Oral Presentations Integration of Metabolism and Survival OP-1 The ankyrin repeat and SOCS box-containing protein Asb-9 targets creatine kinase B for degradation M. A. Debrincat 1 , J. G. Zhang 2 , T. A. Willson 3 , B. T. Kile 3 , S. L. Masters 2 , L. M. Connolly 4 , R. J. Simpson 4 , H. M. Martin 2 , N. A. Nicola 2 and D. J. Hilton 3 1 Cancer and Haematology/Molecular Medicine, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, 2 Cancer and Haematology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, 3 Molecular Medicine, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, 4 The Joint Proteomics Laboratory of the Walter and Eliza Hall Institute of Medical Research and Ludwig Institute for Cancer Research, Melbourne, Australia. E-mail: debrincat@wehi.edu.au The suppressors of cytokine signalling (SOCS) proteins inhibit cytokine signalling by direct interaction with Janus kinases (JAKs) or activated cytokine receptors. In addition to the N-ter- minal and SH2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. Evidence suggests SOCS box-containing proteins act as part of an elongin-cullin- SOCS (ECS) E3 ubiquitin ligase complex, marking target pro- teins for degradation. The specificity of the complex is deter- mined by the protein interaction motif located upstream from the SOCS box. A number of other protein families that possess a SOCS box have been identified, the largest of which are the ank- yrin repeat and SOCS box-containing Asbs. While it is known that the SOCS proteins are involved in the negative regulation of cytokine signalling, the biological and biochemical functions of the Asbs are undefined. To understand the functional role of Asb proteins, a proteomic approach was implemented and creatine kinase B (CKB) was identified as a specific binding partner of Asb-9. Transfection of increasing concentrations of a tagged Asb-9 construct into 293T cells increased the polyubiquitination of CKB and resulted in a concomitant decrease in total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was entirely SOCS box-dependent. The interaction has been confirmed in vivo and suggests that Asb-9 may act as a specific ubiquitin ligase-regulating CKB abundance. OP-2 Signal transduction of cytokinine M. Gilmanov 1 , S. Ibragimova 1 , Sv. Sadykova 1 , Zh. Basygaraev 1 and A. Sabitov 2 1 Laboratory of Enzyme Structure and Regulation, Aithozhin’s Institute of Molecular Biology and Biochemistry, 2 Department of Chemistry, Al-Faraby’s Kazakn National University. E-mail: baltakay@mail.ru The cytokinine the most important phytohormone, which is con- trolling the division of the plant, cells. We carried out the investi- gation of cytokinine signal transduction. We were discovered the most important participant of cytokinine signal transduction. This participant was purified from germinated wheat grains by hydrophobic chromathography on octyl-sepharose 4B CL and by reversed-phase chromatography on RP-18 type column. Thus we were named the isolated substance as secondary cytokinine hor- mone or shorter mediator of cytokinine (MC). The MC is very powerful phytohormone as it shows the physiological activity at concentration 1000 times less than cytokinine. In contrast of cytokinine the MC appears own biochemical activities. For exam- ple, MC is activated NADP-glutamate dehydrogenase and H+ATP-ase, while the cytokinine not be able to activate this enzymes. The purified MC is competitive repressed the binding of tritium-labeled fusicoccine with fusicoccine receptors on plas- matic membrane winch were isolated from roots of Zea mays sprouts. It was shown that the binding of MC with fusicoccine receptors led to increasing the level of cytoplasmic calcium ions. Then calcium ions by participation of inositides is activated the proteinkinase C. Proteinkinase C was isolated by gel chromatog- raphy on sephacryle S-300 column and by ion-exchange chroma- tography on DE-52 column. This enzyme is the last participant of the signal transduction of cytokinine. OP-3 Skeletal muscle elongation factor 2 phosphorylation during contraction: mechanism of regulation A. J. Rose, J. B. Kobberø, T. J. Alsted, T. E. Jensen and E. A. Richter Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise & Sport Sciences, Copenhagen, Denmark. E-mail: arose@ifi.ku.dk Very little is known about the effect of exercise on the molecular regulation of polypeptide synthesis in skeletal muscle. Here, the effect of contractions on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation and eEF2 kinase activity was investigated. In response to contractions in situ, there was a rapid (i.e. 15s) fivefold increase in eEF2 phosphorylation at Thr56 in the contracting gastrocnemius muscle of rats that was maintained at this level during 30 min of contractions, with no change in the non-stimulated contralateral muscle. Furthermore, eEF2 phos- phorylation was higher in both soleus and extensor digitorum longus muscles of mice when contracted ex vivo, indicating that the mechanism behind this increase is related to local factors. No change in in vitro eEF2 kinase activity was observed in the con- tracted rat muscles at any time-point or when measured at pH 6.8 versus 7.2. Furthermore, the increase in eEF2 phosphoryla- tion occurred at a time before any change in AMPK activity was observed and was normal in contracting muscles of mice expres- sing non-functional AMPK. However, Ca 2+ -calmodulin potently increased the activity of skeletal muscle eEF2 kinase when meas- ured in vitro. Taken together, these data indicate that the inhibi- tion of protein synthesis in contracting muscle may arise from phosphorylation of eEF2 via a Ca 2+ -calmodulin-eEF2 kinase cascade. Abstracts 42 Integration of Defence and Survival OP-4 Investigation of multidrug resistance in docetaxel and doxorubicin-resistant MCF-7 cell lines O ¨ . Darcansoy _ Is¸ eri 1 , M. Demirel Kars 1 , U. Gu ¨ ndu ¨ z 1 and F. Arpacı 2 1 Department of Biological Sciences, Middle East Technical University, Ankara, Turkey, 2 Department of Oncology, Gu ¨ lhane Military Academy School of Medicine, Ankara, Turkey. E-mail: dozlem@metu.edu.tr Ineffectiveness of anticancer drugs during chemotherapy or recur- rence of malignancy after therapy is a frequently observed situation in cancer chemotherapy. Multidrug resistance (MDR) phenomenon is defined as the resistance of tumor cells to various cytotoxic drugs. It is a major impediment to successful treatment of breast cancer using chemotherapy. Cancer cells either strengthen the already pre- sent systems necessary for the removal of toxins from cells or acquire resistance to cytotoxic drugs. Members of the ATP-binding cassette (ABC) transporter superfamily have an important role in MDR. Among these, proteins coded by the ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (BCRP) genes are the most important trans- porters related to MDR phenotype. In this study, effects of expres- sion levels of the MDR1, MRP1, BCRP genes on the development of docetaxel and doxorubicin resistance using a model MCF-7 breast carcinoma cell line is evaluated. Docetaxel and doxorubicin were applied to cell culture in dose increaments and resistant sub- lines were developed. Cytotoxicity analysis of drugs was performed in wild type and developed resistant sublines to test development of resistance. Total RNA was isolated from cells, converted to cDNA and amplified by using gene (MDR1, MRP1, and BCRP)- specific primers by RT-PCR. Western blot analysis and immuno- staining were performed to determine the related protein levels. OP-5 GSK3: identification of a novel mechanism controlling inflammation in the brain E. Beurel 1 , S. Michalek 2 and R. Jope 1 1 Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA, 2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA. E-mail: beurel@uab.edu Controlling inflammation is a major challenge in the brain where inflammation has devastating consequences because, since dam- aged neurons cannot be replaced, neuroinflammation contributes to neurodegenerative diseases. In response to lipopolysaccharide (LPS), brain microglia, and astrocytes activate cytokines produc- tion, such as IL-6. Glycogen synthase kinase-3 (GSK3) regulating transcription factors, was studied as a regulator of neuroinflam- mation. In mouse primary astrocytes, we examined if GSK3- regulated IL6 production stimulated by LPS and its amplified production caused by co-administered interferon-c. Both LPS- induced IL-6 production and also its potentiation by interferon-c are highly dependent on GSK3. With both IL-6 production was abolished by GSK3 inhibitors, demonstrating the key role of GSK3 in neuroinflammation. The mechanism of LPS-induced IL-6 production potentiated by interferon-c was due to GSK3 activation and nuclear depletion of GSK3. This suggests that interferon-c plus LPS potentiates IL-6 production by activating cytosolic GSK3 which can control the activation of transcription factors that activate IL-6 expression. Chromatin immunoprecipi- tation is being used to identify the transcriptional targets of GSK3, such as NF-jB that regulate IL-6 production. This study identified GSK3 as a key regulator of neuroinflammation, estab- lishing that GSK3 inhibitors provide a new strategy to counteract the devastating effects of neuroinflammation. Rhythmic Signals: the Setting of Biological Time OP-6 Computational search of the interaction between melanopsin and cryp tochrome-2 proteins E. B. Unal 1 , B. Erman 2 and I. H. Kavakli 2 1 Computational Sciences and Engineering, Koc University, Istan- bul, Turkey, 2 Chemical and Biological Engineering, Koc University, Istanbul, Turkey. E-mail: evunal@ku.edu.tr Circadian rhythms are the biological processes that oscillate in the biochemical, physiological and behavioral functions of organisms with a periodicity of approximately 24 h without any external cues. In mammals, circadian rhythm is generated by molecular clock, which is located at suprachiasmatic nuclei (SCN) part of brain. Circadian rhythm is reset by external factors such as light. The cryptochromes,which was first discovered in Arabidopsis,are the blue-light photoreceptors. They absorb light and transmit the elec- tromagnetic signal to blue-light dependent of signal transduction. In mammals, the cryptochromes and melanopsin have been pro- posed as circadian photoreceptor pigments that exist in the inner retina to transmit signal to the SCN to tell the time of day. Both humans and mice have two cryptochrome proteins; CRY1 and CRY2. CRY2 is mostly expressed in retinal cells. Based on current evidence we propose that CRY2 may interact with melanopsin to mediate the light dependent signal-transduction in mammals.We have taken both computational and experimental approaches in order to show possible interaction between them during the circa- dian photoreception. First, we have predicted 3-D structure of cryptochrome using EsyPred, Robetta programs.Then, we have shown that both cryptochrome and melanopsin may interact in silico using various computational softwares, such as AUTO- DOCK, HEX.We are currently verifying our computational data taking experimental approach, specifically the FRET. OP-7 Mitochondrial electron transport chain reactivity in the brain and eye tissues under circadian rhythm alterations M. B. Yerer 1 , M. P. Alcolea Delgado 2 , S. Aydog ˘ an 1 , F. J. Garcia-Palmer 2 and P. R. Salom 2 1 Faculty of Medicine, Department of Physiology, University of Erciyes, 38039 Kayseri, Spain, 2 Department of Biochemistry and Molecular Biology, University of Balearic Islands, Mallorca, Spain. E-mail: eczbetul@yahoo.com Mitochondria plays a central role in energy-generating processes within the cell thorough the electron transport chain (ETC), the primary function which is ATP synthesis via oxidative Abstracts 43 phosphorylation (OXPHOS) which is shown to be related to age- ing and apoptosis when this balance is destroyed under different circumstances. This study is performed to investigate the effects of alterations in the physiological melatonin levels via the circa- dian rhythm changes, on the mitochondiral ETC in brain and eye and how these changes are correlated to the pineal gland melatonin receptor expressions. Fifty Sprague–Dawley male rats weighing 200–250 g were used in five groups of different circa- dian rhythms. The control group was 12/12 h of light/dark (L/D) cycle. Different circadian rhythms of 24/0 h L/D, 0/24 h L/D, 16/8 h L/D and 8/16 h L/D cycles were applied to the groups for 1 week, respectively, in special cages where the duration of the light and the climate can be adjusted. The melatonin receptors, MEL1 and MEL2 expressions were determined by real-time PCR in the pineal gland. The mitochondria of the brain and eye tis- sues were isolated from the homogenates and the activation of the mitochondrial OXPHOS complexes were determined by spec- trophotometric micro-methods described before. Plasma melato- nin levels were also determined by ELISA kit (IBL, Turkey). Related to circadian rhythms, the plasma melatonin levels were the highest in the 0/24 L/D group compared to the other groups (p < 0.05) and the MEL1 and MEL2 receptor expressions were also altered significantly by the circadian rhythms (p < 0.05). The Complex I activity is found to be decreased significantly in the 24/0 L/D group compared to the control and the 0/24 h L/D group (p < 0.05) in the brain mitochondria whereas it was sig- nificantly higher in the eye mitochondria compared to the control (p < 0.05). Complex III activities were slightly lower in the 24/ 0 h L/D group, whereas there was a significant increase in the eye mitochondria in all the groups compared to the control (p < 0.05). Furthermore, there was a significant increase in the Complex IV and V activities in the brain mitochondria were sig- nificantly higher 24/0 L/D group compared to its control (p < 0.05) whereas they were found to be unaffected in the eye mitochondria. As a consequence, this is the known first report to show the MEL1 and MEL2 receptor expressions by real-time PCR under different circadian rhythms. These alterations both in the receptors and the plasma melatonin levels are found to be correlated with the mitochondrial respiratory chain complexes which are directly related to the energy metabolism in the cells during the ageing process and the apoptosis. This study was granted by NATO Science Fellowships A2 Programme of TUBI- TAK. Signaling and Cancer: Nuclear Receptor Connection OP-8 Dysregulated Msx and Dlx gene expre ssion in epithelial odontogenic tumors S. Ghoul-Mazgar 1 , B. Ruhin 2 , D. Hotton 3 and A Berdal 3 1 Laboratoire de Biologie Oro-Faciale et Pathologie, INSERM U714-IFR-58, Universite ´ s Paris 7 and Paris 6 Laboratoire d’Histologie-Embryologie, Faculte ´ de Me ´ decine Dentaire de Monastir, Tunisia, 2 Stomatology and Maxillofacial Surgery Department, Pitie ´ Salpe ˆ trie ` re University Hospital, Paris Cedex 13, France, 3 Laboratoire de Biologie Oro-Faciale et Pathologie INSERM U714-IFR-58, Universite ´ s Paris 7 and Paris 6, France. E-mail: ghoulsonia@yahoo.fr Odontogenic tumors are rare pathologies, mostly benign, located in maxillary area. The most frequently observed benign epithelial odontogenic tumors is called ameloblastoma and may although give rise to the extremely rare malignant epithelial odontogenic tumors, usually named odontogenic carcinomas.The differential diagnosis between these tumors is so difficult regarding the diverse clinical prognosis and therefore management. Homeodo- main proteins comprise transcription factors that are essential in many developmental processes. Homeodomain is encoded by a highly conserved 60 amino acid sequence called homeobox that is responsible for specific interactions with DNA. Non-clustered ho- meobox genes are called non-HOX and include the Msx and Dlx gene family. In this study, we examined the Dlx and Msx gene expression by RT-PCR and in situ hybridization in recurrent 13 benign ameloblastomas and one malignant clear cell odontogenic carcinoma (CCOC). Our data show specific expression pattern of Msx and Dlx gene in the CCOC compared with benign amelobl- astomas. Furthermore, exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be inactivated in the carcinoma, but not Bmp4. Malignancy of epithelial odontogenic carcinoma seems to be a multistep and highly heterogeneous pro- cess requiring activation and deactivation of multiple and specific genes suggesting exploration of homeogene expression to discrim- inate benign ameloblastomas and odontogenic carcinomas. OP-9 Ligand-specific dynamics of the androgen receptor on its target promoter in living cells T. I. Klokk 1 , P. Kurys 1 , C. Elbi 2 , A. K. Nagaich 2 , A. Hendarwanto 2 , T. Slagsvold 1 , C. Y. Chang 3 , G. L. Hager 2 and F. Saatcioglu 1 1 Department of Molecular Biosciences, University of Oslo, Oslo, Norway, 2 Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, USA, 3 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, MD, USA. E-mail: t.i.klokk@imbv.uio.no Androgen receptor (AR) mediates the action of androgens, which are important in the development and maintenance of the male reproductive system and in pathologic conditions such as pros- tate cancer. This is the basis for the routine use of antiandrogens to block AR function in disease states, but little is known on the mechanisms involved. We studied ligand-dependent AR interac- tion with a target promoter in vivo, using photobleaching micros- copy, kinetic modeling, FRET analysis, and in vitro chromatin remodeling. The interaction of agonist-bound AR with the MMTV promoter was rapid and transient. In the presence of antagonist, and with a transcriptionally impaired AR mutant, an even faster interaction was seen due to decreased residence time on the promoter. The short residence times seen for AR in response to all ligands support the ‘hit-and-run’ model and three- dimensional genome-scanning hypothesis of transcription factor action. Furthermore, agonist and partial antagonists, but not pure antagonists, induced the recruitment of a chromatin-re- modeling complex to the HRE. Finally, FRET analysis in vivo demonstrated both intermolecular and intramolecular interac- tions between the N- and C- termini of AR at the HRE. Thus, three-dimensional scanning of the genome space, ligand-depend- ent modulation of AR kinetic properties, recruitment of chroma- tin remodeling complexes and proper intermolecular and intramolecular interactions are all critical for the in vivo function of AR on its target promoter. Abstracts 44 Cell Surface Receptors and Downstream Targets OP-10 Mutations of the growth hormone receptor gene in Turkish patients with Laron-type dwarfism A. Arman 1 , N. Yordam 2 , A. Ozon 2 , P. Isguven 3 , A. Coker 4 and I. Peker 1 1 The Faculty of Engineering, Marmara University, Istanbul, Turkey, 2 The Department of Pediatrics, Division of Endocrinology, Hacettepe University, Ankara, Turkey, 3 The Department of Endocrinology, Goztepe Government Hospital, Istanbul, Turkey, 4 The Department of Biology, Art and Sciences, Marmara University, Istanbul, Turkey. E-mail: aarman@eng.marmara.edu.tr Growth hormone (GH) mediates its growth, fat and carbohy- drate metabolism through insulin-like growth factor-I (IGF-I). Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of IGF-I that mediates GH actions. Mutations in the GHR cause severe postnatal growth failure and the disorder is an autosomal recessive genetic disease called Laron-type dwarfism, characterized by elevated serum GH associated with low levels of IGF-I. In this research, our purpose was to analyse the GHR gene for mutations in eight patients with Laron-type dwarfism. Eight patients were selected based on their phenotypic characteristics and their genomic DNAs were isolated from bloods of eight laron children. Exon 2–9 specific polymerase chain reactions (PCRs) and their flanking splice sites were amplified PCR. The PCR products were purified and se- quenced. We defined three missense mutations (S40L, V125A, I506L), one nonsense mutation (W157X), one sense mutation (G168G), one exon 3 deletion (exon 3 deletion) and one frame- shift mutation (G insertion in exon 2) located in the extracellular domain of GHR in eight patients. Acknowledgment: This project was supported by Turkish Republic State Planning Organization. OP-11 Human XLas signals more efficiently than the a-subunit of the stimulatory G protei n (GSa) in vitro A. Linglart 1 , M. J. Mahon 2 , T. Dean 2 , T. J. Gardella 2 , H. Jueppner 2 and M. Bastepe 2 1 Pediatric Endocrinology and INSERM U561, Saint Vincent de Paul Hospital, Paris, France, 2 Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. E-mail: bastepe@helix.mgh. harvard.edu XLas is partly identical to the a-subunit of the stimulatory G pro- tein (Gsa) with an extended N-terminus. Using adenoviral expres- sion and cells that endogenously lack Gsa and XLas (GnasE2–/– cells), we investigated human XLas (hXLas). Immunofluorescence microscopy showed membrane and punctate perinuclear staining for hXLas. On metal affinity chromatography, hXLas co-purified with a histidine-tagged Ga1. Furthermore, a PTH(1–15) analog, which normally prefers binding to a Gsa-coupled form of PTHR1, bound to membranes from GnasE2–/– cells co-expres- sing PTHR1 and hXLas. Also, hXLas was able to mediate basal and agonist-induced cAMP accumulation. However, while hXLas was expressed at lower levels than Gsa in transduced cells, basal cAMP level in cells expressing hXLas was ~twofold higher than in cells expressing Gsa. Similarly, basal ERK1/2 phosphorylation in GnasE2–/– cells transiently expressing hXLas was markedly enhanced. Isoproterenol treatment also resulted in significantly higher levels of cAMP accumulation in hXLas-expressing cells than Gsa-expressing cells, whereas PTH-induced cAMP accumu- lation appeared similar in cells co-expressing PTHR1 and either hXLasorGsa. These findings indicate that hXLas can act as a more potent signaling protein than Gsa, which may have implica- tions in responses mediated typically by Gsa. OP-12 Growth inhibition of c6 glioma cells in monolayer and spheroid cultures by the combination of carvedilol and imatinib M. Erguven 1 , A. Bilir 2 , S. Tuna 2 and N. Akev 1 1 Department of Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey, 2 Department of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul, Turkey. E-mail: merguven@istanbul.edu.tr Rat C6 glioma is a chemoresistant experimental brain tumour, which is difficult to treat with a combination of drugs. A new tyrosine kinase inhibitor, imatinib (Gleevec), has recently been found efficacious in the pre-clinical trials for glioblastoma (GBM). Carvedilol (Dilatrend), antihypertensive drug, has been demon- strated to reverse multidrug resistance (MDR) to anticancer drugs in several tumor cell lines in vitro. However, any possible modula- tory effect of carvedilol on brain tumours and on the efficacy of imatinib has not yet been evaluated experimentally. In the present study, we have investigated whether carvedilol provides synergistic or antagonistic effect on imatinib-induced cytotoxicity in mono- layer and spheroid cultures of malignant C6 glioma cells. C6 glioma cells in monolayer and spheroid cultures were treated with the combination of carvedilol and imatinib in concentration of 10 lM. The cell proliferation, morphology, spheroid volumes, bromodeoxyuridine-labelling index (BrdU-LI) were evaluated. The expression levels of caspase-3, caspase-9, hypoxia inducible factor-1 (HIF-1), Bcl-2 and platelet-derived growth factor receptor alpha (PDGFRa) were examined by Western blot analysis. The statistical significance was analysed by using the Student’s t-test. The results demonstrated that carvedilol and imatinib in combina- tion display enhanced antitumour activity in vitro against experi- mental rat C6 glioma in monolayer and spheroid cultures. OP-13 Non-traditional ways of B lymphocyte regulation: receptors for acetylcholine and thrombin S. Komisarenko 1 , R. Grailhe 2 , Y. Petrova 1 , J. P. Changeux 2 , W. Bahou 3 and M. Skok 1 1 Palladin Institute of Biochemistry, Kiev, Ukraine, 2 Pasteur Institute, Paris, France, 3 State University of New York, Stony Brook, NY, USA. E-mail: svk@biochem.kiev.ua Immune system is highly specialized to recognize and destroy the invading pathogens and transformed self-cells. For this purpose, it is equipped with unique antigen-specific receptors and employs specific ways of cell-to-cell communication. However, both the development and functioning of immune cells are regulated by various external factors including those of nerve and blood coagu- lation systems. Correspondingly, lymphocytes express receptors for non-immune mediators, such as acetylcholine and throm- bin. We demonstrated the presence of nicotinic acetylcholine Abstracts 45 receptors (nAChRs) and protease-activated receptors type 3 (PAR3) on mouse B lymphocytes using specific antibodies gener- ated to functionally important parts of these receptors: compo- nents of acetylcholine-binding site or thrombin-cleavage site. It was shown that signaling through nicotinic receptors stimulated proliferation of B lymphocyte-derived cell lines, supported B-lym- phocyte survival in the bone marrow, but limited its activation in mature state. In contrast, PAR3 activation inhibited B-cell line proliferation, but enhanced CD40-mediated B-lymphocyte prolif- eration. It is concluded that signaling through nAChR and PAR3 affects basic vital functions of B lymphocytes, such as prolifer- ation and survival, and interferes with their activation pathways. These data show the ways, by which acetylcholine, nicotine or thrombin can regulate the humoral branch of immunity. OP-14 Identification and characterization of a novel transcriptionally active domain in the linker region of the TGFb-regulated Smad3 protein M. Siderakis, V. Prokova, S. Mavridou, P. Papakosta and D. Kardassis Department of Medicine, University of Crete and IMBB-FORTH, Heraklion, Crete, Greece. E-mail: kardasis@imbb.forth.gr Our structure–function analysis of human Smad3 protein, a key mediator of TGFb signaling in mammalian cells revealed that the middle, non-conserved, linker domain has an autonomous and potent transactivation function. The region with the maximal transactivation capacity was the 143–248 that consist of almost the entire linker domain and the first 18 amino acids of the MH2 domain. The corresponding regions in Smad4 as well as in Smad1, which is a key mediator in Bone Morphogenetic Protein (BMP) signaling, are also transcriptionally active in mammalian cells further supporting the important role of this domain for Smad function. Smad3 mutants bearing an internal deletion of the 200–230 region or single amino acid substitutions in two highly conserved residues of this region (Q222A and P229A) had severe defects in oligomerization and transcriptional activation of target promoters. In contrast, mutagenesis of a non-conserved amino acid residue in the same region (N218A) did not affect any Smad function examined. Using a protein–protein interaction assay based on biotinylation in vivo, we were able to show that the Smad3 mutant with the internal deletion of amino acids 200– 230 is unable to interact physically and functionally with the his- tone acetyltransferase p/CAF. Our data support an essential role of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interac- tion with Smad coactivators Signaling Through Ion-channels OP-15 Sigma1 receptor/beta1 integrin complex: a novel target site for breast cancer cell adhesion R. Mahen, C. Palmer, C. Edwards, M. Djamgoz and E. Aydar Imperial College London, Divisions of Biology, Cell & Molecular Biology and Molecular Biosciences, Sir Alexander Fleming Building, South Kensington Campus, London, UK. E-mail: e.aydar@imperial.ac.uk The sigma receptor is a novel protein that is highly expressed in cancer cells and tissues, and has been shown to modulate proliferation and adhesion of breast cancer cells. The mechan- ism of action of the sigma1 receptor in these processes; how- ever, has not been elucidated. Using the Single Cell Adhesion Measuring Apparatus (SCAMA; confocal microscopy, sigma1 receptor RNAi, immunoprecipitation, surface biotinylation and lipid raft fractionation, the function of sigma1 receptor in adhe- sion of MDA-MB-231 breast cancer cells was investigated. Functional studies using SCAMA revealed that disruption of lipid rafts eliminated sigma receptor modulation of adhesion. Moreover,immunoprecipitation experiments in MDA-MB-231 cells,demonstrated that sigma1 receptor and beta1 integrin are associated. Furthermore, both confocal microscopy experiments and surface biotinylation experiments indicated that both appli- cation of sigma receptor drugs and knock-down of the sigma1 receptor increased the beta1 integrin expression in the mem- brane. Lipid raft fractionation experiments in MDA-MB-231 cells demonstrated that both application of the sigma receptor drugs and the knock-down the sigma1 receptor levels, beta1 in- tegrin protein in lipid rafts fraction of MDA-MB-231 cells were altered. All these data suggest that sigma1 receptor is associated with beta1 integrin and is likely modulate beta1 integrin levels. Therefore, sigma1 receptor is likely to be a novel target for breast cancer metastasis. OP-16 HERG potassium channels and heart disease S. Kuyucak School of Physics, University of Sydney, NSW, Sydney, Australia. E-mail: serdar@physics.usyd.edu.au The heartbeat is controlled by electrical signals mediated by the flow of ions through specialized ion channels. Of the channels that contribute to cardiac electrical activity, potassium channels encoded by the Human ether-a-go-go-related gene (HERG) have been of particular interest for many reasons. First, mutations in HERG are the cause of one-third of cases of congenital long QT syndrome, an inherited cause of sudden cardiac death. Secondly, HERG is the molecular target for the vast majority of drugs that cause drug-induced long QT syndrome, the commonest cause of drug-induced arrhythmias and cardiac death. Thirdly, HERG channels have very unusual biophysical properties, which suggest that they may act as an endogenous antiarrhythmic agent. There- fore, understanding the operation of HERG channels has become an important goal-post in medicine, physiology and pharmacol- ogy. While there is no crystal structure for the HERG protein yet, homology models based on the crystal structure of bacterial potassium channels provide a promising avenue for progress. In this talk, a survey of advances made in the field using a com- bined experimental/simulation approach will be given. This involves using homology models of HERG to find the important residues on the protein that are involved in channel dynamics (e.g. toxin binding, fast inactivation) and then test these hypothe- ses via mutagenesis experiments. This information is then used to refine the model, followed by further tests. Abstracts 46 Signaling and Apoptosis OP-17 Mechanism of pancreatic acinar cell apoptosis induced by crambene S. Adhikari 1 , Y. Cao 1 , A. D. Ang 1 , M. V. Clement 2 , M. Wallig 3 and M. Bhatia 1 1 Department of Pharmacology, National University of Singapore, Yong Loo Lin School of Medicine, Singapore, 2 Department of Biochemistry, National University of Singapore, Yong Loo Lin School of Medicine, Singapore, 3 Department of Pathobiology, University of Illinois at Urbana Champaign, Urbana, IL, USA. E-mail: sharmila@nus.edu.sg We investigated the molecular mechanisms that regulate the apoptosis of acinar cells induced by crambene (1-cyano-2-hydrox- y-3-butene-CHB), a plant nitrile. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apop- tosis but not necrosis of pancreatic acini. Caspase 3, 8 and 9 activity in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase 3, 8 and 9 inhibi- tors inhibited annexin V staining, as well as caspase 3 activity, pointing to an important role of these caspases in crambene- induced acinar cell apoptosis. The mitochondria membrane potential was collapsed in crambene-treated acini than in untreated that displayed polarized mitochondria. Also, the treat- ment of acini with crambene induced the release of cytochrome C by mitochondria than in untreated acini. Neither TNF-a nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induc- tion of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis. OP-18 Androgen inhibition of apoptosis in prostate cancer cells is due to downregulation of JNK activation P. I. Lorenzo and F. Saatcioglu Department of Molecular Biosciences, University of Oslo, Oslo, Norway. E-mail: petri@imbv.uio.no Androgens have a significant role in normal prostate physiology, as well as in prostate cancer. Removal of androgens during the initial stages of prostate cancer causes tumor regression via a combination of reduced cellular proliferation and increased apop- tosis. We have studied the molecular mechanisms of apoptosis in prostate cancer cells, focusing on the inhibition of apoptosis by androgens. We show that androgen treatment protects LNCaP prostate cancer cells from thapsigargin (TG)- and 12-o-tetradeca- noyl-13-phorbol-acetate (TPA)-induced apoptosis through a mechanism that involves the inhibition of c-Jun N-terminal kin- ase (JNK). The inhibitory effect of androgens on JNK activation is dependent on the androgen receptor (AR) since it is blocked by the androgen antagonist bicalutamide. The inhibition of JNK by androgens is independent of the stimulus that activates JNK, since it occurs readily when the JNK pathway is activated in response to TG, TPA or ultraviolet irradiation (UV). The inhibi- tory effect of androgens on JNK activation and apoptosis requires new gene expression, which is consistent with the time required to observe this effect. ATP depletion experiments indi- cate that inhibition of JNK activation by androgens is mediated, at least in part, through an increase in phosphatase activity. This crosstalk between AR and JNK signaling pathways may have important implications for both normal prostate physiology, as well as prostate cancer progression. OP-19 Enhancement of peptidylarginine deiminase 4 enzymatic activity assists apopt osis C Y. Lin 1 , Y F. Liao 2 , P C. Hsu 3 , H C. Hung 2 and G Y. Liu 1 1 Institute of Immunology, Chung Shan Medical University, Tai- chung, Taiwan, 2 Department of Life Sciences, National Chung- Hsing University, Taichung, Taiwan, 3 Department of Medicine, Da Chien General Hospital, Taiwan. E-mail: mt229.tw@yahoo.com.tw PAD4 post-translationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apop- tosis. A haplotype of single nucleotide polymorphism (SNP) in PAD4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Inducible PAD4 causes haematopoietic cell death (Liu et al. 2006) apopto- sis. Herein, we further investigate whether the increase of PAD4 enzymatic activity induces apoptosis. In Tet-On Jurkat T cells, ionomycin (Ion) only treatment did not induce apoptosis; how- ever; it promoted inducible PAD4-decreased cell viability and enhanced apoptosis. In vitro PAD enzyme activity assay, we dem- onstrated PAD4 enzyme activity of SNP relative to RA was higher than wild type (WT) relative to non-RA following Ca++ treatment. The effect of PAD4 SNP-induced apoptosis was superior to PAD4 WT. In addition, both Ion and PAD4 SNP sy- nergistically provoked apoptosis compared with both Ion and PAD4 WT. Western blotting data showed apoptosome activation during the programming cell death. Concurrently, the expression of Bcl-xL was downregulated remarkably and Bax upregulated in Ion treatment cells. These data demonstrated that increasing PAD4 enzyme activity could enhance apoptosis through mitoch- ondrial pathway and provide a conceivable explanation in the pathogenesis of RA following the upregulation of PAD4 activity. OP-20 Glucose-induced impairment of the insulin-signaling path way in mouse pancreatic beta cells E. Tsilibary 1 , P. Venieratos 1 , A. Charonis 2 and P. Kitsiou 1 1 Institute of Biology, National Center for Scientific Research ‘Demokritos’, Greece, 2 Foundation for Biomedical Research of the Academy of Athens, Athens, Greece. E-mail: effie@bio.demokritos.gr Type 2 diabetes is characterized by progressive pancreatic b-cell dysfunction and apoptosis. Recent reports provided evidence for an autocrine role of insulin on the signalling cascade in b-cell growth, function and survival are concerned. We examined the effects of chronic glucose stress on insulin signalling. Exposure of bTC-6 cells to high glucose resulted in impairment of insulin-sti- mulated phosphorylation of IRS-2. These changes were accom- panied by significant impairment of IRS-2-associated PI3-kinase activation, and substantially decreased activation of Akt. We also examined mTOR kinase, a downstream effector of Akt, which stimulates protein synthesis in response to Akt phosphorylation. High glucose abolished insulin-induced activation of mTOR without affecting mTOR expression, thus protein synthesis should also be impaired. A mechanism by which Akt promotes cell survival includes phosphorylation of the pro-apoptotic Abstracts 47 protein BAD, which keeps the pro-apoptotic protein BAX engaged to Bcl-XL. Exposure of cells to high glucose also resul- ted in suppression of insulin-stimulated phosphorylation of BAD without affecting BAD expression. In conclusion, we demonstra- ted that chronic exposure of pancreatic b-cells to increased glu- cose concentration resulted in impaired activation of the IRS-2/ PI3-kinase/Akt signalling pathway in response to insulin. The observed defects in insulin signalling may eventually have a neg- ative effect on b-cell function and survival. OP-21 Caspase-dependent and geldanamycin- enhanced cleavage of co-chaperone p23 in leukemic apoptosis G. Gausdal 1 , B. T. Gjertsen 2 , K. Fladmark 1 , H. Demol 3 , J. Vandekerckhove 3 and S. O. Døskeland 1 1 Department of Biomedicine, University of Bergen, Bergen, Norway, 2 Department of Medicine, Haukeland University Hospital, Bergen, Norway, 3 Department of Biochemistry, University of Ghent, Ghent, Belgium. E-mail: gro.gausdal@biomed.uib.no The co-chaperone p23 is a component of the Hsp90 multiprotein complex and is an important modulator of Hsp90 activity. Hsp90 client proteins involved in oncogenic survival signaling are often found to be mutated in leukemia, and the integrity of the Hsp90 complex could therefore be important for leukemic cell survival. We demonstrate here that the Hsp90 co-chaperone p23 is cleaved in leukemic cell lines treated with commonly used che- motherapeutic drugs. The cleavage of p23 paralleled the activa- tion of procaspase 3 and was suppressed by z-DEVD-FMK. Interestingly, p23 cleavage was also observed in caspase 3-defici- ent MCF-7 cells, and in vitro translated 35S-p23 was cleaved by both caspase 3 and 7. Two caspase target sites were identified in the C-terminal sequence EVD142GAD145, and only Asp to Ala mutagenesis of both sites (D142/145A) completely blocked p23 cleavage. The Hsp90 inhibitor geldanamycin, which inhibits p23 binding to Hsp90, did not induce cell death or p23 cleavage on its own, but enhanced anthracycline-induced caspase activation, p23 cleavage and apoptosis. This implies that Hsp90 inhibition amplifies caspase activation. Geldanamycin also enhanced caspase cleavage of 35S-p23 in vitro, indicating that the associ- ation of p23 to Hsp90 protects against cleavage. These findings underscore the importance of the Hsp90-complex in antileukemic treatment, and suggest that p23 may have a role in survival signaling. OP-22 Overview how colorectal cancer cells avoid cell elimination; mechanisms of immune- and chemo-resistance B. Pajak 1 , H. Engi 1 , J. Molnar 1 and A. Orzechowski 2 1 Institute of Medical Microbiology and Immunology, Faculty of Medicine, University of Szeged, Szeged, Hungary. E-mail: bepaj@wp.pl, 2 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland The resistance of cancer cells to deletion allows tumors to grow and develop. In the past, several tactics were shown how colon a- denocarcinomas avoid cell deletion and maintain cell viability. In particular, colorectal cancer cells resist death ligands-induced apoptosis by expressing antiapoptotic proteins, including FLIP. By direct interaction with FADD, FLIP inhibits the signal des- cending from death receptors in COLO 205 cells. Colorectal can- cer cells also stimulate own survival by the cytoplasmic retention of proapoptotic protein clusterin. In contrast, in normal cells clus- terin translocates to the nucleus and induces cell death. We found that apoptotic activity of clusterin is dependent on calcium ions, and depletion of intracellular calcium caused extensive death of COLO 205 cells. Other type of strategy of chemotherapy-induced cell death is the activity of multidrug resistance proteins (MDR). These cell membrane efflux pumps actively expel the drugs from the cell interior to prevent their action on intracellular targets. Upon phenothiazine derivatives rhodamine 123 accumulated within cells interior, which indicates the reversal of Pgp efflux pump in chemoresistant COLO 320 cell line. The variety of antia- poptotic mechanisms found in colorectal cancer cells and the knowledge how complex they are renders the anticancer therapy a challenge but the more we know how to sensitize cancer cells to death signals the more likely promise to eliminate them. Embryonic Stem Cells OP-23 Expression of the cholinergic system components during mouse embryonic stem cell differentiation L. Paraoanu, G. Steinert and P. Layer Developmental Biology, Darmstadt University of Technology, Darmstadt, Germany. E-mail: paraoanu@bio.tu-darmstadt.de Expression of cholinesterases during phases of embryonic devel- opment is a general phenomenon. However, no precise function for cholinesterases could be described during early developmental stages. We have examined the expression of cholinesterases and other cholinergic components during in vitro differentiation of CGR8 cells, an embryonic stem cell line. Undifferentiated mono- layers of these cells can be maintained in culture, or embryoid bodies can be generated in vitro. The embryoid bodies were allowed to differentiate in the absence of further additives or treated with retinoic acid, and collected at various times for fur- ther analysis. The cholinesterase expression was analyzed by reverse transcriptase polymerase chain reaction, histochemistry, and activity measurement. Low levels of cholinesterase activity and transcripts could be detected already in undifferentiated cells. The phases of differentiation are accompanied by increased ace- tylcholinesterase transcripts. Butyrylcholinesterase expression was initially constant, decreasing at later differentiation stages. All transcripts of muscarinic cholinergic receptors were present; how- ever, their expression did not vary throughout the differentiation steps. Choline-acetyltransferase was also detected, indicating that the cells are possibly able to produce acetylcholine. With these results we could show that mouse embryonic stem cells express a primitive cholinergic system. Its function remains to be deci- phered. Abstracts 48 Differentiation of Stem Cells OP-24 Human umbilical cord strom a cells differentiate into neurons in vitro S. Karahuseyinoglu 1 , O. Cinar 2 , F. Kara 3 and A. Can 1 1 Department of Histology-Embryology, Ankara University School of Medicine, Ankara, Turkey, 2 Department of Infertility, Etlik Maternity and Women’s Health Research Training Hospital, Ankara, Turkey, 3 Department of Obstetrics, Zubeyde Hanim Maternity Hospital, Ankara, Turkey. E-mail: alpcan@medicine. ankara.edu.tr A recently isolated source for stem cells is the mesenchymal cells of human umbilical cord stroma. Although neuronal differenti- ation has been demonstrated in human umbilical cord stroma cells (HUSCSs), there is a still discordance among studies about the potential of these cells to differentiate into neurons. The aim of this study was to differentiate HUSCSs into neurons and investi- gate the temporal development potency of newly differentiated neuronal cells. The spatio-temporal distribution and the quantifi- cation of various proteins during differentiation, immunofluores- cent single and multi-immunolabeling techniques were performed using a series of antibodies raised against major cellular markers for neurons, such as MAP1, MAP2, GFAP, tau, NSE, NFM, b- III tubulin, NeuN, and thyrosine hydroxylase. MAP1, a specific structural protein in the cytoplasm, MAP2, specific for dendrites and NFM were all positive during the course of the entire differ- ention period whereas tau and b-III tubulin were detected at only certain time-points of differentiation. While NeuN stained faintly, GFAP and thyrosine hydroxylase were both negative since no dif- ferentiation toward astrocytes and dopaminergic neurons was accomplished. Conclusively, it appears that throughout the differ- entiation of HUCSCs into neurons, expression of neuronal mark- ers present a diversity which follows a similar progress as seen in embryologic development of neurons of the body. Gene Therapy OP-25 Cationic nanoparticle synthesis and their use in gene transfer G. Guven 1 , M. Turkoglu 1 and E. Piskin 2 1 Hacettepe University Chemical Eng. Dept, 2 Hacettepe University Chemical Eng. & TUBITAK-BIOMEDTEK. E-mail: gguven@ hacettepe.edu.tr Polymer microspheres would be synthesized by use of an emulsi- fier-free emulsion polymerization, which gives more pure, and monodisperse samples. In this method the particle formation occurs when the growing radicals reach to a certain critical chain length depending upon the solvency of the continous phase. In this study, monosized non-functional poly(styrene/N-[3-dimethyl- amino)propyl]methacrylamide/poly(ethyleneglycol)ethylethermetha- crylate) [poly(St/PEG-EEM/DMAPM)] and functional group carrying poly(styrene/N-[3-dimethylamino)propyl]methacrylamide/ poly(ethyleneglycol) methacrylate) [poly(St/PEG-MA/DMAPM)] nanoparticles were synthesized by emulsifier-free emulsion copo- lymerization in the presence of a cationic initiator 2,2’-azobis(2- methyl propionamidine) dihydrochloride (V-50). The spherical particles with diameters in the nanometer range and cationic surface charge were prepared. Generally, the polymeric nanoparticle dimen- sions are below 100 nm. The smallest particle size (71 nm) with very low polydispersity index and very high surface charge was obtained by increasing water content. This particle size was achieved by using both of the functional (PEG-MA) and non-functional (PEG-EEM). Nanoparticles with such a high amount of surface charge make these materials useful for the gene transfer especially. OP-26 Peripheral pool of CD8+ lymphocytes contains T cells that recognize syngeneic MHC class II molecules E. S. Zvezdova, T. S Grinenko, L. A Pobezinsky, E. L. Pobezinskaya and D. B. Kazansky Russian N.N. Blokhin Cancer Research Center. E-mail: katyazvezdova@yahoo.com A number of publications appeared, which assume that autoim- mune cells exist in the pool of CD8+ T lymphocytes with the potential to recognize both MHC class I and MHC class II mole- cules. To test this, we generated autoreactive T-cell hybridomas with the ability to recognize both allogeneic MHC class I mole- cule and MHC class II molecules: the LN CD8+ T cells from C57BL/6 mice were activated in the presence of allogeneic MHC class I molecules Kbm3 and fused with BW5147 thymoma cells, expressing CD4 coreceptor. The percentage of autoreactive hy- bridomas in CD8+ T cells was 9.3%. One of these hybridomas expressed two a-chains. One of these a-chains help to recognize syngeneic MHC class II molecules, while the other allogeneic MHC class I molecule Kbm3. To elucidate would the same situ- ation be observed in TCRa +/0 mice capable to express single a-chain of TCR, we obtained hybridomas from their CD8+ T cells. The frequency of self MHC class II reactive hybridomas was lower in contrast with that of wild type 1.3%. We have shown that in the peripheral pool of wild-type CD8+ T cells the autoreactive T cells exist that recognize foreign MHC class I and self MHC class II molecules. One part of these cells expresses the TCR with dual specificity to MHC class I and MHC class II molecules, while the other expresses two a-chains, one defines the specificity to MHC class I molecule and the other to syngeneic MHC class II molecule. OP-27 Salmonella typhimurium aroB-encoding murine IL-18 or CD40L: evaluation for gene therapy D. Aydin 1 , A. Gunel-Ozcan 2 , N. Menager 3 , G. Esendagli 1 , M. O. Guc 4 , M. Hayran 5 , E. Kansu 1 and D. Guc 1 1 Institute of Oncology, Department of Basic Oncology, Hacettepe University, Ankara, Turkey, 2 Faculty of Medicine, Department of Medical Biology and Genetics, Kirikkale University, Kirikkale, Turkey, 3 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK, 4 Faculty of Medicine, Department of Pharmacology, Hacettepe University, Ankara, Turkey, 5 Institute of Oncology, Department of Preventive Oncology, Hacettepe University, Ankara, Turkey. E-mail: didemaydin@gmail.com Attenuated Salmonella typhimurium (ST) strains have being investigated as promising vectors for gene therapy. In our study, Abstracts 49 STaroB strain was evaluated as a gene delivery vehicle for the first time. Eukaryotic expression vectors, pTARGET and pcDNA3, carrying murine IL-18 or CD40L genes were construc- ted to enhance immune responses of the host for further cancer therapy studies. mRNAs used to synthesize cDNAs to amplify IL-18 and CD40L DNAs were obtained from lipopolysaccharide- induced adherent peripheral blood mononuclear cells, and Phor- bol 12-Myristate 13-Acetate and Ionomycin-induced splenocytes respectively. A bioactive IL-18 molecule consisting of IFN-b sig- nal and mature IL-18 sequences was constructed by overlap extension method. The plasmids were transformed into STaroB by electroporation. While in vitro stability of pTARGET con- structs was very low, pcDNA3 constructs were highly stable. HEK293 cells were transfected with pcDNA3 constructs by a cat- ionic liposome and mRNA expressions of the molecules were shown by RT-PCR. STaroB carrying pcDNA3 constructs were injected to mice intravenously. Plasmids carrying insert DNAs were found statistically more stable than vector only control in vivo; however, in vivo stability of all was below 1% and mRNA expressions in liver and spleen could not be detected by RT- PCR. Our preliminary results showed that STaroB carrying pTARGET or pcDNA3 may not be suitable to investigate the effects of a therapeutic gene in a disease model. OP-28 Kanamicin-induced adaptive mutation in E. coli cells harbouring plasmids with direct repeats S. C. Ribeiro, P. H. Oliveira, D. M. F. Prazeres and G. A. Monteiro Departamento de Engenharia Quı ´ mica e Biolo ´ gica, Instituto Superior Te ´ cnico, Lisbon, Portugal. E-mail: pcoliveira@mail.ist.utl.pt This study describes a kanamycin stress-induced adaptive mutation in Escherichia coli cells harbouring either a candidate plasmid DNA vaccine (pGPV-PV) or its mammalian expression vector backbone (pCI-neo) [1]. The mutation, which was shown to occur due to the presence of a 28 bp direct repeat flanking 1.6 (pCI-neo) and 3.2 kb (pGPV-PV) intervening sequences, was responsible for the acquisition of a kanamycin resistance pheno- type in different E. coli strains (DH5a, Top 10F’, HB101 and JM109). This could be explained as follows: as a result of slip- page events, the Neo r gene falls under the control of a promoter- like sequence located at the origin of replication, thus enabling cells to strive in kanamycin. The Poisson distribution of mutants determined by Luria-Delbru ¨ ck fluctuations tests and reconstruc- tion experiments surprisingly, but unequivocally, indicates that this plasmid slippage event behaves adaptively. This study high- lights the need to carefully design plasmid vectors avoiding, for example, repetitive sequences that are genetically unstable and may give rise to unforeseen problems during plasmid DNA pro- duction and subsequent utilization. Reference: 1. Bahloul et al.Vaccine 1998; 16: 417–425. Therapeutic Enzymes OP-29 Engineering of nucleoside kinases to improve the activation of nucleoside analog prodrugs M. Konrad 1 , S. Ort 1 , C. Monnerjahn 1 and A. Lavie 2 1 Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany, 2 Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, IL, USA. E-mail: mkonrad@ gwdg.de The objective of our study is to improve therapeutic enzyme-pro- drug systems. Compounds, such as AZT for the treatment of HIV infections, ACV and GCV used against Herpes virus, or the antican- cer compounds AraC and gemcitabine, enter cells only in the un- phosphorylated form (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with DNA replication. Based on crystal struc- ture analyses of various enzyme–nucleotide complexes we first designed mutants of the human TMP kinase (hTMPK) that phos- phorylate AZTMP up to 200-fold faster than wild type. Expression of this enzyme in human cells leads to 10-fold higher intracellular concentrations of AZTTP and to enhanced HIV inhibition. Sec- ondly, the prodrugs ACV and GCV are not phosphorylated by human kinases, but are converted to their monophosphates by HSV1-TK, which has been used in enzyme/prodrug-dependent can- cer suicide gene therapy trials. With the aim of improving this thera- peutic system we generated enzyme variants, which show selective and efficient phosphorylation of GCV. Thirdly, an engineered human dCK variant catalyzes more efficiently the activation of the prodrugs AraC and gemcitabine. Thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intra- cellular protein transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. OP-30 Effect of N2A connectin/titin on disassembly of p94/capn3 caused by autolysis in IS2 Y. Ono 1 , F. Torii 2 , K. Ojima 3 , N. Doi 3 , K. Yoshioka 2 , D. Labeit 4 , S. Labeit 4 , K. Suzuki 5 , K. Abe 2 , T. Maeda 6 and H. Sorimachi 1 1 Department of Enzymatic Regulation for Cell Functions, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo, Japan, 2 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 3 CREST, Japan Science and Technology (JST), Saitama, Japan, 4 University of Mannheim, Mannheim, Germany, 5 New Frontiers Research Laboratories, Toray Industries Inc., Kanagawa, Japan, 6 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan. E-mail: yakoono@rinshoken.or.jp p94/calpain3 is the skeletal muscle-specific member of calpains, Ca 2+ -regulated cytosolic cysteine protease family. Defective p94 protease activity originated from gene mutations causes muscular Abstracts 50 dystrophy called calpainopathy, indicating p94s’ indispensability for muscle survival. One of unique properties of p94 is its very rapid and exhaustive autolytic activity, which is proposed to be regulated by its binding protein connectin/titin. In vitro analysis of p94 autolysis revealed that autolytic cleavage in each p94-spe- cific insertion sequence, NS, IS1, and IS2, causes different impact on molecular integrity of p94 as a protease; autolysis in IS2, but not in IS1, causes disassembly of autolyzed fragments. Using proteinase trapping system to semiquantitatively assay p94 auto- lysis, where p94 was expressed in yeast as a hybrid protein between DNA binding and activation domains of the yeast tran- scriptional activator Gal4, N2A connectin was shown to suppress p94 autolytic disassembly. Proximity of IS2 autolytic and connec- tin-binding sites in p94 suggested that N2A connectin interferes with IS2 autolysis. It was also shown that N2A connectin with mdm mutation that is causative of dystrophy in mice and abol- ishes connectin–p94 interaction was incapable of suppressing p94 autolytic disassembly. These data indicate that p94–connectin interaction plays an important role in the control of p94 func- tions by regulating autolytic decay of p94. OP-31 Prevention of thermal aggregation of yeast alcohol dehydrogenase by arginine and a-cyclodextrin A. Barzegar and A. A. Moosavi-Movahedi Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. E-mail: barzegar@ibb.ut.ac.ir Yeast alcohol dehydrogenase (YADH) is a tetrameric enzyme and is a widely studied as a metaloenzyme for its well-known biotechnological importance. Arginine (Arg) and a-cyclodextrin (a-CD) are the universal reagents that are effective in assisting re- folding of recombinant proteins from inclusion bodies. The results showed that YADH (0.5 mg/ml) initiate to aggregation after 15 min at 45 °C and 5 min at 50 °C in sodium phosphate buffer (pH 7.5). In the presence of Arg (100 mM) aggregation phenomenon completely suppressed at 50 °C until 2 h and a-CD suppressed aggregation until 40 min (aggregation initiates after 40 min) in the same condition. The fluorescence and UV–Vis data show that enzyme structure does not change in the presence of these additives. The residual activity of YADH at 50 °C after 50 min was very low and negligible (£5%) and in the presence of 100 mM Arg its activity completely inhibit whereas maintained at G30% in the presence of 100 mM a-CD. We propose Arg has a major factor for diminishing the electrostatic forces and a-CD has a role for controlling of the hydrophobicity due to decreasing the aggregation of YADH. RNA Interference OP-32 RNA interference shows critical involvement for NF-lkappaB p65 in the production of Wnt-1 protein S. Tsai 1 , C. Cheng 1 , C. Sun 2 , C. Tzeng 3 and A. H. Wang 4 1 Liver Research Unit, Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan, 2 Division of Hepatogastroentero- logy, Department of Internal Medicine, Chi Mei Medical Center, Tainan, Taiwan, 3 Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan, 4 Core Facilities for Proteomics Research and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. E-mail: sltsai@mail.chimei.org.tw The link of proto-oncogenic protein Wnt-1 with NF-lkappaB activity has been reported in PC12 cells, a rat pheochromocytom- a cell line of neural crest lineage, showing that the Wnt-1-medi- ated survival of these cells is dependent on NF-lkappaB activation, and that stable expression of Wnt-1 increases NF- lkappaB activity. In Wnt-1-mediated breast tumorigenesis, Wnt-1 is reported to be a fundamental signaling event in metastatic pro- gression of human cancers. We have shown that enhanced NF- lkappaB-associated Wnt-1 protein expression is detected in the majority of hepatoma samples from both hepatitis B and C patients. Insight into how NF-lkappaB regulates Wnt-1 protein production may help design highly effective therapeutic agents in treating Wnt-1-producing cancers and prevent their metastatic progression. RNA interference (RNAi) was used in this study to assess the role of p50 and p65 of NF-lkappaB in the regulation of Wnt-1 protein production by Het-1A cells, a human esopha- geal cancer cell line with Wnt-1 protein production. Cultured Het-1A cells were transfected with sequence-specific double-stran- ded small interfering RNAs (siRNAs) of P50, P65, and Wnt-1 respectively. All these siRNAs did not inhibit NF-lkappaB acti- vation in Het-1A cells. The production of Wnt-1 protein was totally abolished in cells transfected with P65 siRNAs and parti- ally reduced in cells transfected with P50 siRNAs, indicating a pivotal requirement for P65 in Het-1A production of Wnt-1 protein. OP-33 Molecular mechanism of paclitaxel-induced apoptosis in ER+/– breast cancer cell lines: MCF-7 and MDA-MB-231 E. D. Arisan, O. Kutuk, A. Verim and H. Basaga Biological Sciences and Bioengineering Program, Faculty of Science and Engineering, Sabanci University, Istanbul, Turkey. E-mail: ebuyuktuncer@su.sabanciuniv.edu Taxanes such as paclitaxel (Pac) and docetaxel are cytotoxic agents, which act by promoting deformation of stable microtu- bules, inhibiting the normal dynamic reorganization of microtu- bule networks required for mitosis. However, the development intrinsic/acquired of resistance against Pac is a major obstacle to successful cancer chemotherapy. Bcl2 is believed to have role in drug resistance since Bcl2 overexpression is very frequent especi- ally in ER+ breast cancer tumors through ER–responsive ele- ment in its promoter. Bcl2 also causes prevention of Pac-induced Abstracts 51 [...]... structural integrity of microtubule Hyperphosphorylation of tau protein observed in AD and several mutations of the tau genes have been considered to result in reduced binding ability of tau to microtubule and resulted in a lack of microtubule dynamic instability Therefore, disruption of its structure leads to impairment of axonal transport and finally loss of synaptic function in neural cells and cell death... between genes of interest, alternative splice forms and proteinencoded sequences function and structure We found some interesting features that are presented here We discuss the results of genes of interest in order to temp establishing of preventive gene Abstracts set They concern the coverage quality, in terms of consistency and reproducible, of the protein-encoded sequences of genes of interest,... knowledge of the role of genes in IS may improve our understanding of the cause of IS, provide new insights in prevention and factors that influence the outcome of IS, as new therapeutic targets when preventive strategies have failed OP-48 Genetic risk factors and early ischemic stroke Fibrinogen, is suggested to play a significant role in the pathogenesis of atherosclerosis and complications of atherothrombotic... strains and selected spots are identified by MALDI-TOF MS (Waters/Micromass) analysis OP-71 UniProtKB/Swiss-Prot: the protein sequence knowledgebase A Stutz1, A Bairoch2, Swiss-Prot Group1 and A Estreicher1 1 Swiss-Prot, Swiss Institute of Bioinformatics, CMU, Geneva, Switzerland,2Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland E-mail: andre.stutz@isb-sib.ch... Vargel1, F Oner3, B Cil4 and Y Erk1 1 Faculty of Medicine, Department of Plastic and Reconstructive Surgery, Hacettepe University, Ankara, Turkey,2Faculty of Medicine, Department of Histology and Embryology, Hacettepe University, Ankara, Turkey,3Faculty of Pharmacy, Department of Pharmaceutical Technology, Hacettepe University, Ankara, Turkey,4Faculty of Medicine, Department of Radiology, Hacettepe... contributions of individual domains to the stability, GDP/GTP binding and GTPase activity of EF-Tu proteins We examined, in functional and structural (CD) tests, the domains of mesophilic, Gram–, Escherichia coli (Ec) EF-Tu, and thermophilic, Gram+, 75 Abstracts B stearothermophilus (Bst) EF-Tu, and six chimaeric forms of EF-Tu constructed by combination of domains of both EF-Tus All three domains of both... concentrations (5% and 20% NaCl) and different temperatures (10 and 40 °C) After growing and cell disrupture, the entire proteins of the organisms are subfractionated as membrane proteins and cytosolic proteins An acidic character of most of the whole cell proteins is detected both in isolates and the reference strains (H salina DSMZ5928 and H halophila DSMZ4770) The 2-DE maps of the new halophiles are compared... differentiation of pre-adipocytes to adipocytes Troglitazone and the ciglitazone had similar effects on PAI-1 expression and decrease the level of PAI-1 in HUVEC, undifferentiated and differentiated 3T3 L1 cells OP-45 Effect of modifications of Grb14 protein expression on insulin signaling and sensitivity ´ N Carre, J Girard and A Burnol Institut Cochin, INSERMU567 CNRS UMR8104 Endocrinology, Metabolism, ... main targets of pharmaceutical research today However, the structural information on these molecules are quite scarce, and ligand specificity for most of them are unknown In this study, we describe the utilization of support vector machines to predict the ligand specificity of a given class of GPCRs, based on total and partial primary sequence information We used three-letter word composition of GPCRs using... consistent and eight proteins were identified by MALDI-TOF-TOF mass spectrometry These proteins were: albumin, a-enolase, calgranulin B, galectin 7, myoglobin, myosin light chain 1 and light chain 2, and tropomyosin b-chain Also, differences in allele frequencies of a Wnt gene family member between aggressive and non-aggressive cases and in alternative splicing isoform expression between normal and metastatic . Oral Presentations Integration of Metabolism and Survival OP-1 The ankyrin repeat and SOCS box-containing protein Asb-9. Demol 3 , J. Vandekerckhove 3 and S. O. Døskeland 1 1 Department of Biomedicine, University of Bergen, Bergen, Norway, 2 Department of Medicine, Haukeland University