session 10. cellular bioenergetics lecture oral ... session 10. cellular bioenergetics 47th congress...

47th Congress of the Polish Biochemical Society, 2012

Lecture

L10.1

Biogenesis of mitochondrial membrane protein complexesPeter Rehling

Department for Biochemistry II, University Medical Center and Max Planck Institute for Biophysical Chemistry, Göttingen, Germanye-mail: Peter Rehling <[email protected]>

Mitochondrial proteins are predominantly encoded in the nucleus and post-translationally imported into the orga-nelle. The translocase of the outer mitochondrial mem-brane (TOM complex) mediates protein transport across the outer membrane. Transport across the inner membrane requires one of two translocases in the inner membrane (TIM complexes). A subset of the mitochondrial proteome however is encoded on mitochondrial DNA. These pro-teins are co-translationally exported across the inner mem-brane by Oxa1 and assemble with newly imported proteins into membrane protein complexes of the respiratory chain. In order to maintain mitochondrial function, the assembly of respiratory chain complexes from imported and mito-chondria-encoded subunits has to be tightly regulated in order to adapted to cellular requirements. However, mal-function of these regulatory processes is linked to human disorders. To understand such regulatory processes, we have focused on the cytochrome c oxidase assembly pro-cess. Here, translational regulation is coupled to the assem-bly state of the enzyme complex. Our analyses provide new insight into the mechanism of complex assembly and how this process signals back to control expression of the core subunit Cox1.

Session 10. Cellular Bioenergetics

Oral presentations

O10.1

Mitochondrial biogenesis of plant mitochondria in stress — from proteomics to physiologyMichał Rurek1, Tomasz Pawłowski2, Jennifer Klodmann3, Włodzimierz Krzesiński4, Hans-Peter Braun3

1Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, Poland; 2Institute of Dendrology, Polish Academy of Sciences, Kórnik, Poland; 3Institute of Plant Genetics, University of Hannover, Germany; 4Department of Vegetable Crops, University of Life Sciences, Poznań, Polande-mail: Michał Rurek <[email protected]>

Mitochondria play important role in abiotic stress response in plant cell. About 22% of stress-responsive organellar proteins comprises mitochondrial proteins (Taylor et al, 2009). There is a growing demand for the characterisation mitochondrial pro-teome of agronomically important plant species (Heazlewood et al, 2003). The aim of our study was to study molecular and physiological responses of cauliflower mitochondria to cold and heat stress, and after post- stress plant recovery.We identified 22 stress- responsive protein spots from 2D (IEF/SDS-PAGE) gels by LC-MS/MS. They included ami-noacid and carbohydrate metabolism enzymes, heat-shock proteins, a chaperonin, translation factor, processing peptidase and ATP synthase subunits. Many proteins of various func-tional classes were involved especially in heat response and heat recovery. In cold stress, significant variations in AOX and photorespiratory enzymes abundance were noticed.Using BN-PAGE, BN/SDS-PAGE DIGE coupled with LC-MS/MS and protein crosslinking, in heat stress and heat re-covery we noticed (1) surprisingly high stability of SCI+III2; (2) some alterations in the abundance of CII and CIV subu-nits; (3) affected ATP synthase biogenesis; (4) desintegration of TCA cycle enzymatic complexes; (5) stable transient inter-actions within SCI+III2 components and during ATP syn-thase dimerisation.By in gel assays, we showed significant decrease of CII, CIVa and b isoform, and ATP synthase activity in stress and stress recovery; in the same conditions we noticed variations in the profile of mitochondrial translation. However, mitochondrial morphology in the apical layer of cauliflower curds was sig-nificantly affected only in cold and heat recovery. In addition, a significant decrease in leaf dark respiration and transpiration rate, accompanied by stomatal closure and decreased pho-torespiratory rate, was evident in stress conditions (especially in heat) and partially lasted after stress recovery.We conclude that the biogenesis of cauliflower mitochondria on various levels is significantly, but not equally affected by cold and heat stress conditions, which suggests the participa-tion of distinct response pathways for diverse stress condi-tions.ReferencesHeazlewood JL et al. (2003) Plant Physiol 132: 230-242.Taylor NL et al. (2009) J Proteomics 72: 367-378.

AcknowledgementsThis work was supported from the state resources for science in 2008-2010 by the Ministry of Science and Higher Education, Poland, grant No. N N303 338835.

Session 10. Cellular Bioenergetics 227

The First Polish-German Biochemical Societies Joint Meeting, 2012

O10.2

Mitochondrial potassium channels in Dictyostelium discoideumMichal Laskowski1, Anna Kicinska2, Wieslawa Jarmuszkiewicz2, Adam Szewczyk1

1Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw, Poland; 2Laboratory of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznań, Polande-mail: Michal Laskowski <[email protected]>

Mitochondria are crucial not only in energy metabolism but also in regulation of cell senescence and apoptosis. The strict control of inner mitochondrial membrane permeabil-ity and selective ion transport is essential for mitochondria functioning. Potassium ions homeostasis is an important process for mitochondrial optimal functioning. Potassium channels such as ATP-regulated, large conductance calcium activated and voltage dependent channels were observed in inner mitochondrial membrane in various mammalian tissues. Recently, we have identified potassium channels in inner mitochondrial membrane of potato Solanum tubero-sum and Acanthamoeba castellanii. Currently we characterize mitochondrial potassium channels from one of Dictyoste-lium species. It is commonly used as a model organism to study cell differentiation, metabolism and programmed cell death. Preliminary experiments are focused on biophysical and pharmacological characterization of mitochondrial ion channels. Purified inner mitochondrial membranes (submi-tochondrial particles) were reconstituted into planar lipid bilayer. To form model membranes asolectin from soybean mixture of phospholipids was used. We observed two types of potassium selective ion channels in submitochondrial particle samples: a large- and small-conductance channels. Experiments were performed both in gradient solution 50/150 mM KCl (cis-trans) and in symmetrical solution 150/150 mM KCl at voltages from -50 to 50 mV. Regu-lation of the channel activity by divalent cations such as Ca2+ and Mg2+ was explored. Additionally, interaction of the ATP with mitochondrial potassium channels was char-acterized. The knowledge on mitochondrial ion channels may contribute to understanding molecular mechanism of Dictyostelium discoideum functioning.AcknowledgementsThis work was supportedby Polish Mitochondrial Network. MitoNet.pl.

O10.3

Kinetic characteristic of NAD(P)-dependent malic enzyme — a key enzyme for herring (Clupea harengus) spermatozoa bioenergeticsNatalia Niedźwiecka, Edward F. Skorkowski

University of Gdansk, Department of Molecular Evolution, Gdańsk, Polande-mail: Natalia Niedźwiecka <[email protected]>

Introduction: Baltic herring (Clupea harengus membras) is an industrially essential fish species with interesting mecha-nism of reproduction. Precise bioenergetics processes tak-ing place in its sperm cells are still unclear.In recent decades Baltic herring has been subjected to over-fishing and habitat pollution (especially high in the coastal waters where spawning takes place) [1].An aerobic organism needs an adequate intracellular con-centration of NADH and NADPH to survive. NADH serves primarily to generate the ATP via oxidative phos-phorylation, while NADPH maintain the reductive envi-ronment necessary for cellular activities [2]..Our previous study showed that malic enzyme (see below) is a key enzyme that enables herring sperm cells to fulfill their requirement for NADH and NADPH. The activity of NAD(P)-dependent malic enzyme was the highest among NADP-dependent enzymes present in herring sperm cell (data not published).Materials and Methods: Herrings were caught in the Gdansk Bay during spawning season (April-May). Two iso-forms of malic enzyme were purified on DEAE-Sepha-rose, 2’,5’-ADP-Sepharose 4B (in case of NADP-specific form) or RED-Sepharose (in case of NAD-preferring form) chromatography and Sephadex G-200 gel filtration. Malic enzyme activity was followed spectrophotometrically by observing the appearance (during decarboxylation) or disappearance (during carboxylation)of NADH /NADPH at 340 nm and 25°C. The malic enzyme activity assay was performed in 50 mM Tris-HCl buffer, containing 20 mM imidazole-HCl, pH 7.0, 0.5 mM NAD or NADP, 1 mM L-malate, 1 mM MnCl2, and 1 μg of enzyme. Enzyme ac-tivities were calculated using EmM/340 = 6.22 for reduced coenzyme in a 1-cm light path cuvette.The localization of malic enzyme in herring spermatozoa was determined us-ing immunofluorescence with anti-herring ME rabbit anti-bodies by confocal microscopy.Results: In this study, two isoforms of ME from herring sperm were characterized after 3-step purification.NADP-specific ME from herring sperm has a molecular weight of about 61 kDa, NAD-preferring ME — 57 kDa.Reversibility of reaction catalyzed by NAD(P)-depend-ent ME was examined. The optimum pH for oxidative decarboxylation was 7.5 for NADP-specific ME and 7.0 for NAD-preferring ME. The optimum pH for reductive carboxylation was 7.5 for NADP-specific ME and 7.0 for NAD-preferring ME.Ratio of decarboxylation to carboxylation reaction in pH 7.0 was 8.4:1 for NAD-preferring ME and 3.7:1 NADP-specific ME.Kinetic characteristics were described for NAD-prefer-ring ME. The Km values determined at pH 7.0 for malate, NAD+, Mn2+ in the decarboxylation reaction were 0.16 mM, 0.21 mM, 0.08 mM respectively. The Km values de-termined at pH 7.0 for pyruvate, NADH, NAHCO3 in the carboxylation reaction were 4.12 mM, 0.06 mM, 16.84 mM respectively.For NADP-specific ME, the Km values determined at pH 7.0 for malate, NADP+, Mn2+ in the decarboxylation reac-

228 Session 10. Cellular Bioenergetics


tion were 0.03 mM, 0.020 mM, 0.25 mM, respectively. The Km values determined at pH 7.0 for pyruvate, NADPH in the carboxylation reaction were 1.53 mM, 0.025 mM re-spectively.Reference1. HELCOM (2010) Balt Sea Environ Proc 120B.2. Rui B et al. (2010) BMC Systems Biol 4:122.

O10.4

The effect of the vitamin E on the morphological picture of kidneys and the activity selected enzymes on the rat intoxicated with sodium fluorideNatalia K. Grzegorzak1, Barbara Stawiarska-Pięta1, Dominika Trocha1, Jolanta Zalejska-Fiolka2, Agnieszka Paszczela1, Ewa Grucka-Mamczar2, Ewa Birkner2

1Department of Pathology, Faculty of Pharmacy and Division of Laboratory Medicine in Sosnowiec, The Medical University of Silesia in Katowice, Sosnowiec, Poland; 2Department of General Biochemistry, Faculty of Medicine in Zabrze, The Medical University of Silesia in Katowice, Zabrze, Polande-mail: Natalia Katarzyna Grzegorzak <[email protected]>

The exposure to increased doses of sodium fluoride has a negative effect on carbohydrate, adipose and protein al-terations and causes pathomorphological changes in many organs. Fluoride also disturbs free radical processes, which may have the important meaning in pathomechanism of observed changes of people and animals.The aim of this study was to investigate the effects of vita-min E on the morphology of the kidneys and as well as to examine of the selected activity of enzymes in kidneys of rats exposed to sodium fluoride.The study was conducted in 18 male rats of Wistar strain. The rats were divided into 3 groups: a control group, which received distilled water and two experimental group, which received sodium fluoride (10 mg/kg of body mass/24 h) in water solution. Animals in the second experimental group received 3 mg of vitamin E/rat/24 h. The experiment last-ed 35 days. In supernatants obtained after homogenization of rat kidneys slices, the activity of the following enzymes was assayed: MDH, SDH, GPx, GST and GR. Pathomor-phological evaluation was conducted on preparations made by standard paraffin method, followed by staining with he-matoxylin and eosin.Our study demonstrated regressive changes of the kidney cells exposed to sodium fluoride. In kidneys occurred cell necrosis in the convoluted parts of the renal tubule. These changes were accompanied by microinfiltrations in the stroma, and also by the hyperemia. The results received in the experiment indicate an interference of fluorine ions in sorbitol change (an decrease in the activity of MDH). No statistically significant changes in GST, SDH, GPX, GST, GR activity were detected in rats treated with sodium fluo-ride.The administration of vitamin E effectively antagonized the changes in the activity of MDH. The advantageous changes in biochemical rates after administration of vita-min E are accompanied by less intensive pathomorphologi-cal changes formed in a kidneys after the intoxication of animals with NaF.



O10.5

Mitochondrial/nuclear responses to the loss of the mitochondrial protease FtsH4 during Arabidopsis seed germinationLukasz Pogorzelec, Iwona Migdal, Malgorzata Czarna, Hanna Janska

Laboratory of Molecular Cell Biology, Faculty of Biotechnology, University of Wroclaw, Wrocław, Polande-mail: Malgorzata Czarna <[email protected]>

Mitochondrial AtFtsH4 protease is one of the inner mem-brane-bound ATP-dependent metalloproteases in Arabi-dopsis, with its catalytic site exposed to the intermembrane space. We found that a loss of the AtFtsH4 protease slight-ly delays Arabidopsis seed germination under the long-day conditions and optimal growth temperature of 22°C. How-ever, when Arabidopsis seeds were exposed to the elevated temperature of 30°C, the germination delay of the ftsh4 was significantly increased. Quantitative RT-PCR analysis revealed an increase in the transcript level of alternative oxidase Aox1a in the ftsh4 beginning from the early hours of germination, while in the wild type its level remained constant during studied period of time. Additionally, us-ing specific antibodies against AOX1/2 we could observe a gradual increase in the alternative oxidase expression level in germinating ftsh4 seeds, reaching the highest level af-ter 24 hours of germination. The expression of AOX in the wild type was only detected after 24 hours and at the lower level than in the ftsh4. Measurements of a total cel-lular respiration with a Clark-type electrode revealed that significant oxygen uptake by the wild type and ftsh4 begins after 6 hours of germination. However, the ftsh4 showed slightly lower rate of total oxygen consumption during studied period of time than the wild type. The estimated contribution of the cytochrome pathway in the ftsh4 total respiration was also lower (16–18%). By contrast, in the ftsh4 alternative oxidase exhibited higher activity and con-tribution in a total respiration, with remarkably increased AOX activity after 12 hours of germination. Interestingly, at this time of germination the wild type AtFtsH4 protease showed the highest level of expression. That higher alter-native oxidase activity correlates with increased levels of Aox1a transcript and AOX protein in the ftsh4 germinating seeds. When the cytochrome pathway was inhibited with antimycin A, we observed a strong reduction of COX ac-tivity in the wild type after 18 hours of germination. No-ticeable, antimycin A had only slight effect on the activity of cytochrome pathway in germinating ftsh4 seeds. Here we propose that the loss of Arabidopsis FtsH4 protease affects mitochondrial biogenesis during early embryo ger-mination, while AOX may be more than just an energy dis-sipating oxidase but it can also act as an initiator of a cellu-lar reprogramming allowing for the ftsh4 seeds to become more tolerant and germinate.

O10.6

Mitochondrial uncoupling protein of Acanthamoeba castellanii is activated by lipid peroxidation end product, hydroxynonenalAndrzej M. Woyda-Ploszczyca, Wieslawa Jarmuszkiewicz

Adam Mickiewicz University, Department of Bioenergetics, Poznań, Polande-mail: Andrzej Woyda-Ploszczyca <[email protected]>

Uncoupling proteins (UCPs) function in the mitochondrial inner membrane and represent mitochondrial anion carrier protein (MACP) family. UCPs mediate an inducible, purine nucleotide-inhibited proton leak that dissipates the proton electrochemical gradient generated during the oxidation of respiratory substrates.Non-enzymatic and self-propagating process of membrane lipid peroxidation is induced by reactive oxygen species (ROS). 4-Hydroxy-2-nonenal (HNE) is identified as the most abundant end product of lipid peroxidation as well as specific activator of vertebrate and plant UCPs to decrease mitochondrial ROS production through mild uncoupling.We studied the effect of exogenously generated superoxide and exogenous HNE on the activity of UCP of unicellular organism such as amoeba A. castellanii. Superoxide-generat-ing xanthine/xanthine oxidase system was ineffective but exogenously added HNE did induce specific and GTP-sensitive mitochondrial uncoupling mediated by A. castel-lanii uncoupling protein (AcUCP). In non-phosphorylating mitochondria, AcUCP activation by HNE was demon-strated by increased oxygen consumption accompanied by a decreased membrane potential and ubiquinone reduction level. In phosphorylating mitochondria, the HNE-induced AcUCP-mediated uncoupling decreased the yield of oxi-dative phosphorylation. The efficiency of GTP to inhibit HNE-induced AcUCP-mediated uncoupling was regulated by the endogenous ubiquinone redox state.Our results provide an evidence for the universality of HNE-induced UCP-mediated mitochondrial uncoupling among eukaryotes. This study also supports the idea that the first and ancestral function of UCPs could be the atten-uation of mitochondrial ROS production through a nega-tive feedback loop involving membrane lipid peroxidation products as a late response to the elevated ROS level. The rapid response through the membranous ubiquinone redox state could directly regulate the sensitivity of the HNE-in-duced (and free fatty acid-induced) UCP activity to purine nucleotides.AcknowledgementsThis work was supported by the grant from the Polish Ministry of Educa-tion and Science (N N 301 636440).



O10.7

Polyunsaturated fatty acids and mitochondrial potassium channelsAnna Kajma, Adam Szewczyk

Nencki Institute of Experimental Biology, Laboratory of Intracellular Ion Channels, Warsaw, Polande-mail: Anna Kajma <[email protected]>

Polyunsaturated fatty acids (PUFA) and their metabolites, have been implicated in the modulation of a variety of bio-chemical processes in the cell, and also in prevention of various diseases. They were able to interact with the mem-brane proteins including ion channels in normal and path-ological conditions. The final goal of the project was to develop a description of the mechanism, that is responsible for activity modulation of neuroprotective mitochondrial potassium channels by selected PUFAs. The aim of the ex-periments was to determine whether the neuroprotective mechanism of the large-conductance calcium activated po-tassium (mitoBKCa) channel from the inner mitochondrial membrane can be explained by mutual interaction between the channels and polyunsaturated fatty acids.The study was performed using patch-clamp technique and mitochondria isolated from rat astrocytes. We analyzed ef-fect of arachidonic acid (AA), eicosatetraynoic acid (ETYA) a non-metabolizable analog of AA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The open prob-ability of the channel did not change significantly after ap-plication of 10 μM ETYA, but after adding 10 μM AA, which suggest that channel is affected by metabolites of AA rather than fatty acids itself. Application of 30 μM DHA, the same as 30 μM EPA increased open probability of the channel. Also number of the open channels in the patch increased in the presence of 30 μM EPA.Summarising, our results indicate that neuroprotective PU-FAs, like DHA and EPA activate mitoBKCa channel, while proapoptotic and proinflamatory AA had no effect on mi-toBKCa channel.AcknowledgementsGrant support DEC-2011/01/N/NZ1/04311.

Posters

P10.1

The unbalance between small and large subunits of mitoribosomes changes the pattern of proteins synthesized in Arabidopsis mitochondriaMalgorzata Kwasniak, Aleksandra Adamowicz, Hanna Janska

Laboratory of Cell Molecular Biology, Department of Biotechnology, University of Wroclaw, Wrocław, Polande-mail: Aleksandra Adamowicz <[email protected]>

Biogenesis of ribosomes that are competent to protein synthesis is an essential cellular process. We have found that silencing of the nuclear Rps10 gene encoding the mito-chondrial S10 protein, a component of the small subunit of mitoribosome in Arabidopsis, altered the stoichiometry between the small (SSU) and large (LSU) subunits of mi-toribosmes due to small subunits deficit. However, despite this unbalance, the level of mitochondrial rRNAs and ribo-somal proteins showed that abundance of both mitoribo-somal subunits was significantly higher in rps10 than in wild type plants. It seems conceivable that the silencing of Rps10 results in the lower level of the S10 protein and the small subunits lacking S10 are unstable and prone to degradation, which explains their deficit in rps10. On the other hand, cells somehow sense the ribosome defect and attempt to compensate for it by up-regulation of the mitoribosomal subunits biogenesis.To investigate whether the unbalance between the small and large subunits of mitoribosomes in rps10 could affect the mitochondrial translation, we tested the capacity for in organello protein synthesis in isolated mitochondria. Surpris-ingly, the pattern of de novo synthesized proteins differs between rps10 and wild-type plants. The autoradiogram of the labeled translation products in wild type plants resem-bles the pattern known from literature, where the major bands corresponds to the OXPHOS proteins, whereas in rps10 mutant majority of these OXPHOS proteins are hardly visible. The most intensive bands in rps10 pattern, according to the electrophoretic mobility, represent ribo-somal proteins encoded by mitochondrial genome. Taken together, the aforementioned results suggest that the syn-thesis of the majority of mitoribosomal proteins was in-creased while OXPHOS proteins were translated at a lower level by ribosomal-defective rps10 mitochondria. In a con-sequence, we speculate that the almost entire rate of pro-tein synthesis in rps10 mutants is shifting to the production of mitoribosomal proteins. This finding implies that the changes in the ratio between small and large subunits of mitoribosomes could have an ample effect on mitochon-drial protein synthesis in plants.



P10.2

Uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratusN. Antos-Krzeminska1, M. Slocinska2, G. Rosiński2, W. Jarmuszkiewicz1

1Department of Bioenergetics, 2Department of Animal Physiology and Development, Adam Mickiewicz University, Poznań, Polande-mail: Nina Antos-Krzeminska <[email protected]>

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling and thereby energy dissipation. The sequence alignment pre-dicts the existence of UCP4 in several insects. The aim of our study was to molecularly identify and elucidate the developmental expression pattern of UCP at the protein and mRNA levels in the larval and pupal fat bodies of the holometabolous insect, Zophobas atratus. We also function-ally characterised Z. atratus UCP (ZaUCP) in isolated fat body mitochondria under phosphorylating and non-phos-phorylating conditions. Because the fat body undergoes developmental remodelling during insect metamorphosis, it is an interesting experimental model to study the role of UCP. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. A lower expres-sion of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atra-tus pupal fat body compared with the larval fat body. The different expression pattern and activity of ZaUCP4 dur-ing the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.AcknowledgementsThis work was partially supported by the grant from the Polish Ministry of Education and Science (N N 301 636440).

P10.3

The metabolism of carbohydrates and lipid peroxidation in occupational lead-exposureAleksandra Kasperczyk, Michał Dobrakowski, Alina Ostałowska, Jolanta Zalejska-Fiolka, Ewa Birkner

Medical University of Silesia, Department of Biochemistry, Zabrze, Polande-mail: Michał Dobrakowski <[email protected]>

Due to the fact that there have only been few reports on the influence of lead on carbohydrate metabolism in lead-exposed workers, the present study was undertaken to es-timate the effect of occupational exposure to lead on the blood concentrations of glucose and several enzymes in-volved in glycolysis, the citric acid cycle, and the pentose phosphate pathway. To estimate the degree of lipid per-oxidation, the concentrations of conjugated dienes were determined.The examined group consisted of 145 healthy male em-ployees of lead-zinc works. By the degree of lead exposure, the exposed group was subdivided into two subgroups. The control group was composed of 36 healthy male ad-ministrative workers.The markers of lead exposure (blood levels of lead and zinc-protoporphyrin) were significantly elevated in the ex-posed group compared with the control group. There were no significant changes in glucose concentration in the study population. The concentration of conjugated dienes was significantly higher in both subgroups, whereas the activ-ity of malate dehydrogenase was significantly higher only in the group with higher exposure. However, the activity of lactate dehydrogenase was significantly decreased in the examined subgroups. The activity of glucose-6-phosphate dehydrogenase decreased significantly only in the group with higher exposure. Levels of lead and zinc protoporphy-rin in the blood were negatively correlated with glucose-6-phosphate dehydrogenase activity.In conclusion, lead interferes with carbohydrate metabo-lism. However, compensatory mechanisms seem to be ef-ficient, as glucose homeostasis in lead-exposed workers was not disturbed. Lead-induced decreases in the activity of glucose-6-phosphate dehydrogenase may have decreased the availability of reduced glutathione, which may have in-creased lipid peroxidation.



P10.4

Expression of hypoxia-inducible factor-1α (HIF-1α) in thyroid lesionsPaweł Jóźwiak1, Anna Krześlak1, Magdalena Bryś1, Lech Pomorski2, Anna Lipińska1

1University of Łódź, Department of Cytobiochemistry, Łódź, Poland; 2Medical University of Łódź, Department of General and Oncological Surgery, Zgierz, Polande-mail: Paweł Jóźwiak <[email protected]>

The hypoxia-inducible factor-1 is a transcription factor that is activated under low oxygen tensions (hypoxia). HIF plays a pivotal role in adaptation of tumors cells to survival by activating and repressing multiple genes that promote tumor proliferation and invasion. HIF-1 is a heterodimer composed of oxygen-labile HIF-α and constitutively ex-pressed HIF-β. Both subunits are members of the basic helix-loop-helix (bHLH) proteins of the PER-ARNT-SIM (PAS) family of transcription regulators. Under intratumor-al hypoxia condition HIF-1α heterodimerises with HIF-1β through the PAS domains, and translocates to the nucleus. The complex then binds to the hypoxia responsive element (HRE) in the promoter region of genes and controls their transcription. Hypoxia occurs in majority of solid tumors, thus HIF-1 plays an important role in cancer signaling pathways. Up to now, a little is known about the role of HIF-1 in thyroid cancer. Since, the HIF-1 activity depends on availability of the HIF-1α subunit, we analyzed its ex-pression at the mRNA and protein levels. Surgical speci-mens were obtained from 73 patients who underwent sur-gery for nodular thyroid disease. The histological diagnosis report was obtained for each patient. Quantitative real-time PCR method with commercially available primers and fluo-rescent probes (TaqMan Gene Expression Assay, Applied BiosystemsTM, CA., U.S.A.) was employed to detect HIF-1α mRNA expression in pathological thyroid samples. The endogenous control was a housekeeping gene, i.e., glycer-aldehyde-3-phosphate dehydrogenase (GAPDH). HIF-1α protein expression level was determined by Western blot method and analyzed densitometrically using the GelPro Analyzer software. Statistically significant differences were found in HIF-1α protein levels between well differentiated thyroid carcinomas: papillary carcinoma (PC) and follicular carcinoma (FC) and non-neoplastic lesions. However, the relative HIF-1α mRNA level was lower in FCs in compari-son with the other types of lesions. A tendency towards an increased expression of HIF-1α was observed in the PC group with an elevated tumor size or tumor stage. No ob-vious differences were noted regarding other histological features, such as the lymph node status. The results indicate that HIF-1α expression level in thyroid carcinoma corre-lates with poor prognosis and may play a significant role in adaptation of tumor cells to hypoxia.

P10.5

An inception report on huntingtin interaction with VDACA. Karachitos1, O. Stobienia1, J. Jordan2, K. Sobczak3, V. De Pinto4, H. Kmita1

1 Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland; 2 Neuropharmacology Group, Department of Medical Sciences, Albacete College of Medicine, University of Castilla-La Mancha, Albacete, Spain; 3Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland; 4 Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, Italy; National Institute for Biomembranes and Biosystems, Section of Catania, Catania, Italye-mail: Andonis Karachitos <[email protected]>

Huntington’s disease (HD) is an autosomal-dominant neu-rodegenerative disorder caused by an abnormal increase in amount of CAG codon in exon 1 of the gene encoding the protein huntingtin (Htt). Studies concerning HD patients as well as studies using HD models including transgenic animals, cell cultures and the yeast Saccharomyces cer-evisiae indicate that profound mitochondrial impairment is an early and important event in HD pathogenesis. It is also evident from some of these studies that the severity of mitochondrial abnormalities correlates with increasing number of glutamine repeats in mutated form of hunting-tin (mHtt), suggesting mitochondria as a major target of mHtt. However, till now the mechanisms of mHtt effect on mitochondria have not been clearly defined. Interesting-ly, our preliminary pulldown assay with purified and GST tagged Htt and mHtt constructs obtained from M.J. Mon-teiro and S. cerevisiae mitochondria suggests that VDAC (voltage-dependent anion-selective channel) interacts both with Htt and mHtt. Accordingly, the proposed “mitochon-drial targets” of mHtt include processes that are known to be affected by VDAC; i.e. the respiratory chain, tran-scriptional regulation and protein import, calcium balance, oxidative stress and apoptosis, or might be affected due to interaction between VDAC and the involved proteins; i.e. mitochondrial trafficking and fusion/fission. Moreover, our studies point at VDAC involvement in cytoprotection including neuroprotection. Thus, studies concerning direct interaction between VDAC and Htt/mHtt appear a logical step in studies of HD etiology and their results could be important for development of new therapeutic strategies concerning HD.Since experimental approaches have shown that N ter-minus fragments of mHtt recapitulate several aspects of the fulllength mutant protein's toxicity we applied only the first exon of Htt and mHtt encoding gene. First we meas-ured the effect of Htt on reconstituted VDAC. We applied VDAC isoform mix isolated from human neuroblastoma cells and the most abundant isoform of human VDAC iso-forms, i.e. VDAC1 expressed and isolated from S. cerevisi-ae cells. We observed a strong effect of Htt on the channel conductance, particularly in the case of purified VDAC1. Accordingly, our pulldown experiments indicate direct in-teraction between Htt and human VDAC1. Moreover, Htt did not handicap growth of S. cerevisiae cells expressing human isoforms of VDAC.



P10.6

Is VDAC a direct target of minocycline?Andonis Karachitos1, Anna Kasprowicz2, Joaquin Jordan3, Hanna Kmita1

1Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poland; 2Laboratory of Molecular and Cellular Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poland; 3Grupo de Neurofarmacología, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spaine-mail: Hanna Kmita <[email protected]>

Minocycline (MC) belongs to tetracycline family of antibi-otics and displays broad-spectrum of antibacterial activity as it is able to block bacterial protein synthesis. Moreover, cytoprotective properties of MC have been shown to be beneficial in treatment of different diseases, including neu-rodegenerative and cerebrovascular diseases although the underlying mechanism is still a matter of debate. However, there are also data pointing at detrimental effects of MC. Correspondingly, MC is regarded as an anticancer agent and proapoptotic factor. Thus, detailed description of cel-lular and molecular mechanism(s) triggered by MC appears to be reasonable topic of study.It is widely accepted that mitochondrial dysfunction con-tributes to cell death, that makes mitochondria an impor-tant target of cytoprotective drugs. This, in turn, makes the effect of MC on mitochondria an interesting field of stud-ies. Unfortunately, the available data indicated discrepant or even detrimental mechanism(s) of MC effect on isolated mitochondria. Despite the differences, proposed explana-tions point at changes at the level of the inner membrane permeability resulting in uncoupling or/and inhibition of the respiratory chain as a direct or indirect consequence of MC interaction with mitochondria. To establish the mean-ing of the observed phenomena for cell functioning we decided to compare MC effect on respiratory coupling in mitochondria after isolation and in intact cells. Using the yeast Saccharomyces cerevisiae as a model system we have shown that cytoprotective activity of MC against H2O2 ap-pears to result from an improvement of energy coupling within cells and the effect of MC depends on the presence of functional VDAC (voltage-dependent anion-selective channel) in the mitochondrial outer membrane and MC concentration in the vicinity of mitochondria. Accordingly, we have shown that MC modulates channel properties of VDAC reconstituted in artificial membranes.To further study the possibility of direct interaction be-tween VDAC and MC we measured the channel proper-ties in the presence of different MC concentrations and cations, known to modulate MC effect on mitochondria. Interestingly, we observed qualitative differences in effects of MC, dependently on its concentration and the presence of bivalent cations. Moreover, initial bioinformatic analysis as well as MC fluorescence binding studies suggest direct interaction between VDAC and MC.

P10.7

Oxidative function in human endothelial EA.hy926 cellsAgnieszka Koziel, Wieslawa Jarmuszkiewicz

Adam Mickiewicz University, Department of Bioenergetics, Poznań, Polande-mail: Agnieszka Koziel <[email protected]>

The endothelium is not considered to be a major energy-requiring organ, but nevertheless endothelial cells have an intensive mitochondrial network. Healthy endothelium is essential for homeostasis of cardiovascular system, while endothelial dyfunction leads to cardiovascular diseases including atherosclerosis, diabetes and heart failure. En-dothelial dysfunction is tightly linked to the overproduction of reactive oxygen species, development of oxidant stress and inflammatory response of endothelium. Mitochondria of the vascular endothelium seem to be an important play-er in these processes.It is considered that, in contrast to numerous cell types, synthesis of ATP in endothelium occurs mainly via a gly-colytic pathway and endothelium seems to be relatively in-dependent of the mitochondrial energy supply. In the pre-sent work, we study mitochondrial function in the human umbilical vein endothelial cultured cells (line EA.hy926) by measuring cell oxygen consumption in media with various energy fuels, i.e., with different levels of glucose, pyruvate, glutamine or fatty acids. The highest oxygen consumption rate was observed with pyruvate alone or glutamine and the lowest with 25 mM glucose alone. Glucose decreased the oxygen consumption rate with pyruvate and glutamine in a concentration-dependent manner.AcknowledgementsThis work is supported by the European Union from the resources of the European Regional Developmental Fund under the Innovative Economy Programme (POIG.01.01.02-00-069/09).



P10.8

Pentosofosphate way condition in the liver of rats at chronic alcohol and morfine intoxicationS. V. Lelevich, V. V. Lelevich, A. G. Vinitskaya

Grodno State Medical University, Grodno, Belaruse-mail: sergei lelevich <[email protected]>

The functional condition of a pentosofosphate way (PFP) in a liver of rats is investigated at long introduction of alco-hol and morfine. In a hepatic fabric were defined activity of key enzymes of this metabolic way: G-6PHDG, 6-PHG-DG and transketolase. Alcohol introduction within 29 days led to decrease in speed of transketolase reaction (for 33 %, p<0.05). At introduction morfine with 14 days of activity of enzymes of an oxidizing branch of PFP (G-6PHDG and 6-PHGDG) didn't differ from control, and speed of transketolase reaction decreased. Three-week introduction of drug led to further inhibition of activity ransketolase at invariable speed dehydrogenases PFP.

P10.9

PCr depletion and hydrogen ion accumulation in human calf muscles during dynamic exerciseJoanna Majerczak1, Justyna Zapart-Bukowska1, Piotr Kulinowski2, Sylwia Heinze-Paluchowska2,3, Marcin Grandys1, Bernard Korzeniewski 4, Jerzy A. Zoladz1

1Department of Muscle Physiology, Faculty of Rehabilitation, University School of Physical Education, Kraków, Poland; 2Institute of Nuclear Physics PAN, Kraków, Poland; 3Cancer Institute, Kraków, Poland; 4Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Polande-mail: Joanna Majerczak <[email protected]>

Introduction: Little is known concerning the relationship between the degree of muscle PCr decrease and an increase in muscle hydrogen ion concentration ([H+]m) during ex-ercise. Therefore, in the present study, we aimed to estab-lished the relationship between a decrease in muscle PCr and accumulation of [H+]m in the working calf muscles during moderate exercise intensity (up to 50% decrease in muscle PCr) and during heavy exercise intensity (above 50% of a decrease in PCr-until muscle fatigue). Subjects and Methods: Fourteen young, healthy, untrained men participated in this study (mean±SE: body mass 79.5±4.2 kg; height 181±2 cm; BMI 24.2±1.1 kg·m-2). All subjects performed two 6-minutes plantar flexion exercises using prototype ergometer placed in the NMR system (4.7 T MR scanner with MARAN DRX console). 31P NMR spec-tra were recorded from the calf muscles thought 6-min-utes rest period, 6-minutes of moderate intensity exercise, 6-minutes recovery, 6-minutes of heavy intensity exercise. This system allowed us to measure ATP, Pi, PCr indepen-dently. Results: At rest [H+]m, PCr, PCr/ATP amounted to 85±2 nmol·L-1, 33.6±0.9 mmol·L-1, 4.09±0.11, respective-ly. During exercise of moderate intensity (up to a decrease in PCr not exceeding 50%) a decrease in PCr concentration was accompanied by a slight and linear increase in [H+]m. During exercise of high intensity (above 50% decrease in PCr) [H+]m in relation to a decrease in muscle PCr followed also linear pattern, but a several times higher rate of in-crease in [H+]m was observed. Conclusions: The present study shows that even during very light exercise intensity there is a slight linear increase in [H+]m in relation to PCr decrease, however, at the exercise intensities exceeding 50% decrease in muscle PCr [H+]m increases at faster rate, therefore the relationship between [H+]m accumulation and PCr depletion is curvilinear.AcknowledgementsThis work was supported by the Ministry of Science and Higher Educa-tion, grant No NN404196637.



P10.10

The plant Atp23 homologue-a novel component of the mitochondrial proteolytic systemRenata Skibior, Iwona Migdal, Hanna Janska

Wroclaw University, Laboratory of Molecular Cell Biology, Wrocław, Polande-mail: Renata Skibior <[email protected]>

The yeast mitochondrial Atp23 metalloprotease is associ-ated with the inner membrane and has dual activities. It mediates the maturation and assembly of mitochondrially encoded F1F0-ATP synthase subunit 6 (Atp6). Moreover, together with the i-AAA type protease Yme1 plays a role in turn-over of Ups1, which is an intermembrane space pro-tein regulating mitochondrial phospholipid metabolism.In A. thaliana, homologue of Atp6 is also synthesized with cleavable presequence. Up to now the proteases involved in processing of the subunit 6 precursor has not been identi-fied.Atp23 is conserved among of all higher eukaryotes. In A. thaliana genome, there is only one gene coding for Atp23 (At3g03420), which product shows approximately 38% identity with yeast Atp23.To study if the function of the plant Atp23 homologue can be similar to the yeast counterparts, functional comple-mentation assay in yeast was performed. Yeast cells lacking Atp23 protease will become respiratory-deficient and fail to grow on nonfermentable carbon sources, such as glycerol. However, the plant Atp23 homologue could not restore the respiratory growth of Δatp23 mutant suggesting a plants-specific function.To determinate the physiological role of the plant Atp23 homologue a reverse genetic approach has been under-taken. Two independent Arabidopsis lines with a T-DNA insertion in the At3g03420 gene were selected. One of the mutant lines with the T-DNA insertion in 5’-UTR, shows no phenotype under long day conditions (LD). Interest-ingly, in the mutant line with the T-DNA in the first exon, developmental and morphological abnormalities in LD can be observed. Homozygous plants have loss of apical dominance and remarkably reduced seeds production. Our further experiments are underway to understand molecular bases of that phenotype.

P10.11

Comparative activity of gamma-aminobutyric acid catabolism in liver during different types of alcohol intoxicationHanna Vinitskaya, Vladimir Lelevich, Sergej Lelevich, Elena Golovenko

Grodno State Medical University, Department of Biochemistry, Grodno, Belaruse-mail: Hanna Vinitskaya <[email protected]>

Interrupted alcohol ingestion followed by alcohol free pe-riods is the common pattern of alcohol abuse in human population. It has been shown early that chronic alcohol in-toxication evokes changes in NAD/NADH ratio, and leads to the deficiency of some Krebs’ cycle substrates in brain and liver. Gamma-aminobutyric acid (GABA) metabolism was detected in brain and liver, and is likely to play a role in ethanol-induced liver injury.The study was aimed to investigate the state of GABA ca-tabolism in the liver of rats undergone different types of alcohol intoxication.Male heterogeneous stock rats (150-200 g) have been used. Interrupted alcohol intoxication (IAI) was evoked by in-tragastrical (i.g.) infusion of 25% ethanol solution in the dose of 3.5 g/kg of body weight, twice daily, within 4 days. Then the alcohol infusions were followed by the 3 days’ al-cohol-free periods. In sum 2 and 4 cycles of IAI were used, and rats were sacrificed 3 days after the last alcohol inges-tion. In chronic model rats had been preliminary treated by chronic i.g. administration of 25% ethanol solution in the dose of 5 g/kg of body weight, twice daily, within 5 days. Animals were sacrificed 3 hours, and 1 day after the last alcohol administration. In the control groups the rats were treated i.g. by saline solution according to the above-mentioned schemes. The activities of the GABA-transam-inase (GABA-T), succinic semialdehyde dehydrogenase (SSA-DH), succinate dehydrogenase (SDH), the contents of GABA and glutamate were assayed in rat liver after the last alcohol, or saline infusion.Interrupted alcohol administration resulted in the reliable enhancement of GABA catabolism in liver, followed by in-crease in the GABA and glutamate levels, compared to the control group. However chronic alcohol intoxication ex-erted decrease in the GABA-T activity in rat liver, whereas other indices were unchanged.It was proposed that the changes observed may occur due to non-specific adaptation of hepatocytes to the excessive alcohol consumption and further withdrawal. The results obtained on disturbances of energy and GABA metabo-lism in liver evoked by different types of alcohol intoxica-tion might be useful for working up of new effective meth-ods of alcoholism treatment.



P10.12

Biochemical and molecular aspects of cytoplasmic male sterility (CMS) in beets (Beta vulgaris L.)Wojciech Wesołowski, Judyta Słowińska, Michał Szalonek, Marek Szklarczyk

Agricultural University of Kraków, Department of Genetics, Plant Breeding and Seed Science, Kraków, Polande-mail: Wojciech Wesołowski <[email protected]>

Cytoplasmic male sterility is a maternally inherited inability of plants to produce viable pollen. Extensive studies on many plant species show that genetic determinants of this trait are located in the mitochondrial genome and have a form of chimeric open reading frames (ORFs) which may contain fragments of essential mitochondrial genes. The molecular similarity between the protein products of the chimeric ORFs and the standard mitochondrial proteins may impair the proper functioning of mitochondria leading to pollen abortion. Expression of CMS is also controlled by specific nuclear genes which either maintain or suppress the deleterious effect of the mitochondrial determinants. In the latter case these nuclear genes are referred to as restorers of fertility (Rf).The first experimental series was aimed at identification of differences between the mitochondrial proteomes of the normal and Owen-type sterile cytoplasms in sugar beet and beetroot. For this purpose the mitochondrial fractions were subjected to two-dimensional electrophoresis — Blue Native/SDS-PAGE. From over 100 detected proteins six showed cytoplasm-dependent distribution. The 30 kDa protein was observed only in the CMS plants of both sugar beet and beetroot. Gel localization of this protein sug-gested its correspondence to the translation product of the preSatp6 gene and this assumption was confirmed with the use of mass spectrometry. Another detected protein was characteristic for plants with restored fertility. Moreover, the restored plants resembled the plants with the normal cytoplasm in decreased accumulation of the 8 kDa protein.Secondly, a series of zymography assays was performed in order to elucidate whether the observed differences in mitochondrial protein composition were associated with the altered activity of the selected mitochondrial protein complexes. The analyses showed that in male-sterile plants activity of complex V (ATP synthase) is distinctly lower than in plants carrying the normal cytoplasm. These data indicate that in the CMS plants ATP production may not be sufficient to meet the high energy demand of pollen development.The last series of experiments was aimed at identification of molecular markers which were linked to the restorer genes of sugar beet. One population of plants segregating with respect to the fertility/sterility phenotype was geno-typed with the use of DArT markers. In total, 16 DArT markers co-segregated with the male-fertile phenotype on the level higher than 80%.

P10.13

First insight into the Dictyostelium discoideum TOM complex Monika Antoniewicz, Andonis Karachitos, Michał Rogosz, Hanna Kmita, Małgorzata Wojtkowska

University of Adam Mickiewicz, Institute of Molecular Biology and Biotechnology, Department of Bioenergetics, Poznań, Polande-mail: Małgorzata Wojtkowska <[email protected]>

The vast majority of proteins found in mitochondria are encoded by nuclear genes and synthesized in the cytosol. These proteins must be imported into mitochondria, di-rected to the proper mitochondrial subcompartments, and properly assembled at their final destination. These pro-cesses are mediated by wide variety of proteins forming translocases localized in both mitochondrial membranes. One of these translocases is the TOM complex (translo-case of the outer membrane) which recognize most of mi-tochondrial precursor proteins in the cytosol and imports them into the organelle. Thus, undoubtedly the TOM com-plex is fundamental for mitochondrial functioning.The subunit organization of the TOM complex has been shown to be characteristic for a given phylogenetic line-age. Beside common subunits the complex may contain subunit(s) that is(are) not present in representatives of other phylogenetic lineages. Till now the complex has been described for representatives of fungi and animals (termed Opisthokonta in the recent eukaryotic classification system involving the six supergroups) as well as for plants (Ar-chaeplastida). However, for representatives of Amoebo-zoa, Chromalveolata, Excavata and Rhizaria (former Pro-tista) the amount of data concerning the complex is indeed small. Therefore we decided to study the TOM complex of the slime mold Dictyostelium discoideum, an important model in medical studies, classified as a representative of Amoe-bozoa.By expression of his-tagged form of D. discoideum Tom7 protein and using affinity and ion-exchange chromatogra-phy we isolated a protein complex of MW about 450 kD that displays channel activity characteristic for the TOM complex. The complex was then analyzed by protein elec-trophoresis and mass spectrometry that allowed for identi-fication of Tom40, a channel forming subunit of the TOM complex. Thus, we proved that the isolated complex is the TOM complex and therefore it can be used for identifica-tion of its other subunits.



P10.14

Effect of endurance training on muscle metabolic stability in human calf musclesJerzy A. Zoladz1, Joanna Majerczak1, Justyna Zapart-Bukowska1, Piotr Kulinowski2, Tomasz Banasik2, Sylwia Heinze-Paluchowska2,3, Marcin Grandys1, Bernard Korzeniewski4

1Department of Muscle Physiology, Faculty of Rehabilitation, University School of Physical Education, Kraków, Poland; 2Institute of Nuclear Physics PAN, Kraków, Poland; 3Cancer Institute, Kraków, Poland; 4Faculty of Biochemistry, Biophysics and Biotechnology Kraków, Polande-mail: Justyna Zapart-Bukowska <[email protected]>

Introduction: Muscle fatigue is accompanied by a pro-nounced disturbances in muscle metabolic stability.It has been shown on animal model that training/conditioning increases muscle metabolic stability, as reflected by a less-er decrease in muscle PCr and ΔGATP as well as a smaller increase in ADPfree, Pi, and H+ during exercise of given power output. Little however is known concerning the ef-fect of training on muscle metabolic stability in humans. In the present study we have hypothesized that 16-weeks of endurance training will increase muscle metabolic stability during moderate and heavy exercise in humans. Subjects and Methods: Nine young, healthy, untrained men par-ticipated in this study (mean±SE: body mass 76.6±5.6 kg; height 179.6±2.5 cm; BMI 23.6±1.2 kg·m-2). All subjects performed two 6-minutes plantar flexion exercises using prototype ergometer placed in the NMR system (4.7 T MR scanner with MARAN DRX console). 31P NMR spec-tra were recorded from the calf muscles thought 6-min-utes rest period, 6-minutes of moderate intensity exercise, 6-minutes recovery, 6-minutes of heavy intensity exercise and 12-minutes of recovery. This system allowed us to measure ATP, Pi and PCr independently. The measurement protocol in the magnet was repeated twice i.e. before and after 16-weeks of the endurance training (cycling or run-ning) performed 4–5 times per week for about 40 minutes. Results: The sixteen weeks of the endurance training re-sulted in a significant attenuation of an exercise-induced decrease in PCr concentration both during moderate (P=0.03) and heavy intensity exercise (P=0.004). Moreo-ver, a lesser decrease in ΔGATP during moderate (P=0.02) and heavy intensity exercise (P=0.01) was observed. A sig-nificantly smaller increase (P<0.05) in ADPfree, Pi, and H+

concentrations after the training was observed only during heavy intensity exercise. Conclusions: Sixteen weeks of regular endurance training significantly improves metabolic stability in human calf muscles during exercise. This mus-cle adaptive response leads to an enhancement of exercise tolerance and fatigue resistance.AcknowledgementsThis work was supported by the Ministry of Science and Higher Educa-tion, grant No NN404196637.

P10.15

Role of mitochondria in endothelial cells inflammatory processesAgata Zgrzywa1, Agnieszka Łukasiak2, Antoni Wrzosek1, Adam Szewczyk1

1Laboratory of Intracellular Ion Channels, Departament of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; 2Department of Biophysics, Warsaw University of Life Science SGGW, Warsaw, Polande-mail: Agata Zgrzywa <[email protected]>

It is well documented that inflammation is a key factor that accelerates the onset and progression of atherosclerosis. The endothelial cells play prominent role in this disease. Recent reports suggest that despite of the glycolytic metab-olism of endothelial cells, mitochondria perform regulatory functions in these cells and are involved in apoptosis. The aim of our study was to determine the role of endothe-lial mitochondria in regulation proinflammatory response. Therefore we compared two cell types: the immortalized human endothelial cell line EA.hy926 and the ρ0 cell type, which derived from EA.hy926 cells and was developed by long-term exposure to ethidium bromide until oxidative phosphorylation was completely stopped. The respiratory control of EA.hy926 and ρ0 cells was performed. Addition of FCCP to EA.hy926 cells increased the oxygen con-sumption of about 300% compared to baseline. FCCP was ineffective to ρ0 cells. It indicates that the ρ0 culture can be treated as cell type without active respiratory chain.Inflammation was stimulated with tumor necrosis factor α (TNF-α) in dose and time dependent manner. Expression of intercellular adhesion molecule 1 (ICAM-1), which is one of the inflammatory marker, was detected using flow cytometry. In control cells there was a residual expression of ICAM-1. After addition of TNF-α we observed a large increase in ICAM-1 expression in EA.hy926 and ρ0 cells. We demonstrated that rotenone, inhibitor of complex I of mitochondrial electron transport chain, modulates inflam-matory response as measured as ICAM-1 expression on the surface of EA.hy926. Rotenone in the concentration between 1-300 nM in the presence of 5 ng/ml of TNF-α activates expression of ICAM-1 in EA.hy926 and there are no activation in ρ0cells.Summarising, our results suggest that mitochondria can be involved in expression of proinflammatory molecules in endothelial cells.AcknowledgementsThis work was supported by the European Union from the resources of the European Regional Development Fund under the Innovative Econo-my Programme (POIG.01.01.02-00-069/09)

session 10. cellular bioenergetics lecture oral ... session 10. cellular bioenergetics 47th congress...

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