1, a plant defensin from the flowers of the ornamental tobacco, Nicotiana alata, has potent antifungal activity and is able to protect transgenic cotton from fungal infection in the field. The antifungal activity of NaD1 involves a multi-step mode of action whereby NaD1 interacts specifically with the cell wall, permeabilises the plasma membrane and ultimately enters the cytoplasm. Unlike some other antifungal molecules, NaD1 requires an intact cell wall for its antifungal activity. The fungal cell wall is composed of three layers; a thin layer of chitin adjacent to the plasma membrane, a layer of (1-3)-β-glucan and an outer layer of glycoproteins. We are currently investigating how NaD1 interacts with each of these components in an attempt to understand why cell wall binding is essential before NaD1 can permeabilise the plasma membrane and enter the cell.

DYNOLE 34-2 IS A POTENT INHIBITOR OF DYNAMIN THAT INDUCES CELL DEATH FOLLOWING CYTOKINESIS FAILURE AND SUPPRESSES GLIOBLASTOMA TUMOUR GROWTH IN VIVOChircop M.1, Perera S.1, Ma M.P.C.1, Lau H.2, Gilbert J.3, Jones N.C.2, Dabacco G.2, Gordon C.P.4, Young K.A.4, Morokoff A.2, Sakoff J. 3, O’Brien T. 2, McCluskey A. 4 and Robinson P.J. 1 1Children’s Medical Research Institute, The University of Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia. 2Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville, 3050, Victoria, Australia. 3Senior Hospital Scientist, Department of Medical Oncology, Newcastle Mater Misericordiae Hospital, Edith Street, Waratah, NSW 2298, Australia. 4Chemistry, School of Environmental & Life Sciences, The University of Newcastle, Callaghan NSW 2308, Australia. Glioblastoma brain tumours are one of the most lethal and difficult forms of cancer to treat. With optimal therapy the median survival period is 40-60 weeks. Several inhibitors of mitotic proteins (e.g. Aurora kinase, Plk) have emerged in preclinical or early clinical development for the treatment of cancer. Cytokinesis is the final stage of mitosis resulting in the generation of two independent daughter cells. Our recent discoveries highlight the endocytic protein dynamin II (dynII) as a novel cancer therapeutic target. DynII is required for the abscission phase of cytokinesis – a stage in mitosis not yet targeted for cancer treatment. One of the difficulties in treating brain cancers is the ability of small molecules to cross the blood brain barrier. The dynole class of dynamin inhibitors represent such molecules. Here, we reveal that analogous to MiTMABs, the five most potent dynoles, 25, 26, 33, 34-2 and 35, induce cytokinesis failure at the point of abscission, consistent with inhibition of dynamin. Dynoles inhibited cell proliferation (MTT and colony formation assays) with dynole 34-2 being the most potent and the SMA-560 glioblastoma cells the most sensitive, compared to ten other cancer cell lines screened. Dynole 34-2 also induced apoptosis as evident by PARP cleavage. Cell death was specifically induced in cells following cytokinesis failure, suggesting that dynole 34-2 selectively targets dividing cells. Moreover, this compound inhibited glioblastoma tumour volume by >80% in an in vivo othotopic glioblastoma mouse model with no adverse toxicity affects at clinically relevant doses. We conclude that dynamin inhibitors are a new class of anti-mitotic agents with a novel mechanism of action – targeting cytokinesis. Thus, dynamin inhibitors are a new approach for the treatment of cancers, particularly brain tumours that require the rare ability of chemotherapeutic agents to cross the blood brain barrier.

OLIGOMERIC STRUCTURE OF DAP DECARBOXYLASE FROM DIVERSE BACTERIAL SPECIES

Peverelli M.G., Dogovski C. and Perugini M.A. Bio21 Institute, 30 Flemington Road, Parkville VIC 3010 Australia, Department of Biochemistry and Molecular Biology, University of Melbourne.

Diaminopimelate decarboxylase (DAP-DC) plays a critical role in the lysine biosynthesis pathway of bacteria and plants. The enzyme catalyses the final step in the pathway, namely, the decarboxylation of meso-diaminopimelate to form L-lysine. The structure of the enzyme has been characterised from several bacterial species, including, E. coli, M. jannaschii, and M. tuberculosis. The enzyme forms different oligomeric structures in the crystal form of these species. Moreover, DAP-DC from the Gram-negative eubacterium E. coli is monomeric; the enzyme from the Gram-negative archaea M. jannaschii is dimeric; whilst DAP-DC from the eubacterium M. tuberculosis forms a tetramer in the crystal form. We hypothesise that the diversity of the enzyme’s quaternary structure is linked to species diversity and/or environment. Accordingly, the aim of this study is to characterise the quaternary structure of DAP-DC in solution and crystal forms from diverse eubacterial and archaeal species. We have thus cloned, expressed, and purified DAP-DC from Bacillus anthracis and are currently conducting structural studies comparing B. anthracis DAP-DC with the enzyme from E. coli, M. jannaschii, and M. tuberculosis.

DISTINCT PARTITIONING OF POLYQ AND POLYA-EXPANDED PROTEINS IN CELLS

Polling S., Ramdzan Y.M., Hill A.F. and Hatters D.M. Biochemistry and Molecular Biology, University of Melbourne, VIC 3010.

Nine human congenital disorders result from polyalanine (polyA) sequence expansions in nine different host proteins. In addition, nine other neurodegenerative diseases, including Huntington’s, are caused by polyglutamine (polyQ) expansions in different host proteins. While polyQ-expansions are well-understood to lead to the aggregation of the host proteins into amyloid like fibrils, which is predicted as a major source of toxicity to cells, the corresponding influence of polyA on proteins remains largely unknown. Here, we examined the biophysical properties of model proteins containing polyA expansions and compared them to proteins with polyQ expansions. In cells co-expressing polyA and polyQ-containing proteins, we found that polyA-expanded proteins formed punctate structures, predicted to contain aggregated protein, that accumulated into mutually exclusive cellular locations relative to polyQ-containing protein aggregates. In contrast, different proteins that contained polyA expansions, and likewise with the polyQ expansions, always accumulated together within the same punctate structures in cells. The differential partitioning of polyA proteins to polyQ proteins in cells, suggest the presence of multiple cellular processes for the trafficking and handling of protein aggregates formed by different proteins.





SYNTHESIS OF THE RECOMBINANT CHIMERIC TRANSTHYRETIN FOR ITS FUNCTIONAL STUDY

Poodproh R., Leelawatwattana L. and Prapunpoj P. Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.

Transthyretin (TTR) is a homo-tetrameric protein that plays major role in distribution of thyroid hormones (THs), and transport of retinol via binding to retinol binding protein (RBP). Besides, its proteolytic catalysis was recently demonstrated and has been proved to associate to human pathology. However, the amino acid residues participate in the catalysis could not be identified. To obtain more insight the underlying mechanism, the comparative effect of changes in structure on the catalysis of TTR could be a useful tool. In this research, cDNAs coding for TTRs having altered amino acid sequence particular in the N- and C-terminal regions were constructed and produced in Pichia pastoris. These include Crocodylus porosus TTR with N-terminal sequence of Xenopus TTR and C-terminal sequence of pig TTR, human TTR with N- and C-terminal sequences of Xenopus and pig TTR, respectively, and human TTR with the C-terminal segment of pig TTR. The recombinant TTRs were successfully synthesized and extracellularly secreted into culture medium, and all of them had general physicochemical properties such as subunit mass, tetrameric formation and the electrophoretic mobility under native condition similar to TTRs found in nature. The cleavage ability of the chimeric TTRs was examined on either universal substrate such as casein or natural substrate such as apolipoprotein A-I. By comparing the catalysis of the chimeric TTRs to that of the unaltered TTRs, the results revealed the effect and confirmed the relationship between these structural segments and function of TTR.

DEVELOPMENT OF A PEPTIDE INHIBITOR OF MATRIPTASE FOR IN VIVO TESTING

Quimbar Dominguez P.M.1, Daly N.L.2, Dunse K.M.1, Hulett M.D.1, Craik D.J.2 and Anderson M.A.1 1Department of Biochemistry, La Trobe University, Melbourne, 3086, Australia. 2Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, 4072, Australia.

Matriptase, a member of the type II transmembrane serine protease family, has been implicated in the progression of some epithelial cancers. Matriptase is thought to aid tumour progression through activation of hepatocyte growth factor which regulates the proliferation and migration of epithelial cells. Therefore discovery of potent inhibitors of tumour associated matriptase activity presents the potential to develop anti-cancer drugs. We are investigating the ability of a group of cyclic peptides to inhibit matriptase in order to develop inhibitors that block metastasis. Cyclic peptides are ideal candidates for cancer therapeutics due to their proteolytic stability and small size. Preliminary tests have shown that squash trypsin inhibitor MCoTI-II has a higher binding affinity for matriptase than the cyclic sunflower trypsin inhibitor 1 (SFTI-1). Comparison of these two inhibitors may aid in the development of more potent inhibitors of matriptase. We are currently investigating the potential of these inhibitors using in vitro cell migration assays.

MITOGENIC AND METABOLIC SIGNALLING VIA THE INSULIN RECPTOR ISOFORM-A

Rajapaksha K.H., Alvino C.L., Wallace J.C. and Forbes B.E. School of Molecular and Biomedical Sciences, University of Adelaide.

There are two insulin receptor isoforms (IR-A and IR-B) which arise from alternative splicing. The IR-B is responsible for metabolic control more commonly associated with IR action. The IR-A isoform lacks 12 amino acids normally encoded by exon 11 and it binds insulin and IGF-II with high affinity. Interestingly, IGF-II is able to stimulate mitogenic responses via the IR-A. The IR is a heterodimer of 2α and 2β subunits. The ligand binding site is found in the extracellular α subunits whereas the kinase domain and phosphorylation sites involved in signalling are found on the intracellular portions of the β subunits. Phosphorylation of the IR β chain in response to insulin has previously been detected on 7 of the possible 14 Tyr residues, 12 of the 32 Ser residues and a single Thr. The total pattern of phosphorylation of the IR-A in response to IGF-II has never been reported. We are aiming to define receptor mitogenic and metabolic phosphorylation barcodes using an innovative mass spectrometry approach. Cells expressing IR-A were treated with insulin and IGF-II. Phosphorylation barcodes were defined in response to these two proteins by purifying stimulated receptors from cell lysates and labelling using Isotope-Coded Protein Labelling (ICPL) methodology. The intracellular domain was trypsin digested and phosphorylated and nonphosphorylated peptides were quantitated by mass spectrometry.

ULTRACELLULAR ALTERATIONS OF THE HEPATOCELLULAR CARCINOMA CELL LINE INDUCED BY Hep88 mAb: A NOVEL HARMFUL mAb

Rojpibulstit P.1, Manochan S.1, Puthong S.3, Gamnarai P.1, Kittisenachai S.2, Kangsadalampai S.1 and Roitrakul S.2 1Faculty of Medicine, Thammasat University (Rangsit Campus), Klong-Luang, Pathumthani, 12121 Thailand. 2Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phahonyothin Road, Klong Luang, Pathumthani 12120 THAILAND. 3Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Patumwan, Bangkok, 10330.

Abstract Hepatocellular carcinoma (HCC) is the most leading cause of cancer- death in the world. Nowadays many targeted therapy particularly an immunotherapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is continually explored. In this regard, Hep88 mAb was established and subsequently revealed its cytotoxicity on hepatocellular carcinoma cell line (Hep G2 cell line) and normal liver cell line (Chang liver). We are then further focused on the localization of Hep88 mAb’s specific protein from hepatocellular carcinoma Hep G2 cell line by western blot analysis. Moreover, 3 days incubation of Hep88 mAb on Hep G2 cell line was also investigated by using a transmission electron microscope. The result demonstrated that the recognizing proteins of molecular weights about 53-60 kDa and 70-90 kDa have been found not only on cell membrane but also on cytoplasmic compartment. The cytotoxicity’s result at its IC50 concentration against HepG2 cell line showed its tumoricidal effect on hepatocellular carcinoma cell line (Hep G2 cell line) with harmful-less to the normal liver cell line (Chang liver). Furthermore, the transmission electron micrograph illustrated an intracellular vacuolization with mitochondria and endoplasmic reticulum dilatation. These fascinating results from this study imply that Hep88 mAb might be a promising tool to a development of an effective treatment of HCC in the next decade.





THE EXTRACURRICULAR ACTIVITIES OF UPSTREAM OPEN READING FRAMES

Friend L.1, Hillman K.1, Skarshewski A.1, Wang X.Q.1, Gorman J.1, Smith R.1 and Rothnagel J.A.1 1School of Chemistry and Molecular Biosciences, University of Queensland, Qld, 4072, Australia.

Many eukaryote transcripts contain at least one AUG codon upstream (uAUG) of the main start codon. In mammals, approximately 30% of these are followed by open reading frames (uORFs) of 20 or more codons. We developed a web-based program to rapidly identify conserved uORFs and their putative bioactive peptides. We identified over 200 uORFs that are strongly conserved. An examination of the consensus sequences surrounding their uAUGs found that ~20% were in an optimal context for ribosomal initiation and therefore predicted to be efficiently translated. We hypothesize that these uORFs encode functional peptides which we have termed upstream peptides (uPEPs). Several uPEP sequences were prioritized for further analysis based on their predicted likelihood of being expressed. Ten uPEP sequences were amplified by RT-PCR and cloned into a GFP plasmid expression vector. After appropriate quality assurance, these plasmids were transfected into HeLa cells and GFP expression visualised by confocal microscopy. Surprisingly, we found that eight exhibited distinct cellular localisation patterns that can provide insight into their cellular roles. Four uPEPs showed nuclear localisation, four showed cytoplasmic localisation and two were indistinguishable from the GFP alone control. In only one case was the localisation predicted by an examination of the sequence; this uPEP contained 5 positively charged residues reminiscent of a nuclear localisation sequence and was specifically localized to the nucleolus of transfected cells. The use of bioinformatic tools and in vivo cell analysis has provided insights into an over-looked component of the proteome. While not all uORFs encode bioactive peptides, a subset will have important cellular roles and aberrations in their expression will result in disease.

INHIBITION OF MUSHROOM TYROSINASE BY NOVEL MONOFUNCTIONAL AND BIFUNCTIONAL DITHIOCARBAMATES SODIUM SALTS

Saboury A.A. and Amin E. Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

Two novel monofunctional dithiocarbamates, C6H5NHCSSNa (I) and C6H5CH2NHCSSNa (II), and two bifunctional dithiocarbamates, NaSSCNHC6H4NHCSSNa (III) and NaSSCNHCH2C6H4CH2NHCSSNa (IV), were synthesized and characterized by spectroscopic techniques. These water-soluble compounds were examined for their inhibition of both activities of mushroom tyrosinase (MT). L-3, 4-dihydroxyphenylalanine and L-tyrosine were used as substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic studies showed noncompetitive inhibition of I and II and mixed type of III and IV on both activities of MT. The inhibition constant (KI) value of II is smaller that of I. Also, the inhibition constant value of IV is smaller that of III. Moreover, the inhibition constant value of III is smaller that of I, as the same as IV is smaller than of II. It seems bifunctional dithiocarbamates due to two charged head groups chelate active site and are potent MT inhibitors. Raising temperature caused decreasing KI values of I and II and increasing those of III and IV. The binding process for inhibition of monofunctional dithiocarbamates (I and II) is entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for inhibition of bifunctional dithiocarbamates (III and IV) due to the enthalpy driven binding process. In order to find more information about mechanism of inhibition of the enzyme, we studied structural investigations on the effect of our four synthesized compounds on MT. Fluorescence studies showed probably conformation of inhibitor-bound MT is stable and inflexible than of uninhibited MT. Circular Dichroism studies did not show any considerable secondary structure changes of MT.

THE STRUCTURE AND VACCINE POTENTIAL OF AN IMMUNOMODULATORY SALIVARY CYSTATIN FROM A SOFT TICK

Salat J.1, Paesen G.C.2, Rezacova P.3, 4, Kotsyfakis M.1, 5, Kovarova Z.3, 6, Sanda M.3, Andersen J.F.5, Kopacek P.1, Kopecky J.1 and Mares M.3 1Biology Centre ASCR and Faculty of Sciences, University of South Bohemia, Czech Republic. 2Centre for Ecology and Hydrology, Mansfield Road, Oxford, UK. 3Institute of Organic Chemistry and Biochemistry, ASCR, Czech Republic. 4Institute of Molecular Genetics, ASCR, Czech Republic. 5Laboratory of Malaria and Vector Research, NIH, Rockville, USA. 6Department of Biochemistry, Faculty of Science, Charles University, Czech Republic.

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines, and proliferation of antigen-specific T-cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of ticks in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

CHARACTERISATION OF OXIDIZED RECOMBINANT HUMAN GALECTIN-1 STRUCTURE AND FUNCTION

Scott S.A. and Blanchard H. Institute for Glycomics, Griffith University, Gold Coast Campus, Australia.

Galectin-1 protein is over-expressed by many aggressive human cancers. The lectin mediated apoptotic affects galectin-1 has upon T cells suggests that secretion of galectin-1 into the tumour stroma indirectly contributes to tumour survival and growth by essentially creating a shield from immune surveillance. Futhermore, human galectin-1 lectin activity promotes cell migration and adhesion, two critical processes in angiogenesis and the invasion of metastatic cancer cells. Galectin-1 lectin activity is exhibited by the reduced homodimeric (~ 30 kDa) form of the protein and to maintain this lectin activity the cysteine residues within each 14.5 kDa subunit must be kept from oxidizing to form disulphide bonds. Cysteine oxidation however does not render galectin-1 inactive with respect to all biological functions. An oxidized form of human galectin-1 can heal injured and diseased axons. Given the well-established link between oxidative stress and pathological conditions such as injury and disease, greater understanding of the oxidized human galectin-1 structure and function is required. We have detected two forms of oxidized human galectin-1, an oligomer of 68 kDa and a smaller protein species of apparent molecular weight 17 kDa. The 68 kDa form contains inter- and intra-molecular disulphide bonds, whereas the 17 kDa form contains only intra-molecular disulphide bonds. The oligomer is formed under higher protein concentration and lower salt concentration, whereas the smaller species is formed under opposite conditions. Both forms exhibit lectin-independent activity upon a cancerous immune cell line and reveal affinity for the fatty acid portion of an intracellular binding partner.





IDENTIFICATION AND CHARACTERIZATION OF PROTEIN KINASES THAT BIND TO N-TERMINAL DOMAIN OF DNA METHYLTRANSFERASE 1

Sekiguchi M., Sugiyama Y., Sueyoshi N. and Kameshita I. Department of Life Sciences, Kagawa University, Kagawa 761-0795, Japan.

DNA methylation plays an important role in transcriptional repression of imprinted genes in mammals. DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we explored protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. Dnmt1-binding protein fraction was prepared from mouse brain extract using Dnmt1(1-290)-affinity column. This fraction was further subjected to DNA-cellulose column chromatography and analyzed by Western blotting with kinase-specific antibodies. The protein kinase activity toward Dnmt1(1-290) was also examined using separated fractions by gel filtration column chromatography. These analyses revealed that Dnmt1-binding protein fraction contained two Ser/Thr protein kinases with molecular masses of approximately 110 kDa and 45 kDa. When these proteins were subjected to LC-MS/MS analysis, 110-kDa kinase and 45-kDa kinase were identified as cyclin-dependent kinase-like 5 (CDKL5) and casein kinase 1 (CK1), respectively. Both CDKL5 and CK1 could be coprecipitated with Dnmt1 when they were analyzed by GST pull down experiments. CDKL5 and Dnmt1 were found to colocalize in the nucleus and appeared to interact with each other. Catalytically active form of CDKL5, CDKL5(1-352) phosphorylated the N-terminal domain of Dnmt1 in the presence of DNA. CK1 also phosphorylated this domain much more significantly than CDKL5, and phosphorylation of Dnmt1 by CK1 decreased the affinity for DNA. These results suggest that CDKL5 and CK1 bind to and phosphorylate the N-terminal domain of Dnmt1 and may regulate the functions of Dnmt1.

KNOCKDOWN OF CALMODULIN-DEPENDENT PROTEIN KINASE I-DELTA CAUSES DEVELOPMENTAL ABNORMALITIES IN ZEBRAFISH

Senga Y., Nagamine T., Nimura T., Sueyoshi N. and Kameshita I. Department of Life Sciences, Kagawa University, Kagawa 761-0795, Japan.

Ca2+/calmodulin-dependent protein kinase I-delta (CaMKIδ), a fourth isoform of CaMKI, is known to be expressed ubiquitously in various tissues. However, the functional roles of this enzyme in vivo have not been fully clarified yet. To investigate the biological significance of CaMKIδ during zebrafish embryogenesis, we cloned and characterized two splice variants of zebrafish CaMKIδ, zCaMKIδ-L (392 AA) and zCaMKIδ-S (368 AA). Although recombinant proteins expressed in Escherichia coli showed weak protein kinase activities, they were activated when phosphorylated by mouse Ca2+/calmodulin-dependent protein kinase kinase alpha. Activated CaMKIδ significantly phosphorylated various proteins including CREB, histones, and myelin basic protein in a Ca2+/CaM-dependent manner. In COS7 cells, transiently expressed zCaMKIδ-L and zCaMKIδ-S were localized in the cytoplasm as in case of mouse CaMKIδ. During zebrafish embryogenesis, zCaMKIδ-L and zCaMKIδ-S were detected in the embryos after 36 hpf and 60 hpf, respectively, by Western blotting using specific antibodies. Western blotting analysis in adult zebrafish demonstrated wide expression of zCaMKIδ-L in the brain, eyes, ovary, and skeletal muscle. In contrast, zCaMKIδ-S specifically localized in the brain. The knockdown of the zCaMKIδ genes with morpholino-based antisense oligonucleotide resulted in an increase of abnormal embryos with small heads. These results clearly show the significance of zCaMKIδ-L and zCaMKIδ-S during zebrafish embryogenesis.

FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS OF THIOREDOXIN IN CANCER CELLS UNDER DIFFERENT OXYGEN CONDITIONS

Shah F.L.1, 2, Di Trapani G.1, Karlenius T.C.1, 2, Clarke F.M.1, 2 and Tonissen K.F.1, 2 1School of Biomolecular and Physical Sciences, Griffith University, Nathan QLD 4111, Australia. 2Eskitis Institute for Cell and Molecular Therapies, Griffith University.

Thioredoxin is a redox capable protein that is integral to the functioning of cells. It performs several roles within the intracellular environment, including the scavenging of reactive oxygen species or ROS. Oxidative stress is caused when the cell contains excessive levels of ROS, which can lead to inflammation, ischemic tissue damage, carcinogenesis and cancer progression, or even cell death. Thioredoxin expression increases under conditions of oxidative stress, which can also cause thioredoxin to be secreted into the extracellular environment. Another condition that causes an increase in thioredoxin levels is hypoxia, which is defined as a loss of oxygen in cells and tissues and is widespread in solid tumours. While it is not known how hypoxia leads to an increase in thioredoxin expression, there is evidence that ROS has roles to play within the hypoxic cellular environment, which may be related to the increased thioredoxin levels. There is not much known about the increase of thioredoxin in hypoxic conditions, or whether intracellular thioredoxin increase corresponds to increased thioredoxin on the surface of cells. Therefore, the aim of this project is to quantitatively look at the levels of thioredoxin both within and on the surface of cancer cells under different oxygen conditions (oxidative stress, normoxia, hypoxia) via FACS analysis. Immunoflourescence was performed as a visual complement to the FACS analysis.

QUASI-IRREVERSIBLE INSULIN-RECEPTOR COMPLEXES: EVIDENCE FOR THE FORMATION OF DISULPHIDE BONDS BETWEEN INSULIN AND THE INSULIN RECEPTOR?

Shelton B.P.1, 2 and Helmerhorst E.1 1Western Australian Biomedical Research Institute & Curtin Health Innovation Research Institute, School of Biomedical Sciences, Curtin University of Technology, Bentley Western Australia 6012. 2Lung Institute of Western Australia, QEII Medical Centre Nedlands Western Australia 6009.

The binding of insulin to its cell surface receptor initiates a signalling cascade that mediates insulin’s cellular effects. While it is well established that insulin binding induces a conformational change in the insulin receptor, the exact mechanism involved in insulin binding and receptor activation is still poorly understood. Complex binding kinetics, which are not fully described by current models of biomolecular interactions, typify the interaction between insulin its receptor. A negative cooperativity model has been widely reported to describe the ligand-induced enhancement of insulin dissociating from its receptor. Further, evidence from our laboratory and others demonstrates a reduced dissociation of insulin from its receptor as a function of increased association time, suggesting a time-dependent transition from a rapid to a slow dissociating complex. Careful analysis of data in our laboratory shows that this reduced dissociation is only partially explained by changes in the dissociation rates per se. Instead, these effects appear dependent on the amount of complex available for dissociation, with a population of complexes displaying binding that does not dissociate even after 24hrs. This quasi-irreversible population of ligand- receptor complexes is sensitive to reducing, oxidising and alkylating agents, suggesting that disulphide bonds may be involved. This quasi-irreversible population is also sensitive to changes in ionic strength, independent of the salt used, suggesting that key ionic interactions may be involved in the formation of the quasi-irreversible state. The existence of a non-dissociating population of receptor complexes may have significant physiological implications and must be considered when modelling the insulin receptor interaction.





THE BIOMOLECULAR CHANNELLING OF A REACTIVE INTERMEDIATE

Smith N.E., Vrielink A., Corry B. and Attwood P. University of Western Australia, M310, 35 Stirling Highway, Crawley WA 6009.

The channelling of intermediates through buried molecular tunnels in multi-enzyme subunits is a topic of considerable interest as it is thought to occur in many different enzymes. This process shields reactive or poisonous intermediates from the rest of the organism and ensures direct, rapid transport from one active site to the other. Many of these molecular channels have been identified using techniques such as X-ray crystallography, but little is known about the internal mechanisms they use to transport their intermediates. In this case, computational and kinetic methods have been applied to study the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase-aldehyde dehydrogenase (acylating) (DmpFG) and its proposed channelling activity. Previous crystallographic studies have found that DmpFG has a 29 Å channel linking its two active sites. This raises questions about whether it functions as a conduit for the poisonous intermediate acetaldehyde. In the case of this project computational techniques, including both standard molecular dynamics and metadynamics, have been used to investigate whether it is energetically feasible for the intermediate to be transported in this way between the two active sites and to verify the role of proposed checkpoints at the entry and exit of the channel. The kinetic assays on wild type DmpFG are designed to determine the kinetic parameters of both the active site 1 and 2 reactions and the channelling event. Both computational and kinetic data will be presented.

CROSS-REACTIVITY BETWEEN LUPIN AND PEANUT

Soo L.1, Smith P.M.C.1, Mir G.A.1, Smith W.B.2, Prescott S.L.3, Soutter V.4 and Loblay R.H.4 1School of Biological Sciences, University of Sydney. 2School of Paediatrics & Child Health Research, UWA, Princess Margaret Hospital Perth, WA. 3Royal Adelaide Hospital, South Australia. 4Allergy Unit, RPA Hospital Sydney.

The number of immunoglobulin E (IgE) mediated allergic reactions to lupin has increased incrementally with the increasing inclusion of lupin products in human foods. Although some individuals are allergic to only lupin, there is evidence that some individuals who are allergic to peanuts are more likely to be allergic to lupin. This suggests that lupin and peanut allergens may share common IgE epitopes ie. they cross-react. We have conducted lupin food challenges on individuals who are allergic to peanut. Approximately 6.5% of peanut allergic individuals also react to lupin. In an inhibition assay we used peanut proteins to inhibit binding of IgE to lupin proteins suggesting that they share cross-reactive epitopes. Further characterization identified that the peanut protein extract inhibited binding to proteins of 25 and 34 kDa. We are currently expressing the major seed storage proteins of lupin, that have been identified as allergenic, in a cell-free protein synthesis system. The recombinant proteins will be used in inhibition assays to identify the cross-reactive proteins. A site-directed mutagenesis will then be used to characterize the shared IgE epitopes. Findings from this work will contribute to the understanding of lupin allergy and the cross-reactivity between lupin and peanut allergens, and possibly other legumes. These results will also assist in designing better diagnostic and therapeutic approaches for food hypersensitivity reactions. This work was funded by Grain Food CRC.

PLASMODIUM SERINE HYDROXYMETHYLTRANSFERASE IS DIFFERENT IN SUBSTRATE SPECIFICITY FROM ITS HOMOLOGUES - A POTENTIAL ANTIMALARIAL TARGET

Sopitthummakhun K.1, Leartsakulpanich U.2, Yuthavong Y.2 and Chaiyen P.1 1Center for Excellence in Protein Structure & Function, Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, 10400, Thailand. 2National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Road, Pathumthani 12120, Thailand.

Abstract World malaria report in 2008 estimated the widespread of malaria as 247 million cases among 3.3 billion people with nearly a million deaths and most of cases were in children less than 5 years. Plasmodium falciparum (Pf) was among the leading causes of death worldwide from a single infectious agent, whereas P. vivax (Pv) was the cause of chronic malaria. The effective control remains chemotherapy. However, increasing numbers of reports of drug resistance in parasites press the need to find new effective anti-malarial compounds. Deoxythymidylate synthesis pathway is vital for Plasmodium which consists of the bi-functional enzyme, dihydrofolate reductase-thymidylate synthase, and serine hydroxymethyltransferase (SHMT). The latter is less well-characterized. We previously showed that both recombinant Pf- and PvSHMT are PLP enzyme that catalyze the conversion of serine and tetrahydrofolate to glycine and methylene tetrahydrofolate. Here, the ability of PvSHMT to use other substrates in THF-independent reaction was studied. The results showed that PvSHMT can catalyze the retro-aldol cleavage toward 3-hydroxy amino acids, L-threonine, allo-threonine and β-phenylserine. Moreover, the enzyme catalyzed the aminotransferase reaction of D-alanine and pyridoxal phosphate to pyruvate and pyridoxamine. Interestingly, PvSHMT can use D-serine as substrate in addition to L-serine which is unlike that of the eukaryotic enzymes. Based on the distinct property, selective inhibitors against Plasmodium enzyme such as D-amino acid analogs can be designed. We currently are developing an assay to increase the throughput for inhibitor screening. Inhibition study with various derivatives of folate and D-amino acid analogs will be reported.

A PROPHAGE BASED GENETIC SELECTION FOR USE IN PROTEIN STRUCTURE FUNCTION STUDIES

Murchland I., Schubert R.A., Dodd I.B. and Shearwin K.E. School of Molecular and Biomedical Science, The University of Adelaide.

Using the principles of synthetic biology, we have developed a modular genetic selection system designed to allow the isolation of mutant proteins defective in particular types of interactions. The system is based on the observation that an E. coli strain carrying an integrase deficient (int-) lysogen of bacteriophage 186 shows a very high level of cell death upon induction (derepression) of the lysogen. By customised construction of genetic circuits which interface with those of the resident int- lysogen, we have adapted the system to isolate collections of mutants for a number of different proteins, in each case targeting a specific function of that protein. For example, we have isolated repressor protein mutants defective in self association at particular interfaces, transcriptional activator proteins defective in their interaction with RNA polymerase, and anti-repressor mutants unable to interact with their target repressor. In each case, the genetic selection also contains a modification to ensure that only full length mutant proteins are isolated. The various sets of mutants have provided valuable data for deciphering the molecular mechanisms by which these proteins carry out their biological actions.





ENAMIDE ANALOGUES OF PANTOTHENATE (VITAMIN B5) INHIBIT PROLIFERATION OF THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM

Spry C.1, Villa M.V.J.3, Marquez R.3 and Saliba K.J.1, 2 1Research School of Biology, The Australian National University, Canberra, Australia. 2Medical School, The Australian National University, Canberra, Australia. 3Department of Chemistry, University of Glasgow, Glasgow, Scotland.

Survival of the virulent human malaria parasite Plasmodium falciparum during intraerythrocytic development is dependent on an extracellular supply of pantothenate (vitamin B5), a precursor of the fundamental enzyme cofactor coenzyme A (CoA). Previously, a naturally occurring pantothenate analogue called CJ-15,801 was shown to inhibit proliferation of P. falciparum in vitro (IC50 of 39 μM). This analogue differs in structure from pantothenate only by containing a carbon-carbon double bond in place of a carbon-carbon single bond, which, by virtue of its position relative to an amide group, forms part of an enamide moiety. CJ-15,801 was shown to exert its antiplasmodial effect specifically by interfering with parasite pantothenate utilisation and to be less toxic to mammalian cells. Inspired by the activity of CJ-15,801, we synthesised and tested 43 novel fully-synthetic enamide-bearing pantothenate analogues for antiplasmodial activity. Twenty-eight antiplasmodial analogues were identified, including two with improved potency relative to CJ-15,801 (IC50 values of 4 and 14 μM). The effect of pantothenate on the antiplasmodial activity of the enamides was investigated to identify those exerting an antiplasmodial effect by competitively inhibiting pantothenate utilisation. Inhibition of pantothenate utilisation by the enamides was explored further by testing their activity against P. falciparum pantothenate kinase(s)-catalysed pantothenate phosphorylation (the first step in the metabolism of pantothenate to CoA). The differential activity of the analogues in this series has shed light on the key structural features required for inhibition of pantothenate utilisation and parasite growth, which should facilitate the future development of pantothenate utilisation inhibitors with improved antiplasmodial activity.

ACCUMULATION OF ENDOGENOUS LOW MOLECULAR WEIGHT CRYSTALLIN PEPTIDES IN THE HUMAN LENS: A KEY EVENT IN THE AGING PROCESS

Su S.-P.1, 2, Truscott R.J.W.2, McArthur J.D.1 and Aquilina J.A.1 1School of Biological Sciences, University of Wollongong, NSW 2522. 2Save Sight Institute, University of Sydney, NSW 2001.

Age-dependent modification of crystallin proteins is a major contributor to cataract in human lenses. Recent studies have shown that endogenous low molecular weight (LMW) peptides, derived from the breakdown of crystallins, accumulate in old and cataractous lenses, with some possessing natural anti-chaperone abilities against α-crystallin - the molecular chaperone in the lens. Furthermore, during aging and/or the onset of cataract, α-crystallin has been shown to associate increasingly with the lens fibre cell membrane. To investigate structural and functional changes of crystallins in the aging lens, we characterized and quantified crystallin-derived LMW peptides present in water-soluble and water-insoluble extracts of human lenses aged 20 to 86 years using MALDI-MS and nanoLC-ESI-MS/MS. The prominent LMW peptides (αB1(2)-18, αA66(67)-80, γS2-22 and βA3189-215) present in the lens were mapped using MALDI-imaging MS. Our results indicate that LMW peptides emerged during early middle-age and are confined to nuclear fibre cells. Interestingly, in older lenses these peptides were present in both cortical and nuclear regions. Using fluorescently-tagged (Alexa350) α-crystallin subunits in direct membrane-binding assays, we showed that the presence of the LMW crystallin peptides (αB1(2)-18 and αA66(67)-80) can influence the binding of α-crystallin subunits with lens fibre cell membranes. Taken together, these results demonstrate that LMW crystallin peptides accumulate in the human lens during the aging process and can affect the molecular chaperone function of α-crystallin and its interaction with fibre cell membranes. This process can directly contribute to the aggregation of water-insoluble lens protein complexes, leading to the opacification commonly observed in old lenses.

CRYSTAL STRUCTURE OF IMPORTIN α COMPLEXED WITH THE DNA REPAIR PROTEIN KU70 NLS PEPTIDE

Takeda A.A.S.1, 2, Kobe B.2 and Fontes M.R.M.1 1Departamento de Física e Biofísica, Instituto de Biociências, UNESP - Univ Estadual Paulista, Botucatu, SP, Brazil. 2School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Centre for Infectious Disease Research, University of Queensland, Brisbane, Queensland 4072, Australia.

Importin α (Impα) plays a role in the classical nuclear import pathway, binding to cargo proteins with activities in the nucleus. Those proteins contain nuclear localization sequences (NLS), which are characterized by one or two clusters of basic amino acids (monopartite and bipartite NLSs, respectively). In this work we present the expression, purification and the crystal structure of Impα from M. musculus (residues 70-529, lacking the autoinhibitory domain) and the NLS peptide from the DNA repair protein Ku70 to provide more information about the NLS recognition by Impα. Impα (70-529) sample was obtained in E. coli expression system and purified by affinity chromatography followed by an ion exchange chromatography. A single crystal of Impα-Ku70NLS complex was grown by hanging drop method. The data were collected at 100K using a Synchrotron Radiation Source (Laboratório Nacional de Luz Synchrotron, LNLS, Brazil) and processed to 2.6 Å. Molecular replacement techniques determined the crystal structure. Results of the Impα-Ku70NLS complex indicated an electron density available only in the major binding site corresponding to the Ku70 NLS peptide. It bounded to the ImpA similarly to the phosphorilated version of the simian virus 40 (SV40) large tumour (T)-antigen NLS showing the possibility of this peptide being a monopartite type in spite of being a bipartite as suggested in previous studies. Keywords: Importin α, nuclear import, NLS, X-ray crystallography, Ku70. Supported by: FAPESP and CNPq.

PURIFIED MOUSE CYP27B1 CAN HYDROXYLATE 20,23-DIHYDROXYVITAMIN D3 PRODUCING 1α,20,23-TRIHYDROXYVITAMIN WHICH HAS ALTERED BIOLOGICAL ACTIVITY

Tang E.K.Y.1, Janjetovic Z.2, Li W.3, Nguyen M.N.1, Slominski A.2 and Tuckey R.C.1 1The University of UWA (School of Biomedical, Biomolecular and Chemical Sciences). 2University of Tennessee Health Science Center (Department of Pathology and Laboratory Medicine). 3University of Tennesse Health Science Center (Department of Pharmaceutical Sciences, College of Pharmacy).

20,23-Dihydroxyvitamin D3 is a biologically active metabolite produced via the action of cytochrome P450scc (CYP11A1) on vitamin D3. It inhibits keratinocyte proliferation, stimulates differentiation and inhibits NF-κB activity working as a vitamin D receptor agonist. We have tested the ability of purified mouse 25-hydroxyvitamin D3 1α -hydroxylase (CYP27B1) to add a 1α-hydroxyl group to this vitamin D analogue and whether this altered its biological activity. 20,23-Dihydroxyvitamin D3 incorporated into phospholipid vesicles was converted to a single product by CYP27B1 which was confirmed to be 1α,20,23-trihydroxyvitamin D3 by NMR. The 20,23-dihydroxyvitamin D3 was a relatively poor substrate for CYP27B1 compared to the normal substrate, 25-hydroxyvitamin D3, displaying a 5-fold higher Km and 8-fold lower kcat value. Like 20,23-dihydroxyvitamin D3, 1α,20,23-trihydroxyvitamin D3 decreased keratinocyte proliferation. It caused little stimulation of the expression of the vitamin D receptor at the mRNA level compared to the 30-fold induction observed with the same concentration (100 nM) of 1α,25-dihydroxyvitamin D3 at 24 h. Addition of the 1α-hydroxyl group to 20,23-dihydroxyvitamin D3 greatly enhanced its ability to stimulate the expression of the CYP24 gene but not to the extent seen with 1α,25-dihydroxyvitamin D3. This study shows that purified CYP27B1 can be used to enzymatically add a 1α-hydroxyl group to 20,23-dihydroxyvitamin D3 with the product showing altered biological activity, especially for the stimulation of CYP24 gene expression. Supported by NIH grant R01AR052190.





ANS FLUORESCENCE STUDIES ON FLEXIBILITY AND SUBSTRATE-INDUCED CONFORMATIONAL CHANGES OF THE PSYCHROTROPHIC AND MESOPHILIC ACETATE KINASES

Tang A.K.1, 2, 3, Motoshima H.1 and Watanabe K.1, 2 1Department of Applied Biochemistry and Food Science, Faculty of Agriculture, Saga University, 1-Honjo-machi, Saga 840-8502, Japan. 2The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan. 3Department of Applied Nutritin and Food Technology, Islamic University, Kushtia, Bangladesh.

The acetate kinase from the Antarctic psychrotroph Shewanella sp. AS-11 (SAK) has a significantly higher activity at low temperatures when compared with the mesophilic homologue from Escherichia coli K-12 (EAK). The SAK and EAK were investigated by fluorescence spectroscopy of the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) to compare their flexibility and substrate-induced conformational changes. The fluorescence maximum wavelength of ANS with SAK and EAK were observed at 458 and 480 nm, respectively, when excited at 360 nm. The maximum ANS fluorescence intensity with SAK was 5.8-fold higher than that with EAK. The binding of ANS had no effect on the kinase activity of both enzymes. The fluorescence intensity with EAK increased with decreasing temperatures, but that with SAK decreased. When ATP bound to SAK, almost all ANS fluorescence was quenched, whereas bound to EAK, only 60% was quenched. These data suggest that SAK has a more hydrophobic ANS binding site with a more flexible structure when compared with EAK. The bindings of ATP to free enzymes resulted in substantial conformational changes, which can be interpreted as the transition from an open to closed form. This substrate-induced conformational change in SAK appears to be larger than that in EAK. It is likely that these structural features of SAK may contribute to its high activity at low temperatures.

MOLECULAR BASIS OF DEATH DOMAIN INTERACTIONS THAT REGULATE ASSEMBLY OF PROINFLAMMATORY CASPASE-1 ACTIVATING COMPLEXES

Vajjhala P.R., Kaiser S., Mirams R.E. and Hill J.H. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Qld 4072, Australia.

The assembly of proinflammatory caspase-1 activating complexes, known as inflammasomes, is initiated in response to intracellular pathogens and various cellular stress conditions. These complexes process proinflammatory cytokines such as pro-interleukin-1β into active forms that trigger an inflammatory response. Inflammasome assembly is mediated by interactions between death domains present in components of these complexes. Death domains consist of six antiparallel α-helices and typically mediate homotypic interactions between members of the same subtype. Two subtypes of death domains are present in inflammasomes; the pyrin domain (PYD) and the caspase activation and recruitment domain (CARD). Despite the importance of PYD domains in both inflammasome assembly and regulation, nothing is known about the molecular details of their interactions. We have identified molecular determinants important for interaction of the PYD domain of ASC, a critical adaptor protein for inflammasome assembly. In addition to interacting with inflammasome components, ASC interacts with several regulatory proteins via its PYD. Moreover, ASC is a dimer and dimerisation is proposed to be mediated via the PYD. To identify residues that are important for ASC PYD interactions, we have individually mutated 17 surface exposed residues on the ASC PYD and tested the effect of these mutations on ASC self-association and interaction with inflammasome-related proteins. We have done this using a range of biochemical, biophysical and cell-based techniques. We have also identified the complementary binding surface on the ASC-interacting protein, pyrin. Here we will present our data and the implications for inflammasome assembly and regulation.

EXPRESSION, PURIFICATION AND CHARACTERIZATION OF CLONING-GRADE ZINC FINGER NUCLEASE

Tovkach A.1 and Tzfira T.2 1CSIRO Plant Industry, Canberra, ACT, Australia. 2University of Michigan, Ann Arbor, MI, USA.

The limited number of naturally occurring rare-cutting restriction enzymes and the slow and tedious engineering of existing restriction enzymes for novel specificities have prompted the design of new strategies for the development of restriction enzymes with specificities for long DNA sequences. One possibility is using zinc finger nucleases (ZFNs) - synthetic restriction enzymes that are custom-designed to target and cleave long DNA sequences and which have been recently shown useful for DNA cloning. Here we report on the purification and biochemical analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and gel-filtration purification methods are sufficient to produce a cloning-grade enzyme. We show that ZFN-10 can function as an accurate and reliable ZFN using the same reagents and protocols used for naturally occurring and commercially available recombinant restriction enzymes. We also show that ZFN-10 tolerates a set of target-site substitutions which can be predicted from the specificities of recognition helices incorporated into the structure of its DNA-binding domain. The relative simplicity of ZFN-10 design, expression, purification and analysis suggests that novel ZFNs can potentially be designed and applied for various recombinant DNA applications.

INHIBITORS OF VIRAL PROTEIN NUCLEAR IMPORT AS ANTI-VIRAL AGENTS

Wagstaff K.M., Rawlinson S.M. and Jans D.A. Dept. Biochemistry and Molecular Biology, Monash University, Clayton, Vic 3800 Australia.

Infectious diseases, such as those caused by human immunodeficiency virus (HIV) and Dengue Virus (DENV) remain highly significant health burdens world-wide. Due to a lack of effective treatments and the ability of many viruses to develop resistance to anti-viral agents, there is an urgent need for the identification/development of new anti-viral drugs. Significantly, a number of viral proteins, even from cytoplasmically replicating viruses such as HIV or DENV, localise in the host cell nucleus as an essential part of the infectious cycle, making viral protein nuclear import an attractive target for therapeutic intervention. We have developed a novel high-throughput ALPHAScreen-based assay to identify inhibitors of important biological protein-protein interactions from compound library screens. Since nuclear import of HIV Integrase protein (IN) is critical to HIV function we conducted a pilot screen to identify inhibitors of IN binding to its nuclear import receptor protein Importin (Imp) α/β. Through a series of counter/cross-screen we were able to identify several potent and specific IN:Imp α/β inhibitors, which significantly inhibit IN nuclear accumulation in transfected cells, but have no effect on the nuclear accumulation of other nuclear proteins. In addition we have identified several general inhibitors, which potently inhibit all nuclear import of proteins mediated by Imp α/β, but do not effect nuclear import mediated by alternative pathways. Significantly, several of these compounds have potent anti-viral action. Identifying inhibitors of nuclear transport through our approaches is clearly a viable approach to tackle many of the world’s significant viral pathogens.





CHARGED REPETITIVE PROTEINS UNITE DIVERSE EUKARYOTES

Waller R.F., Gould S.B. and McFadden G.I. School of Botany, University of Melbourne.

Human parasites of the phylum Apicomplexa (e.g. the malarial parasite Plasmodium) belong to the supergroup Alveolata that also includes ciliates (e.g. Tetrahymena thermophila)) and dinoflagellate algae. This diverse group of important organisms share a common cell pellicular organization of flattened membrane sacs (alveoli) beneath the plasma membrane and associated with a membrane-anchored cytoskeleton. Despite diverse functional requirements of the pellicles of the different Alveolata members, a common family of proteins (called “Alveolins”) that associate with these alveoli have recently been identified and are unique to Alveolata. To further our knowledge of the organization and makeup of this Alveolata-specific cell feature we have performed a proteomic analysis of purified cell pellicles from the model ciliate Tetrahymena thermophila. Purified pellicle fractions were analysed by electrospray ionisation on a QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer, peptides searched against the T. thermophila genome and 1178 proteins identified by two or more peptides. 272 of these proteins were of unknown function, but contain similar proteins when searched against Apicomplexan genomic data, implying a broad suite of common pellicular proteins. A conspicuous feature of almost two thirds of these common proteins is the presence of highly charged repetitive motifs. Through localization of such proteins we have identified novel components of pellicular structures involved in host invasion in apicomplexan parasites and the oral apparatus of ciliates. Thus it appears that despite the different roles of the cell pellicular structures of Alveolates, a similar suite of repetitive proteins contribute to their diverse functionality.

CHARACTERISATION OF A RECOMBINANT PLANT-MADE SOLUBLE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS (SRAGE)

Webster D.E.1, Mulcair M.1, Madhavan S.2, Bialkowski K.3, Talbo G.3, Premaratne R.1, Cooper M.E.3, Forbes J.M.3 and Thomas M.C.3 1School of Biological Sciences, Monash University, Vic 3800, Australia. 2Burnet Institute, Vic 3004, Australia. 3Baker IDI Heart and Diabetes Institute, Vic 3004, Australia.

Advanced Glycation End-products (AGEs) play a key role in the development and progression of human disease. The pathogenic activity of AGEs is partly mediated by activation of AGE receptors, the best known of which is the Receptor for AGEs (RAGE), a multi-ligand glycoprotein receptor from the immunoglobulin superfamily. Activation of RAGE can be prevented by a truncated ‘dummy receptor’ (soluble RAGE, sRAGE) and this strategy has been successfully used to prevent disease in vivo. The development and optimisation of an efficient method of sRAGE production and its detailed characterisation represents an important step in the practical application of sRAGE. Plants represent a viable production system, which is safe, scalable, affordable and produces high-quality recombinant protein. Plant-made proteins are functionally and structurally comparable to native protein, with similar patterns of glycosylation. Our research with plant-based production systems has shown that sRAGE can be expressed by stable transgenic plants and hairy root cultures. Plant-made sRAGE is stable, appropriately folded and glycosylated and retains its ability to form polymeric complexes required for ligand binding. In addition, we have shown that plant-made sRAGE can bind a range of putative RAGE-ligands in a specific manner and inhibit activation of RAGE in cell-based assays. Further studies have optimised a purification protocol and undertaken secondary structure determination by CD spectroscopy. The results will be discussed in the context of in planta protein engineering with the aim of improving the bioactivity and half-life of sRAGE.

SWITCHING THE CATALYTIC PROPERTIES OF C3 AND C4 PLANT RUBISCOS

Whitney S.M. Plants Sciences, Research School of Biology, The Australian National University.

The catalytic properties of the photosynthetic CO2-fixing enzyme, Rubisco, have a profound impact on photosynthetic carbon assimilation. In C4 plants the protein machinery and leaf physiology have evolved to append to C3 photosynthesis systems that concentrate CO2 around Rubisco to enhance its activity as well as plant productivity, water and nitrogen use efficiency. Despite sharing the same complex catalytic chemistry, improvements in the carboxylation rate (vc

max) of Rubisco from C4 plants have generally come at the expense of CO2 affinity. The homologous-recombination mechanism of chloroplast genomes has been used to identify the structural switches that lead to the higher vc

max of Rubisco from C4-plants. The catalytic Rubisco large (L) subunit gene (rbcL) in tobacco has been replaced with the rbcL from either C3 or C4 Flaveria species. Sequence and catalytic analysis of the resulting hybrid L8S8 Rubiscos (comprising eight Flaveria L-subunits and eight native tobacco small (S) subunits) have identified the L-subunit residues responsible for the catalytic disparity between these C3 and C4 Rubiscos. Introducing these changes into the tobacco L-subunit also endow this C3-Rubisco with C4 catalytic features, that is, an improvement in vc

max and a higher Km for CO2. Identifying structural switches that can increase the catalytic turnover of C3-Rubiscos may assist ongoing CO2 transgenic approaches (i.e. engineering strategies to concentrate CO2 around Rubisco in C3-plants to minimize wasteful photorespiration) aimed at improving yield potential and resource use efficiency.

BIOPHYSICAL CHARACTERISATION OF PROTEIN-RNA INTERACTIONS

Wilce J.A.1, Kim H.1, Yoga Y.1, Cowieson N.2, Scanlon M.J.3, Headey S.J.3, Gorospe M.4, Williams B.5 and Wilce M.C.J.1 1Biochemistry and Molecular Biology, Monash University, Clayton, Australia. 2Australian Synchrotron, Clayton, Australia. 3Monash Institute of Pharmaceutical Sciences, Parkville, Australia. 4NIH, Baltimore USA. 5Monash Institute of Medical Research, Clayton, Australia.

The control of mRNA stability is a key factor in the regulation of gene expression. The competing events of translation and mRNA decay ultimately dictate the level of protein expressed and are readily affected in response to changes in cellular conditions. mRNA transcripts bearing AU-rich sequences in their 3’UTR, in particular, appear to be under the control of many factors that determine whether the transcript is destined for translation, degradation or held in a translationally repressed state. These factors include RNA-binding proteins such Hu proteins which are protective against degradation and TIA proteins which are involved in sequestering mRNA into stress granules under conditions of cellular stress. Exactly how these proteins recognise and interact with AU-rich sequences is only partially understood, including whether they actually compete for the same target mRNAs. We have focused on two of these proteins, HuR and TIAR which are both triple RNA recognition motif (RRM) proteins that bind AU-rich RNA with nM affinity. We report our studies using SPR that reveal similar mRNA target sequence preferences, yet differences in their stringency of interaction. We also explore the importance of the separate RRMs, as well as the protein regions beyond the RRMs, for the mRNA interaction using NMR spectroscopy and differences in molecular shape of the protein/RNA complex using SAXS. Together these data bring us closer to an understanding of the dynamic interplay that takes place in the cell in the regulation of gene expression at the level of mRNA.





CHARACTERISATION OF SUBSTRATE SPECIFICITY OF THE OXYGEN-DEPENDENT ASPARAGINYL HYDROXYLASE FIH-1

Wilkins S.E.1, Hyvarinen J.2, Koivunen P.2 and Peet D.J.1 1School of Molecular and Biomedical Science, University of Adelaide, South Australia. 2Department of Medical Biochemistry and Molecular Biology, University of Oulu, Finland.

FIH-1 (Factor Inhibiting HIF-1) is an oxygen-dependent asparaginyl hydroxylase that plays a role in cellular oxygen homeostasis. It functions as an oxygen sensor and regulates the activity of essential mediators of the chronic hypoxic response, the hypoxia-inducible transcription factors (HIFs), which have been implicated in a number of human diseases including cancer, vascular disease, and cerebral and myocardial ischemia. In normoxia, FIH-1 hydroxylates the transactivation domain of HIF-α, resulting in transcriptional repression. FIH-1 has also been found to hydroxylate numerous ankyrin domain-containing proteins, and work by our laboratory has identified members of the Notch receptor family as substrates of FIH-1. We present data that identify Notch1-3, but not Notch4, as substrates of FIH-1. Binding studies indicate that FIH-1 has a much higher affinity for Notch substrates than for HIF-1α, although HIF-1α demonstrates a higher efficiency of hydroxylation. Interestingly, FIH-1 is able to bind to Notch4, even though it is not a substrate, suggesting a possible role for FIH-1 that is independent of hydroxylation. Analyses of chimeric substrate proteins indicate that secondary structure is an important determinant of both binding affinity and hydroxylation efficiency, providing an explanation for observed differences in hydroxylation of the Notch ankyrin domain compared with HIF. Site-directed mutagenesis shows that substrate recognition is substantially influenced by the amino acids directly adjacent to the target asparagine, a surprising result considering the observed variation in these residues between different substrates. Characterisation of substrate specificity will aid in identification of the full array of substrates and is essential to predict and interpret the functional consequences of therapeutic targeting of FIH-1.

NEW TRICKS TO MEASURE HOW GENETIC MODIFIERS OF MUTANT HUNTINGTIN TOXICITY AFFECT HUNTINGTIN AGGREGATION IN DROSOPHILA

Wong Y.Q.1, 2, 3, Quinn L.M.2, Bottomley S.P.3 and Hatters D.M.1 11 Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne. 2Department of Anatomy and Cell Biology, University of Melbourne. 3Department of Biochemistry, Monash University.

Huntington’s Disease (HD) is caused by mutations that expand a polyglutamine (polyQ) sequence within the first exon of the huntingtin protein. Mutated, polyQ-expanded Huntingtin forms visible aggregates, known as inclusions, within neurons as a key marker of pathology. While in vitro studies have provided insight into the kinetics of aggregation, how aggregation occurs in cells and relates to cellular dysfunction remains poorly understood. This problem is compounded by studies indicating inclusions are actively formed by cells as a protective response to sequester smaller diffuse forms of huntingtin such as monomers and submicroscopic oligomers. We have developed new methods to quantitatively measure the size, localization and heterogeneity of all huntingtin molecules in cells and are using them to examine how previously validated genetic modifiers of mutant huntingtin toxicity affect in-cell aggregation directly in vivo in Drosophila models of HD. In a work in progress, we describe our new methodology and their implementation in Drosophila. We describe preliminary studies on candidate genes in the Wnt pathway known to modulate huntingtin toxicity and bind to huntingtin on their role in inclusion formation.

INTERACTION OF DENGUE VIRUS ENVELOPE PROTEIN WITH ENDOPLASMIC RETICULUM-RESIDENT CHAPERONES FACILITATES DENGUE VIRUS PRODUCTION

Wongwiwat W.1, 2, Limjindaporn T.1, 2, 3, Noisakran S.2, 4, Yenchitsomanus P.2, 4 and Malasit P.2, 4 1Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 2Office for Research and Development (Medical Molecular Biology Unit), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 3Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 4Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand.

Dengue virus infection is an important mosquito-borne disease and a public health problem worldwide. A better understanding of interactions between human cellular host and dengue virus proteins will provide insight into dengue virus replication and cellular pathogenesis. The glycosylated envelope protein of dengue virus, DENV E, is processed in the endoplasmic reticulum of host cells and therefore reliant on host processing functions. The complement of host ER functions involved and nature of the interactions with DENV E has not been thoroughly investigated. By employing a yeast two-hybrid assay, we found that domain III of DENV E interacts with human immunoglobulin heavy chain binding protein (BiP). The relevance of this interaction was demonstrated by co-immunoprecipitation and co-localization of BiP and DENV E in dengue virus-infected cells. Using the same approach, association of DENV E with two other chaperones, calnexin and calreticulin was also observed. Knocking-down expression of BiP, calnexin, or calreticulin by siRNA significantly decreased the production of infectious dengue virions. These results indicate that the interaction of these three chaperones with DENV E plays an important role in virion production, likely facilitating proper folding and assembly of dengue proteins.

STRUCTURE OF A DIMERIC ENZYME FROM A BACTERIAL PSYCHROPHILE PROVIDES INSIGHT INTO MOLECULAR EVOLUTION AT THE QUATERNARY LEVEL

Wubben J.M.1, 2, Dogovski C.1, 2, Dobson R.C.J.1, 2, Codd R.3 and Perugini M.A.1, 2 1Department of Biochemistry and Molecular Biology, The University of Melbourne, VIC 3010, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC 3010, Australia. 3Department of Pharmacology, School of Medical Sciences, The University of Sydney, NSW 2006, Australia.

Shewanella benthica (Sb) is a psychrophilic bacterium that inhabits cold ocean environments. Studying enzymes from this bacterium thus offers great potential to probe the molecular evolution of quaternary structure of proteins, including the tetrameric bacterial enzyme dihydrodipicolinate synthase (DHDPS). Recent studies show that tetramerisation of DHDPS in mesophilic bacteria, such as Escherichia coli and Bacillus anthracis, is essential for stability and also necessary for enzymatic activity. We have cloned, expressed and purified milligram quantities of recombinant DHDPS from Shewanella benthica (Sb-DHDPS). By employing a series of enzyme kinetic, biophysical and structural analyses, we show that DHDPS from the cold bacterium Shewanella benthica (Sb-DHDPS) exists as an active dimer in solution and crystal form at biologically-relevant temperatures (< 13 °C). However, Sb-DHDPS aggregates at higher mesophilic-like temperatures (20-37°C) and is shown to possess significantly less enzymatic activity relative to cooler temperatures. This finding provides evidence for an ancestral dimer of DHDPS and offers tremendous insight into the evolution of the quaternary structure of bacterial dihydrodipicolinate synthase.


POSTERS WEDNESDAY & THURSDAYY



DEVELOPMENT OF A NEW SUITE OF POLYCYCLIC-BASED CONJUGATES OF DESFERRIOXAMINE B FOR IRON OVERLOAD DISEASE

Liu J.1, 2, Obando D.1, 2, Schipanski L.G.1, 2, Groebler L.K.1, 2, Witting P.K.1, 2, Kalinowski D.1, 2, Richardson D.R.1, 2 and Codd R.1, 2 1University of Sydney. 2Bosch Institute.

The hydrophilic nature of the ‘Gold standard’ for iron chelation therapy, desferrioxamine B (DFOB), means that the drug is unable to cross lipophilic cell membranes. This results in the retention of intracellular pools of iron, leading to the continuous generation of reactive oxygen species. Although DFOB coordinates Fe(III) effectively, the drug is not orally available and has a short plasma half-life, which translates as an arduous subcutaneous treatment regimen for iron overload patients. The aim of this project is to use a semi-synthetic approach to modulate the properties of DFOB. A library of DFOB conjugates has been synthesised using peptide coupling methods [1]. All DFOB conjugates maintained their iron binding abilities. Lipophilicity was determined as log P values (octanol/water partition coefficient) as calculated from RP-HPLC retention times. The compounds were more lipophilic than DFOB (log P ~2.1). Iron(III)-loaded compounds had a shorter retention time than the non-ferrated compound. This suggests that prior to metal binding, the lipophilic conjugates may cross the lipid bi-layer of the cell, bind to excess intracellular iron and become sufficiently hydrophilic to allow for easy urinary excretion. This new class of iron chelating drugs combines the affinity of DFOB to Fe(III) and the lipophilic nature of the ancillary ligands, forming potentially superior iron chelators. 1.Liu, J., et al., Conjugates of Desferrioxamine B (DFOB) with Derivatives of Adamantane or with Orally Available Chelators as Potential Agents for Treating Iron Overload. J. Med. Chem., 2010. 53(3): p. 1370-1382.

PAIN PHENOTYPING OF INTERSTITIAL CYSTITIS/PAINFUL BLADDER SYNDROME PATIENTS TO TARGET BIOMARKER STUDIES

Mayer R. University of Rochester,Rochester, NY, USA.

Introduction: Interstitial Cystitis/ Painful Bladder syndrome is a chronic debilitating idiopathic condition characterized by urinary frequency and discomfort with bladder filling. Previous reports utilizing biomarkers to characterize patients have been problematic due to likely heterogeneity. Attempting to sub classify these patients may allow planning of future molecular studies leading to effective therapy. We compared a cohort of patients using pain severity scores to evaluate differences as a guide for future investigations. Methods: The records of 186 patients with IC who completed an intake form were reviewed under approval by our institutional human subjects review board. Patients with mild pain score 0-2/10 (n=75) were compared patients with severe pain 8-10/10 ( n=32 ) with SPSS software / chi square analysis (p< 0.05)used to determine associations of significance. Results: There was no difference between the groups with age or duration of symptoms. Anxiety and irritable bowel syndrome were relatively frequent in both groups but were not significantly different. Severe pain patients did report a higher frequency of pelvic surgery, UTI, dysmenorrhea and/ or endometriosis in addition to increased frequency of migraine and asthma suggestive of systemic hypersensitivity. A history of abuse and increased contemporary psychological stress were also more frequent in patients with severe pain. Conclusions: Patients with Painful Bladder syndrome/Interstitial Cystitis show differences in pain severity which correspond to different patterns of clinical history. Estrogen related mechanisms of pain, signaling pathways common to asthma, migraine, and anxiety/coping may be fruitful targets of molecular research especially for those in severe pain.

OPTIMIZATION OF THE HORSERADISH PEROXIDASE CATALYSED BIODEGRADATION OF PENTACHLOROPHENOLMartinez-Ruiz J.1, Tomas V.2, Martinez-Gutierrez R.3, Garcia-Canovas F.1 and Tudela J.1 1GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Dept. Bioquimica y Biologia Molecular-A, Facultad de Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de Quimica Analitica, Facultad de Quimica, Universidad de Murcia. 3NOVOZYMES SPAIN S.A. (www.novozymes.com), Madrid, Spain. Pentachlorophenol (PCP CAS Registry No. 87-86-512) is a major industrial and agricultural biocide that has been used primarily as a wood preservative. Its worldwide usage, up to 90000 tons, and relative stability make PCP a ubiquitous environmental pollutant that has contaminated air, soil, and water and resulted in persistent, widespread exposure through drinking water and food. The major target organs for PCP toxicity include the liver, kidneys, hematopoietic system, pulmonary system, and central nervous system. Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme with Fe(III) in the hemin group of its active site, and with two Ca(II) cations in the proximal and distal dominia of HRP. There are few studies about the enzymatic biodegradation of PCP by HRP, mainly focused on the identification of 2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ) and other oxidation products, by MS and NMR. The aim of this work is the kinetic optimization of the assay conditions for this enzymatic reaction, with determination of the PCP breakdown by HPLC-DAD (Agilent 1200 UHPLC-RR). The enzymatic activity and stability, lead to the best assay conditions for the uniexponential biodegradation in 10 minutes of 260 micromolar PCP, which are 90 nM HRP, 130 micromolar hydrogen peroxide and 10 mM pH 4.0 sodium citrate buffer at 25 ºC. The stoichiometry PCP:H2O2 = 2:1 was established. HRP shows low affinity towards PCP, but generates in each catalytic cycle two molecules of PCP-radical, which evolves generating several biodegradation products, as reported by other authors. Experimental data were fitted by non linear regression to equations of the kinetic analysis for the reaction mechanism proposed. This work has been partially supported by grants from several Spanish organizations. Projects BIO2009-12956 (MICINN, Madrid), BIO-BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowship JMR BES-2007-16208 (FPI, MICINN, Madrid).

THE PLANT DEFENSIN NAD1 ACTS SYNERGISTICALLY WITH FUNGICIDES TO INHIBIT THE GROWTH OF FUNGI IN VITRO

McKenna J.A.1, Van Der Weerden N.L.2, Anderson M.A.2 and Heath R.L.1 1School of Botany, The University of Melbourne, VIC 3010 Australia. 2Department of Biochemistry, La Trobe University, VIC 3086 Australia.

Plant defensins are small, basic, cysteine-rich proteins that are members of the innate plant defence mechanism. The plant defensin NaD1 was isolated from the floral tissues of ornamental tobacco (Nicotiana alata) and inhibits a range of filamentous fungi in vitro. NaD1 inhibits fungal growth by the destabilising and permeabilising the fungal plasma membrane. Members of the triazole and strobilurin families are highly valued as agricultural fungicides. Together these classes of fungicide are the dominant form of chemical protection of crop plants worldwide. Strobilurins inhibit fungal growth by blocking mitochondrial respiration while triazoles inhibit growth by blocking the production of ergosterol. We have demonstrated that combinations of NaD1 and fungicides act synergistically to inhibit the in vitro growth of F. graminearum and F. oxysporum, dramatically reducing the concentration of fungicide and NaD1 required for fungal control.





REMAZOL BRILLIANT BLUE ROYAL BIODEGRADATION CATALYSED BY HORSERADISH PEROXIDASEMendez P.1, Parra M.1, Tomas V.2, Martinez-Gutierrez R.3,Garcia-Canovas F.1 and Tudela J.1 1GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Dept. Bioquimica y Biologia Molecular-A, Facultad de Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de Quimica Analitica, Facultad de Quimica, Universidad de Murcia. 3NOVOZYMES SPAIN S.A. (www.novozymes.com), Madrid, Spain. Remazol Brilliant Blue Royal (RBBR, Reactive Blue 19, C.I. 61200) is an anthraquinone dye related with the reactive dyes group, which attains the 12 % of worldwide production. It has been estimated that 4 % of dyes are discharged into the environment, with harmful effects on biodiversity and humans. Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme with Fe(III) in the hemin group of its active site. There are few studies about the biodegradation of RBBR by oxidative enzymes, mainly focused on structural identification of their oxidation products. The aim of this work is the kinetic optimization of assay conditions for the enzymatic oxidation of RBBR by hydrogen peroxide with HRP as biocatalyst, with separation and determination of RBBR by HPLC-DAD (Agilent 1200 UHPLC-RR) and by UV-visible spectrophotometry. The absorbance decrease at 595 nm was recorded in scan (Perkin Elmer Lambda-35) and kinetic (Molecular Devices SpectraMax-340PC) assays. The enzymatic activity and stability, lead to the best assay conditions for the uniexponential biodegradation in 20 minutes of 50 micromolar RBBR, which are 30 nM HRP, 250 micromolar hydrogen peroxide and 10 mM pH 5.0 sodium citrate buffer at 25 ºC. The stoichiometry RBBR:H2O2 = 1:1 was established. HRP generates in each catalytic cycle two molecules of RBBR-radical, which react between them with regeneration of one molecule of RBBR, to originate only one molecule of a transient oxidation product, which evolves towards further products, as reported by other authors. Experimental data were fitted by non linear regression to equations of kinetic analysis for the reaction mechanism proposed. This work has been partially supported by grants from several Spanish organizations. Projects BIO2009-12956 (MICINN, Madrid), BIO-BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowship MPC 09378/FPI/08 (Fundacion Seneca, CARM, Murcia).

ROSMARINIC ACID OXIDATION AND ITS ANTIOXIDANT CAPACITY

Munoz-Munoz J.L.1, Garcia-Molina F.1, Garcia-Molina M.1, Ros E.1, Varon R.2, Tudela J.1, Garcia-Canovas F.1 and Rodriguez-Lopez J.N.1 1GENZ: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de Química-Física. Escuela de Ingenieros Industriales de Albacete. Universidad de Castilla la Mancha. Avda. España s/n. Campus Universitario, E-02071, Albacete, Spain.

Rosmarinic acid is a fundamental compound of Rosmarinus officinalis. In this work, this compound has been kinetically characterized as substrate of polyphenol oxidase and peroxidase (PPO and POD). In addition, polyphenol oxidase inactivation under aerobic conditions (suicide inactivation) has been studied. However, under anaerobic conditions, polyphenol oxidase does not inactivate by this compound. The chemical structure of rosmarinic acid predicts its capacity to act as antioxidant, which has been characterized as a primary and secondary antioxidant capacity. Prooxidant capacity of this compound has been determined. This work has been partially supported by grants from several Spanish organizations. Ministerio de Educación y Ciencia (Madrid, Spain) Project BIO2009-12956, from the Fundación Séneca (CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08, from the Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004 and from the Consejería de Salud y Bienestar Social de la Junta de Comunidades de Castilla La Mancha, Project FISCAM PI-2007/53. JLMM has a fellowship from the Ministerio de Educación y Ciencia (Madrid, Spain) Reference AP2005-4721. FGM has a fellowship from Fundación Caja Murcia (Murcia, Spain).

INVESTIGATION OF MHC HAPLOTYPING WITH PEPTIDE NUCLEIC ACIDS USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTIONMurphy N.M., Pouton C.W. and Irving H.R. Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaThis study reports on the modelling and assessment of a novel haplotyping strategy using peptide nucleic acids (PNAs) to physically separate haploid material in a diploid DNA sample. Alleles of the human leukocyte antigen DRB1 gene were targeted by two biotinylated PNAs synthesised to target the HLA-DRB1*01 and DRB1*03 alleles containing either Alexa Fluor 488 or Alexa Fluor 532 respectively. Stocks of two plasmids lines with the second exon of the HLA-DRB1*01 and the DRB1*03 alleles were generated using the pDONR 201 Gateway system. PNAs were hybridised to DNA and incubated on a neutravidin bound 96-well microplate (Pierce). Samples were washed to remove unbound PNA and any DNA remaining in solution, and fluorescence levels were monitored on an Envision 2101 microplate fluorometer. Following elution, allele-specific quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the level of specific and non-specific binding. Non-biotinylated PNAs were eluted following a single wash once the first supernatant was removed. Biotinylated samples remained bound to the plate and decreased in fluorescence by ~ 2 % with each succeeding wash. The [Alexa Fluor 488]-PNA*01-Biotin probe hybridised with the HLA-DRB1*01 plasmid construct had a yield significantly higher than controls (P < 0.05). The [Alexa Fluor 532]-PNA*03-Biotin probe hybridised with the HLA-DRB1*03 plasmid had a significantly higher yield than controls (P < 0.01). In conclusion, the haplotyping model assay presented provides a foundation for creating a haplotyping assay by hybridising PNAs to genomic DNA and physically separating the chromosomes.

DEVELOPMENT OF A MHC CLASS II PEPTIDE BINDING PREDICTION METHOD WITH HIGH-POPULATION COVERAGE FOR EFFICIENT VACCINE DESIGN

Oyarzun P.1, 2, Ellis J.1 and Kobe B.1 1School of Chemistry and Molecular Biosciences, Institute for Molecular Biosciences and Centre for Infectious Disease Research, The University of Queensland, Australia. 2Universidad San Sebastian, Chile.

Vaccines have being historically obtained from living attenuated organisms. Nowadays, the interest is turning to peptide vaccines rationally designed using immunoinformatics. The peptides are small fragments of antigenic proteins containing a molecular structure (epitope) that is presented by MHC molecules on antigenic presenting cells (APC) to T cell receptors (lymphocytes), potentially triggering an immune response. Exogenous peptides bind MHC class II proteins through a number of interactions with amino acids lying along a highly polymorphic groove, leading to over 1200 MHC class II alleles. This high variability pose serious difficulties for vaccine design. We developed the method Predivac for prediction of peptide binding to MHC class II proteins. The method is based on the so-called specificity-determining residues (SDRs), which contact the side chains of the peptide ligand in the binding groove. A similar approach has being used by our group for prediction of kinase substrates (Predikin). The analyses of crystal structures revealed that the SDRs are highly conserved throughout the MHC class II molecules. This allowed us to establish a predictive correlation between SDR patterns and binding specificity for experimentally characterized and non-characterized alleles. The bioinformatics tool is being written in Perl. Thus far, a database has being constructed including high-affinity binding peptides for 28 MHC class II alleles (DRB allotype). The database accounts for 500 alleles for which binding predictions are possible. The performance of the method was assessed by leave-one-out cross-validation in terms of the area under ROC curve (AROC). The AROC values range from 0.647 to 0.938, which is comparable or better than available state-of-the-art methods. Our method will help designing vaccines that effectively protect genetically diverse human populations.





INDIGO CARMINE BIODEGRADATION CATALYSED BY METHYLSYRINGATE-LACCASE MEDIATOR SYSTEM

Parra M.1, Jimenez L.1, Tomas V.2, Martinez-Gutierrez R.3, Garcia-Canovas F.1 and Tudela J.1 1GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Dept. Bioquimica y Biologia Molecular-A, Facultad de Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de Quimica Analitica, Facultad de Quimica, Universidad de Murcia. 3NOVOZYMES SPAIN S.A. (www.novozymes.com), Madrid, Spain.

Indigo Carmine (IC) is a pH and redox indicator used as denim dye and in the manufacturing of capsules. IC is harmful to the respiratory tract if swallowed, and irritant to the skin and eyes. Laccase (EC 1.10.3.1) catalyses the oxidation by molecular oxygen of substrates up to radical products. The broad substrate selectivity and the high oxidation potential of LAC, have lead to be used in biotechnological applications. This selectivity is enhanced by the use of mediators, true substrates of LAC which radical products oxidize molecules that are not direct substrates of the enzyme. There are few studies about the biodegradation of IC by oxidative enzymes, mainly focused on the identification of the oxidation product, isatin-5-sulfonic acid (ISA), by MS and NMR. The aim of this work is the kinetic optimization of the assay conditions for the oxidation of IC by a methylsyringate-laccase mediator system as biocatalyst, with separation and determination of IC and ISA by HPLC-DAD and by UV-visible spectrophotometry. The enzymatic activity and stability, lead to the best assay conditions for the biodegradation in 10 minutes of 98 micromolar IC, which are 63 nM LAC, 6 micromolar methylsyringate and 10 mM pH 5.0 sodium acetate buffer at 25 ºC. The reaction mechanism consists of the enzymatic oxidation of the mediator to a radical, coupled to the non enzymatic oxidation of IC by methylsyringate-radical, with regeneration of the mediator. A kinetic equation is proposed to explain the experimental data and to predict the kinetic behaviour of the methylsyringate-laccase mediator system. This work has been partially supported by grants from several Spanish organizations. Projects BIO2009-12956 (MICINN, Madrid), BIO-BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowship MPC 09378/FPI/08 (Fundacion Seneca, CARM, Murcia).

DELIVERY OF MULTIPLE PROTEINS USING MULTI-GENE EXPRESSION VEHICLES

Poon S.1, Connelly A.A.1, Rainone M.1, Guarino R.2, McKenna J.A.1, Carroll K.T.2, Anderson M.A.2 and Heath R.L.1 1School of Botany, The University of Melbourne, VIC 3010. 2Department of Biochemistry, La Trobe University, VIC 3086.

We have developed Multi-Gene Expression Vehicles (MGEVs) based on the modification of multi-domain proteinase inhibitors from Nicotiana alata. The N. alata proteinase inhibitors (NaPIs) are produced as precursor proteins which form a circular conformation via disulfide linkage between the amino- and carboxyterminal peptide domains. Proteolytic processing of the precursor protein occurs in a linker region between each domain resulting in the release of mature 6 kDa proteinase inhibitors which have inhibitory activity against chymotrypsin or trypsin. We have produced MGEVs which are either circular or linear in conformation. Insertion of additional proteins such as plant defensins or different PIs, or the replacement of the native NaPI domains has allowed us to produce precursors that contain from two to potentially eight individual proteins. We demonstrate the expression and processing of multiple proteins from single MGEV constructs using stable and transient gene expression systems and show that MGEVs are a promising strategy for protein stacking in transgenic plants.

THE AGILENT TECHNOLOGIES SURESELECT TARGET ENRICHMENT SYSTEM FOR NEXT-GENERATION SEQUENCING : ENRICHMENT AND DEPLETION APPLICATIONS FOR TRANSCRIPTOME ANALYSIS

Roberts D.1, Happe S.2, Lin E.1, Pabon C.1, Giuffre A.2, Fullmer-Smentek S.1, McInnes R.3, Joshi S.2, Novak B.1 and Leproust E.1 1Agilent Technologies, Santa Clara, California. 2Agilent Technologies, Cedar Creek, Texas. 3Agilent Technologies, Melbourne, Victoria.

The discovery of rare polymorphisms, structural variants, and novel transcripts has been accelerated dramatically by next-generation sequencing technologies. However, it remains cost-prohibitive to sequence entire genomes in large cohort studies. To enable larger sample sets, Agilent Technologies has developed the SureSelect platform, allowing focused analyses on specific genomic loci at considerable cost savings. Agilent is continuing to expand its portfolio to increase the number of applications available to users. We demonstrate high performance of DNA capture of several focused areas as measured by capture efficiency, uniformity, reproducibility, and SNP detection. We also describe performance of new SureSelect panels such as the mouse exome. We then describe the adaptation of SureSelect to whole transcriptome analysis applications and introduce methods to deplete highly abundant transcripts such as ribosomal RNAs. Finally, we reveal the use of custom libraries to interrogate specific subsets of transcripts for abundance and variation. SureSelect applications will continue to expand as users harness the power of next-generation sequencing.

VITAMIN D INTERACTIONS WITH P-GLYCOPROTEIN EXPRESSION AND FUNCTION IN INTESTINAL LS174T CELLS

Kota B.P.1, Tran V.H.1, Allen J.2 and Roufogalis B.D.1 1Faculty of Pharmacy, University of Sydney, NSW 2006. 2Centenary Institute, University of Sydney, NSW, 2006.

Intestinal P-glycoprotein limits bioavailability of drugs by inhibiting absorption of orally administered drugs from the intestine. Vitamin D receptor (VDR) is a transcriptional factor that mediates induction of P-glycoprotein (P-gp). The aim of the current study was to investigate the role of VDR on P-gp expression and function in LS174T intestinal cells. In addition, the effect of ketokonazole, a pregnane X receptor (PXR) inhibitor, on VDR mediated P-gp expression and function was examined to determine the nature of the interaction between VDR and PXR mediated P-gp regulation. Intestinal LS174T cells were treated with 1α-25(OH)2D3 (DHC), the active metabolite of Vitamin D3. DHC was found to significantly increase P-gp expression in LS174T cells after two days of treatment. The increase in P-gp expression correlated with decreased accumulation of Rh123 (used to monitor P-gp function) in LS174T cells. DHC treatment reduced the cytotoxic effects of colchicine, a P-gp substrate (determined by MTS cytotoxicity assay). Interestingly, ketoconazole, in combination with DHC, enhanced P-gp protein expression above that of DHC alone. However, the effect on P-gp expression by KTZ and DHC treatment was also observed in PXR deficient Caco-2 cells, and was most likely due to the observed enhanced VDR protein expression. We conclude that DHC increases P-gp expression and reduces cytotoxicity in an intestinal cell line, which can be useful for in vitro drug pharmacokinetic studies. The interaction observed between ketaconozole and DHC may have implications in decreasing oral drug bioavailability of P-gp substrates.





GROWTH HORMONE GENE MANIPULATION IN CHANNA STRIATUS

Shrestha T.R.1 and Majumdar K.C.2 1Tribhuvan University, Department of Zoology, Bhaktapur M. Campus, Bhaktapur, Nepal. 2Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India.

The GH gene manipulation of Channa striatus was undertaken in order to engineer autotransgenic construct to produce transgenic fish. Out of 32 different species of Channa present in the world, this is the first report on molecular biological study of GH gene and three constitutive promoters (histone 3, β actin and myosin) of five Channa species, viz, C. striatus, C.punctatus, C. gachua, C.marulius and C. diplogramma. For autotransgene constructs, the three constitutive promoters were isolated, cloned, sequenced and characterized from C. striatus. The functional assays of these promoters were done in vivo in zebrafish by monitoring the green fluorescence. The cDNA and chromosomal growth hormone genes including 5’ and 3’UTR regions were isolated, cloned, sequenced and characterized from pituitary and blood of Channa striatus by using RACE and PCR strategies. Thus GH cDNA transcript has 848bp with a coding region of 615bp, 54bp of 5UTR and 159bp of 3’ UTR regions whereas total GH gene has 2089 including 5’ and 3’ UTR regions. In all these species the GH gene has six exons and five introns and is single copy. The two autotransgene constructs of C. striatus, CSH3-GHcDNA and CSβ-GHcDNA were transferred into the eggs of H. fossilis and P. suchi through sperm electroporation. Detection of transgene, CSH3-GHcDNA by PCR in 18 months old putative transgenic H. fossilis and P. suchi revealed their presence in 66.6% and 61.1% individual response respectively. The growth analysis in our preliminary study revealed that transgenic fishes grew faster than non-transgenic individuals confirming the functionality of the transgenic DNA prepared from Channa striatus.

PRODUCTION OF NON-NATURAL SIDEROPHORES FROM SHEWANELLA PUTREFACIENS AND ALTEROMONAS HALOPLANKTIS

Soe C.Z.1, 2, Pakchung A.A.H.1, 2 and Codd R.1, 2 1Universiity of Sydney. 2Bosch Institute.

Iron is a vital element for life, being necessary in metalloproteins, such as cytochromes and ribonucleotide reductase, which are key to fundamental cellular processes. In oxic, aqueous environments, the majority of Fe(III) exists in the form of insoluble oxyhydroxides (Ksp ~ 10-39). Microorganisms have evolved different strategies to overcome this problem. Under iron-deprived conditions, selected bacteria produce iron-chelating molecules called ‘siderophores’, classified as hydroxamate-, catacholate- or mixed- type chelates. Polyamines, such as putrescine, cadaverine and spermidine, are fundamental precursors of siderophore assembly pathways. The current research is focused on the macrocyclic dihydroxamate-based siderophores, due to their potential as therapeutic lead compounds as anti-tumour and anti-infective agents. The known members of this class are bisucaberin, alcaligin and putrebactin isolated from Alteromonas haloplanktis, Bordetalla pertussis and Shewanella putrefaciens, respectively. Based on the genetic data on bacterial siderophore biosynthesis, this present study will explore the potential of S. putrefaciens and A. haloplanktis as biosynthetic machines to produce novel siderophores via augmenting culture media with non-natural substrates. Bacterial cultures were grown in the presence of a range of diamine precursor substrates and ornithine decarboxylase (ODC) inhibitors for 6 d at room temperature. Optical density (OD) and the Chrome Azurol S (CAS) assay were used for siderophore detection and monitored at 600 and 630 nm, respectively. The novel siderophores will be isolated from the culture supernatant using chromatographic steps and the structure will be determined using HPLC/MS and NMR spectroscopy.

ANTIMICROBIAL PEPTIDES PRODUCED BY PEPSIN DIGESTION OF THE FRUIT PROTEIN OF BRUCEA AMARISSIMA DESV

Sornwatana T.1, Roytrakul S.2, Wetprasit N.3 and Ratanapo S.4 1Biochemistry department, faculty of science, Kasetsart U., Bangkok, Thailand 10900. 2Genome institute, National Center for Genetic engineer and Biotechnology, National Science and Technology Development Agency(NSTDA), Pathumthani, Thailand 12120. 3Biotechnology department, faculty of science, Ramkhamhaeng U., Bangkok, Thailand 10240. 4Biochemistry department, faculty of science, Kasetsart U., Bangkok, Thailand 10900.

Peptides exhibiting antimicrobial activity has gained scientific interest as components in functional food for their potential in disease prevention and health improvement. In this study, antimicrobial active peptides encrypted in the dried fruit protein of Brucea amarissima Desv was released by in vitro digestion with pepsin for 12 h at enzyme/protein ratio of 1:25. The hydrolysate was purified by reverse phase HPLC followed by LC-MS analysis. The antimicrobial activity of each purified fraction was performed by agar dilution technique. Two potent peptide fractions with 2418.076 m/z and 2022.462 m/z showed broad-spectrum inhibition against 7 strains of human-pathogenic bacteria and a fungi with the most potent inhibitory activity against Streptococcus pyogenes at MIC value of 2.6 μg/ml. S. pyogenes is the cause of many important human diseases ranging from superficial skin infections to life-threatening systemic disease. The result suggested that the pepsin digestion of the fruit protein of B. amarissima generated bioactive peptides which might provide health benefits in reducing the risk of developing the disease caused by S. pyogenes.

BACILLUS CEREUS WHICH CAN GROW IN THE PRESENCE OF BENZENE

Tsugeno Y. Department of Environmental Management, School of Health Sciences, University of Occupational and Environmental Health, Japan.

The environment where the higher organism cannot live is called “Extreme environment”, and the microorganism that can be grown under such an extreme environment is said, “Extremophile” usually. Most organic solvents are generally biotoxic and prohibit growth of microorganisms even at low concentrations. Thus, interest stems from the fact that many organic solvents are classified as environmental pollutants and their degradation by microorganisms is the focus of the emerging bioremediation industry. I isolated the benzene-resistant bacterium from Japanese soil by selecting for microbial growth in a nutrient medium containing benzene. To isolate benzene-resistant microorganisms,a small amount of soil was suspended in sterile water and 0.2ml of this suspension transferred to test tubes containing 5ml of a nutrient medium comprising 0.1% glucose, 0.25% yeast extract and 0.5% polypeptone, at pH7.0. Benzene was then added to a final concentration of 5%, 15% and 30% (v/v), the test tubes were sealed with a cap, and the cultures incubated at 37°C for 120h in a test tube shaker. I report here the discovery of microorganisms which is capable of growing in all media culture containing of 5%,15% and 30% benzene. Cell growth was determined by measuring the optical density at 660 nm. 16S rRNA gene sequencing date of microorganisms indicated Bacillus cereus, Bacillus mycoides,and Bacillus thuringiensis respectively at the possibility of 99%. In addition, Bacillus cereus strain isolates were identified by morphological, physiological and biochemical analysis, and representative above-mentioned strains were identified at the molecular level using 16S rRNA sequence analysis. This time, I succeeded in the discovery of “Extremophile” that was able to be grown under benzene that was the organic solvent.





EXPRESSION ANALYSIS OF MINCLE ON HUMAN PERIPHERAL BLOOD CELLS

Vijayan D.1, 4, Radford K.2, Bellette B.1, Beckhouse A.1, Ashman R.3, Nair R.4 and Wells C.1 1Eskitis Institute for Cell and Molecular Therapies, Griffith University, Australia. 2Mater Medical Research Institute, Australia. 3University of Queensland, Australia. 4School of Dentistry and Oral Health, Griffith University, Australia.

C-type lectins play key roles in antigen recognition, uptake and initiation of signalling cascades that influence the balance between tolerance and immunity. Mincle, a class II C-type lectin receptor is induced in mouse macrophages responding to Candida albicans. Mice lacking Mincle are susceptible to oral and systemic Candida infections. Understanding the role of Mincle in different cell compartments will provide important information about how the body recognises and fights fungal infection. In the present work, we aim to determine the cellular distribution of Mincle on human peripheral blood. These findings form preliminary investigations into examining the role of this lectin in patients with chronic oral candidiasis. Blood from healthy Red Cross volunteers were analyzed by flow cytometry for the presence of Mincle on leukocyte populations. Mincle expression was identified on human monocytes, granulocytes, circulating dendritic cells and lymphocytes. Mincle expression on monocytes directly correlated to its expression on macrophages differentiated subsequently in culture. Mincle is expressed on a wide range of immune cell types, and is particularly abundant on professional antigen presenting cell including B-lymphocytes. This study provides a first essential description of the distribution of a major fungal receptor, which provides further insight into the molecular basis for defence against chronic oral infections.

DEVELOPMENT OF A LUCIFERASE REPORTER CGMP RESPONSIVE PROMOTER SYSTEM SUITABLE FOR USE IN PLANT PROTOPLASTS

Wheeler J.I. and Irving H.R. Monash Institute of Pharmaceutical Sciences.

cGMP is a second messenger molecule associated with plant responses to hormones, light, nitric oxide and both biotic and abiotic stress. Currently cGMP levels can be assessed using antibody kits designed for mammalian cells or mass spectrometry analysis. Maathuis (2006) used micro-array analysis of Arabidopsis roots to identify genes that are differentially expressed 90 mins after a 30 min exposure to membrane permeable cGMP. Three genes that were up-regulated were NTL1 (At1g33440), CHX21 (At2g31910) and SOS3 (At5g24270). We have shown that these three genes are also expressed in Arabidopsis leaves and protoplasts derived from mesophyll cells. Promoters of these genes were cloned into the pLUCTRAP3 vector (Calderron-Villalobos et al., 2006) to generate luciferase fusions. Results of cGMP induced expression of each promoter luciferase clone in freshly isolated Arabidopsis mesophyll protoplasts show slight variations in luciferase expression after cGMP treatment. The NLT1 promoter fragment contains the mammalian cGMP responsive element (RE) identified by Hum et al (2004). We are currently inserting four additional cGMP RE into the NTL1 promoter with the aim of enhancing the promoter response to cGMP. Bastian and colleges (2010) identified a common GA responsive element (GARE) in the promoters of genes that are up regulated in response to cGMP and down regulated in a GA insensitive mutant. We have inserted six copies of the GARE into the NTL1 promoter fragment and report here the effect on luciferase activity in our protoplast system. The development of a cGMP sensitive luciferase reporter system would then allow us to assess receptors such as AtBRI1, AtPSK1, AtRPK and AtPepR1 with guanyl cyclase activity by measuring differences in luciferase activity. Maathuis (2006) The Plant Journal 45: 700-711. Calderron-Villalobos et al. (2006) Plant Physiology 141: 3-14. Hum et al. (2004) Hypertension 43: 1270-1278. Bastian et al. (2010) Plant Signalling and Behaviour 5: 1-9.

IMPLEMENTATION OF SPLICE SWITCHING THERAPIES FOR DUCHENNE MUSCULAR DYSTROPHY

Wilton S.D. and Fletcher S. CNND University of Western Australia, 35 Stirling Highway, Crawley, WA 6009.

The exquisitely precise and co-ordinated process of gene transcript splicing, that is, intron removal and joining of exons, not only greatly increases genetic plasticity through alternative splicing, but also offers a point of therapeutic intervention for some genetic disorders. Protein truncating mutations in the 2.4Mbp, 79 exon dystrophin gene lead to Duchenne muscular dystrophy (DMD), the most common and severe form of childhood muscle wasting. The size and complexity of the dystrophin gene poses major challenges to gene replacement therapy for DMD, but antisense oligomer induced exon skipping has progressed from a concept demonstrated in vitro to clinical trials in just over a decade. Proof-of-concept was demonstrated by restoration of localised dystrophin synthesis after injection of a morpholino oligomer, designed to excise exon 51 from the dystrophin mRNA, into the extensior digitorum brevis of non-ambulant DMD patients with a common genomic deletion sub-type. Preliminary results from a systemic antisense oligomer, dose-escalating study over 12 weeks indicate that the compound is well-tolerated and showed that dystrophin synthesis was restored in patient muscle. Specific skipping of dystrophin exon 51 will only be relevant to about 10% of DMD individuals, however, we have developed a panel of oligomers to skip different dystrophin exons and allow treatment for other dystrophin mutations as a form of personalized genetic therapy. We now report that subtle DNA changes in the target exon influence the exon skipping efficiency of splice switching oligomers, further emphasizing the individual nature of this potential treatment. DMD is regarded as an orphan disease, and additional stratification of patients according to the nature of their primary gene lesion will pose great challenges to the application of this therapy.

STRESS INDUCIBLE EXPRESSION OF TADREB4 INCREASES DROUGHT TOLERANCE OF TRANSGENIC WHEAT AND PREVENTS UNDESIRABLE CHANGES IN PLANT DEVELOPMENT

Yadav M.K.1, 2, Eini O.1, Nataliya K.1, Tatiana P.1, Bazanova N.1, Singh R.1, Ismagul A.1, Eliby S.1, Langridge P.1 and Lopato S.1 1ACPFG, University of Adelaide Waite Campus Urrbrae Adelaide SA 5064. 2SVP University of Agriculture & Technology Modipuram Meerut INDIA 250110.

Wheat is the most widely grown crop in the world covering around 16% of the arable lands and staple food of about 35% of the world population. Global environmental warming, followed by increasing drought threaten the world’s food supply. One of the sustainable and economically viable solutions for increasing crop stability and productivity is crop genetic improvement for higher tolerance to abiotic stresses by the introgression of agronomically important genes. Transcription factors (TFs) have been shown to mediate stress signal transduction and response to different stresses in plants. The full length cDNA of a new drought response element binding factor, designated TaDREB4, was isolated in the Y1H screen of the cDNA library prepared from flag leaves and spikes of drought tolerant wheat cultivar subjected to drought/heat stress. The DRE TACCGACT was used as bait. With the aim to increase drought tolerance, we over-expressed TaDREB4 in transgenic barley and wheat. Transgenic plants with strong constitutive over-expression of these factor lead to little negative changes in growth and flowering time. However, expression of this factor under stress inducible ZmRab17 promoter shows no undesirable phenotypes in wheat plants. Transgenic wheat T1 plants in the first experiment showed increased drought tolerance at the seedling stage as compared with control plants and quick recovery after re-watering. However, no frost tolerance improvement has been demonstrated for barley plants with constitutive expression of TaDREB4 in several independent experiments. Analysis of the expression of potential downstream genes is currently in progress.





PLANT-BASED APPROACH TOWARDS UNDERSTANDING THE INFLUENZA A VIRUS ANTIGENIC DRIFT

Yamato H., Cueno M.E., Omagari K. and Okamoto T. Nagoya City University, Nagoya City, Aichi, Japan.

Influenza A virus has been an elusive human pathogen mainly due to its ability to rapidly mutate and evade antibodies specific for its attachment protein, the hemagglutinin (HA). Current influenza vaccine strategies target the HA protein, however, annual mutations within the HA region occurs allowing the new viral strain to be elusive to previously made vaccines. Several attempts had been made to understand the HA antigenic drift in hopes to predict the next influenza strain and develop appropriate vaccines in advance. To date, this approach has been unsuccessful. In this study, we determined the possible role of the conserved H1N1 HA1 and HA2 nucleotide regions in antigenic drift. Codon-optimized FLAG-HA1-HA2-GUS genes were fused accordingly. H1N1 HA1 and HA2 conserved regions were determined through multiple sequence alignment. The gene construct was ligated into pBI121 expression vector, bombarded into leaf calli and grown in appropriate growth medium. Immunoblot assays using antibodies against FLAG, the universal epitope found in HA2 and GUS were used. Using FLAG antibody, we detected a 36 kDa protein expressed during the callus stage and two proteins, 36 and 30 kDa, expressed from transgenic tomato leaves. Interestingly, when we used a GUS antibody to confirm fusion protein expression, we only detected the 36 kDa protein from callus and none form the transgenic leaves. In addition, when we used the universal epitope antibody, no band was detected in both callus and leaves. We verified the sequence of the gene fusion expressed in tomato and found variations from the original sequence emphasizing the significance of the HA conserved regions in causing antigenic drift.

NMDA RECEPTOR ANTAGONIST ADMINISTRATION LEADS TO DOWN-REGULATION OF DISC1 EXPRESSION AND OVER MIGRATION OF NEWLY GENERATED NEURONS IN THE ADULT HIPPOCAMPUS

Namba T.1, 2, Uchino S.2, Kohsaka S.2 and Kaibuchi K.1 1Dept. of Cell Pharmacology, Nagoya Univ. Grad. Sch. of Med., Aichi, Japan. 2Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.

New neurons are continuously generated throughout life in the hippocampus. Newly generated neurons migrate short distance and are integrated into pre-existing neuronal circuit. However, the extra cellular signals which regulate the neuronal migration are not fully understood. Here we report that NMDA receptor (NMDA-R) signaling is involved in cellular migration and proper positioning of newly generated neurons in the adult hippocampus. An intraperitoneal injection with NMDA-R antagonists into adult male mice caused aberrant positioning of doublecortin-positive immature neurons. BrdU labeling analysis suggested that NMDA-R antagonist caused an over migration of newly generated neurons. Using a quantitative real-time PCR analysis, we examined the expression of Disrupted-In-Schizophrenia 1 (DISC1) that has been identified as a candidate susceptibility gene for major psychiatric disorders such as schizophrenia, because it has been known that DISC1 is one of the important molecules regulating migration of newly-generated neurons in the adult hippocampus. The expression of DISC1 in the dentate gyrus was reduced by NMDA-R antagonist-injection in a dose-dependent manner. In contrast, the expression of the DISC1-interacting molecules, Lis1, NDEL1 and Girdin, was not affected by the NMDA-R antagonist-injection. Furthermore, the over migration phenotype by NMDA-R antagonists was partially rescued by lentiviral-mediated DISC1 expression. These results suggest that the NMDA-R signaling affects the migration of newly generated neurons via regulating the expression of DISC1.

ADIPOSE-DERIVED MESENCHYMAL STEM CELLS ENHANCE TISSUE REGENERATION BY INDUCING GROWTH OF BLOOD VESSELS AND NERVES

Kalinina N.I., Lopatina T.V., Efimenko A.Y.U., Parfyonova E.V. and Tkachuk V.A. Faculty of Fundamental Medicine, Lomonosov Moscow State University.

Transplantation of adipose-derived stem cells (ASCs) induces tissue regeneration by accelerating the growth of blood vessels and nerve sprouts. Previously we demonstrated that ASCs promote the formation and growth of blood vessels by producing angiogenic growth factors as well as enhancing vessel maturation. However mechanisms of their action on the growth of nerve fibers are only partially understood. Here we show that transplantation of ASCs stimulates a repair of motor and sensory nerves as well as induces nerve sprouts growth in matrigel implants. Transcriptome analysis has revealed that ASCs express mRNAs for growth factors and extracellular matrix proteins specific for the outgrowth of nerve sprouts and myelination. Exposure of ASCs to a combination of retinoic acid with 5-azacytidine up-regulates BDNF production in these cells as well as their ability to induce nerve fiber growth in matrigel implants. BDNF neutralizing antibodies have abrogated stimulatory effect of ASCs on the growth of nerve sprouts. These data suggest that ASCs induce nerve repair and growth via BDNF production and this stimulatory effect can be further enhanced by cell exposure to neural differentiation medium prior to transplantation.

HYPEROSMOTIC AND OXIDATIVE STRESS INDUCES INTRACELLULAR CA2+- DEPENDENT APOPTOSIS THROUGH THE CATEPSIN B AND CASPASE 3-MEDIATED PATHWAYS IN SKIN EPIDERMAL HUMAN CELL LINE

Silva R.A.1, Machado D.1, Souza A.C.S.1, Shishido S.M.1, Ferreira C.V.1, Paredes-Gamero E.J.2 and Justo G.Z.1, 2 1Departamento de Bioquímica, IB, UNICAMP, Campinas, SP. 2Disciplina de Biologia Molecular, Departamento de Bioquímica, UNIFESP, São Paulo, SP.

The epidermis is confronted with multiple environmental stressors that affect its redox state and osmotic balance. One of the main factors by which hyperosmotic and oxidative stress influence skin condition is the modulation of intracellular Ca2+.Increase in cytosolic Ca2+ is an essential element in the control of many physiological processes. However, sustained increases in Ca2+ concentration may contribute to oxidative stress and activation of different cell death mechanisms. Several other events are related to the increase in Ca2+, including regulation and activation of a number of enzymes such as Ca2+/phospholipid-dependent protein kinase C (PkCs) and proteases. The aim of this study is to examine the involvement of calcium homeostasis in the apoptotic death induced by hyperosmotic and oxidative stress in human skin epidermal cell line, HaCaT cells. This study shows that sorbitol and H2O2 induce keratinocyte death, displaying an IC50 of 1,0 mol/L and 2.0 mmol/L, respectively. The increase of cytosolic Ca2+ induced the production of reactive oxygen species (ROS). PepChip and Western blot analysis demonstrated that the activity of different Ca2+-dependents PKCs was modulated by hyperosmotic and oxidative stress. It was also observed permeabilization of the lysosomal-membrane followed by release of cathepsin B into the cytosol. Activation of caspase 3 was also detected. Our findings clearly indicate that apoptosis occurs in a time and dose-dependent manner and is the predominant type of cell death, as proven by increase of Annexin V positively stained cells. Therefore, our research suggests that control of calcium homeostasis may be a key factor in protecting skin against environmental stress.





ATF3 IS REQUIRED FOR TLR MEDIATED SURVIVAL SIGNALLING IN MURINE BONE MARROW DERIVED MACROPHAGES VIA ITS REPRESSION OF PRO-APOPTOTIC BAK AND BAX

Thompson M.R., Xu D. and Williams B.R.G. Monash Institute of Medical Research - Centre for Cancer Research.

Macrophages play an essential role in innate immune responses by producing pro-inflammatory mediators and through phagocytic clearance of pathogens or apoptotic cells. Activated macrophages, are bestowed with resistance to apoptosis, allowing prolonged function in inflammatory responses. However, excessive macrophage activation contributes significantly to chronic inflammatory diseases. The toll-like receptor (TLR) family are key regulators of innate immune responses through their ability to induce pro-inflammatory cytokine production and elicit survival responses. Recently the adaptive response gene, activating transcription factor 3 (ATF3), has been shown to be induced by TLR signalling. Following its induction, ATF3 is able to negative regulate TLR signalling through its ability to repress transcription of pro-inflammatory cytokines, including IL-6 and IL-12p40. ATF3 has also been suggested to play various roles in the regulation of cell cycle progression and apoptosis, so we were interested in investigating whether ATF3 could elicit similar responses in macrophages following TLR stimulation. Here we show that TLRs elicit survival of bone marrow derived macrophages (BMM), in the absence of colony stimulating factor 1 (CSF-1). Importantly, the induction of survival by TLRs in ATF3-

/- BMMs is significantly reduced. Compared to WT BMMs, ATF3-/- BMMs fail to down regulate apoptosis in response to LPS. In contrast, cell cycle transition did not change between the genotypes, indicating that it is a defect in the apoptotic response that contributes to decreased overall survival of ATF3-/- BMMs. In this regard, we demonstrate that ATF3 lies downstream of c-Jun N-terminal kinase (JNK), an important regulator of apoptosis, following TLR activation and that induction of ATF3 by TLR signalling allows for the transcriptional repression of pro-apoptotic mediators BAK and BAX. The deregulation of BAK and BAX in ATF3-/- BMMs may be responsible for skewed responses towards apoptosis. Therefore ATF3 is as an important factor for the maintenance of innate immune homeostasis.

PHENOTYPIC ANALYSIS OF SOYBEAN ROOT AND NODULE TISSUES AFTER RNAI INDUCED SILENCING OF THE MEMBRANE BOUND TRANSCRIPTION FACTOR GMSAT1

Mohammadi-Dehcheshmeh M.1, Ebrahimie E.2, Chiasson D.1, Tyerman S.D.1 and Kaiser B.N.1 1Plant Research Center, School of Agriculture, Food and Wine, Waite Campus, University of Adelaide, Australia. 2Department of Crop Production & Plant Breeding, College of Agriculture, Shiraz University, Iran.

GmSAT1 is membrane bound basic helix-loop-helix transcription factor (TF) expressed in soybean nodules and localized to the peribacteroid membrane surrounding nitrogen-fixing bacteroids and cell membrane (Kaiser et al 1998). GmSAT1 was initially characterized as a putative ammonium transporter based on its ability to complement growth of an ammonium transport deficient yeast mutant 26972c. However, recent studies have demonstrated GmSAT1 behaves as a TF in both yeast and in soybean (Loughlin et al unpublished results). We are examining the functional activity of GmSAT1 using a reverse genetics approach where SAT1 has been silenced using RNAi technology in Agrobacterium rhizogenes induced adventitious hairy-roots and nodules. We have further developed this protocol using a combination of in vitro and in vivo conditions where the efficiency of hairy root transformation has been greatly improved. Analysis of silenced tissues reveals the loss of SAT1 impacts upon root nodule development resulting in predominantly ineffective white nodules. Moreover, light microscopy studies have also indicated that silencing of SAT1 influences the vascular cylinder of the root where xylem vessel and endosperm development are altered relative to the roots containing the empty vector control. These studies will provide insight into the overall function of GmSAT1 in both roots and nodules and its relationship to the development and or maintenance of the nitrogen-fixation symbiosis in soybean.

ANALYSIS OF SHARPIN MUTANT MICE AND THE ROLE OF SHARPIN IN THE TNF RECEPTOR SUPER-FAMILY PATHWAYS

Gangoda L.R.1, 2, Hasbold J.3, Gerlach B.4, Carter H.1, Silke J.1 and Vaux D.L.1 1Department of Biochemistry, La Trobe University, Kingsbury Drive, Bundoora, VIC 3086, Australia. 2Cooperative Research Centre for Biomarker Translations (CRC), La Trobe University Bundoora, VIC, 3086 Australia. 3Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. 4Tumour Immunology Unit, Imperial College London, London, UK.

Sharpin (Shank-associated RH domain-interacting protein) is a 45 kDa protein abundantly expressed in brain, spleen and lung. Sharpin binds to the HOIL-1 and HOIP, which are two components of the TNF-RSC (Tumour necrosis factor receptor signaling complex) and regulate their activity. It is also associated with the CD40 receptor signaling complex where it is recruited and function together with HOIL-1 and HOIP to enhance the downstream signaling which results in activation of many genes leading to proper development of lymphoid tissues, B cell activation, proliferation, survival and up-regulation of surface markers involved in antigen presentation. The research aims are to discover the role of Sharpin in the TNF-R1 and CD40 signaling pathways and its putative role in other TNF superfamily (TNFSF) signaling pathways such as BAFF and APRIL mediated pathways. Mice with spontaneously occurring chronic proliferative dermatitis mutation (cpdm) of the Sharpin gene have a phenotype that is entirely consistent with deregulated TNF signaling, characterized histologically by severe inflammation and defects in secondary lymphoid organ development.TNFSF signaling impacts many human diseases, including tumorigenesis and auto-immune diseases. Therefore defining the role of sharpin in these signaling pathways could eventually lead to therapeutic applications. Ultimately cpdm mice will also provide a unique opportunity to study the development of lymphoid tissues and signals that regulate the humoral response.

RECEPTOR INTERACTING PROTEIN KINASE 1 - INTERACTIONS IN TNF RECEPTOR 1 SIGNALLING

Evans J.M., Gentle I.E., Vaux D.L. and Silke J. La Trobe University, Bundoora.

Receptor interacting protein kinase 1 (RIPK1) is the founding member of the RIP kinase family. It has multiple roles, and can promote both cell survival and cell death following ligation of tumour-necrosis factor receptor 1 (TNFR1). RIPK1 has been reported to interact with many proteins, including TNFR1, TNF Receptor 1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2) and 5 (TRAF5), and another RIP kinase family member, RIPK3. Putative TRAF2 binding regions of Mus musculus (mm) RIPK1 were characterised. The EE354AA mutant appeared to bind mmTRAF2 less efficiently than wild-type RIPK1, however this finding was not consistent. The DE367AA putative TRAF2 binding mutant was able to bind mmTRAF2 readily, indicating this mutation did not cause a loss of function. A form of RIPK1 lacking its death-domain (ΔDD RIPK1) retained the ability to bind TRAF2, indicating that this region is not required for TRAF2 binding. RIPK1-/- murine embryonic fibroblasts (MEFs) were reconstituted with wild-type RIPK1, the putative TRAF2 binding mutants, or the ΔDD RIPK1. Although the cells bearing the EE354AA mutant responded less to TNF-α+cycloheximide (CHX) than MEFS with wild-type RIPK1, MEFs bearing either TRAF2 binding mutant behaved the same as those with wild-type RIPK1 in response to treatment with TNF-α and the Smac mimetic drug, Compound A (CA). RIPK1-/- cells reconstituted with the ΔDD RIPK1 responded the same way as RIPK1-/- cells in the presence of TNF-α+CHX or TNF-α+CA, indicating that RIPK1 needs the death domain for its functionality under these conditions. Cells reconstituted with a RIPK1 mutant that could not to bind RIPK3 acted similarly to those reconstituted with wild-type RIPK1 in response to both TNF-α+CHX and TNF-α+CA, indicating that engagement of RIPK3 by RIPK1 is not necessary for responses to TNF-α under those conditions.


CELLULAR INHIBITOR OF APOPTOSIS PROTEINS AND MOUSE EMBRYONIC FIBROBLAST PROLIFERATION

Rickard J.A.1, 2, Moulin M.1, Feltham R.1, Anderton H.1, Vaux D.L.1, 2 and Silke J.1, 2 1Department of Biochemistry, La Trobe University, Kingsbury Drive, Bundoora, VIC, 3086 Australia. 2Cooperative Research Centre for Biomarker Translations (CRC), La Trobe University Bundoora, VIC, 3086 Australia.

Genetic deletion or drug-induced degradation of cIAP (cellular inhibitor of apoptosis protein) 1 and 2 has been shown to increase murine embryonic fibroblast (MEF) proliferation. Multiple myeloma has been found to select for biallelic deletions of the BIRC2/BIRC3 locus encoding cIAPs which may promote oncogenesis due to loss of inhibition of nuclear factor kappa B signalling. This phenomenon is cell and cancer type specific as cIAPs are over-expressed or implicated in the development of other cancers. cIAP antagonism holds chemotherapeutic promise with pre-clinical testing of novel compounds that induce cIAP degradation underway. Characterisation of pro-proliferative effects of cIAP loss may yield mechanistic insights into cancers like multiple myeloma whilst concurrently having implications for treatment of other cancers with IAP antagonists. This study shows wild type but not cIAP1/2 double knockout (DKO) SV40T transformed MEFs proliferate more rapidly when treated with a novel Smac (second mitochondria-derived activator of caspases) mimetic compound that induces cIAP degradation (p ≤ 0.01). TNF-receptor associated factor (TRAF) 2 and/or 5 may be important for this increase in proliferation which is intriguing since a lack of cIAP1 and TRAF5 interaction was found using co-immunoprecipitation experiments. Reconstitution of cIAP DKO MEFs with over-expressed levels of cIAP1 slowed proliferation by an average of 44% (p ≤ 0.01), however no significant reduction was seen with wild type levels of reconstitution. This study provides additional evidence that cIAP loss increases SV40T MEF proliferation and establishes methods that may be used to further characterise this phenomenon.




IMPROVEMENT OF WHEAT AND BARLEY TOLERANCE TO DROUGHT BY MODULATED EXPRESSION OF TRANSCRIPTION FACTORS

Eini O.1, Pyvovarenko T.1, Singh R.1, Eliby S.1, Sivasankar S.2, Simmons C.2, Williams B.3, Tingey S.3, Langridge P.1 and Lopato S.1 1Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Urrbrae, SA-5064, Australia. 2Pioneer Hi-Bred, a DuPont Business, Johnston, IA 50131. 3DuPont Crop Genetics Research, Wilmington, DE 19880.

Wheat and barley grown in Australia are subjected to intermittent periods of drought from winter through spring that can significantly affect yield. The expression of numerous plant genes involved in plant protection and adaptation is regulated by drought stress. Promoter regions of stress-inducible genes contain cis-acting elements involved in stress-responsive gene expression. These elements act as binding sites for transcription factors (TFs). The aim of our work was to identify and isolate drought related transcription factors using cis-acting elements as bait and to improve plant tolerance to drought by modulated expression of selected stress-inducible transcription factors from drought-tolerant varieties. We have prepared 12 different yeast expression cDNA libraries using leaf, spike, developing grain and root tissues collected from unstressed and drought, drought/heat, cold/frost stressed wheat, barley and maize plants. These libraries were used to support yeast one-hybrid (Y1H) and two-hybrid (Y2H) screens. More than 20 different known and computer predicted cis-elements were used to prepare bait yeast strains, over 10 of these elements were successfully used in the Y1H screen. About 35 drought related TFs from 6 different families were cloned either in the Y1H screen using cis-elements as baits or in the Y2H screen using known TFs as baits. Selected candidates were over-expressed under constitutive and stress-inducible promoters in wheat, barley and maize varieties. Analysis of the developmental phenotypes, drought tolerance and grain yields under drought conditions are currently in progress.

IN VIVO DYNAMICS OF HUNTINGTIN PROTEIN AGGREGATION

McCoey J.M.1, 2, Wong Y.Q.1, 2, Hatters D.M.2 and Quinn L.M.1 1Department of Anatomy and Cell Biology, University of Melbourne. 2Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne.

Huntington’s disease occurs due to mutation that leads to the expansion of a poly-glutamine sequence in the Huntingtin protein. In the disease state, the mutant Huntingtin protein coalesces into large intracellular protein agglomerates, known as inclusions, through a largely unknown mechanism. Although inclusion formation is a key feature of the disease, it is not understood how the Huntingtin protein is trafficked and aggregates in cells. In particular, the identity of the cytoskeletal proteins controlling inclusion formation and trafficking has remained poorly defined. We have developed an in vivo Drosophila model to investigate aggregate formation in live cells. Using time-lapse confocal microscopy, we have observed the dynamic process of actin-cytoskeleton interactions with aggregate formation directly in living tissue. We aim to use this model combined with the powerful genetic tools available in Drosophila to investigate the role of cytoskeletal proteins in the aggregation pathway of Huntingtin and how this relates to cellular dysfunction.

DECIPHERING THE STEPS INVOLVED IN THE TRAFFICKING OF PFEMP1, A KEY VIRULENCE PROTEIN IN THE MALARIA PARASITE PLASMODIUM FALCIPARUM

Mwesigye E.E.1, McMillan P.J.1, Maco B.1, Hanssen E.2 and Tilley L.1 1Department of Biochemistry and Centre of Excellence for Coherent X-ray Science, La Trobe University, VIC, Australia. 2Bio21 Institute, Melbourne University, VIC, Australia.

The malaria parasite Plasmodium falciparum is responsible for one of the most prevalent infectious diseases world wide. The most severe symptoms of malarial infection are caused by the adhesion of parasite infected erythrocytes to endothelial cells in the circulatory system. The adhesion phenotype is a result of the parasite exporting proteins into, and trafficking proteins across the erythrocyte cytoplasm into the erythrocyte membrane. However, as the mature erythrocyte has no trafficking machinery, the parasite must therefore develop its own trafficking network in the erythrocyte cytoplasm. Maurer’s clefts (MCs) are parasite derived membranous organelles present in the erythrocyte cytoplasm and are known to be involved in trafficking of the major virulence factor PfEMP1 as well as other proteins to the erythrocyte membrane. Despite their importance in virulence, the mechanisms by which MCs are formed and traffic PfEMP1 are not well understood. Using highly synchronised cultures we will investigate the sequential steps involved in the formation of Maurer’s cleft and PfEMP1 trafficking to the surface of the erythrocyte. We will use P. falciparum transfectants expressing Maurer’s cleft proteins (tagged with fluorescent proteins) to visualise and investigate the processes involved in protein trafficking in vitro. By combining immunofluorescence, live cell confocal microscopy and immunoelectron microscopy we hope to gain a better understanding of the interactions between these trafficking organelles and their cargo proteins over the 48 hour asexual life cycle of the parasite.





MYOSIN II AND RAB6 ARE INVOLVED IN GOLGI VESICLE FORMATION/RELEASE AND CISTERNAL MAINTENANCEMicaroni M.1, Storrie B.2, Morgan G.P.1, 3 and Marsh B.J.1, 3, 4 1Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia. 2Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. 3Centre for Microscopy & Microanalysis, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia. 4ARC Centre of Excellence in Bioinformatics, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia. Bi-directional protein transport between spatially and functionally distinct compartments of the biosynthetic/secretory pathway in mammalian cells requires the formation and fission of membrane carriers from the compartment where they originate, their processive movement along cytoskeletal filaments and the subsequent docking and fusion of the carrier with the compartmental membrane at their destination. Many of these critical steps in protein transport are tightly coordinated by small Rab GTPases and their cognate effectors, which together act to modulate the specificity, directionality and efficiency of membrane trafficking events. The Golgi-associated GTPase Rab6 has been shown to play an important role in regulating membrane traffic at the Golgi to maintain cisternal homeostasis, and a wide variety of proteins have been identified as specific Rab6 effectors. Most recently, Miserey-Lenkei et al. (Nat Cell Biol, 2010, 10.1038/ncb2067) have demonstrated that myosin II also acts as a Rab6 effector and contributes to its localization at the Golgi as well as regulating the fission of Rab6-positive carriers; siRNA-mediated depletion of either protein was reported to yield a similar Golgi phenotype whereby the fission of Rab6-positive carriers was inhibited. Here, we report that selective depletion of Rab6 or myosin II manifests in distinctly different structural phenotypes at the Golgi; silencing of both proteins impairs the trafficking of anterograde as well as retrograde Golgi traffic. Whereas Rab6 knockdown results in the significant accumulation of vesicular traffic at the Golgi and an increase in cisternal number, size and uniformity, myosin II depletion induces fragmentation of the Golgi ribbon per se accompanied by a significant inhibition of vesicle fission from Golgi cisternal membranes. These results suggest that rather than acting as a direct effector of Rab6, myosin II and Rab6 have related but not mutually dependent roles in Golgi vesicle formation/release and cisternal maintenance.

TBC1D24 REGULATES NEURONAL DIFFERENTIATION: CELLULAR MECHANISMS BEHIND A NOVEL GENETIC CAUSE OF FOCAL EPILEPSY AND INTELLECTUAL DISABILITY

Jolly L.A.1, Corbett M.A.1, Bahlo M.2, Afawi Z.3, Gardner A.E.4, Oliver K.5, Shalata A.6, Korczyn A.D.7, Samuel S.F.5 and Gecz J.1, 4, 8 1Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia. 2Walter and Eliza Hall Research Institute, Melbourne, Australia. 3Department of Neurology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel. 4Women’s and Children’s Health Research Institute, Adelaide, Australia. 5Epilepsy Research Centre, University of Melbourne, Austin Health, West Heidelberg, Australia. 6Ginatuna Association, Sakhnin city, West Galilee, Israel. 7Sieratzki Chair of Neurology, Tel-Aviv University, Tel-Aviv, Israel. 8Department of Paediatrics, The University of Adelaide, Adelaide, Australia.

We have characterised a novel syndrome of focal epilepsy, dysarthria and mild to moderate intellectual disability in a consanguineous Arab-Israeli family and mapped the gene to a 3.2 Mb interval within 16p13.3. We used the new technology of targeted sequence enrichment and massively parallel sequencing of the entire linkage interval to identify a pathogenic mutation in the uncharacterised TBC1D24 gene. This gene encodes a TBC domain and is predicted to function as a Rab GTPase activator. Consistent with the clinical features, we show that murine Tbc1d24 mRNA is expressed in neuronal populations. Further analysis revealed that the mutant TBC1D24 protein could be stably expressed, and like the wild-type protein, was found enriched at peri-nuclear and growth-cone structures of cultured hippocampal neurons. We discovered novel roles for TBC1D24 in the regulation of neuronal morphology, with over-expression promoting neurite aborisation, axonal growth and ectopic axonal specification. That these effects were not observed upon over-expression of the mutant protein suggests a loss-of-function. These cell culture data not only support the pathogenesis of the TBC1D24 mutation, but also identifies candidate processes of neuronal differentiation disrupted in patients.

ROLE OF CYSTEINE PROTEASES IN THE MECHANISM OF ACTION OF THE ANTICONVULSANTS LEVETIRACETAM AND CARBAMAZEPINE AND THE CALPAIN INHIBITOR CALPASTATIN IN PENTENYLTETRAZOL-KINDLED RATS

Mansour M.1, Albaker A.1 and Alhaider A.2 1King Saud University-College of Pharmacy-Pharmacology. 2King Saud University-College of Medicine-Pharmacology.

Atypical antiepileptic drugs like levetiracetam (LEV) shows antiepileptic activity in animals and human and their actions do not seem to be similar to the traditional antiepileptic drugs. The present study investigated the possible role of cysteine proteases in the mechanism of action of levetiracetam and the traditional antiepileptic carbamazepine (CBZ) in kindled rats and the role of these enzymes in seizure. Materials and Methods: The effect of increasing doses of CBZ (50, 100, and 200 mg kg-1, p.o), LEV (13, 27, and 54 mg kg-1, p.o) and the calpain inhibitor calpastatin (CS) (1.59, 3.18 and 6.36 mg kg-1, i.p) on calpain, caspase 3 and cathepsin B were studied in normal and in kindled rats. Results: Seizure induced by pentylenetetrazole increased the level of calpain, caspase 3 and cathepsin B (8.44+/-1.4 vs. 40.42+/-0.47 μmol min-1 mg-1 protein, P<0.05), (87.50+/-0.36 vs. 495.91+/-3.51 µmol min-1 mg-1 protein, P<0.05) and (88.89+/-0.38 vs. 500.66+/-2.51 µmol min-1 mg-1 protein, P<0.05), respectively. Two weeks pretreatment and treatment of fully kindled rats with CBZ and LEV produced a significant inhibition (P<0.05) in the level of these cysteine proteases in a dose dependent manner besides CS returned calpain activity values to the level in saline treated group (p < 0.05). Conclusions: These results indicate that both antiepileptic drugs carbamazepine, levetiracetam and the calpain inhibitor calpastatin act partially through the inhibition of these proteases and also that their antagonism by calpastatin might has an important role in the treatment of epilepsy.

SONIC HEDGEHOG AND NOTCH SIGNALING CAN COOPERATE TO REGULATE NEUROGENIC DIVISIONS OF NEOCORTICAL PROGENITORS

Dave R.K., Ellis T., Toumpas M.C., Robson J.P. and Wainwright B.J. 1The Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.

Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors. Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex. Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.





MICRORNAS ARE REQUIRED FOR THE PROPER CORTICAL LAYERING OF THE ANTERIOR ZONE CEREBELLUM

Constantin L., Dave R.K. and Wainwright B.J. Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, Australia, 4072.

Multiple studies have implicated microRNAs (miRNAs) as important regulators of cerebellar function. For example, they have been shown to modulate the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 1, act as tumour suppressors in malignant tumours of the cerebellum (medulloblastoma) and their loss in Purkinje cells has been shown to lead to neuronal degeneration. Canonical miRNA processing is regulated by Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor (GCP) development, we ablated mature miRNA production in GCPs by conditionally inactivating Dicer1 via the GCP-specific Math1-Cre-recombinase transgene. Here, we show that loss of Dicer1 function in GCPs resulted in a cortical layering defect in the anterior zone of the cerebellum, due to an increase in GCP differentiation rates during early and mid postnatal cerebellum development. The increase in GCP differentiation rates led to a reduction in mature granule neurons in the anterior zone only, and mice presented with a high-frequency tremor. These findings have identified a novel role for miRNAs in the compartmentalization of the mammalian cerebellum.

CONSTITUTIVE REDUCTION IN NEURITE GROWTH BY TRPM2 UNDERLIES THE ETIOLOGY OF BIPOLAR DISORDER

Oh U.1, 2 and Jang Y.1 1Seoul National University, College of Pharmacy. 2Seoul National University, Department of Molecular Medicine & Biopharmaceutical Sciences.

Genetic linkage studies strongly suggest that TRPM2, a Ca2+-permeable cation channel is associated with a mental disorder, but the nature of this linkage is not known. Here we show that TRPM2 negatively regulates neurite growth. Knock-down of TRPM2 or treatment with its pharmacological inhibitors markedly increased neurite growth, whereas its up-regulation had a suppressive effect. Furthermore, lysophosphatidic acid, a well established suppressor of neurite growth, was found to act by enhancing TRPM2 activity. More importantly, lithium, which is currently used to treat the mental disorder, blocked the lysophosphatidic acid-induced activation of TRPM2. One putative single nucleotide polymorphism of TRPM2 in the mental disorder patients showed an augmented TRPM2 activity, and led to the constitutive suppression of neurite growth. In addition, amphetamine-induced hyperactivity, an animal model for the mental disorder, was markedly reduced by chronic TRPM2 inhibitor treatment in mice. Furthermore, the reduction by lithium of amphetamine-induced hyperactivity was completely eliminated in TRPM2 deficient mice. These results strongly suggest that prolonged suppression of neurite growth by hyperactive TRPM2 mutant accounts for the pathology of the mental disease. Furthermore, these results provide the link between the mental disease and TRPM2 and delineate the mechanism underlying the therapeutic effect of lithium.

CONSTRUCTION OF DENGUE VIRUS-2 DOMAIN III ENVELOPE PROTEIN

Ahmad Rashidi H.S., Yusof R. and Abdul Rahman N. Drug Design and Development Research Group, University of Malaya, 50603, Kuala Lumpur, Malaysia.

The dengue virus, a flavivirus belonging to the Flaviridae family is a single stranded RNA positive-strand virus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It is currently one of the leading public health threats throughout tropical and subtropical regions and the existence of four closely related, but antigenically distinct serotypes (DEN-1, 2, 3, 4) has been the major factor that has hampered vaccine development efforts. The envelope (E) protein domain III of dengue virus has been proposed to be the possible binding sites on the post-fusion E trimer to host cell and acts as an attractive target for prevention of infection or inhibition of virus spread in an ongoing infection. Thus, we aim to construct a clone of domain III envelope protein of dengue virus serotype-2 (DEN2EIII) to study the interaction of this domain to host cell surfaces. The construction of recombinant DEN2EIII was prepared through directional cloning into pQE9 and plasmid harboring DEN2EIII-pQE9 was transformed into Escherichia coli [strain XL-1]. Bacterial cells grown in LB medium were induced with isopropyl-β-D-thiogalactopyranose (IPTG) to allow high level expression of N-terminal hexahistidine-tagged recombinant proteins and these proteins were then subjected to an affinity purification using Ni charged resin column. The integrity of the EIII protein was confirmed by performing Western blot analysis using a monoclonal antibody against the DEN2 envelope protein and the functional expressed EIII protein will be further employed in studying the involvement of domain III in binding to the host cell.

SILENCING SUPPRESSION IN THE VIRAL FAMILY LUTEOVIRIDAE

Fusaro A.F.1, Correa R.L.3, Kawchuk L.4, Jackson C.S.1, McLean H.1, Vaslin M.F.S.3 and Waterhouse P.M.1, 2 1University of Sydney, Australia. 2CSIRO- Plant Industry, Australia. 3Federal University of Rio de Janeiro, Brazil. 4Agriculture and Agri-Food Canada, Canada.

Gene silencing is a conserved mechanism for controlling gene expression and in plants is associated with viral defense. Sequence specificity is provided by small RNAs (sRNAs) made by the RNase III Dicer enzyme that bind to effector proteins called Argonautes (AGO). The proliferation and diversification of silencing-related genes during evolution allowed the specialization of small RNA-directed pathways in controlling different biological fates, like development, chromatin structure and defense against viruses and repetitive sequences. In the model plant Arabidopsis thaliana there are 4 Dicer-like (DCL1 to DCL4) and 10 AGO (AGO1 to AGO10) genes, giving rise to at least five silencing pathways. Exogenous invading sequences, like viruses, can be degraded by DCL4, DCL2 and DCL3, giving rise to 21-, 22- and 24-nucleotide short interfering RNAs (siRNAs), respectively. Virus-derived siRNAs can both drive viral RNA degradation through AGO1-containing complexes or recruit RNA-dependent RNA polymerase proteins for de novo dsRNA synthesis. Viruses, as a counter defense, developed diverse and unrelated strategies to block the plant RNA degradation machinery. Binding to long or short dsRNAs is a common strategy for silencing suppression and it seems to have evolved independently many times. A different and curious mode of silencing suppression is observed in some members of the viral family Luteoviridae. The family is composed by positive single-stranded RNA viruses that are divided in three different genera: Luteovirus, Polerovirus and Enamovirus. The P0 gene from viruses belonging to the Polerovirus group encodes an F box-like protein that is able to suppress silencing by targeting Argonaute proteins for degradation. Luteovirus and Polerovirus are similar in their biology, but surprisingly, the Luteovirus completely lack a P0 gene. Here we explored the existence of different modes of silencing suppression in the family Luteoviridae.





A SENSITIVE ASSAY TO QUANTITATE O-GLYCANS IN CRUDE SAMPLES

Bhavanandan V.P., Abdul-Rahman P.S. and Hashim O.H. Department of Molecular Medicine and University of Malaya Centre for Proteomics Resaerch, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Mucins are large glycoproteins containing many clustered glycosylated serines and threonines in tandem repeat regions. Mucins and mucin-type glycoproteins, collectively referred to as O-glycans, are implicated in many important biological functions and pathological conditions, specifically malignancy. It is well established that in cancer patients a repertoire of mucins synthesized by the tumor cells enters the circulation by active secretion and by shedding during cell turnover. Currently there are no reliable and sensitive methods to measure the total O-glycan content particularly of crude samples such as tissue extracts or body fluids. One of the reasons for this is the heterogeneous nature of the saccharides in O-glycans. However, in contrast to the high variability of the peripheral saccharides in O-glycans the sugar-amino acid linkage is a unique and constant feature. Previously, we had developed an assay based on the specific chemistry of the GalNAc-Ser/Thr linkages for the estimation of O-glycans (Bhavanandan et al, Anal. Biochem.188, 142-148). However, the sensitivity of the colorimetric assay is limited. Therefore, to measure trace amounts of O-glycans in samples we have developed an alternate assay. The first step of this assay involves the removal of the peripheral sugars of O-glycans by chemical and or enzymatic techniques. In the second step the exposed linkage GalNAc residues are estimated by competitive enzyme-linked lectin assay to provide a measure of the total O-glycan content of the sample. The assay was evaluated using porcine gastric mucin, bovine submaxillary mucin, fetuin and orosomucoid as well as normal human serum spiked with these glycoconjugates. [Supported by grant RG210/10HTM from the University of Malaya].

INHIBITORY EFFECTS OF PES3520 ON PDGF-BB-INDUCED PROLIFERATION OF VSMCS THROUGH BLOCKING PDGF-RB PHOSPHORYLATION

Park, E.S.1, Lim, Y.2, Tudev, M.1, Yoo, H.S.1 and Yun, Y.P.1 1College of Pharmacy, CBITRC, Research Center of Bioresource and Health,. 2Research Institute of Veterinary Medicine, Chungbuk National University, Korea.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is an important pathogenic factor for vascular disorders such as atherosclerosis and restenosis after angioplasty. The present study was designed to investigate the inhibitory effects of PES3520 on FBS- and PDGF-BB-induced VSMC proliferation as well as their molecular mechanisms. PES3520 significantly inhibited the proliferation of FBS- and PDGF-BB-stimulated VSMCs and suppressed the DNA synthesis. In accordance with these findings, PES3520 blocked of the FBS- and PDGF-BB-induced progression through G0/G1 phase of the cell cycle in synchronized cells. PES3520 also decreased the expressions of cyclin-dependent kinase (CDK)2, cyclinE, CDK4 and cyclinD1 as well as retinoblastoma (Rb) protein and proliferative cell nuclear antigen (PCNA) from PDGF-BB-stimulated VSMCs. Preincubation of VSMCs with PES3520 significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt as well as phospholipase C (PLC)-γ1 activation. Consisted with these findings, PES3520 at the dosage of 10, 20 and 40 μM suppressed the PDGF-Rβ phosphorylation induced by PDGF-BB in a concentration-dependent manner. These results suggest that the inhibitory effect of PES3520 on the FBS- and PDGF-BB-induced proliferation of VSMCs may be mediated by blocking PDGF-Rβ phosphorylation. Thus, PES3520 may be a potential antiproliferative agent for the therapy of atherosclerosis and angioplasty restenosis.

DECURSIN INHIBITS HISTONE DEACETYLASE ACTIVITY BY ACTIVATING SPHINGOSINE KINASE TYPE II

Shin, K.O., Kim, S.M., Seo, C.H., Lee, E.S. and Lee, Y.M. College of Pharmacy, CBITRC, Chungbuk National University, Chongju, Korea.

Inhibition of angiogenesis is an attractive approach for the treatment of angiogenic diseases, such as cancer. Sphingosine-1-phosphate(S1P) is one of the most important activators of angiogenesis. The pyranocoumarin compound decursin isolated from the herb, Angelica gigas, are known to possess potent anti-inflammatory activities. However, little is known about their antiangiogenic activity Here, we show the antiangiogenic effects of decursin using in vitro assays. Decursin inhibited sphk -induced angiogenic processes in vitro, including proliferation, migration of human umbilical vein endothelial cells(HUVEC). The sphk type1 activity in HUVEC treated with decursin for 24hr was significantly decreased (43%) compared to control group. However, sphk type 2 activity was mildly increased (1.6 fold) in HUVECs. According to the increase of a sphk2, the histone deacetylase is inhibited by the accumulated S1P. Our data suggests that decursin might be a potent anti-angiogenic agent regulating sphk type1 and sphk type2 signaling pathway. “This work was supported by the grant of the Korean Ministry of education science and technology” ( The Regional Core Research Program / Chungbuk BIT Research-Oriented University consortium).

ROLE OF ANTI-APOPTOTIC BCL-2 FAMILY MEMBER MCL-1 IN LYMPHOMA DEVELOPMENT

Grabow S.1, 2, 3, Kelly P.N.1, Bouillet P.1 and Strasser A.1 1The Walter and Eliza Hall Institute of Medical Research, Australia. 2University of Melbourne, Department of Medical Biology, Australia. 3Cancer Therapeutics CRC, Australia.

The transcription factor Myc is mainly involved in the control of cell growth, proliferation, differentiation and, under conditions of stress (e.g. cytokine deprivation) also apoptosis. Myc function is abnormally increased in ~70% of human cancers. Over-expression of c-myc under control of the IgH enhancer Eµ causes abnormal accumulation of pro-B and pre-B cells during the pre-leukaemic state and ultimately results in monoclonal pre-B or B lymphoma in all animals. Myc-induced lymphomagenesis is impeded by apoptotic death of pre-leukaemic pre-B/B cells and, accordingly, over-expression of anti-apoptotic Bcl-2 greatly accelerates tumorigenesis in this model. We are interested in determining which pro-survival Bcl-2 family members are essential for Eμ-myc induced lymphoma development. Previous studies using gene-targeted mice have shown that, surprisingly, endogenous Bcl-xL, but not Bcl-2, is required for Eµ-myc-induced B lymphomagenesis. It remains, however, unclear whether Bcl-xL is the sole pro-survival Bcl-2 family member required for tumorigenesis in this experimental model. Mcl-1, another pro-survival member of the Bcl-2 family, is highly expressed during the early stages of B lymphopoiesis (HSC, CLP, pro-B), and we therefore decided to examine its role in pre-B/B lymphoma development. Mice that are deficient in Mcl-1 die around embryonic day 4 prior to implantation. We therefore generated gene-targeted mice in which the mcl-1 gene has been flanked with loxP sites to facilitate its deletion by Cre recombinase in a lineage-specific manner. We introduced onto the mcl-1loxP background a CD19-Cre transgene, in which allows specific deletion of loxP flanked genes in B lymphoid cells, and the Eμ-myc transgene to promote lymphoma development. Analysis of Eμ-myc/CD19-Cre/mcl-1loxP/loxP mice will provide valuable insight into the role of Mcl-1 in the survival of pre-leukaemic B cell progenitors and lymphoma development.





NRF2 DYSFUNCTION IN PATIENT-DERIVED CELL LINES IN A NEW MODEL OF SPORADIC PARKINSONS DISEASE

Cook A.L.1, 2, Vitale A.M.2, Ravishankar S.2, Matigian N.2, Mellick G.D.2, Wells C.A.2, Mackay-Sim A.2 and Wood S.A.2 1School of Human Life Sciences, University of Tasmania, Launceston, Tasmania, Australia. 2National Centre for Adult Stem Cell Research, Griffith University, Brisbane, Queensland, Australia.

We recently reported a novel patient-derived cellular model of Parkinsons disease generated from biopsies of the olfactory mucosa, the organ of smell in the nose. Olfactory neurosphere-derived (hONS) cells from Parkinsons disease patients had reduced metabolic functions already associated with disease, including reduced levels of the important antioxidant molecule glutathione. Transcriptomic analysis of patient and Control hONS cells identified the signalling pathway surrounding NRF2, the major transcriptional regulator of the antioxidant response, as being highly differentially expressed in Parkinsons disease. We have shown that activation of the NRF2 pathway in Parkinsons disease hONS cultures restored metabolic function to Control levels, thereby validating NRF2 as a genuine therapeutic target. Paradoxically, transcriptomic analysis of hONS cells after NRF2 pathway activation revealed increased dysregulation of the NRF2 pathway in treated Patient-derived cells. We are now investigating the functional consequences of dysregulated NRF2 activation in Patient-derived hONS cells. Specifically, we are pursuing the recently described link between NRF2 and p62, an important regulator of autophagy, a cellular process implicated in many neurodegenerative diseases. Our results demonstrate that Patient cells can respond to NRF2 activation but that this may elicit a different cellular outcome than that of Control cells. This may be an important factor when developing new therapeutics.

HOMOZYGOUS FRAME SHIFT MUTATION IN ECM1 GENE IN TWO SIBLINGS WITH LIPOID PROTEINOSIS

Azhar A.1, Samdani A.J.2, Shahid S.M.1, Nawab S.N.1, Sheikh R.1, Qader S.A.1 and Ismail M.3 1The Karachi Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi, Karachi. 2Department of Dermatology, Jinnah Postgraduate Medical Center, Karachi. 3Institute of Biomedical & Genetic Engineering (IBGE), Islamabad, Pakistan.

The extracellular matrix protein 1 (ECM1) is a glycoprotein, expressed in skin and other tissues. Loss-of-function mutation in ECM1 causes a rare, autosomal recessive disorder, Lipoid Proteinosis (LP). LP is presented by varying degrees of skin scars, beaded papules along the eyelid margins, variable signs of hoarseness and respiratory disorders. Over 250 cases of this disorder have been described in the literature, but occurrence of LP in siblings is very rare. In this study, two siblings of a Pakistani family are reported. Two (12 and 9-years-old) sisters were presented with scaly itchy lesions on the whole body, hoarse voice and macroglossia. Their deceased father had similar clinical manifestations but mother and younger brother were unaffected. Blood samples from affected and clinically unaffected family members were collected with informed consent. The coding region of ECM1 gene containing 10 exons were amplified and sequenced. Both the affected siblings were shown to have homozygous frame shift mutation by deletion of the nucleotide T at 507, codon 169, exon 6. This resulted in a frame shift from codon 169 and the appearance of stop codon at 177. The normal ECM1 protein contains 540 amino acids. The frame shift mutation has resulted in appearance of a mutated protein containing 176 amino acids. A case of homozygous 62-bp insertion in ECM1 causing LP has been reported in a Pakistani family. The current study presents a novel homozygous frame shift mutation supporting an unusual function of ECM1 protein and broadens the spectrum of disease-linked mutations in this rare case of genodermatosis in this region.

NEURODEVELOPMENTAL PATHWAYS ALTERED IN A NOVEL, PATIENT-DERIVED CELL MODEL OF SCHIZOPHRENIAFan Y.1, Abrahamsen G.1, Mills R.2, Cooper-White J.2, McGrath J.J.1, 2 and Mackay-Sim A.1 1National Centre for Adult Stem Cell Research, Griffith University, Brisbane, Qld, Australia. 2The University of Queensland, Brisbane, Qld, Australia.

Schizophrenia is considered a disease of brain development, based on post-mortem brain and epidemiological analyses. Genetic and environmental risk factors are tested in animal models but their clinical relevance is not clear. Our novel model uses patient-derived cells from the olfactory mucosa, the organ of smell, which regenerates throughout life from a neural stem cell. Olfactory neurosphere-derived (hONS) cells were generated from biopsies of olfactory mucosa from healthy controls and patients with schizophrenia. Gene expression analysis identified 1700 genes whose expression was dysregulated in 9 hONS cell lines from schizophrenia patients compared to 9 control cell lines. Among the down-regulated genes were RGS4, RLN, ERBB3 and GSK3b, which are all down-regulated in post-mortem brain in patients. Pathway analysis identified core neurodevelopmental functions that were dysregulated in schizophrenia including cell cycle and focal adhesion. Subsequent functional studies demonstrated a faster rate of proliferation in schizophrenia hONS cells due to altered cell cycle regulation. After cell cycle synchronisation the schizophrenia cells had earlier and larger increases in cyclin D, cyclin A, and phospho-ERK expression preceding S-phase and a larger proportion of cells entering S-phase in the first 30 hr after synchronisation. Focal adhesion kinase protein levels were significantly lower in the schizophrenia hONS cells and numerous genes in this signalling pathway had altered mRNA expression. This was associated with significantly lower adhesion of the schizophrenia cells to fibronectin and collagen substrates. In addition, schizophrenia hONS cells migrated twice as quickly as control hONS cells. This difference was eliminated by inhibition of focal adhesion kinase. This novel approach identifies for the first time significant alterations in neurodevelopmental signalling pathways in patient-derived cells in schizophrenia.

APOPTOTIC GENES EXPRESSION IN MICE HEPATOCYTES DURING MALARIA INFECTION

Alkahtani S. King Saud University.

Apoptotic genes expression in mice hepatocytes during malaria infection Saad Alkahtani Department of Biology, Teachers College, King Saud University, Riyadh, Saudi Arabia. Abstract: Malaria represents a major global health problem by causing multi-cellular dysfunctions in the infected host. The aim of the present study was to investigate the Apoptotic gene expression in liver of mice during malaria disease by mRNA expression of three genes involved in apoptosis; Bax, Bcl-2 and Caspase-3 and other parameters at different time points after infection with Plasmodium chabaudi in the liver cells of female C57BL/6 mice. Mice were injected intraperitoneally (ip) with 106 P. chabaudi-infected erythrocytes and then scarified at days (0, 1, 4 and 8 respectively). Quantitative real time PCR and immunoblotting were used to quantify apoptotic genes and protein kinetic, respectively. The levels of Bax, Bcl-2 and Caspase-3 were significantly (P<0.05) increased only at days 1 and 8 compared with day 0 in both the liver cells and protein. The levels of parasitemia was significantly (P<0.01) increased at day 8 compared with day 0 in the blood. These results have shown that P. chabaudi induced apoptosis in the liver cells. Thus, this study suggest that the induced apoptotic gene expression was due to the out come of malaria.





LILRA2 SELECTIVELY REGULATES LPS-MEDIATED CYTOKINE PRODUCTION AND PHAGOCYTOSIS IN PERIPHERAL BLOOD MONOCYTES

Lu H.K.1, Endoh Y.1, Huynh O.1, Mitchell A.1, Hampartzoumian T.1, Geczy C.1, Bryant K.1, Arm J.P.2, Borges L.3 and Tedla N.1 1Inflammation and Infection Research Center, School of Medical Sciences, University of New South Wales, NSW, Australia. 2Harvard Medical School, Boston, MA. 3Amgen, Seattle, WA, USA.

The activating immunoglobulin-like receptor A2 (LILRA2) is expressed on the surface of cells that play key role in innate immune responses such as monocytes, macrophages and neutrophils. LILRA2 cross-linking on these cells induces pro-inflammatory cytokine production. However, whether the functions of this molecule differ to the effects of the primary receptors involved in the innate immunity including responses to LPS remain unknown. In this study, we show optimal activation of monocyte by LILRA2 cross-linking failed to induce IL-12 and MCP-1 production that were strongly up regulated by LPS. Interestingly, LILRA2 cross-linking on monocytes induced a similar level of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ to those stimulated with LPS. In addition, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads, while had no effect on phagocytosis of Escherichia coli. Unexpectedly, simultaneous co-stimulation of monocytes through LILRA2 and LPS, or pre-activation of monocytes via LILRA2 followed by LPS treatment led to significantly lower levels of TNFα, IL-1β and IL-12 production as compared to LPS alone. However, there was no effect on the levels of IL-6, IL-10 and IFNγ production. Furthermore, LILRA2 cross-linking on monocytes caused significant down-regulation of TLR4 and CD14 mRNA and protein, indicating LILRA2-mediated suppression of LPS responses might be in part through regulation of these receptors. Taken together, we provide evidence that LILRA2 strongly and selectively regulates LPS-mediated monocyte activation and IgG-dependent phagocytosis.

ATRX IS A SERTOLI CELL SURVIVAL FACTOR AND REGULATOR OF SPERMATOGENESIS VIA INTERACTION WITH ANDROGEN RECEPTOR

Bagheri-Fam S.1, Argentaro A.1, Svingen T.2, Combes A.2, Koopman P.2 and Harley V.R.1 1Prince Henry’s Institute of Medical Research, Melbourne, VIC Australia. 2Institute of Molecular Bioscience, UQ, Brisbane, QLD, Australia.

The ATR-X syndrome, (α-thalassemia mental retardation, X-linked) is a developmental disorder affecting males. ATR-X syndrome individuals display a range of genital abnormalities, including small testes. ATRX shows nucleosome remodeling activity in vitro, suggesting an involvement in gene regulation but no target genes have been defined. We inactivated Atrx specifically in testicular Sertoli cells (ScAtrxKO mice) from embryonic day 14.5. Fluorescence-based three-dimensional modeling at E17.5 revealed that testis cord volume in ScAtrxKO mice is ~30% of wildtype, with discontinuous or isolated testis cords apparent. Sertoli cell apoptosis is increased 10-fold with no difference in Sertoli cell proliferation. One third of ScAtrxKO tubules contain germ cells arrested in late meiosis or at the round spermatid stage. Sertoli cells, upon androgen stimulation of the androgen receptor (AR) transcribe genes necessary for the completion of germ cell meiosis and spermatid development, such as Rhox5. While gene expression of AR itself was unchanged in ScAtrxKO testes, expression of three AR-dependent genes including Rhox5 was significantly down-regulated. Moreover ATRX and AR proteins interact in vivo and in the TM4 Sertoli cell line to co-operatively activate the Rhox5 promoter. In summary, ATRX plays an important role in Sertoli cell survival during fetal testis development and in adult testis function, where ATRX protein interacts with AR to regulate the transcription of AR-dependent genes that control spermatogenesis, such as Rhox5, the first ATRX target gene identified.

DIFFERENTIAL METHYLATION OF HNRNP A2/B1 PARALOGS DETECTED IN NEURONAL CELLS AND CELL LINES BY MASS SPECTROMETRY

Friend L.R.1, Landsberg M.J.2, Nouwens A.S.1, Rothnagel J.A.1 and Smith R.1 1School of Chemistry and Molecular Biosciences, The University of Queensland. 2Institute for Molecular Biosciences, The University of Queensland.

The heterogeneous nuclear ribonucleoprotein paralogs, A1, A2/B1 and A3 (hnRNPs A/B) share a high degree of sequence similarity and may have closely related roles in processes such as RNA packaging, alternative splicing, telomere maintenance and cytoplasmic trafficking. However, in a previous study, the hnRNPs A/B were shown to exhibit different patterns of sub-nuclear localization, suggesting that these paralogs have diverged to perform functionally distinct roles. Additional functional diversity is created by post-translational modifications, coupled with the generation of alternatively spliced isoforms. In the work reported here, we have isolated hnRNP A2 using pulldowns on immobilized RNA-trafficking elements or telomeric ssDNA repeat oligonucleotides, followed by reverse-phase HPLC. We investigated hnRNP A2 post-translational modifications in native rat brain, rat B104, human HeLa and human SH-SY5Y transformed cells using mass spectrometry and Edman degradation. Our results show that hnRNP A2 is NG,NG-dimethylated on a single arginine residue, Arg254, encoded within the alternatively spliced exon 9. This is in contrast to hnRNP A1, which has four of its five RGG motifs NG,NG-dimethylated on the arginine residues. In native rat brain, Arg254 is almost completely dimethylated, while in rat and human cell lines, a greater proportion of this arginine residue is either unmethylated or monomethylated. This conserved modification of Arg254 in hnRNP A2, suggests that hnRNP A2 is functionally regulated by dimethylation in a manner that distinguishes it from hnRNP A1.

STRUCTURAL/FUNCTIONAL PROPERTIES OF ISOLATED HUR-HNRNP COMPLEXES OF MAMMALIAN CELL ORIGIN

Papadopoulou C., Ganou V., Patrinou-Georgoula M. and Guialis A. Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, GREECE.

Post-transcriptional processes that include splicing, polyadenylation, transport, stability and translation of mRNA are important events in regulating gene expression in higher eukaryotes. These are brought about by the extensive network of interactions among a large number of RNA-binding proteins (RBPs) operating within diverse multifactorial RNP complexes. We have recently focused on RBP proteins; hnRNPs having a major role in RNA splicing, and HuR an AU-rich element-binding protein (ARE-BP) with a so far established role in stability/translation of target mRNAs. Both hnRNPs and HuR have a major nucleoplasmic localization and ability for nuclear/ cytoplasmic shuttling. Recently we reported on an extensive range of hnRNP-HuR interactions within immunopurified hnRNP and mRNP complexes (Papadopoulou et al., Biochem Biophys Acta, 2010,1804:692-703). We have now extended our study in directly immunoselected HuR complexes from HeLa nuclear extracts. With respect to associated protein species, these HuR complexes were strikingly similar with immunopurified hnRNPs via the anti-hnRNP C1/C2 (4F4) antibody. The authenticity of the HuR-associated proteins was tested using a mouse embryonic cell line (MEF) completely devoid of HuR. Presently, we combine biochemical analysis with confocal microscopy on intact as well as on nuclear matrix preparation of HeLa cells to look at cellular localization of HuR and of selected hnRNP proteins under several growth conditions. In particular, results concerning the functional implications of the identified HuR-hnRNP interactions under stress conditions, as well as under inhibition of protein phoshorylation, will be presented.





CELL POLARITY COMPLEXES REGULATE GROWTH VIA THE SALVADOR/WARTS/HIPPO (SWH) PATHWAY

Parsons L.M., Grzeschik N.A., Allott M.L., Harvey K.F. and Richardson H.E. Peter MacCallum Cancer Centre, 2 St Andrew’s Place East Melbourne, Victoria 8006, Australia.

Inactivating mutations in Drosophila tumour-suppressor genes result in tissue overgrowth. It has long been recognized that loss-of-function mutations in the junctional-scaffold neoplastic tumour suppressor gene, lethal-2-giant-larvae (lgl) result in cellular overproliferation, ultimately resulting in the development of lgl- tumours. We have shown that in the developing Drosophila eye, loss of lgl activity results in ectopic proliferation and suppression of developmental cell death (apoptosis), without loss of cell polarity. In this system, targets of the SWH pathway are increased upon lgl inactivation. This occurs by decreased phosphorylation and re-localization of the SWH pathway transcriptional activator, Yorkie (Yki). A critical role for the SWH pathway and Yki activity in the development of lgl- tumours is supported by genetic evidence that shows Yki is required for the development of lgl- tumour phenotypes. The SWH pathway is proposed to modulate Yki activity by two distinct mechanisms: 1) Fat/Dachs proteins which regulate the level of Warts protein (Warts kinase is responsible for phosphorylating Yki and inhibiting its activity) and 2) Ex protein which binds Yki, sequestering it within the cell cortex thus preventing it from entering the nucleus. We have genetic and biochemical evidence that lgl activity does not regulate Yki activity by regulating Warts protein levels. Our immuno-histochemistry data shows that a second kinase (Hippo, Hpo) that also regulates Yki activity is mis-localised in lgl mutant tissue. Thus, we propose a novel mechanism whereby the apical-basal polarity regulator Lgl regulates the SWH pathway by correctly localizing components of the SWH pathway. These data provide the first mechanistic links between the neoplastic and hyperplastic tumour suppressor pathways in Drosophila.

QUINIC ACID METABOLISM IN KIWIFRUIT

Marsh K.B.1, Boldingh H.2 and Laing W.1 1Plant and Food Research, Private Bag 92-169, Auckland, 1142, NZ. 2Plant and Food Research Ruakura, Private Bag 3123, Hamilton 3249, NZ.

Kiwifruit are unique in containing high concentrations of quinic acid (1-2% w/w), which contribute to the flavour, and health-giving properties of the fruit. In a study of quinic acid storage and metabolism in three kiwifruit species (Actinidia chinensis, A. deliciosa and A. arguta), quinic acid accumulation was shown to occur principally in the early stages (<60 days after anthesis; (DAA)) of fruit development. We studied the genes responsible for quinic acid metabolism, including DAHP (Dehydroquinate synthase) and two iso-enzymes of a DHQ (bi-functional dehydroquinate dehydratase/shikimate dehydrogenase) by quantitative reverse transcriptase PCR and by over-expression of ESTs in recombinant systems. RT-qPCR results for DHQ synthase showed that there was a high level of expression in the early season, which was sustained through the mid-season. A kiwifruit EST that had homology to chloroplastic isoforms of SDH showed an induction in the middle to late season but could not explain the high shikimate dehydrogenase activity in the early season. The activity in the early season (<30 DAA) may have been due to the expression of a cytosolic isoform of the enzyme. This suggests that dehydroquinate is a key substrate, particularly for a bifunctional dehydroquinate dehydratase /shikimate dehydrogenase at the branch point in the shikimate and quinate pathways. We have been studying the genes that control the synthesis of Dehydroquinate using a recombinant DAHP synthase, and a DHQ synthase, and also attempting to recombinantly express cytosolic and chloroplastic versions of the bifunctional dehydroquinate dehydratase/shikimate dehydrogenase.

BETA-SITOSTEROL REGULATES ADIPOCYTE GENES INVOLVED IN GLUCOSE UPTAKE, ADIPOGENESIS AND LIPID MOBILIZATION

Kanthimathi M.S., Chai J.W. and Kuppusamy U.R. Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

The nutraceutical benefits of β-sitosterol (SIT) are well documented. The present study investigated the in vitro effects of SIT on adipogenesis, glucose transport and lipid mobilization in rat adipocytes. Primary cultures of rat preadipocytes and differentiated adipocytes were used in this study. Glucose uptake was measured by the uptake of radio-labeled glucose. Adipogenesis and lipolysis were measured by oil-red-O and glycerol quantification methods, respectively. The expression of protein kinase B (Akt), glucose transporter 4 (GLUT4), hormone sensitive lipase (HSL) and phosphatidylinositol-3-kinase (PI3K) genes in SIT-treated adipocytes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). The data showed that SIT induced glucose uptake in adipocytes. It also stimulated adipogenesis in differentiating preadipocytes. Interestingly, although SIT displayed general insulin-mimetic activity by stimulating glucose uptake and adipogenesis, it also induced lipolysis in adipocytes. Furthermore, the SIT-induced lipolysis was not attenuated by insulin and co-incubation of SIT with epinephrine improved epinephrine-induced lipolysis. GLUT4 gene expression was highly down-regulated in SIT-treated adipocytes, compared to insulin-treated adipocytes, which was up-regulated. Insulin- and SIT-treated adipocytes showed similar levels of Akt, HSL and PI3K gene down-regulation. These observations suggest that the elevation of glucose uptake in SIT-treated adipocytes was unrelated to de novo synthesis of GLUT4 and that the SIT-induced lipolysis is associated with the down-regulation of Akt and PI3K genes. The unique effects of SIT on the regulation of glucose uptake, adipogenesis and lipolysis in adipocytes show that it has potential to be utilized in diabetes and weight management.

A CRITICAL ROLE FOR THE PROTEIN PHOSPHATASE 2A B’α REGULATORY SUBUNIT IN DEPHOSPHORYLATION OF SPHINGOSINE KINASE 1

Pitman M.R.1,3, Barr R.K.1,3, Magarey A.M.1, Moretti P.A.B.1 and Pitson S.M.1, 2 1Molecular Signalling Laboratory, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia 5000, Australia. 2School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia. 3These authors contributed equally to this work.

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localisation and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B’α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B’α was found to interact with the C-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. Furthermore, siRNA-mediated knockdown of B’α increased SK1 phosphorylation, activity and membrane localisation of endogenous SK1. Thus, the PP2A-B’α holoenzyme appears to function as an endogenous regulator of SK1.





SUMOYLATION OF IGF-1R IS MEDIATED THROUGH SUMO-E3 LIGASE RANBP2

Sjostrom S.1, Warsito D.1, Sehat B.1, 2 and Larsson O.1 1Karolinska Institute, Department of Oncology-Pathology, CCK, R8:04, SE-17176 Stockholm, Sweden. 2McGill University, Goodman cancer center, 1160 Ave Des Pine Quest,Montreal, QC H3A 1A3, Canada.

The insulin-like growth factor-1 receptor (IGF-1R) is a cell surface receptor belonging to a class of receptors known as receptor tyrosine kinase (RTK). It is known that the IGF-1R is vastly expressed in malignant tissues and plays a crucial role in growth and survival of cancer cells. Inhibition of IGF-1R has been demonstrated to cause apoptosis. However, in many cases patients treated with drugs aimed to inhibit the IGF-1R have developed resistance against the drug, and the explanation behind this is not fully understood.Recently, our laboratory reported that IGF-1 stimulation promotes SUMO-1 modification of the IGF-1R, followed by nuclear translocation of the receptor. Nuclear IGF-1R associates with enhancer-like elements of the genomic DNA and increases transcription. Malignant cells, e.g. breast cancer cells, have more than a 20-fold increase in nuclear IGF-1R comparing to normal cells, but the total amount of IGF-1R is only 5-fold increased in malignant cells. In this present study we show that the IGF-1R is co-localized with Importinβ, which is known to transport proteins into the nucleus through the nuclear pore complex (NPC). Further, we found that IGF-1R interacts with RanBP2, a SUMO-E3 ligase enzyme, also located at the NPC. By over-expressing the 32 kDa active SUMO-E3 ligase fragment of RanBP2, we can show that there is enhanced SUMOylation of the IGF-1R, confirming that RanBP2 is a significant part of the IGF-1R SUMOylation chain. Further investigations in how the IGF-1R enters the nucleus would provide novel drug targets and enable the development of novel cancer therapies.

EVALUATION OF P16 PROTEIN AS A BIOMARKER IN PENILE CARCINOMA

Kagohara L.T.1, Guimaraes G.C.2, Lopes A.2, Cunha I.W.1, Coudry R.A.1 and Soares F.A.1 1Hospital A.C. Camargo - Anatomic Pathology Department. 2Hospital A.C. Camargo - Pelvic Surgery Department.

Penile carcinoma is an uncommon disease and the most frequent subtype is squamous cell carcinoma. This cancer represents less than 1% of all cases in developed countries. However, in some regions of developing countries it represents 20% of all male cancers. The main risk factors associated to this tumor are poor hygienic habits, low quality of life, HPV infections and high number of sexual partners. Very little is known about penile carcinoma, including its pathogenesis, etiology and gene expression patterns. To identify the role of the protein p16 in penile carcinoma progression, we evaluated its expression pattern by immunohistochemistry in 301 samples displayed in tissue microarrays. This protein is known to have reduced levels in many tumor types, is associated to patient’s clinical outcome and is involved in the tumorigenesis process. The p16 protein has an important role in the cell cycle control. It is a cyclin-dependent kinase (CDK) inhibitor that inhibits retinoblastoma protein phosphorilation. The stained slides were analyzed at the ACIS-III (DAKO) equipment, which digitalizes images and quantifies frequency and intensity based on the brown areas of the samples. We observed loss of expression of p16 in 65.1% of the cases. The statistical analysis showed an association between the presence of protein p16 and low histologic grade tumors, while loss of expression of p16 is associated to presence of perineural and vascular invasion. These results indicate that p16 loss of expression is an important event in penile carcinoma tumorigenesis and it may be a biomarker for this tumor type as perineural and vascular invasion are clinical markers of bad prognosis.

A NOVEL 4,6-DISUBSTITUTED 2,2-DIMETHYLCHROMAN INDUCES APOPTOSIS IN HUMAN CERVICAL CARCINOMA HELA CELL VIA INHIBITION OF DNA SYNTHESIS

Zhang X., Hwang J. and Kim D.-K. Department of Biomedicinal Chemistry, Inje University, 607 Aubang-dong, Gimhae 621-749, Korea.

1-((3S,4R)-4-(2-Ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy-2,2-dimethylchroman-6-yl)-3-phenylurea (S24), a novel 4,6-disubstituted 2,2-dimethylchroman was synthesized and its apoptotic activity was investigated on the in vitro growth of human cervical carcinoma HeLa cells. Significantly decreased rates of proliferation and viability (IC50 ~150 μM) as well as evidence of apoptosis were observed with S24. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. The results from Annexin V/PI dual staining and the cell cycle arrest assay indicated that S24 induced an earlier apoptosis of HeLa cells in a time-dependent manner. S24 also induced the activation of a protease cascade involving caspase-9 and -3 by western blotting assay. Furthermore, [3H]thymidine incorporation and flow cytometry analysis revealed that S24 treatment decreased DNA synthesis and arrested the cells at the G0/G1phase of the cell cycle. In addition, the very sensitive alkaline microgel electrophoresis technique (comet assay) was used for estimation of the S24-induced DNA single- and double-strand breaks. Taken together, we suggest that S24-induced apoptosis through caspase pathway and the apoptotic effect may correlate with its inhibition of DNA synthesis.

TISSUE MICROARRAY STUDY OF MATRIX METALLOPROTEINASES IN SOFT TISSUE SARCOMAS

Muto N.H.1, Cunha I.W.1, Carvalho K.C.2, Malagoli R.1, Buim M.E.C.1, Nascimento C.F.1, Ferreira S.S.1 and Reis L.F.L.3 1Hospital AC Camargo, Sao Paulo, Brazil. 2Faculdade de Medicina - USP, Sao Paulo, Brazil. 3Hospital Sirio Libanes, Sao Paulo, Brazil.

Soft tissue tumors are a heterogeneous group of mesenchymal tumors with variable clinical behavior and histological presentation. Tumor size, histologic grade, depth, and status of surgical margins have been identified as prognostic factors. However, these prognostic variables do not explain biologic differences in aggressiveness between sarcomas. Given that human soft tissue sarcoma is a highly lethal malignancy in which control of metastasis determines survival, a marker of more aggressive and metastatic behavior could be valuable. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that, collectively, are capable of degrading almost all ECM components. MMPs can contribute to local and distant tumor invasion through degradation of basement membrane and stromal invasion. In this study we investigated the immunohistochemical expression of a panel of MMPs (-2, -9, -13 and -19) in 3 tissue microarrays (TMAs) containing a total of 266 sarcoma samples, including 118 sunovial sarcomas, 37 liposarcomas, 45 pleomorphic sarcomas, 18 leiomyosarcomas, 12 myxoid type sarcomas, 5 malignant peripheral nerve sheet tumor, 6 fibrosarcoma, 18 fibromatoses, 7 benign tumors and 20 other type sarcomas. Images were digitalized and quantified using ACIS® III Tissue Microarray Analysis (DAKO, CA) and data were statistically analyzed using the software PRISM 4. In general, we found a higher expression of MMP-2 and -9 in synovial sarcomas compared to other samples, and a higher expression of MMP-13 and -19 in synovial and pleomorphic sarcomas. Finnancial support: CEPID-FAPESP.





INDUCTION OF APOPTOSIS AND DNA DAMAGE BY AN ANALOGUE OF METHYL JASMONATE

Zhao J.1, Zhang X.1, Kang S.1, Hwang J.1, Jung J.2 and Kim D.-K.1 1Department of Biomedicinal Chemistry, Inje University, 607 Aubang-dong, Gimhae 621-749, Korea. 2College of Pharmacy, Busan National University, Busan 609-735, Korea.

The current study was undertaken to investigate the effects of methyl 5-chloro-4, 5-didehydrojasmonate (J7), an analogue of methyl jasmonate, on the in vitro growth of human cervical carcinoma HeLa cells. Significantly decreased rates of viability (IC50 ~15 μM) as well as evidence of apoptosis were observed with J7. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. The results from Annexin V-FITC/PI double staining and the cell cycle arrest assay further supported that J7 was able to induce apoptosis of HeLa cell and influence cell cycle process resulting in S phase arrest. By Western blot assay, a decrease in Bcl-2 level followed by an increase of a protease cascade involving caspase-9 and -3 was observed. Furthermore, an activation of SAPK/JNK and p38 MAPK was also detected. The single-cell gel electrophoresis (comet assay) and [3H]dTTP incorporation assay indicated that J7 induced DNA damage and inhibited DNA replication. Taken together, our studies suggest that J7 induces HeLa cell apoptosis though activation of both caspase pathway and the sub-MAPK pathways. Moreover, the apoptotic effect is associated with DNA damage. Therefore, J7 may be a candidate compound to be developed into an anticancer agent.

COPY NUMBER VARIATION IN INDIVIDUALS WITH CLINICAL DIAGNOSIS OF LI-FRAUMENI OR LI-FRAUMENI LIKE SYNDROME AND WITHOUT GERMLINE ALTERATIONS IN TP53 GENE

Silva A.G.S.1, Achatz M.I.1, Brentani R.1, Rosenberg C.2 and Krepischi A.C.1 1Hospital A.C. Camargo, Sao Paulo, Brazil. 2Laboratory of Human Genetics, Department of Genetics and Evolutionary Biology, University of Sao Paulo, Brazil.

Li-Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a remarkably increased risk of early-onset and multiple cancers, including breast, brain tumors and other neoplasms. Constitutional copy number variation of DNA segments (CNV) are likely associated to cancer, either as a susceptibility factor or as a result of a genetic instability. It has been recently reported that individuals carrying TP53 mutations show about 3-fold increase in CNV frequency compared to wild type individuals, raising the possibility that the number of CNVs could be larger in individuals with high cancer risk. We used a 180K oligoarray platform from Agilent to estimate the CNV frequency in 63 affected and unrelated patients with clinical diagnosis of Li-Fraumeni and Li-Fraumeni-like syndrome. We compared the resulting frequencies between these individuals to a population based control group (n=100). We have defined as rare genomic alterations those located in coding regions and covered by ≤3 individual CNVs described in DGV (Database of Genomic Variants). The initial results point to a slight increase in the average frequency of CNVs per individual in the cancer patients samples (9.3) when compared to the control sample (6.8). Although the sample is still small, the category so-called rare alterations are approximately twice as frequent among cancer patients (average of 1.36±1.69 per individual) than among controls (average of 0.42±0.67 per individual). Some of the genes encompassed by these CNVs have been previously implicated in tumors, but not reported in germline.

ACTIN FIBERS AND FIBRONECTIN NETWORKS REMODELING DURING EARLY ADIPOCYTE DIFFERENTIATION

Nobusue H.1, Oki Y.1, Shimizu T.2, Onishi N.2, Saya H.2 and Kano K.1 1Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University. 2Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine.

Adipocyte differentiation at an early stage is directly regulated by gene expression of a master regulator such as peroxisome proliferator-activated receptor-γ2 (PPARγ2). During adipogenesis, extracellular matrix (ECM) remodeling defines important events of the differentiation process. This is characterized by conversion from the fibronectin (FN)-rich stromal matrix of a preadipocyte to the basement membrane of an adipocyte. Adipocyte differentiation has been reported to result in the conversion of filamentous actin from stress fibers and lamellipodia to cortical actin structures. However, the order of the following events has not been clarified: (a) PPARγ2 expression, (b) ECM remodeling, and (c) actin cytoskeleton remodeling. In this study, we aimed to elucidate the remodeling of the actin cytoskeleton and ECM during early adipocyte differentiation. Before adipogenic induction, preadipocytes showed fibroblastic morphology with a well-developed actin stress fiber pattern and formed FN networks in connection with their actin cytoskeltons. After differentiation for 24 h, the actin fibers were disassembled and disorganized, and as a result, the FN networks were also disorganized. After 48 h of differentiation, the fibers disappeared and a cortical actin structure was formed. Coincidentally, the FN networks degraded and disappeared. After 60 h of differentiation, lipid droplets accumulated in the cytoplasm and laminin-8 expression was detected on the plasma membrane. Matrix metalloproteinase (MMP)-2, a catabolic enzyme essential for ECM remodeling, was activated after 48 h of adipocyte differentiation, whereas MMP-9 was not detected. And PPARγ2 increased significantly after 48 h of differentiation. Furthermore, the remodeling of the actin fibers and the FN networks occurred after 24 h of differentiation even when depleted expression of PPARγ was achieved by retrovirus-mediated shRNA. These results indicate that remodeling of the actin fibers and the FN networks precedes PPARγ2 expression during adipocyte differentiation, and therefore, we suggest that this remodeling acts as a trigger for adipocyte differentiation.

A WINDOW OF OPPORTUNITY: THE MECHANISM OF PERFORIN AND GRANZYME SYNERGY

Lopez J.A., Trapani J.A. and Voskoboinik I. Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC 3002.

Perforin is a pore forming member of the Membrane-Attack-Complex-Perforin (MACPF) family of proteins. It is stored in the secretory granules of cytotoxic lymphocytes (CLs) represented by natural killer cells and cytotoxic T lymphocytes. CLs seek out and bind to virally infected or transformed target cells and form a transient immunological synapse. Formation of the synapse results in cell polarisation and exocytosis of secretory granules containing monomeric perforin molecules together with pro-apoptotic serine proteases (granzymes) into the synaptic cleft. At the target cell, perforin facilitates the delivery of granzymes into the cytosol to initiate cell apoptosis. The precise mechanism of perforin-mediated granzyme delivery has remained elusive. To investigate the mechanism of perforin mediated granzyme delivery, we have performed a detailed kinetic analysis of perforin function on target cells. Sublytic doses of perforin were shown to elicit a transient calcium influx into target cells. Restoration of intracellular calcium concentrations was attributed to the induction of a membrane repair response by the target cell. Inhibition of membrane repair through temperature restriction resulted in a block in intracellular calcium restoration and the induction of cell death through cell lysis/necrosis. Membrane repair of perforin pores formed at the plasma membrane resulted in a rapid loss of synergy with granzymes. Collectively, these data suggest that perforin acts primarily at the plasma membrane of target cells to facilitate granzyme entry, leading to cell death through apoptosis. Concomitantly, the repair of perforin lesions acts to prevent cell death through necrosis.





CONSERVED EXPRESSION OF VASA IN MARSUPIALS AND MONOTREMES

Hickford D.1, Frankenberg S.1, Pask A.2, Shaw G.1 and Renfree M.B.1 1The University of Melbourne, Australia. 2The University of Connecticut, USA.

VASA is a widely-used marker of germ cells across all animal phyla, but little is known of its distribution and expression in non-eutherian mammals (marsupials and monotremes). VASA was cloned from a marsupial, the tammar wallaby, and a monotreme, the platypus. Tammar VASA has 21 exons encoding a protein with 726 amino acids; platypus VASA has 22 exons encoding 743 amino acids. VASA protein sequence is highly conserved among mammals, especially within the DEXDc and HELICc domains, which contain nine conserved sequence motifs. The N terminus of VASA is less conserved and the human, mouse, rat, wallaby and platypus all have at least two transcripts for VASA, one encoding a full length protein and the other missing an exon within the N terminus. The exon missing varies between species. Curiously, the platypus has an extra exon not present in other mammals. In contrast to eutherians, wallaby germ cells do not express VASA protein until ~4-5 days after they reach the gonads. In females, germ cells remain VASA-positive throughout their life cycle and in some oocytes VASA appears to be concentrated in a distinct paranuclear patch, as occurs in humans but not mice. In both wallaby and platypus adult testes, VASA protein is present in spermatocytes and round spermatids, with weaker staining in spermatogonia and elongating spermatids and none in spermatozoa. The spermatocytes of both species exhibited distinct VASA-positive granules within their cytoplasm, similar to in eutherians. In summary, the high sequence conservation and similarity in VASA distribution in marsupials and monotremes suggest conservation of the role of VASA in germ cell development across all mammals.

EPIGENETIC REGULATION OF OCT4 AND PDX-1 IN MOUSE EMBRYONIC STEM CELLS AND MIN6 CELLS

Wong J.C.Y.1, Jack M.M.2 and O’Neill C.1 1Kolling Institute of Medical Research, The University of Sydney, Sydney, Australia. 2Department of Endocrinology, Royal North Shore Hospital, Sydney, Australia.

The differentiation of embryonic stem cells (ESCs) to insulin producing β-cells involves a complex series of transcriptional changes such as repression of pluripotency associated with the Oct4 gene and activation of Pdx-1, a critical transcription factor that is considered the earliest specific marker of pancreatic endoderm. These changes may be regulated by epigenetic factors such as DNA methylation and covalent histone modifications. This study aimed to determine the role of these mechanisms in the regulation of Oct4 and Pdx-1 in the D3 mouse ESC line in comparison to the insulinoma cell line, MIN6. The presence of histone modifications was examined by chromatin immunoprecipitation assays using anti-histone antibodies and DNA extracts from D3 and MIN6 cells. Specific modifications at the Oct4 and Pdx-1 promoter were then quantitated by real-time PCR. The DNA methylation status of CpG rich areas of Oct4 and Pdx-1 was determined by bisulfite sequencing. The study demonstrated that Oct4 was hypomethylated in D3 and hypermethylated in MIN6. The histone marks found to be present also corresponded to the Oct4 expression status in each cell line. In contrast, Pdx-1 was hypomethylated in both D3 and MIN6 despite it being repressed in ESCs. D3 cells also possessed a bivalent domain of both the active H3K4Me3 and repressive H3K27Me3 histone marks at the Pdx-1 promoter. Together, the results suggest a means by which Pdx-1 is maintained at a transcription-ready state in ESCs. It also provides further insight into identifying the mechanisms that are necessary to activate and repress the key homeotic genes that are required for β-cell specification.

THE ROLE OF NEDD4 IN EMBRYONIC BLOOD AND LYMPHATIC VASCULAR DEVELOPMENT

Secker G., Kazenwadel J., Boase N., Kumar S. and Harvey N. Division of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia.

The cardiovascular system is the first organ network to develop in the vertebrate embryo. Assembly of the initial blood vascular plexus occurs via the aggregation of endothelial progenitor cells and is known as vasculogenesis, while the subsequent sprouting growth and remodeling of the primary vascular network is known as angiogenesis. Lymphatic vessels are a critical, but often overlooked component of the cardiovascular system. These specialised vessels return interstitial fluid and protein to the bloodstream, absorb lipids from the digestive tract and traffic cells of the immune system. Formation of the lymphatic vasculature is initiated once the major arteries (dorsal aortae) and veins (cardinal veins) have been established, originating from the cardinal veins following the onset of expression of the Prox1 transcription factor in a polarized population of venous endothelial cells. Our current work is focused on the identification of genes and molecular events critical for generation of the vasculature during embryogenesis. In particular, we are investigating the role of Nedd4, an E3 ubiquitin ligase, in this process. Ubiquitination of a protein can lead to its degradation, stabilisation, changes in subcellular localization or modification of biochemical properties. As such, protein modification by ubiquitination has crucial roles in regulating many signalling pathways and misregulation of this process is associated with numerous human pathologies. Here we present data that demonstrates loss of Nedd4 function leads to impaired development of the blood and lymphatic vasculature. Future studies aim to define the mechanism by which Nedd4 regulates signalling involved in blood and lymphatic vascular development.

IDENTIFICATION OF EPIGENETIC COMPONENTS REQUIRED FOR CELL-TO-CELL MOVEMENT OF AN RNA SILENCING SIGNAL IN ARABIDOPSIS

Searle I.R.1, 2, Smith L.2 and Baulcombe D.C.2 1Research School of Biology, ANU, Canberra. 2Department of Plant Sciences, University of Cambridge, UK.

RNA silencing is a sequence-specific RNA degradation process conserved in fungi, animals and plants that is associated with cell-to-cell movement of a mobile silencing signal. We developed a cell-to-cell movement of RNA silencing system in Arabidopsis using an inverted repeat transgene under the control of a phloem-specific promoter such that the spread of RNA silencing was manifested in regions around veins. We have previously identified epigenetic components; Polymerase IV, RNA-Dependent-RNA-polmyerase 2, and CLSY (a SNF2chromatin remodelling factor) that are required for mobile RNA silencing. In addition we also identified a putative histone H3 lysine 4 trimethyl demethylase, JMJ14, that is required for mobile RNA silencing. In addition to an effect on mobile silencing the jmj14 mutants also had reduced CHH DNA methylation, increased abundance of endogenous transposon transcripts and they flowered earlier than wild type. We placed the activity of JMJ14 at a downstream point in RNA silencing pathways because the subcellular locations of upstream components RNA-dependent RNA polymerase (RDR2) and Argonaute (AGO4) were not perturbed in jmj14 mutants. These results illustrate the potential for a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream of the Argonaute effector complex to demethylate histone H3 lysine 4 trimethyl residues at the target of RNA silencing.





PAP SIGNALS FROM THE CHLOROPLAST REGULATE EXORIBONUCLEASES, GENE SILENCING AND STRESS RESPONSES IN ARABIDOPSISCrisp P.A.1, Estavillo G.M.1, Pornsiriwong W.1, Wirtz M.2, Carrie C.3, Hell R.2, Whelan J.3 and Pogson B.J.1 1ARC Centre of Excellence in Plant Energy Biology,The Australian National University, Canberra, ACT, Australia, 0200. 2University of Heidelberg, Heidelberg Institute for Plant Sciences, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany. 3ARC Centre of Excellence in Plant Energy Biology,The Universuty of Western Australia, Crawley, 6009, WA, Australia.

Chloroplasts regulate nuclear gene expression during plant development and in response to environmental stresses via retrograde signalling pathways. Here we report a novel chloroplast-nucleus signal that regulates RNA metabolism and gene silencing in the nucleus. In a genetic screen to identify components of the chloroplast stress signalling pathways we isolated the Arabidopsis mutant altered APX2 expression 8 (alx8) [1]. The alx8 mutant exhibits constitutive up-regulation of stress inducible genes, including APX2 and ELIP2, and drought tolerance. alx8 is a mutation in the SAL1 phosphatase [2], which we now show to be localised to the chloroplast, not the nucleus as recently proposed [3]. We have demonstrated that the loss of chloroplastic SAL1 leads to a 10 fold elevation of its substrate, 3-phosphoadenosine 5-phosphate (PAP), which is an inhibitor of 5’-3’ exoribonucleases (XRNs) in yeast [4]. Building on an earlier report linking SAL1 to the activity of the exoribonucleases in plants [5], we show that stress-inducible genes elevated in the SAL1 knockouts – alx8 and fiery1 – are similarly up-regulated in the xrn2,xrn3 double mutant. Furthermore, we show that the alx8 mutation restores sensitivity to ABA in stomata and seeds of abi1 and ost1 (ABA insensitive mutants). Thus, these results implicate exoribonucleases in stress response and gene regulation, and establish a compelling link between chloroplast and stomatal signalling, RNA metabolism and gene expression. [1] Rossel et al. (2006) Plant Cell Environ. [2] Wilson, Estavillo et al. (2009) Plant J. [3] Kim and von Arnim (2009) Plant J. [4] Dichtl et al. (1997) EMBO. [5] Gy et al. (2007) Plant Cell. Research supported by the ARC Centre for Plant Energy Biology (CE0561495); and GRDC Scholarship (GRS184).

MECHANISMS OF SMALL, NON-CODING RNA-DIRECTED GENE SILENCING IN FISH

McFarlane L.J., Fitzner A., Koopman P. and Wilhelm D. Institute for Molecular Bioscience; The University of Queensland, Brisbane, Australia.

RNA interference (RNAi) has emerged as a powerful platform technology for investigating gene function. Although widely used in the major model organisms, the use of RNAi in teleost fish has proven unreliable. We are exploring the reasons for this unreliability. Using a comparative genomics approach, we have identified that the teleost clade has, uniquely among the vertebrates, undergone an expansion of the AGO protein sub-family, the key effector proteins of RNAi and other small non-coding RNA regulatory pathways. Although previous studies have suggested that the vertebrate AGO homologues are largely redundant, our approach identified numerous features that suggest these proteins may have specialised functions. To explore their function, we have isolated the five teleost Ago genes from the zebrafish Danio rerio for use in a variety of assays. We have also developed an in vitro system for biochemically dissecting the RNAi response to determine whether it is analogous with the other more well-characterised model systems. Our results will illuminate the mechanisms of small RNA-directed gene regulation in the teleosts, as well as identifying potential strategies to enable the use of RNAi in fish.

FIELD EVALUATION OF TRANSGENIC PERENNIAL RYEGRASS (LOLIUM PERENNE L.) PLANTS FOR ENHANCED FRUCTAN ACCUMULATION

Dimech A.M.1, 3, Badenhorst P.E.2, 3, Panter S.1, 3, Liu Z.1, 3, Ramage C.1, 3, Stewart A.V.4, Smith K.F.2, 3, Mouradov A.1, 3, 5 and Spangenberg G.C.1, 3, 5 1Department of Primary Industries, Victorian AgriBiosciences Centre, Bundoora, Victoria 3083, Australia. 2Department of Primary Industries, Hamilton Centre, Hamilton, Victoria 3300, Australia. 3Molecular Plant Breeding Co-operative Research Centre, Bundoora, Victoria 3083, Australia. 4PGG Wrightson Seeds, Lincoln, New Zealand. 5La Trobe University, Bundoora, Victoria 3086, Australia.

Fructans, a class of water-soluble storage carbohydrates, play an important role in the forage quality of pasture grasses, providing an energy source for livestock. Up to 279 independent transformation events in elite perennial ryegrass (Lolium perenne L.) germplasm were evaluated for increased fructan accumulation and enhanced biomass yield under field conditions in Hamilton, Victoria, between September 2008 and January 2010. Chimeric fructosyltransferase genes, Lp1-SST and Lp6G-FFT, were expressed in transgenic plants as single genes or as a Lp1-SST::Lp6G-FFT translational fusion under the control of constitutive and light-regulated promoters. Quantitative real-time PCR was used to measure transgene expression and High-Performance Anion-Exchange Chromatography was used to determine fructan accumulation throughout the growing season. Multiple independent transformation events were identified with fructan levels in whole tillers and leaf blades that were up to 80% higher than levels in corresponding tissues of non-transgenic plants of the same genotype. Elite transformation events were selected based on enhanced biomass production, low transgene copy number and consistently high fructan accumulation for transgenic germplasm development.

REGULATION OF THE HUMAN CHOP GENE PROMOTER BY THE STRESS RESPONSE TRANSCRIPTION FACTOR ATF5 VIA THE AARE1 SITE IN HUMAN HEPATOMA HEPG2 CELLS

Yamazaki T., Ohmi A., Kurumaya H., Kato K., Abe T., Okuyama R., Umemura M., Kaise T., Takahashi S. and Takahashi Y. The Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.

Activating transcription factor (ATF) 5 is a member of the cAMP response element binding protein (CREB)/ATF family of transcription factors. We have shown that ATF5 is a stress response transcription factor that responds to amino acid limitation, arsenite exposure, or cadmium exposure. In this study, we used a transient transfection system to express ATF5 and showed that ATF5 activates the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) gene promoter in human hepatoma, HepG2, cells. Both deletion analysis and point mutations of the promoter revealed that amino acid response element (AARE) 1 is responsible for ATF5-dependent promoter activation. Furthermore, the existence of either AARE1 or activating protein-1 (AP-1) is sufficient for transcriptional activation of the CHOP gene promoter by arsenite exposure, although complete induction requires the existence of both elements. We also demonstrated that knockdown of ATF5 reduced arsenite-induced CHOP protein expression. These results demonstrated that besides the AP-1 site, the AARE1 site is also important for arsenite-induced CHOP gene promoter activation. Taken together, these results suggested that the CHOP gene is a potential target for ATF5, and that ATF5 raises the arsenite-induced CHOP gene expression level via the AARE1 site in HepG2 cells.





INVESTIGATING THE ROLE OF KLF3 IN ERYTHROPOIESIS AT THE PHYSIOLOGICAL AND MOLECULAR LEVEL

Norton L.J.1, Mak K.1, Funnell A.P.W.2, Pearson R.C.M.1 and Crossley M.1 1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia. 2School of Molecular and Microbial Biosciences, University of Sydney, Australia.

Development is governed by transcription factors which orchestrate the ordered expression of genes. A major family of transcription factors are the Krüppel like factors (Klfs) which play a role in a wide variety of processes including stem cell biology, adipogenesis, skin development and erythropoiesis. Klf1 was the first Klf to be discovered and is a master regulator of erythropoiesis and driver of β-globin expression. Klf1 has been shown to induce the expression of a related family member, Klf3, which indicates a possible role for this transcription factor in erythropoiesis. This research aims to investigate the role of Klf3 in erythropoiesis in vivo through the investigation of a Klf3 knock out mouse model. An examination of the peripheral blood of the Klf3 KO has revealed extensive defects in haematopoiesis. In particular, we have observed a significant increase in the number of reticulocytes, indicative of anaemia. In agreement with this, the spleen of the KO mouse is enlarged and tissue sections have revealed a dramatic increase in the presence of red pulp. Flow cytometry has confirmed noticeable compensatory erythropoiesis in the spleens of the KO mouse. We have also used flow cytometry to study erythroid differentiation in the fetal liver and adult bone marrow and have observed impaired development. Taken together, this evidence supports a role for Klf3 in normal red blood cell development. We have now collected and purified erythroid tissue from the Klf3 KO mouse at various time points and are using a microarray approach to determine the role of Klf3 in erythropoiesis at the molecular level.

DOES THE EXTRACTION OF CELL CONTENTS DURING DIGESTION OF GRASSES BY THE AUSTRALIAN PLAGUE LOCUST OCCUR VIA PLASMODESMATA?

Armour W.J., Clissold F.J., Barton D.A., Overall R.L. and Simpson S.J. School of Biological Sciences, The University of Sydney, Sydney, NSW Australia.

Barbehenn (2005) has proposed nutrients are extracted from plant tissue via plasmodesmata during digestion by insects. We investigated this in the Australian plague locust (Chortoicetes terminifera, hereafter locusts) and its two natural plant hosts the C4 grasses, Dactyloctenium radulans and Astrebla lappacea. Typically C4 grasses have large, chloroplast rich, bundle sheath (BS) cells that appear to remain intact during digestion by grasshoppers. We found many plasmodesmata pitfields between BS cells and the surrounding ring of cells of both grasses when stained using aniline blue. We investigated if locusts could use these plasmodesmata for nutrient extraction. Leaf fragments taken from locusts four min after ingestion had almost no callose when stained with aniline blue. Thus to test if locust gut enzymes can degrade callose, transverse sections of both grasses were induced to form callose by plasmolysis and then treated with phosphate buffered saline (PBS) or locust enzymes. As there was no significant change in the number of BS cells with callose at plasmodesmata in either treatment after 30 min it suggests locust enzymes cannot degrade callose. We hypothesised plant tissue ingested by locusts had almost no callose because the cells were dying. We tested membrane integrity of BS cells in ingested tissue by loading leaves with the membrane impermeant dye, carboxy-fluorescein (CF). After feeding, leaf fragments taken from locusts four min after ingestion had less than 6% of BS cells retaining CF compared to 70% for controls, suggesting most BS cells lost membrane integrity during ingestion. Thus it appears nutrient extraction via plasmodesmata is unlikely as the cells die early during ingestion.

WASH-DEPENDENT ACTIN DRIVES VESICLE NEUTRALISATION: A REQUIREMENT FOR EXOCYTOSIS

Carnell M.J. and Insall R. The Beatson Institute for Cancer Research.

WASH, a recently identified member of the WASP protein family, is an activator of actin polymerisation around endocytic vesicles. Utilising the model organism Dictyostelium discoideum we demonstrate a novel role for actin in controlling vesicle identity. Here we show that WASH is essential for the formation of actin-coats around late endocytic compartments. The generation of WASH null cells has revealed that these actin-coats are essential for driving lysosome neutralisation, and that failure to do so results in a complete block in the exocytosis of indigestible material. Our data suggests an important role for both WASH and actin in vesicle maturation by driving the removal of the H+ V-ATPase from internal vesicles prior to fusion with the plasma membrane.

EXENDIN-4 ENHANCES ADULT NEUROGENESIS AFTER FOCAL CEREBRAL ISCHEMIA IN MICE

Kang H.M., Jin J. and Park C. Department of Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea.

Cerebral ischemia induces neurogenesis. Neurogenesis occurs mainly in two area of adult mammalian brain including of humans : the subgranular zone (SGZ) of the dentate gyrus (DG) and the anterior part of the subventricular zone (SVZ), along the ventricle. In addition newly generated neurons in the SVZ migrated to the ischemic boundary region. Several factors regulated proliferation, migration, differentiation of neuronal precursor cells. Exendin-4 is peptide hormone, stable analog of glucagon-like peptide-1 (GLP-1), and that selectively binds GLP-1 receptor. Recently, It has been demonstrated that GLP-1 receptor promotes conservation dopaminergic neurons in rodent Parkinson’s diseases model, Exendin-4 markedly reduced cortical infarction induced by transient Middle Cerebral Artery occlusion (MCAo). However, Exendin-4 is still unknown whether it is enhanced neurogenesis. In this study, we used photothrombotic focal ischemia model in mice and observed that systemic administration of Exendin-4 significantly increased not only 5-bromo-2-deoxyuridine (BrdU), proliferation marker, but also Ki-67, necessary cellular marker for proliferation in SVZ. Furthermore immature neurons marker, doublecortin (DCX), increased in ischemic penumbra region. These findings suggest that Exendin-4 may be increases proliferation of neural stem cells through activation of GLP-1 receptor.





HYDROXYSTEROID DEHYDROGENASE TRANSFORMATIONS OF 5β-SCYMNOL AND IDENTIFICATION OF OXOSCYMNOL TRANSFORMATION PRODUCTS BY LCMSMacrides T.A.1, Hodges L.D.1, Wynne P.M.2, Glowacki L.L.1, Kalafatis N.1 and Wright P.F.A.3 1Natural Products Research Group, RMIT University, Bundoora, VIC. 3083 Australia. 2Shimadzu Scientific Instruments (Oceania) Pty Ltd, Mt Waverley, VIC. 3149 Australia. 3Applied & Nutritional Toxicology Key Centre, RMIT University, Bundoora, VIC. 3083 Australia.

Our interest in 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol] stems from the therapeutic potential of this natural marine shark bile alcohol. However, the xenobiotic transformation of scymnol in mammals is unknown. Hydroxysteroid dehydrogenase (HSD) reactions are fundamental to the in vivo transformations of mammalian bile acids, and the presence of 3α-, 7α- and 12α-hydroxyls on scymnol support an investigation of scymnol oxidation products in HSD reactions. Mixtures of scymnol, respective HSD enzyme and NAD+ cofactor were incubated for 5 min (25 °C, pH 8.9) before the addition of methanol. Precipitated protein was removed by centrifugation, and the supernatant analysed for scymnol metabolites by means of a Zorbax SB-C18 column using either an Agilent 1090M liquid chromatograph interfaced with a Thermo Quest LCQ Duo mass spectrometer, or an Agilent 1050 liquid chromatograph interfaced with a Thermo Quest LCQ Classic mass spectrometer. The authentic scymnol standard yielded a protonated molecular ion at m/z 469 Da, and higher mass adduct ions attributed to M+NH4

+, M+CH3OH2+

and M+CH3COOH2+. 3-Oxoscymnol [(24R)-(+)-5β-cholestan-3-one-

7α,12α,24,26,27-pentol, m/z 467 Da, relative retention time 0.89] was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. 7-Oxoscymnol [(24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol, m/z 467 Da, RRT 0.79] and 12-oxoscymnol [(24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol, m/z 467 Da, RRT 0.81] were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Confirmation that 5β-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated LCMS techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.

CHARACTERIZING THE CONFORMATION OF MONOMERIC HUNTINGTIN

Scicluna B.J.1, 2, Ramdzan Y.M.1, 2, Mok Y.F.1, 2, Mulhern T.D.1, 2 and Hatters D.M.1, 2 1Department of Biochemistry & Molecular Biology, The University of Melbourne, VIC 3010, Australia. 2Bio21 Molecular Science & Biotechnology Institute, The University of Melbourne, VIC 3010, Australia.

Huntington’s disease is one of nine neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) region. Recent research has suggested that the expanded polyQ causes the huntingtin monomer to misfold into abnormal collapsed structures that are toxic to the cells. Using small angle x-ray scattering (SAXS) and sedimentation velocity (SV) analysis, we determined the size and shape of the huntingtin monomer and compared non-pathogenic forms (25Q) with pathogenic forms (46Q). Both 25Q and 46Q huntingtin species had similar size and radius of gyration, despite 46Q being 21 residues longer than 25Q, which suggests the polyQ region is unusually collapsed. Shape reconstruction from SAXS data of 25Q and 46Q huntingtin exon 1 fusions with MBP (N-terminal) and Cerulean (C-terminal) depicted both huntingtin regions as extended rod-like shapes, likely due to the averaging of heterogeneous mixtures of structures with a length of ~65 Å. Shape heterogeneity of monomeric huntingtin exon 1 fusions was also observed by SV analysis, which is consistent with the monomer existing as multiple rigid structures.

NEUROPROTECTIVE EFFECTS OF ETHYL PYRUVATE ON PARKINSON’S DISEASE MODEL

Choi J.S. and Jeong J.W. Department of Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea.

Ethyl pyruvate (EP), a simple derivative of endogenous pyruvate, has an anti-inflammatory function. Recently, the protective neurological effects of EP have been reported in cell culture and animal models of neurological diseases. The present study investigates the protective effects of EP on dopaminergic cell death in Parkinson’s disease models. The selective death of dopaminergic neurons in substantia nigra was prevented by EP in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. EP also suppressed the 1-methyl-4-pyridinium-induced cell death of SH-SY5Y cells and restored the phosphorylation of extracellular signal-regulated kinase. Thus, EP has neuroprotective effects of EP in Parkinson’s disease and its related signaling pathways.

THE ROLE OF PLZF IN SHAPING ONCOGENIC CHARACTERISTICS OF CANCER CELLS

Suliman B.A.1, 2, Xu D.2 and Williams B.R.G.2 1College of Applied Medical Sciences, Taibah University, Madinah, Saudi Arabia. 2Monash Institute of Medical Research, Monash University, Melbourne, Australia.

Despite the advances over the past decade in our comprehension of cancer cell biology and the role played by the accumulation of different genetic mutations in numerous proto-oncogenes and tumour-suppressor genes resulting in aggressive activation of many key signalling pathways that delineate the nature of cancerous cells, it is still unclear how transformed cells acquire certain oncogenic characteristics, including enhanced proliferation, lack of contact inhibition, anchorage-independent cell growth, and low apoptotic potential, which are common features linked to the cell’s cytoskeleton. PLZF is a transcriptional repressor belonging to POZ-Krupple (POK) family of transcription factors with critical roles on oncogenesis development and stem cell maintenance. Microarray data suggest that PLZF expression is low or completely lost in many cancer cell lines. PLZF expression is also associated with growth inhibition and cell cycle arrest through its ability to repress the expression of a number of growth promoting and proto-oncogenic genes. Recently, we found that overexpression of PLZF in PC3 (prostate cancer cell line with a high metastatic potential) diminished their motility, cytoskeleton integrity and viability. These results may indicate a potential connection between PLZF induced changes to cytoskeleton architecture and PLZF tumour suppression in oncogene-driven cellular transformation. Cytoskeleton remodelling by PLZF will be further characterized during epithelial to mesenchymal transition (EMT) in prostate and other cancer cells to understand the important roles played by PLZF in precluding the provoked proliferation and invasive nature of these cancers.





TRPC6 WAS NEUROPROTECTIVE IN CEREBRAL ISCHEMIA

Du W.L., Huang J.B., Yao H.L., Zhou K.C. and Duan B. Institute of Neuroscience, Chinese Academy of Sciences.

Neurons are the most vulnerable cells upon ischemic insults. Preservation of neuronal survival is thus important for prevention of ischemic brain injury. We report that the transient receptor potential canonical (TRPC) 6 was neuroprotective in focal cerebral ischemia. TRPC6 protein level in the neurons was greatly reduced in ischemia. This downregulation was specific for TRPC6 in the members of TRPC subfamily and mediated by calpain proteolysis. A peptide that contains a sequence spanning calpain cleavage site on TRPC6 protein specifically blocked TRPC6 degradation. Moreover, this peptide was neuroprotective against ischemic insults in both cultured neurons and animals. The level of phosphorylated cAMP-response element binding protein (pCREB) was maintained by the peptide, suggesting that the CREB signaling pathway was involved in the neuroprotective effect. Our results indicated that suppression of calpain-mediated TRPC6 degradation preserved neuronal survival and prevented ischemic brain damage.

STREPTOCOCCUS PNEUMONIAE: CHOOSING RATIONAL TARGETS FOR STRUCTURE DETERMINATIONRiboldi-Tunnicliffe A. Australian Synchrotron. Streptococcus pneumoniae (the pneumococcus) is a significant pathogen, responsible for high levels of morbidity and mortality worldwide. Most affected by this organism are children with immature immune systems, the elderly and individuals with compromised immune systems, such as those with diabetes or acquired immunodeficiency syndrome [1, 2]. As a human pathogen S. pneumoniae is the most common bacterial cause of acute respiratory infection and otitis media and is estimated to cause 3 million deaths in children from pneumonia, bacteraemia or meningitis every year [3] accounting for 9% of deaths in underdeveloped countries [4]. These diseases are not limited to the developing world, despite the availability of a vaccine and a wide range of antibiotics, S. pneumoniae remains one of the top ten causes of death in the US, where one third of isolates are resistant to penicillin [5]. Since 1990 the resistance to penicillin has increased from 1-5% (isolated strains) to 25-80%, with many strains having resistance to multiple antibiotics [6, 7]. Streptococcus pneumoniae is a bacterium commonly found in the nasopharynx (back of the nose) of 30 –50% of individuals, albeit asymptomatically. Occasionally S. pneumoniae will spread from the nasopharynx of a colonised person into other parts of the body and cause diseases including otitis media (ear infection), sinusitis (sinus infection) and pneumonia (lung infection) [1]. In addition S. pneumoniae can sometimes get into normally sterile places in the body such as the blood, where it causes sepsis and/or bacteraemia, or the lining of the brain and spinal cord, where it causes meningitis. The gene regulatory mechanisms involved in the transition between, and survival in, these distinct host niches are poorly understood [8]. Modulating bacterial virulence provides an array of molecular processes such as adherence factors, toxins and lytic enzymes, which can be targeted to provide new therapies for disease prevention. This proposal aims to provide a thorough understanding of the essential underlying molecular mechanisms contributing to Streptococcus virulence and survival. Use of this systematic functional and structural approach provides a first step to the characterization of new therapeutic molecular targets for drug discovery. 1.Musher, D.M., Clin Infect Dis, 1992. 14: p. 801-7. 2.AlonsoDeVelasco, E., A.F. Verheul, J. Verhoef, and H. Snippe, Microbiol Rev, 1995. 59: p. 591-603. 3.Greenwood, B., Philos Trans R Soc Lond B Biol Sci, 1999. 354: p. 777-85. 4.Poland, G.A., Vaccine, 1999. 17: p. 1674-9. 5.Thornsberry, C., et al., and D.F. Sahm, Antimicrob Agents Chemother, 1999. 43: p. 2612-23. 6.Tomasz, A., N Engl J Med, 1995. 333: p. 514-5. 7.Doern, G.V., A.B. Brueggemann, H. Huynh, and E. Wingert, Emerg Infect Dis, 1999. 5: p. 757-65. 8.LeMessurier, K.S., A.D. Ogunniyi, and J.C. Paton, Microbiology, 2006. 152: p. 305-11.

ANTIPROLIFERATIVE ACTIVITY AND PROTEOMIC ANALYSIS OF GENE EXPRESSED IN HEPATOCELLULAR CARCINOMA (HEPG2) CANCER CELL LINES TREATED WITH PHYTIC ACID

Saad N.1, Norhaizan M.E.2, 3, Hairuszah I.1, 2, Sabariah A.R.1, 4 and Shafie N.H.2 1UPM-MAKNA Cancer ResearchLaboratory, Institute of Bioscience,Universiti Putra Malaysia. 2Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia. 33Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. 4Department of Pathology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia.

Phytic acid or IP6 is a naturally occurring polyphoshorylated carbohydrate, present ubiquitous in plants and animals. Phytic acid is not only a natural antioxidant, but may also be the precursor/storage of intracellular inositol phosphates, important for various cellular functions and potential as anticancer compound. A prominent anticancer action of phytic acid has been demonstrated both in vivo an in vitro in a variety of tumor types, possibly via inhibition of tumor cell growth and differentiation. In this study, the growth inhibitory effect of phytic acid extracted from rice bran on human hepatocellular carcinoma (HepG2) has been tested. Phytic acid proved to induce growth inhibition and differentiation in HepG2. A dose and time dependent growth inhibition of phytic acid was observed as tested by MTT assay. Analysis of flow cytometry was performed for the analysis of cell cycle and apoptosis. Treatment of phytic acid against HepG2 also resulted in cell cycle arrest. Besides, the induction of early apoptosis in a dose- and time dependent manner was confirmed using an annexin V-based assay. In a PCR analyses, phytic acid confirmed the upregulated proapoptotic genes and downregulated antiapoptotic genes. In conclusion, with taken together results from our findings, phytic acid may be an excellent candidate for adjuvant chemotherapy and prevention of cancer.

CHARACTERISATION OF THE MECHANISM BY WHICH THE BTB/POZ ZINC FINGER PROTEIN, ABRUPT MEDIATES TUMOURIGENESIS IN DROSOPHILA

Turkel N.1, 2, 3, Brumby T.1, 2, Goulding K.1, Bolden J.1, Martin-Blanco E.3 and Richardson H.1, 2 1Cell Cycle and Development, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. 2Anatomy and Cell Biology, University of Melbourne, Melbourne, VIC, Australia. 3Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain.

Loss of function of the apico-basal cell polarity regulator, Scribble (scrib-) in patches of cells (clones) leads to over-proliferation, rounding-up of cells and multi-layering in the epithelium of 3rd instar larval eye imaginal discs. However overgrowth of the scrib- clones is prevented by Jun kinase (JNK) signalling induced by the surrounding wild type tissue. Oncogenic versions of Ras(RasACT) or Notch(NotchACT) cooperates with scrib- to block the apoptotic activity of JNK resulting in invasive tumours. In a genetic screen, abrupt was identified as an oncogene, that when over-expressed, cooperates with scrib- to promote tumourigenesis in a similar manner of RasACT or NotchACT. Abrupt encodes BTB/POZ Zinc Finger domain protein. Mammalian homologs of BTB/POZ Zn finger domain proteins are implicated in cancer. We predict that identifying the oncogenic mechanism of Abrupt will increase our understanding of how this class of oncogenes can contribute to tumourigenesis in mammals. Here, we show that over-expression of abrupt in scrib- clones blocks JNK-mediated cell death and results in tumours that massively overgrow and invade the adjacent brain lobes. Blocking JNK signalling in the tumour can prevent invasion, but does not inhibit tumour overgrowth. We found that atypical protein kinase C (aPKC) signaling is responsible for overgrowth in abrupt scrib- tumours. In addition, expression array and chromatin immunoprecipitation coupled with high-throughput sequencing have been applied and revealed that abrupt act as an oncogene by promoting the retention of a progenitor-like state in antenna and eye disc.





BIOFILM FORMATION AS CHARACTER TO SELECT WINE SACCHAROMYCES CEREVISIAE STRAINS

Sidari R.1 and Howell K.S.2 1Department of Scienze e Tecnologie Agro-Forestali e Ambientali, Mediterranea University of Reggio Calabria, Via Feo di Vito, Reggio Calabria, Italy. 2Department of Agriculture and Food Systems, Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria, Australia.

The wine yeasts Saccharomyces cerevisiae are selected according to both technological and quality parameters to obtain starters with oenological characters useful for winemaking. Searching for strains with an ideal combination of oenological characteristics as well as the use of novel oenological traits are two aspects of a continuous process toward the improvement of winemaking. Biofilm growth describes the ability of microorganisms to grow and behave as a multi-cellular community with altered metabolism and reduced susceptibility to chemicals and antiseptics. Early work on S. cerevisiae has identified the Flo11p as protein involved in biofilm formation to various surfaces. The ability to form biofilm is a character well studied in yeasts of medical interest while very little fundamental research has occurred in wine yeasts. Their ability to form biofilm has significant biotechnological relevance; from attachment of yeasts to grape berries in the vineyard, to adhesion and contamination of different surfaces in the winery. The present work aimed to study the behaviour of 50 wine S. cerevisiae strains, already selected for the common oenological characters, toward the aptitude to form biofilm on plastic surfaces. This to give a contribution to the knowledge of the biodiversity in the specie S. cerevisiae but chiefly to propose the biofilm formation as a new possible character of selection due the fact that yeast strains able to form biofilm could remain attached to the various surfaces in winery becoming a problem to the management of the winemaking in purity during years.

REGULATION OF THE ESSENTIAL M. TUBERCULOSIS PEPTIDOGLYCAN-PRECURSOR FLIPPASE

Gee C.L.1, 4, Papavinasasundaram K.G.3, Blair S.R.1, Baer C.E.1, Falik A.M.2, King D.S.2, Sassetti C.M.2 and Alber T.1 1Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, CA 94720-3220. 2Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3200. 3Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 016551. 4Australian Synchrotron, 800 Blackburn Rd, Clayton, Vic 3168 Australia.

Receptor Ser/Thr kinases control broad aspects of physiology in bacteria, but little is known about how the kinases regulate cellular pathways. Among the Mycobacterium tuberculosis (Mtb) proteins essential in an animal model is the flippase that delivers the peptidoglycan (PG) precursor, lipid II, to the cell surface for incorporation into the cell wall. In mycobacteria and several other actinomycetes, the flippase has accessory domains, which presumably add functionality or regulate the activity. We found that the Ser/Thr protein kinase, PknB, efficiently phosphorylates the intracellular pseudokinase domain of the Mtb flippase, and this single modification creates a binding site for the forkhead associated (FHA) domain protein FhaA in vitro and in vivo. To define the mechanisms of recognition, we determined the crystal structures of the flippase extracellular domain and the intracellular pseudokinase alone and in combination with FhaA. The extracellular accessory domain is homologous to a galactose binding domain, while the intracellular domain has a highly diverged, inactive protein-kinase fold. These results suggest support a model in which extracellular PG regulates PG synthesis by controlling assembly of a protein complex containing the lipid II flippase.

EFFECTS OF PROGESTERONE ON CELL GROWTH OF T CELL LEUKEMIA CELLS

Kon A., Sato M. and Toyoda H. Dept. of Clin. Mol. Genet., School of Pharm., Tokyo Univ. of Pharm. and Life Sci.,1432-1 Horinouchi,Hacioji,Tokyo,Japan.

Progesterone (Pg) has been used for the treatment of patients with breast and uterine cancer. It is also known to show antileukemic effects. However, detailed mechanisms are yet to be understood. It has been reported that the long-term survival rate of children and adult of acute lymphoblastic leukemia (ALL) is 80% and 40%, respectively. A serious side effect of current chemotherapy including bone marrow depression is an important issue to improve quality of life (QOL) of patients. In order to understand mechanisms involved in antileukemic effects, two cell lines derived from Jurkat were used to study the effects of Pg on cell proliferation. Two cell lines used in this study were E6-1 (parental cell line) and A3 (sensitive to Fas-mediated apoptosis). We have previously demonstrated that the antiproliferative effects of Pg was most prominent in A3; an increase of cells in S-phase and decrease of those in G2/M accompanied with the increase of cells in subG1 phase; and a high-molecular weight DNA fragmentation was observed. In this study, more than 20% of A3 cells showed Annexin-positive compared with those of 8% of E6-1 cells. No DNA fragmentation was observed on an agarose gel electrophoresis, but TUNEL positive cells were detected only microscopically. An increased activity of caspase-3, -8 and -9 was observed in A3 compared with that in E6-1. Furthermore, our results demonstrated that membrane progesterone receptor α was activated by Pg. Our results demonstrated that Pg administration inhibited cell growth of T-cell derived leukemic cells mediated through membrane progesterone receptor α, following membrane destruction, activation of caspase-8, caspase-9 and caspase-3 along with high-molecular weight DNA fragmentation.

SYNERGISTIC CYTOCIDAL EFFECTS OF DELPHINIDIN WITH ARSENITE ON HUMAN LEUKEMIA CELL LINES

Yoshino Y., Yuan B., Okuzumi S., Takemae F. and Toyoda H. Dept. of Clin. Mol. Genet., Tokyo Univ. of Pharm. and Life Sci., 1432-1 Horinouchi, Hachioji, Tokyo, Japan.

Arsenic trioxide (arsenite, As III ) is effective in the treatment of acute promyelocytic leukemia (APL). Recently, it is demonstrated that As

III induces cell death in malignant cells other than APL. However, the relatively low sensitivity of most malignant cells to As III is shown, suggesting that it may require higher and more toxic doses to show clinical efficacy. Therefore, we initiated to seek reagents that exert synergistic effects with As III to increase the efficacy of As III and reduce its dosage to clinical achievable concentrations. Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, possesses cytocidal effects on various tumor cells including acute myeloid leukemia and solid tumor cells. Moreover, normal cells show low sensitivity to delphinidin-induced cytotoxicity. Our study demonstrated that co-treatment with delphinidin and As III induced synergistic cell death in As III -resistant leukemic cell line, HL-60, but not in human peripheral blood mononuclear cells (PBMCs) from healthy donors. Annexin V staining assay showed that co-treatment with As III and delphinidin induced apoptosis along with necrosis to HL-60. Importantly, the synergistic effect of As III and delphinidin was selectively toxic for leukemia cells, and no combination effect was observed in human PBMCs from healthy donors. Our data suggested that delphinidin may extend the therapeutic spectrum of As III

in the treatment of malignant cells that are less sensitive to As III .





PUTATIVE CIS-REGULATORY ELEMENTS IN HIGHLY EXPRESSING SPERM CELL GENES OF RICE

Sharma N.1, Russell S.D.2, Bhalla P.L.1 and Singh M.B.1 1Plant Molecular Biology and Biotechnology Laboratory, Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria 3010, Australia. 2Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019 USA.

Male germ line augmentation in angiosperms is initiated via asymmetric division within pollen grains, following which the smaller cell becomes totally encased within a much larger vegetative cell. The generative cell subsequently divides to give rise to two non-motile diminutive sperm cells that take part in subsequent developmental advances. The sperm cells have been very difficult to investigate because of their presence within the confines of the vegetative cell. Recent availability of techniques for the isolation of rice sperm cells and the fully annotated rice genome sequence has allowed the transcriptional repertoire of sperm cells to be characterized. Microarray gene expression data has identified a set of genes that show unique or highly preferential expression in sperm cells. This information has made it feasible to identify cis-regulatory elements (CREs) that are conserved in sperm expressed genes and are putatively associated with control of cell specific expression. The aim of our study was to identify CREs associated with rice sperm cell specific gene expression data. Our approach involves analysing one Kb upstream regions of top 40 sperm cell co-expressed genes for over-represented conserved and novel motifs. Analysis of upstream sequences with SIGNALSCAN program on PLACE database, MEME and Mclip tool has identified combinatorial sets of known CREs with two novel motifs putatively associated with co-expression of sperm cell specific genes. These motifs represent likely targets of transcriptional factors regulating sperm cell gene expression and can then be used to design experimental verification of regulatory elements.

A BACTERIAL ELONGATION FACTOR G HOMOLOG EXCLUSIVELY FUNCTIONS IN RIBOSOME RECYCLING IN THE SPIROCHAETE BORRELIA BURGDORFERI

Suematsu T.1, Yokobori S.2, Morita H.3, Yoshinari S.1, Ueda T.3, Kita K.1, Takeuchi N.3 and Watanabe Y.1 1Grad. Sch. of Medicine, University of Tokyo. 2Dept. Mol. Biol., Tokyo University of Pharmacy and Life Sciences. 3Grad. Sch. of Frontier Sciences, University of Tokyo.

Translation elongation factor G (EF-G) in bacteria plays two distinct roles in different phases of the translation system. EF-G catalyzes the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post-termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF-G are distributed over two different EF-G paralogs in human mitochondria. In the present study, we show that the same division of roles of EF-G is also found in bacteria. Two EF-G paralogs are found in the spirochaete Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-G1 is a translocase, while EF-G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF-G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF-3) is present in the reaction. These results indicate that two B. burgdorferi EF-G paralogs are close relatives to mitochondrial EF-G paralogs rather than the conventional bacterial EF-G, in both their phylogenetic and biochemical features.

MOLECULAR CHARACTERIZATION OF THE DUPLICATED TAUROCYAMINE KINASE FROM SCHISTOSOMA MANSONI

Laurent S.1, Awama A.1, Salvador A.2, Gouet P.3 and Marcillat O.1 1Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires -UMR CNRS 8246. 2Université Lyon 1, Laboratoire des Sciences Analytiques (LSA), UMR 5180 CNRS. 3Université Lyon 1, Institut de Biologie et Chimie des Protéines -UMR CNRS 5086.

Guanidino kinases (GK) catalyze the reversible transfer of a phosphoryl group between ATP and a guanidino compound. These enzymes, named according to their specificity towards their guanidino substrate –e.g. creatine kinase, arginine kinase, taurocyamine kinase (TK)-, play a prominent role in energy management in a wide range of eukaryotic cells. Schistosoma mansoni (Sm) is a human parasite responsible for schistosomiasis, a tropical disease that affects about 200 million people worldwide. Sm cercariae express a duplicated TK in which the two structural domains (1-363 and 364-716) show high structure similarity (53% sequence identity). SmTK has an original U-shaped structure, in which the C-terminal side of the first domain interacts with the N-terminal side of the second domain. This arrangement differs from the classical banana-shape observed in dimeric and octameric GKs, in which pairs of monomers show a twofold rotation axis. In spite of this difference, each domain shows the characteristic GK fold with a first α-helical sub-domain (about 100 amino acids) and a second larger one (260 amino acids) with an anti-parallel β-sheet and long α-helices. The enzyme shows its highest activity using taurocyamine (120U/mg) although it is also able to use other guanidino substrates like guanidino propionate. Schistosoma mansoni cercariae contain both taurocyamine and guanidino propionate indicating that both phosphotaurocyamine and phosphoguanidino propionate can form in vivo. The functional implications of the structure will be presented.

IDENTIFICATION OF BENZOPYRAZINE DERIVATIVES AS A NOVEL CLASS OF GRB7 ANTAGONISTS

Ambaye N.D., Gunzburg M., Wilce M.C.J. and Wilce J.A. Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.

Growth factor receptor-bound protein-7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of tumors such as breast, blood, pancreatic, esophageal and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 10 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. It is expected that the identified antagonists will be useful additions to further explore the functional and signaling studies of Grb7 protein.





BIOINFORMATIC ANALYSIS OF SHEEP CORNEODESMOSIN (CDSN) AND ALPACA AGOUTI SIGNALLING PROTEIN (ASIP)

Bottomley S., Subramaniam N.S., Feeley N.L., Morgan E.F., Munyard K.A. and Groth D.M. School of Biomedical Sciences. Faculty of Health Sciences. Curtin University. Western Australia.

There has been an exponential increase in protein sequence information but a corresponding lack of experimental or structural information. This has contributed to an information gap. Single Nucleotide polymorphisms (SNPs), in particular, are the most common sequence variation with non-synonymous SNPs (nsSNPs) most likely affecting protein structure and function. It would be very time consuming, difficult, and costly to experimentally verify the impact of all nsSNPs on protein structure or function. Consequently, bioinformatic methods for predicting potential effects of nsSNPs, or predicting the structure or function of a newly discovered protein sequence, are potentially useful ways to fill the information gap. In this case study we used freely available, and recently developed, bioinformatic methods to predict the potential effect of nsSNPs and aspects of protein structure and function in sheep CDSN and alpaca ASIP. Bioinformatic analyses included protein secondary structure, N-terminal signal peptides, transmembrane helices, mutation analysis, and tertiary structure. The sheep CDSN and alpaca ASIP proteins offered a convenient way to compare and contrast the various bioinformatics methods because they differed in their structure, role, cellular location, and knowledge base. The results suggest that the application of bioinformatic methods can provide useful value-added annotation of newly discovered protein sequences but can also provide some contradictory or ambiguous results. Final interpretation of the results of bioinformatic methods will necessarily involve a subjective assessment based on knowledge of the underlying bioinformatics algorithms, the biology of the protein, and the existing literature.

RHEOLOGICAL BEHAVIOR OF DAIRY DESSERTS PREPARED WITH CAESALPINIA PULCHERRIMA SEED GALACTOMANNAN

Arcanjo S.R.S.1, Holanda M.L.2, Beserra J.F.3, Oliveira A.M.C.3, Paula R.C.M.2, MonteiroMoreira A.C.O.3 and Moreira R.A.3 1Universidade Federal do Piaui. 2Universidade Federal do Ceara. 3Universidade de Fortaleza.

Seed galactomannans, having a polymeric main chain of (1 → 4)-linked beta-D-mannopyranosyl residues, substituted at O-6 by an alfa-D-galactopyranosyl units, are widely used in the food industry as thickening and stabilizing agents. They have lower cost as compared with traditional thickening agents and their synergistic interactions raised their industrial importance. This study evaluated the rheological behavior of dairy desserts prepared with Caesalpinea pulcherrima seed galactomannan blended with starch and milk. From a basic mixture (A) of milk (16.37%), sugar (13.35%), starch (3.35%) and galactomannan (1.71%), different formulations, (B) galactomannan (0.49%), (C) starch (0.65%), (D) sugar (6.63%) and (E) milk (9.63%) were used. The curves obtained at the shear rate: 0.08 to 300 s-1, at 25 °C were used to calculate the flow behavior index (n) and consistency (k). The formulations were adjusted to the Ostwald model-de-Waele (R2 = 0.97-0.99) exhibiting pseudoplastic behavior with n varying from 0.12 to 0.17. The formulation A had k=102.3 Pa sn while the formulations C, D and E showed k values equal to 83.6, 151.5 and 223.4 Pa sn, respectively. Lower concentrations of starch, milk and sugar, caused positive synergism in C and a negative interference of the polymers of milk (protein) and sugar in E and D, respectively, in starch and galactomannan interaction when compared to A. Given these results we may suggest that the Caesalpinea pulcherrima galactomannan shows a pseudoplastic behavior, and variability of their consistency indices in the presence of biopolymers such as starch and protein. Supported by: FINEP, CNPq, RENORBIO, FUNCAP.

CROSSLINKED SEED POLYSACCHARIDES FOR ONE-STEP PURIFICATION OF GALACTOSE-BINDING LECTINS

Teixeira D.M.A.1, Braga R.C.1, Gadelha P.1, Reicher F.2, Moreira R.A.3 and MonteiroMoreira A.C.O.3 1Universidade Federal do Ceara. 2Universidade Federal do Parana. 3Universidade de Fortaleza.

Lectins, a special class of proteins with the particular property of interact, specifically and reversibly, with carbohydrates and glycoconjugates, without any modification in their covalent structure, can bind to cell surfaces producing a series of modifications on their physiology. This interaction is dependent on the OH orientation/substitution in the C2, C3 and C4 of the sugar ring and, thus, can be grouped in Glu-, Man-, Gal-, GlNAc-, GalNAc-binding lectins, and some of them show an even higher specificity, binding to only one of the anomers. Affinity chromatography on sugar containing matrices is the best way to isolate lectins whose specificity dictates the choice of ligand. While commercially available matrices present a mixture of the two anomers, natural glycoconjugates synthesized via enzyme driven reactions, presents only one anomer. This is the case of the endospermic galactomannans [alfa-(1-6)-D-galactose-substituted-mannan] and seed galactoxyloglucans [beta-(1-6)-D-galactose-substituted-xyloglucan]. 12 seed galactomannans and 5 seed xyloglucan were cross-linked by alkaline epichlorohydrin. After exhaustive washing, the cross-linked polysaccharides were packed in a column, to which the excess pure alfa- and beta-linking lectins were applied. After elution of the non-retained proteins, the column was washed with 0.1 M D-galactose. When Artocarpus integrifolia and Artocapus incisa alfa-D-galactose-binding seed lectins were applied to these columns, only the galactomannan columns were able to retain them. When, on the other hand, the Abrus precatorius beta-D-galactose-binding seed lectin was retained only in the xyloglucan columns. These results suggest that these natural polysaccharides can, thus, be used as affinity matrices, not only for the isolation of lectins but, more important, also to determine their fine anomeric specificity. Supported by: FINEP, CNPq, RENORBIO, FUNCAP.

MOLECULAR CLONING AND CHARACTERIZATION OF STRICTOSIDINE SYNTHASE OF MITRAGYNA SPECIOSA

Zainal Z.1, 2, Jumali S.S.1, Baharum S.N.1, Mohd Said I.1 and Ismail I.1 1Institute of System Biology, Universiti Kebangsaan Malaysia. 2School of Biosciences and Biotechnology, Universiti Kebangsaan Malaysia.

Mitragynine is one of the most dominant indole alkaloids present in Mitragyna speciosa leaves, found in most species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative tryptamine and secologanine by the action of Strictosidine synthase (STR). The cDNA clone encoding STR from M. speciosa was cloned through RT-PCR. The clone is a full length cDNA with a size of 1257 bp contained an open reading frame of 1056 bp starting from base pair 19 to 1077 and termed as STRMs1. Sequence analyses showed that STRMs1 (accession HM543187) had high homology with other STRs from some TIA-producing plants. The deduced acid amino sequence is 352 residues with a predicted molecular weight of 30 kDa and isoelectric point of 5.04. Southern blot performed showed that only one copy of STR is present in the genome of M. speciosa. Expression pattern on different tissues were tested using RT-PCR revealed that beside leaf, the expression was also detected in roots, stem and flower. Expression profiles under plant defense signal using salicylic acid (SA) was investigated on leaf tissues and the results showed that the transcript of STRMs1 were detected before and after treatment with salicylic acid. Result obtained from phylogenetic analysis suggested that STRMs1 is the most evolved protein among other STRs. However, the 3D prediction of STRMs1 showed that there are alpha helices and beta propeller structures which remained conserved to other STRs.



POS-WED-357SYNERGISTIC EFFECTS OF ASCORBIC ACID (AA) AND THIAZOLIDINEDIONE (TZD) ON SECRETION OF HIGH MOLECULAR WEIGHT ADIPONECTINWebster, J., Rose, F., Richards, A. and Whitehead, J.Mater Medical Research Institute and University of Queensland

Adiponectin is an insulin-sensitizing/anti-inflammatory adipokine that circulates in a variety of stable multimers (low and high molecular weight (LMW and HMW)). A characteristic of obesity and type 2 diabetes (T2D) is hypoadiponectinaemia, which reflects a specific loss of the more metabolically active HMW form. The efficacy of the TZD class of insulin-sensitizing drugs correlates with increases in HMW adiponectin. Formation of HMW adiponectin is dependent on post-translational modifications including hydroxylation of conserved lysine residues, by Lysyl Hydroxylase (LH). Low levels of AA, a co-substrate for LH, are observed in obesity and T2D raising the possibility that reduced AA may contribute to the specific loss of HMW adiponectin. Our aim was to determine if AA supplementation, alone/or in combination with TZD, would increase production of HMW adiponectin. Human SGBS adipocytes were cultured -/+ AA -/+ TZD for 24 h. AA supplementation increased secretion of HMW adiponectin (1.7 fold) without affecting adiponectin (mRNA) expression or total adiponectin secretion. TZD significantly increased adiponectin expression (3 fold) and total adiponectin secretion (1.4 fold), however it did not increase HMW secretion. In combination, AA + TZD increased adiponectin expression (3 fold) and secretion of total (2 fold) and HMW (5 fold) adiponectin. Similar beneficial effects were observed in cells treated with the pro-inflammatory cytokine TNFα. We conclude that AA supplementation can increase secretion of HMW adiponectin and that this effect is synergistic with TZD. Our findings raise the possibility that differences in AA levels may contribute to (i) the variability in adiponectin multimer profiles and (ii) the efficacy of TZD in humans and (iii) establish a platform for clinical trials.

ozbio2010 poster abstracts

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