S446 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-M.53]

Amelioration of Experimental Autoimmune Myasthenia GravisRats Using 4-Mercaptoethylpyridine-based Adsorbent

Jun Ren 1,2, Ying Bai 1,2, Lan Hao 1,2, Zhiqian Pi 1,2, Li Xu 1,2,Lingyun Jia 1,2,∗

1 School of Life Science and Biotechnology, Dalian University of Tech-nology, China2 Department of Neurology; the Affiliated Xinhua Hospital of DalianUniversity, ChinaKeywords: Adsorbent; Antibody; Myasthenia Gravis; Blood purifi-cation

Immunoadsorption is an important type of blood purificationtherapy normally used in clinic, and has proven effective in treatingmyasthenia gravis (MG) and a range of other autoimmune dis-eases. Protein ligands, such as recombinant Protein A, antibody andautoantigens, are usually employed for immunoadsorbents. Theyhave high specificity for target molecules, but also suffer from thedrawbacks mainly associated with their protein nature, such ashigh cost and low stability. Thus, synthetic compounds are beingexplored for production of specific adsorbents for the treatmentof autoimmune diseases. In this work, 4-mercaptoethylpyridine(MEP)-based adsorbent, an adsorbent previously found to haveaffinity with immunoglobulin (Ren et al., 2009), was investi-gated for its potential effects in treating MG. Animal model ofexperimental autoimmune myasthenia gravis using Lewis ratswas developed, and treated by whole blood apheresis. Adsorptioncolumns were prepared with a sepharose-based adsorbent cou-pling MEP as immobilized ligands. The results showed that, thesymptoms of treatment group rats were apparently improved aftertreatment compared to the controls, with clinic score decreasingfrom 2.75 ± 0.16 to 1.2 ± 0.45. After a treatment using 4 ml adsor-bent for a total 24 ml of blood, the amount of white cells of the ratswas reduced by 43.7 ± 6.4%, while the level of red cells were notsignificant changed. ELISA essays showed that the serum concen-trations of IgG, IgM, acetylcholine receptor antibody, complement3 and IL-17 were reduced by 31.5 ± 7.6%, 24.4 ± 6.1%, 27.5 ± 7.4%,22.3 ± 3.6% and 21.7 ± 5.8%, respectively. This study demonstratedthat MEP-based adsorbent could not only remove pathogenicautoantibodies directly from blood as Protein A adsorbents could,but also further regulate the immune system by eliminating otherinflammation related molecules and cells.

Acknowledgment

This work was supported by the National High Tech-nology Research and Development Program (863 program,2008AA02Z210) from the Ministry of Science and Technology ofChina, and National Natural Science Foundation of China (Project20775011).

Reference

Ren, J., Jia, L.Y., Xu, L., Lin, X., Pi, Z.Q., Xie, J., 2009. Removal of autoantibodies by4-mercaptoethylpyridine-based adsorbent. Journal of Chromatography B 877,1200–1204.

doi:10.1016/j.jbiotec.2010.09.641

[P-M.54]

Generation and Characterization of Novel TautomycetinAnalogs using Streptomyces Targeted Gene Disruption

sisun choi 1,∗, David H. Sherman 2, Eung-Soo Kim 1

1 Inha university, Republic of Korea2 Michigan University, United StatesKeywords: Streptomyces; Tautomycetin; Immunosuppressor;Polyketide

Tautomycetin (TMC), which is produced by Streptomyces sp.CK4412, is a novel activated T cell-specific immunosuppressive andanti-cancer compound with an ester bond linkage between a termi-nal cyclic anhydride moiety and a linear polyketide chain bearingan unusual terminal alkene. Previously, we cloned, sequenced, andcharacterized the entire biosynthetic gene cluster for Tautomycetinfrom Streptomyces sp. CK4412. Sequence information analysis ofan approximately 110 kb DNA region revealed two multi-modulartype I polyketide synthases, type II thioesterase, various proteins fordialkylmaleic anhydride biosynthesis, regulatory proteins and sev-eral tailoring enzymes (Choi et al., 2007; Choi et al., 2008). Althoughboth TMC and its close structural relative called Tautomycin (TM)compounds have similar inhibitory activities against two majorserine/threonine protein phosphatise, PP1 and PP2A, the differ-ent linear polyketide moiety present in TMC and TM is believe toplay a critical role because only TMC exhibits a immunosuppres-sant activity as well as a higher PP1 selectivity. To increase ourunderstanding about the functional specificity, the targeted geneinactivation of various biosynthetic genes involved in the forma-tion of a linear TMC polyketide was performed to generate, severalnovel TMC analogs with different polyketide terminal region. Thedetailed structural elucidations and biological activities of thesenovel TMC-like compounds will be further discussed.

References

Choi, et al., 2007. Isolation of the biosynthetic gene cluster for tautomycetin, a lin-ear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412.Microbiology 153 (4), 1095–1102.

Choi, et al., 2008. Identification of TmcN as a pathway-specific positive regulator oftautomycetin biosynthesis in Streptomyces sp. CK4412. Microbiology 154 (10),2912–2919.

doi:10.1016/j.jbiotec.2010.09.642

[P-M.55]

Overcoming Challenges in the Cryopreservation of Stem Cells in3D Alginate Constructs

S.K. Sambu ∗, X. Xu, H. Ye, Z.F. Cui

University of Oxford, United KingdomKeywords: Stem Cells; DMSO; alginate; cryopreservation

Stem cell banking is critical to regenerative medicine. An effec-tive cryopreservation method should conserve cell viability andphenotype. Current methods suffer from unpredictable viabilityand loss of stemness. Conversely, cryopreservation in 3D scaf-folds may offer better cryoprotection leading to higher survival.However, the exposure of cells to varying cryoprotectant (CPA)concentrations and exposure times in 3D matrices is a signif-icant limitation to this method. This model study investigatedpotential solutions to the size constraints in 3D cryopreserva-tion. It involves the cryopreservation of mouse embryonic stemcells (mESCs) encapsulated in spherical alginate constructs usingdimethyl sulfoxide (DMSO). In 3D experiments, loading times were

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S447

varied between 20–60 minutes while DMSO concentration wasfixed at 10% v/v. Cell viabilities were assessed using trypan bluestaining after recovery from alginate scaffolds. To investigate cellviability in situ, calcein/propidium iodide staining was used fol-lowed by imaging using multiphoton microscopy. Stemness wasassessed via flow cytometry for Octamer-4, Stage Specific Embry-onic Antigen-I and Alkaline phosphatase. Initial results showedvarying cell viabilities across the construct. To overcome the limi-tations of size, a multi-pronged solution involving the modulationof cell location, changing the physical and cryogenic properties ofthe scaffold and modulating cell parameters was adopted yieldingimproved cell viabilities. Baselines for 3D and suspension cryop-reservation were 59 ± 2% and 81 ± 4% respectively. By controllingcell location in constructs, viability improved by 28%. By improv-ing scaffold permeability and lyoprotection, cell viability improvedby 29%. Lastly, by modulating the cell permeability parameters,viabilities were improved by 83%. These promising results are con-comitant with a robust expression of stem cell markers.

Figure 1. (A) Cell survival (in suspension) plotted against exposure time and DMSOconcentration – a single global maximum exists along with a hardly noticeable localmaximum (B) Experimental results for 3D cryopreservation showing maximum sur-vival at about 60% (C) Calcein/PI viability assay results showing varying cell viabilitywithin constructs & (D) Experimental results showing an improvement of cell via-bility (20, 45 & 60mins) when lyoprotection is provided for in the scaffolding andcryopreservative.

doi:10.1016/j.jbiotec.2010.09.643

[P-M.56]

Screening and characterization of Urate oxidase from local iso-lated fungi

Rezvan Parvizi ∗, Abdollah Ghasemian, Younes Ghasemi

Department of Pharmaceutical Biotechnology, Faculty of Pharmacyand Pharmaceutical Sciences Research Center, Shiraz University ofMedical Sciences, Islamic Republic of IranKeywords: Uricase; Fungus; Enzymatic assay; Clear zone

Introduction: Urate oxidase catalyzes the opening of the purinering of urate to yield allantoin, carbon dioxide, and hydrogen per-oxide, in the presence of oxygen. Uricase is useful for enzymaticdetermination of urate in clinical analysis as a diagnostic reagentand plays an important role in nitrogen metabolism. It can bealso used as protein drug for treatment as Rasburicase (a recombi-nant version of urate oxidase enzyme). Rasburicase is a geneticallyengineered, for the treatment and prophylaxis of hyperuricaemiaassociated with tumor lysis syndrome in pediatric patients. Sev-eral forms of uricase from microorganisms are currently used asdiagnostic reagents to detect uric acid. Many organisms includ-

ing higher plants, fungi, yeasts and bacteria are able to produceuricase.

Methods: Isolated fungi were obtained in pure cultures fromsamples collected in various sites of Fars province, Iran. Classi-fication of the isolated fungi was carried out according to themorphological and culture characteristics of the organism. Thesewere plate-screened for their ability to produce uricase enzyme inorder to find new strains for possible application. The fungi wereable to produce an intracellular uricase in a medium mainly con-taining uric acid.

Results: About sixty fungal strains were screened for the useof uricase. Among them, Twenty three have considerable positiveactivity with the appearance of clear zone. Enzymatic assay wasdemonstrated eight strains have apparent high level of enzymaticactivities.

Discussion: In this work, we optimized enzyme assay to screenthe ability of fungi as a novel uricase producer and to maximize theenzyme productivity. Isolated strains with high level of productionof uricase may be use as an alternative source of this expensiveenzyme.

doi:10.1016/j.jbiotec.2010.09.644

[P-M.57]

Synthesis of new thiazolidinedione derivatives as 15-PGDHInhibitor

L.T.M. Hang 1, H. Cho 1,2,∗

1 Dept. of Polymer Science & Engineering, Chosun University, Gwangju501-759, Republic of Korea2 Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Republic of KoreaKeywords: Thiazolidinediones; 15-PGDH; Ulcer healing; Bone for-mation

Prostaglandins have a short life in vivo because they aremetabolized rapidly by oxidation to 15-ketoprostaglandins cat-alyzed by a cytosolic enzyme known as NAD+-dependent15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously,CT-8, a thiazolidinedione analogue, was found to be a potentinhibitor of 15-PGDH. Structure-activity analysis indicated thatthe N-methylation of thiazolidine-2,4-dione, CT-8, abolished theinhibitory activity, whereas the introduction of an ethyl hydroxylgroup at amine in CT-8 still had a good inhibitory effect.Based on the structures of the thiazolidinediones analoguesand inhibitory activity, a range of benzylidene thiazolidine-dione derivatives were synthesized with different substituentson the phenyl ring and their inhibitory activity was evalu-ated. Replacement of the cyclohexylethyl group of CT-8 withthe hetero 5-member ring increased the inhibitory potency.However, replacement of the cyclohexylethyl group with ahetero 6-member ring decreased the inhibitory potency sig-nificantly. It was found that compound 2 (5-(4-(2-(thiophen-2-yl)ethoxy)benzylidene)thiazolidine-2,4-dione) was the mostpotent inhibitor that was effective in the nanomolar range.

doi:10.1016/j.jbiotec.2010.09.645