Báo cáo khoa học: V1–Varia V1-001P Hemolysis of human red blood cells by riboflavin-Cu(II) system: enhancement by azide. ppt

Abstracts V1–Varia V1-001P Hemolysis of human red blood cells by riboflavin-Cu(II) system: enhancement by azide I Ali1 and I Naseem2 Faculty of Pharmacy, Applied Science university, Amman, Amman Jordan, 2Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, UP India E-mail: iyad74@yahoo.com Photoactivated riboflavin in the presence of Cu(II) generates reactive oxygen species (ROS) which can hemolyse human red blood cells (RBC) In the present work we examined the effect of sodium azide (NaN3) on RBC in the presence of riboflavin and Cu(II) The addition of NaN3 to the riboflavin-Cu(II) system enhanced K+ loss and hemolysis The extent of K+ loss and hemolysis were time and concentration dependent Bathocuproine, a Cu(I)-sequestering agent inhibited the hemolysis completely Among various free radical scavengers used to identify the major ROS involved in the reaction, thiourea was found to be the most effective scavenger Thiourea caused almost 85% inhibition of hemolysis suggesting that •OH is the major ROS involved in the reaction Using spectral studies and other observations, we propose that when NaN3 is added to riboflavin-Cu(II) system, it inhibits the photodegradation of riboflavin resulting in increased •OH generation Also, the possibility of azide radical formation and its involvement in the reaction could not be ruled out V1-002P Involvement of thromboxane A2 synthase in the dexamethasone altered response to electrical field stimulation in mesenteric arteries from spontaneously hypertensive rats ´ R M Aras, M Ferrer and G Balfagon ´noma de Madrid, Departamento de Fisiologı´a, Universidad Auto Madrid, Spain E-mail: arasmadrid@wanadoo.es The aim of this study was to analyze whether dexamethasone alters the vasoconstrictor response induced by electrical field stimulation (EFS) in endothelium denuded mesenteric arteries from spontaneously hypertensive rats (SHR) For this purpose, superior mesenteric arteries from male SHR rats were used to analyze whether dexamethasone alters: (i) the vasomotor response to EFS, (ii) the vasoconstrictor response to exogenous noradrenaline (NA), and (iii) the EFS-induced release of [3H]-NA Preincubation with dexamethasone for h decreased the contractile response induced by EFS but did not modify the contraction induced by exogenous NA The EFS-induced [3H]-NA release was increased by dexamethasone preincubation The thromboxane A2 synthase inhibitor, furegrelate, reversed the decreased response to EFS induced by dexamethasone without modifying the unaltered response to NA These results show that preincubation with dexamethasone for h increases the NA release induced by EFS, and indicate decreased thromboxane A2 synthase activity under these conditions Acknowledgment: This work was supported by grants from FIS (PI020335 and C03/01) and DGCYT (BFI2001–1324) 539 Abstracts V1-003P Effects of Nat5 aminotermional acetyltransferase inhibition on HeLa cells A Ametzazurra, E Larrea, M P Civeira, J Prieto and R Aldabe Gene Therapy and Hepatology, FIMA, Pamplona, Navarra Spain E-mail: raldabe@unav.es Alpha aminoterminal acetyltransferases (NAT) acts cotranslationally on the vast majority of eukaryotic proteins In yeast there are at least three NATs: NatA, NatB and NatC being Nat3p one subunit of NATB We have focused on analyzing the effects induced by the inhibition of Nat5, human homologue of Nat3p, in HeLa cells using siRNAs We have observed that there is no induction of apoptosis in the cells that have Nat5 downregulated but there is a clear growth arrest of the HeLa cells that posses Nat5 inhibited that directly correlates with the degree of Nat5 inhibition observed in the cells The cell cycle disruption induced by the downregulation of Nat5 expression is induced by the activation of p53 that induces an increase of p21 mRNA and protein presents in the cell V1-004P Plants as alternative systems for recombinant protein production: Medicago truncatula as an emerging production system R Abranches1, S Marcel2, E Arcalis2, P Fevereiro1 and E Stoger2 Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal, Institute for Molecular Biotechnology, Aachen, Germany E-mail: ritaa@itqb.unl.pt Plants are emerging as a promising alternative to conventional platforms for the large-scale production of recombinant proteins This field of research, known as molecular farming, is developing rapidly and several plant-derived recombinant proteins are already in advanced clinical trials However, the full potential of molecular farming can only be realized if we gain a fundamental understanding of biological processes regulating the production and accumulation of functional recombinant proteins in plants Recent studies indicate that species- and tissue-specific factors as well as plant physiology can have a significant impact on the amount and quality of the recombinant product More detailed comparative studies are needed for each product, including the analysis of expression levels, biochemical properties, in vitro activity and subcellular localization Here we include the first results from an extensive comparative study in which the highly-glycosylated enzyme phytase (from the fungus Aspergillus niger) was produced in different plant species (including tobacco, rice and the model legume Medicago truncatula) Special emphasis is placed on M truncatula, whose leaves accumulated the highest levels of active phytase We discuss the potential of this species as a novel production host V1-005P Role of plasma bilirubin and superoxide dismutase in erythrocytes in elevation of total antioxidant activity of plasma in growing rats intoxicated with acetaminophen A Allameh1, A Dadkhah1, F Fatemi1, Z I Islamifar1 and M Rahmati2 Molecular Biology, Biochemistry, Tarbiat Modarres University, Tehran, Tehran Iran, 2Molecular Biology, Biochemistry, Science and Research Unit- Islamic Azad University, Tehran, Tehran Iran E-mail: allameha@modares.ac.ir Ferric reducing ability of plasma (FRAP) represents the total antioxidant capacity (TAC) of plasma that encompasses different enzy- 540 matic and non-enzymatic antioxidant factors Recently we reported that TAC of plasma, determined as FRAP is remarkably induced in developing rats pretreated with high dose vitamin K1 or menadione Increased FRAP was inversely related to formation of lipid peroxidation products in plasma and erythrocytes In the present study, the role of selected antioxidant factors in the drug-related changes in FRAP was examined Unlike adults, in growing rats treated with a sub-lethal dose of acetaminophen (450 mg/kg b.w) there was about 4–5 fold increase in FRAP Under these circumstances, no significant changes recorded in total protein and uric acid levels in plasma as well as catalase activity in erythrocytes In contrast, increased FRAP value was associated with plasma bilirubin as well as erythrocyte superoxide dismutase (SOD) activities Kinetic studies showed that FRAP is elevated during 1–4 h after acetaminophen treatment Increased FRAP occurred simultaneously with a surge in total bilirubin h after acetaminophen administration However, elevation in SOD in erythrocytes occurred with a delay (12 h after drug administration) These results suggest that in immature rats, plasma bilirubin and erythrocyte SOD play a distinct role in elevation of total antioxidant capacity of plasma leading to suppression in the level of lipid peroxidation products in plasma V1-006P Functional analysis of peptides from the skin extracts of the North African frog Rana saharica F Abassi1, M Amich2, P Nicolas2 and K Hani1 Laboratoire de Biochimie, Departement de Biochimie, Faculte´ de Medecine de Sousse, Sousse, Sousse Tunisia, 2Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Universite de Pierre et Marie Curie, Paris, Paris France E-mail: feten20022002@yahoo.fr During the course of evolution, nature has developed a vast number of peptides in all living and past species that display an exceeding diversity of structure and biological effects, such as hormonal and enzyme-controlling activity, communication between cells, and participation in host defense These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses A few peptides have also been found to be active to sperm and tumor cells These bactericidal, fungicidal, virucidal and tumoricidal properties and the fact that they have potential to overcome bacterial resistance make these peptides promising candidates for therapeutic drugs They serve as research tools and have potential as diagnostic biomarkers and for the development of peptide and peptidometic drugs In the present study, we have used, for the first time, the ‘‘Tunisian frog’’ as a model to isolate, characterize and study the pharmacological properties of new structures, synthesized by the skin of tunisian frogs from the genus Rana These peptides can be used as phylogenetic markers and as probes in the experiments of homologous cloning, to reveal the presence of new peptides with therapeutic interest and to assess the value of amphibian antimicrobial peptides as taxonomic and phylogenetic markers A primary purification of the skin of the frog Rana saharica was done using Sep-Pak C18 cartridges; the fractions obtained were tested for their ability to inhibit growth of pathogenic microorganisms Many fractions inhibited the growth of reference strains of Gram+ bacterium, Gram– bacterium and fungi strains Abstracts V1-007P Detection of H pylori-specific gene in the oral yeasts A Salmanian1, F Siavoshi2, F Akbai3, R Malekzadeh4 and S Massarrat4 National Research Center for Genetic Engineering & Biotechnology, Tehran, Iran, 2M Department Faculty of Science, Tehran University, Tehran, Iran, 3Khatam University, Tehran, Iran, Digest Diseases Research Center, Tehran University of Medical sciences, Tehran, Iran E-mail: fereshteh_mail Background: One reason for the persistence of H pylori in the human stomach might be a stage in the life cycle of H pylori in which the bacterium establishes as an intracellular symbiont in yeast In the previous study H pylori-specific genes, universal prokaryotic 16S rDNA and vacA(s1/s2) were detected in yeasts DNA In this study cagA was targetted Methods: DNA was extracted from 18 yeasts in which the presence of bacterium-like bodies was confirmed by light microscopy H pylori-specific cagA gene was targeted by designing specific primers PCR conditions were optimized and the amplified products were analyzed on agarose gel H pylori and S cerevisiae were used as controls The fragments were cloned for sequencing, then compared with data in Gene bank Results: cagA was amplified from eight of 18 yeasts isolates and H pylori, as a 814bp product, when primer pair No.1 was recruited When seminested-PCR was performed on all of the 18 PCR products of the first reaction cagA gene was amplified, as 316bp band, from 15 samples and H pylori Such product was not amplified from S cerevisiae The PCR product of cagA in H pylori and yeast DNA were sequenced and showed more than 98% homology to the cagA gene of H pylori Discussion: One critical measurement to counteract H pylori is to block the routes of its transmission Universal prokaryotic 16S rDNA, vacA, and cagA genes were detected in oral yeasts, it is concluded that yeasts are important in transmission of H pylori V1-008P Circadian rhythmicity of rPER1 and rPER2 proteins in the rat peripheral tissues ´ ´ ´ Z Bendova, K Laurinova and A Sumova Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences, Prague, the Czech Republic E-mail: bendova@biomed.cas.cz In living organisms, there exists many physiologic and behavioural processes that oscillate in time with circadian period In mammals, these oscillations are controlled by a circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus SCN clockwork mechanism is based on the transcriptional and translational positive and negative feedback loops of clock genes and their corresponding proteins These include the PAS (Per-ARNT-Sim) proteins PER1 and PER2 Several studies revealed that circadian rhythms in the expression of these proteins in the SCN were delayed by about h relative to the mRNA Recently, components of the SCN clockwork system has been found also in multiple peripheral tissues of mammals Several analysis show the circadian rhythmicity in clock genes expression in lung, liver, heart and others but the results are discussed mostly in the context of organization of circadian oscillators within the body In our study, we present the circadian profiles of rPER1 and rPER2 proteins in lung, liver, heart and kidney in rats maintained under LD 12:12 as well as under long (LD 16 : 8) and short (LD 8:16) photoperiod as they were analyzed by Western blots Our data indicate a tissue specific pattern of both rPER proteins expression and the results will be communicated in context of molecular clockwork mechanism in peripheral tissue compared to the SCN Acknowledgment: This work was supported by Grant Agency of the Czech Republic, Grant No: 309/02/D093 V1-009P Reconstitution of human hypoxia inducible factor (HIF-1) in yeast cells: a simple in vivo system to identify hif-1 inhibitors G G Braliou, E Venieris and G Simos Laboratory of Biochemistry, School of Medicine, University of Thessaly, Larissa, Greece E-mail: gbraliou@tee.gr Hypoxia inducible factor (HIF-1) is a transcription factor regulating a number of genes involved in oxygen and energy homeostasis Furthermore, HIF-1 is highly involved in the pathology of various diseases including tumors, angiogenesis-related disorders and preeclamsia Thus, inhibitors of HIF-1 activity are of high interest as potential pharmaceutical or anti-cancer agents HIF-1 is a heterodimer consisting of the hypoxia-regulated subunit HIF-1alpha and the constitutively expressed subunit HIF-1beta (ARNT) To be functional, the two subunits of HIF-1 have to translocate inside the nucleus, dimerize and bind to DNA sequences called Hypoxia Response Elements (HREs), located within promoter or enhancer regions of target genes We show in this work, that these processes can also efficiently occur in S cerevisiae cells, which have been genetically modified to co-express the two human HIF-1 subunits and a suitable reporter gene Therefore, HIF-1 can exert its transcriptional activity in yeast, a finding that has important implications for its function in human cells In addition, this simple and genetically tractable in vivo system can be used for identifying other proteins, peptides or chemical compounds that directly affect the activity of HIF-1 The potential of this system is currently being investigated V1-010P Tyrosine diazoquinone (quinone diazide) and other diazo compounds are the result of NO-dependent protein modifications N Beda and A Nedospasov Institute of Molecular Genetics, Moscow, Russian Federation E-mail: beda@img.ras.ru Nitric oxide (NO) and reactive NO-derived species (RNS) can produce various NO-dependent modifications (NODM) of proteins The essential characteristics of NODM are their relative independence of enzymes, the involvement of very small ions and molecules (NO, NO+, NO-, NO2 etc.) and the possibility of autocatalytic formation of RNS, e.g NO-oxidation products, within protein molecules Micellar catalysis of NO oxidation can maintain uninterrupted fluxes of NO and oxygen to the interior of the protein molecule, formation of RNS inside the molecule, and transfer of the reaction products out of the molecule Tyrosine and tryptophan residues are the main targets for nitration in proteins However, the biochemistry of consequent conversions of nitrated molecules is studied insufficiently The possibility of nitrotyrosine residues reduction in proteins to amino derivatives has been reliably demonstrated Here we show that aminotyrosine residues in proteins can be converted by NODM into tyrosine quinone diazide (Tyr-QD) form, which is in a pH-dependent equilibrium with diazo-, and diazoate forms At physiological pH values these diazo forms are highly reactive and can react with some amino acids and bases of nucleic acids They can be reduced to native tyrosine residues as well Tyr-H fi Tyr-NO2 fi [Tyr-NO fi TyrNHOH] fi Tyr-NH2 fi Tyr-N=N+ fi Tyr-H + N2 In cyclic metabolic paths, stationary concentrations of each product of a 541 Abstracts cycle depend on their formation/decomposition rates ratio Therefore, an increase in the observed concentrations of individual products, for instance, nitrotyrosine or quinone diazide forms of a particular protein, might be a consequence of both their synthesis acceleration and decomposition deceleration Hundreds of proteins are known to undergo nitration and nitrosation in vivo Their diazo forms might be likewise participants of cyclic NO-dependent conversions of these proteins Their distribution and functional significance remain to be elucidated of hemocytes from YHV-infected shrimps by two-dimensional gel electrophoresis were elucidated showing up and down regulated proteins These proteins were in gel digested and analyzed by mass spectrometry Some of the down-regulated proteins were identified as tropomyosin and actin A vast modulation of tropomyosin and actin could clearly explain the transformation of hemocytes morphology observed in an infected shrimp The possible relation of the cytoskeletal-related proteins in mechanism of YHV infection was also investigated and discussed V1-011P Glycan differences between healthy and pancreatic adenocarcinoma serum ribonuclease V1-013P Identification of a novel mitochondrial protein that is essential for peroxisomal membrane synthesis in the yeast Hansenula polymorpha ´ ´ S Barrabes1, G Tabares1, E Fort2, P M Rudd3, P Rosa1 and D L Rafael1 Biochemistry of Cancer, Biochemistry and Molecular Biology Department, University of Girona, Girona, Spain, 2Digestive Department, Hospital Universitari Dr J Trueta, Girona, Spain, Glycobiology Institute, Departament of Biochemistry, University of Oxford, Oxford, UK E-mail: silvia.barrabes@udg.es Pancreatic adenocarcinoma has a poor prognosis because of its high death : incidence ratio, close to Its diagnosis is still difficult due to its low symptomatology and the absence of a specific marker One of the general features of tumour cells consists of changes in their cell surface, which can be also reflected in the glycosylation pattern of their secreted glycoproteins Ribonuclease is a glycoprotein expressed mainly by the pancreas and it is also found in serum RNase from human healthy pancreas presents a different glycosylation pattern when secreted by adenocarcinoma cell lines Capan-1 and MDA-Panc-3 One of the most significative differences is the presence of sialic acid on RNase from tumour cells In order to elucidate whether similar differences in the glycosylation pattern can be found between serum RNase from healthy and pancreatic cancer patients, two-dimensional electrophoresis studies were performed Three healthy patients sera and five pancreatic adenocarcinoma patients sera samples were pretreated by two different chromatographic methods in order to enrich RNase ratio, and analyzed by two-dimensional electrophoresis Some differences due to changes in pI were attributed to different glycosylation patterns In order to get a further insight in these differences, a sandwich Glycosylation ImmunoSorbent Assay (GISA), determining the sialylation potential by Sialyl-Transferase activity, was carried out With the aim to fully characterize the glycan structure of healthy and pancreatic adenocarcinoma serum RNase 1, glycan sequencing is in progress The results presented show differences in glycosylation structures of serum RNase 1, which indicate a possible way for characterize pancreatic adenocarcinoma and could be useful to improve the pancreatic cancer diagnostic V1-012P Yellow head virus infection induces a modulation of cytoskeletal-related proteins in hemocyte from black tiger shrimp A Bourchookarn, P Chongsatja and C Krittanai Institute of Molecular Biology and Genetics, Mahidol University, Nakhon Pathom, Thailand E-mail: mrchookarn@yahoo.com Yellow head virus (YHV), a rod shaped, single-stranded RNA virus, is one of the most severe infectious agents to black tiger shrimp (Penaeus monodon) Rather than lymphoid organ and gills, the hemocytes were previously reported to be infecting by YHV Light microscopy of hemocytes from forty-eight hours post-infection has shown a clear morphological change Proteomic profiles 542 E Bener Aksam1, R Pries1, S Kohlwein2 and I J van der Klei1 Department of Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands, 2SFB Biomembrane Research Center, Institute of Molecular Biosciences, University of Graz, Graz, Austria E-mail: e.bener@biol.rug.nl Peroxisomes are important subcellular organelles, which can function in a variety of metabolic processes, dependent on environmental conditions and the organism in which they occur Yeasts are attractive model organisms to study peroxisome biogenesis and function In recent years, vast studies have aimed at the elucidation of the mechanisms involved in these processes Various genes (PEX genes) have been identified which are essential for peroxisome biogenesis Here we report on the isolation of a mitochondrial protein that functions in the formation/stability of the peroxisomal membrane This protein of yet unknown function has been identified within a novel collection of conditional temperature sensitive (ts) mutants of the yeast Hansenula polymorpha, affected in the utilization of methanol at restrictive temperatures This growth defect appeared to be associated with the destabilization of peroxisomes in the cells due to the disintegration of the peroxisomal membrane Total peroxisome desintegration occurred in a time interval of 2-4 h after the shift of cells from permissive (37 °C) to restrictive (44 °C) temperature The mutant gene was sequenced and appeared to contain a point mutation that changed D407 into N The D407-N mutation was subsequently introduced into a WT H polymorpha strain to confirm the mutant phenotype Fusion of the gene to GFP localized the protein to mitochondria Hence, this protein represents the first known mitochondrial protein involved in peroxisome membrane assembly Mass spec lipid analysis was performed to analyze the phospholipid components affected in the mutant strain V1-014P Phthalocyanine conjugates of oligonucleotides as new reagents for sensitized and catalytic modification of DNA and DNA-binding proteins A A Chernonosov Laboratory of Investigation of Biopolymer’s Modification, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russian Federation E-mail: sandy@niboch.nsc.ru Phthalocyanines (Ptc) is now of interest as a drugs for photodynamic therapy of malignant tumors Their therapeutic effect is based on the ability to sensitize the generation of singlet molecular oxygen 1O2 under irradiation Besides, some metal-Ptc can catalyze in dark conditions the formation of other reactive oxygen species -•O2, H2O2, •OH These properties of Ptc make them very attractive as a reactive group to be linked to antisense oligonucle- Abstracts otides for specific DNA chemical modification We have developed the solid-phase method of synthesis of deoxyribooligonucleotide conjugates carrying Al(III)–, Zn(II)–, and Co(II)tetracarboxy-Ptcs groups The yields of conjugates were 30–40% Thermodynamics of interaction of Ptc-oligonucleotide conjugates with ss- and dsDNA were measured by UV melting method and CD spectroscopy The results demonstrated the stabilizing effect of Ptc moiety on the formation of both duplexes and triplexes DNA Co(II)-Ptc-oligonucleotide conjugate was found to result in modification of target DNA in duplexes and triplexes in the presence of molecular oxygen/reducing agent or hydrogen peroxide The Al(III)- and Zn(II)-Ptc conjugates caused DNA modification under irradiation with Hg-lamp or He/Ne laser lights at 340 and 633 nm, respectively The damages in DNA were revealed by the treatment of products with piperidine or 8-oxoguanine-DNA-glycosylase from E coli The interactions of Ptc-oligonucleotide conjugates with proteins of blood plasma were studied Acknowledgment: This work was supported by grants from the Russian Foundation for Basic Research (05-04-48447) and Ministry of Education and Sciences of Russia However, the activation mechanism for PLD2 is still unclear As reported here, PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation For example, only PLD2 was tyrosine phosphorylated in antigen-stimulated mast cells This phosphorylation was blocked by an Src kinase inhibitor or by small interfering RNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na(3)VO(4) Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation, although two of these mutations were detrimental to PLD2 function PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by coexpression of PLD2 and the Src kinases The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in Fc epsilon RI-mediated signaling V1-015P Bile acids in bile and serum of a mouse model of alpha-methylacyl-CoA racemase-deficiency K Savolainen1, T J Kotti1, W Schmitz2, T I Savolainen1, J K Hiltunen1 and E Conzelmann2 Department of Biochemistry, University of Oulu, Oulu, Finland, Physiologic Chenistry II, Biozentrum, University of Wuerzburg, Wuerzburg, Germany E-mail: conz@biozentrum.uni-wuerzburg.de The enzyme alpha-methylacyl-CoA racemase (AMACR) is required for the degradation of branched-chain fatty acids and is also involved in the synthesis of bile acids from cholesterol The inherited deficiency of the enzyme in humans causes adult-onset sensory and motor neuropathy To study the physiological role of the enzyme, an AMACR-deficient mouse strain was generated Bile acid composition of bile and serum from knockout animals and from their normal litter-mates was quantitatively analyzed by FTICR mass spectrometry The extremely high resolution of this method allowed the simultaneous determination of all relevant compounds without prior chromatographic separation In the bile of enzyme-deficient animals, total concentration of mature (C24) bile acids was reduced to approx half the normal content, while the C27 precur-sors, which are barely detectable in normal controls, made up for most of the difference Similar changes were found in serum and in liver tissue The presence of significant amounts of normal bile acids, in spite of the block of an essential step, indicates the operation of an alternative pathway, with lower capacity V1-016P Src family kinases regulates the activation of phospholipase D2 and degranulation in RBL 2H3 cells W S Choi1, Y M Kim2, E Her1, H Y Lee3, J W Han4, H W Lee4, T Hiragun5 and M A Beaven5 Laboratory of Immunology, College of Medicine, Konkuk University, Chungju, South Korea, 2Department of General Education, Duksung Women’s University, Seoul, South Korea, 3College of Medicine, Konyang University, Nonsan, South Korea, 4College of Pharmacy, Sungkyunkwan University, Suwon, South Korea, Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, MD 20892 USA E-mail: wahnchoi@kku.ac.kr We reported previously that both phospholipase D1 (PLD1) and PLD2 regulate degranulation in RBL 2H3 mast cells V1-017P A systems biology approach of metabolomics applied to design new targets in cancer therapy ´ M Cascante, A Ramos, P Vizan, P de Atauri, J Centelles, L Boros, S Mazurek, W Frederiks, P Lee and J Boren Biochemistry and Molecular Biology, Barcelona, Barcelona, Catalunya Spain E-mail: martacascante@ub.edu In the last few years, science has concentrated their efforts in characterizing the molecular elements that conform cells Several techniques as DNA sequencing, expression arrays, and proteomic and metabolomic experiments have provided us a large amount of new information that cannot be easily interpreted Moreover, the integration of all these information in in vivo models is likely to be the most interesting tool to understand and to complete an overview picture of the cellular processes We must take into account, that metabolic profile is in most cases the end point of the signalling events, where changes caused by diseases like cancer may be reflected Therefore, using bioinformatics – specially a tool as Metabolic Control Analysis- we are able to identify the main steps that control a metabolic pathway after the integration of all the experimental data These control points may be used as new therapeutical targets In this way, we are working in new antitumoral treatments based on the inhibition of the synthesis of DNA and RNA because of their importance for cell proliferation We have focused on ribose-5-phosphate synthesis, which is a component of nucleotides First of all, we characterized the metabolic pathways (utilizing gas chromatography coupled to mass spectrometry) implied in glucose metabolism and ribose synthesis This was followed by the integration of the obtained data in mathematical models using MCA, which led us to identify the main enzymes controlling ribose-5-P synthesis: transketolase and glucose-6-phosphate dehydrogenase Finally, we validated the obtained targets using specific inhibitors and then, we studied the effects produced in cell proliferation as well as in the proteomic profile Consequences of the utilization of these inhibitors on protein-protein interactions and on supramolecular organization of the tumor metabolism have also been studied 543 Abstracts V1-018P Grape seed procyanidins extract and oleoylestrone improve hepatic antioxidant enzyme systems in obese Zucker rats ´ V M Castrillejo1, M Romero2, M Esteve2, A Ardevol1, ´ ´ C Blade1, G Pujadas1, J Fernandez-Larrea1, M Blay1, ´ L Arola1 and M J Salvado1 Biochemistry and Biotechnology Department, University Rovira i Virgili, Tarragona, Spain, 2Nutrition and Bromathology Department, University of Barcelona, Barcelona, Spain E-mail: vanessa.castrillejo@ estudiants.urv.es Recent studies have revealed that pathologies, such as obesity and other metabolic disorders are associated with an imbalance between the rate of free radicals and Reactive Oxygen Species (ROS) production and that of their degradation, known as oxidative stress Using the obese Zucker rat as animal model of oxidative stress, we assayed the effect of procyanidins (GSPE), the most common flavonoids present in red wine, and oleoyl-estrone (OE), a natural hormone with slimming effects, on the hepatic antioxidant enzyme systems Antioxidant activities of Copper,Zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) were assayed by spectrophotometry Glutathione (GSH) content was also determinated spectrophotometrically in order to test the cellular redox status When we compared obese Zucker rats versus lean Zucker rats, the hepatic antioxidant activities of Cu, Zn-SOD, GPx, and GR in obese rats were significantly increased while GSH content was decreased, indicating an imbalance in the redox homeostasis In obese rats, treatment with GSPE and GSPE plus OE resulted in a decrease of antioxidant enzyme activities and in an enhancement of glutathione levels OE treatment decreased GPx activity and also enhanced glutathione levels So, all treatments recovered the redox homeostasis of obese rats GSPE acts as a free radical and ROS scavenger while OE, due to its slimming effects and mobilization of internal fat stores, seems to reduce the organic hydroperoxides formation As conclusion, we showed that GSPE and OE treatments improve the oxidative stress condition in obese Zucker rats by different ways and at different levels V1-019P Elucidation of the biosynthetic pathway of glucosylglycerate Characterization of key-enzymes J C Costa1, N Empadinhas2 and M da Costa2 Microbiologia, Zoologia e Centro de Neurocieˆncias e Biologia Experimental, Coimbra, Coimbra, Portugal, 2Microbiologia, Bioquı´mica e Centro de Neurocieˆncias e Biologia Experimental, Coimbra, Coimbra, Portugal E-mail: jcosta@cnc.uc.pt Glucosylglycerate (GG) is a structural analogue of mannosylglycerate (MG), a compatible solute that occurs predominantly in thermophilic or hyperthermophilic prokaryotes GG has been detected in low amounts in the Methanohalophilus sp FDF1 [1], in E chrysanthemi Strain 3937 [2] and in a H elongate Strain CHR63 [3] We explored public databases for genes similar to those involved in the synthesis of MG in most prokaryotes The ongoing genome sequence of Methanococcoides burtonii, a cold-adapted archaeon, closely related to the GG-accumulating Methanohalophilus sp FDF1, revealed a sequence with high homology to MG phosphatase [4] The analysis of the gene immediately upstream revealed conserved motifs with glucosyltransferases Altogether, these data raised the hypothesis that this glucosyltransferase gene could encode the enzyme responsible for the synthesis of GG The incubation of the cell-free extract carry- 544 ing the recombinant glucosyltransferase with GDP-glucose and 3-phosphoglycerate catalyzed the formation of a compound that was dephosphorylated to GG by alkaline phosphatase These observations allowed us to elucidate the biochemical pathway leading to the synthesis of GG in M burtonii, while proceeds in two steps through a phosphorylated intermediate and by the consecutive action of a glucosyl-3-phosphoglycerate synthase (GpgS) and a glucosyl-3-phosphoglycerate phosphatase (GpgP) The catalytic properties of the two enzymes are presented These results represent an important step towards the elucidation of the real physiological role of GG synthesis References Roberts MF, Lai MC, Gunsalus RP J Bacteriol 1992; 174: 6688–6693 Goude R, Renaud S, Bonnassie S et al Appl Envir Microbiol 2004; 70: 6535–6541 ´ Canovas D, Borges N, Vargas C et al Appl Environ Microbiol 1999; 65: 3774–3779 Empadinhas N, Marugg JD, Borges N et al J Biol Chem 2001; 276: 43580–43588 V1-020P Nitric oxide increases oxidative phosphorylation efficiency P Clerc, X Leverve and E Fontaine Laboratoire de Bioe´nerge´tique Fondamentale et Applique´e, University of Grenoble, Grenoble, France E-mail: pascaline.clerc@ujf-grenoble.fr The yield of the oxidative phosphorylation (P/O = JATP/JO2) can be modified both at short and long term Short-term modifications include activation of UCPs or exogenous uncouplers, which decrease P/O Long-term modifications can be achieved by nutritional modifications or by changing thyroid status, which decrease (hyperthyroidism, polyunsaturated fatty acids deficiency) or increase (chronic ethanol intoxication, hypothyroidism) P/O It has been recently reported that P/O inversely correlates with cytochrome c oxidase content in rat liver mitochondria Because nitric oxide (NO) inhibits cytochrome c oxidase, we have studied the effect of NO on P/O We found that a mild inhibition of the respiratory chain (20% inhibition of basal respiratory rate) using either NO donor DPTA-NONOate or cyanide led to a decrease in the maximal ATP production but to an increase in the P/O Such a mild respiratory chain inhibition did not affect membrane potential in the presence of oligomycin, but decreased the membrane potential for a given respiratory rate in phosphorylating conditions Therefore, the oxygen consumption required for a given membrane potential was always lower when cytochrome oxidase was partly inhibited We propose that NO is an endogenous messenger that may short term regulate the oxidative phosphorylation efficiency V1-021P Expression of hepatic transcription factors is modulated by dietary procyanidins ´ ` J Fernandez-Larrea, J M del Bas, A Ardevol, M Blay, ´ ´ G Pujadas, M J Salvado, L Arola and M C Blade Biochemistry and Biotechnology department, University Rovira i Virgili, Tarragona, Spain E-mail: josepmaria.delbas@estudiants.urv.es Dietary procyanidins, a group of polyphenolic compounds, have shown neuroprotective, cardioprotective and chemoprotective actions Although these beneficial properties were attributed firstly to its antioxidant capabilities, some studies have revealed their aptitude as signalling molecules, able to interact with intra- Abstracts cellular signalling pathways and thus modulating gene expression To study the effect of procyanidins in the gene expression of liver transcription factors and related cofactors, we treated healthy Wistar rats with an oral, high and non-toxic dose of grape seed procyanidins extract (GSPE) Microarray hybridization techniques were used to study the changes induced by GSPE on the liver gene expression profile h after treatment We found significant upregulation of small heterodimer partner (SHP), Max interacting protein (Mxi1), zinc finger protein 354A (Znf354a), Spalt-3, Spi-B, forkhead box E1 (Foxe1) and regenerating liver inhibitory factor-1 (RL-IF-1) in the GSPE group versus the control group On the other hand, the expressions of TRIP-Br2, Hepatocyte nuclear factor beta (HNF3b), High mobility group box (Hmgb1) and hematopoietically expressed homeobox (Hhex) were downregulated in the GSPE treated rats These findings reinforce the results found by our and other groups, which show that procyanidins are able to regulate gene expression Moreover, as transcription factors are key regulators of a wide variety of pathways, the regulation of the expression of these proteins can clarify the different actions that procyanidins exert on diverse processes as lipidic and glucose metabolisms or apoptosis V1-022P Transcriptional repression of human CYP17 by transforming growth factor beta is mediated by Smad signalling pathway N Derebecka-Holysz, T P Lehmann, M Holysz and W H Trzeciak Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznan, Poland E-mail: derebeckanatalia@o2.pl Cytochrome P450c17, encoded by CYP17 gene, is a component of an enzyme complex that catalyses a key reaction in the biosynthesis of glucocorticoids and androgens in the adrenal cortex CYP17 expression is stimulated at the transcriptional level by ACTH acting via cAMP/PKA signalling pathway Both basal and cAMP-induced levels of CYP17 mRNA are decreased by transforming growth factor beta (TGF-b), however the mechanism of TGF-b action on CYP17 expression remains unknown In the present study we have examined this mechanism in human adrenocortical cells H295R Using quantitative PCR we demonstrated that CYP17 mRNA level was decreased in H295R treated with both TGF-b and transcription blocker, actinomycin D in comparison with cells treated with TGF-b or actinomycin D alone Moreover, protein synthesis blocker, cycloheximide increased basal level of CYP17 expression and reduced the inhibitory effect of TGF-b Inhibitor of histone deacetylase, trichostatin A partially abolished stimulatory effect of forskolin on CYP17 expression but did not affect CYP17 inhibition by TGF-b In transient transfection experiments with plasmid containing -57 bp CYP17 promoter fragment and luciferase reporter gene we have demonstrated that transcriptional activity of this region was significantly reduced by TGF-b Moreover, overexpression of Smad7 evoked decrease of inhibitory effect of TGF-b on both -57 bp CYP17 promoter activity and endogenous CYP17 mRNA level We conclude that CYP17 repression by TGF-b is the multi-level process On the one hand, the repression of transcription is histone deacetylase-independent and involves type I TGF-b receptor On the other hand, experiments with actinomycin D imply that CYP17 mRNA level could also be decreased by TGF-b post-transcriptionally V1-023P Recombinational telomere elongation in telomerase deletion mutant of Kluveromyces lactis B Debelec1, G Kantarci1, W McRae2, M J McEachern2 and Z Topcu1 Department of Pharmaceutical Biotechnology, University of Ege, Faculty of Pharmacy, Izmir, Turkey, 2Department of Genetics, University of Georgia, Fred Davison Life Sciences Building, Athens, Georgia USA E-mail: bilged79@hotmail.com Eukaryotic cells lacking telomerase are sometimes capable of maintaining their telomeres via a telomerase-independent mechanism, which is shown to be recombination-dependent (Topcu et al, 2005) In this study we addressed the question whether a single telomere could be used to elongate the other telomeres We transformed telomerase-deficient K lactis cells with a single mutant telomeric repeat bearing URA3 as marker gene that gives rise an AccII recognition site for analyses of telomeric length regulation Transformed colonies were followed by scoring their growth phenotypes from to 0.5; from normal to senescing phenotype, under microscope with serial passaging Upon six successive passaging, the colonies were collected for DNA isolation to estimate their average telomeric lengths using Southern blot and compared to wild type K lactis Our results showed that the transformed K lactis cells manifested a varying cell growth capability One third of the colonies reached near-to-senescing status and around 50% of these cells recovered their growth phenotype following an initial growth decline while the remaining kept their initial phenotypes The deviation among Acc mutants emphasizes the significance of recombinational telomere elongation, an issue to relate with Alternative Lengthening of Telomeres (ALT), manifested in some human cancers Acknowledgment: This study was supported by Turkish Scientific Research Council (Grant number SBAG 2791) V1-024P Protein expression profile of Mesocestoides corti larva submitted to metabolic depression A Esteves, L Canclini and R Ehrlich Laboratory of Biochemistry, Department of Cellular and Molecular Biology, Faculty of Sciences, University of the Republic, Montevideo, Uruguay E-mail: aesteves@fcien.edu.uy Mesocestoides – corti – Cestoda: Cyclophyllidea – despite not being of sanitarian interest, it presents important properties as a model organism The easy manipulation in the laboratory makes of it the selected model to begin the studies of functional genomics in cestodes Metabolic rate depression constitutes an important survival strategy for many animal species submitted to variable conditions The central aim of this work is the study of metabolic pathways of parasite platyhelminths submitted to hypometabolic conditions The molecular comprehension of metabolism in these parasites is scarce; it could be an important source of key molecules with putative use as drug targets or vaccines We focused our attention to the implementation of large range of differential expression techniques to evidentiate changes in the expression profile as a result of the different stimuli response Previously we standardize the metabolic labelling of the larva stage of M.– corti – (tetrathyridia) and the bidimensional electrophoretic analysis Tetrathyridia were aseptically extracted from intraperitoneally infected mice, cultured by different times, and submitted to nutritional starvation, thermal and pH stressỈ Expression profile of each conditions was analyzed by 2D electrophoresis or by auto–radiography Since protein phosphorylation plays a central role in biochemical regulation of metabolic 545 Abstracts rate depression, protein phosphorylation was also analyzed in each situation In a next step, in order to identify the genes responsible of the observed expression profile changes subtraction libraries will be performed Acknowledgment: This work has been supported by CONICYT V1-027P Stannous chloride induces alterations in enzyme activities, lipid peroxidation and histopathology in male rabbit: antioxidant role of vitamin C V1-025P Activation of anticancer prodrugs by human glutathione transferases F M El-Demerdash1, M I Yousef1 and M A Zoheir2 Hormon Lab, Environmental Studies, University of Alexandria, Institute of Graduate Studies & Research, Alexandria, Egypt, Department of Pathology, University of Alexandria, Medical Research Institute, Alexandria, Egypt E-mail: feldemerdash@yahoo.com B Eklund1, A Elfarra2 and B Mannervik1 Department of Biochemistry, Dept of Biochemistry, Uppsala University, Uppsala, Sweden, 2Department of Comparativ Bioscienses, University of Wisconsin, Madison, Wisconsin USA E-mail: Birgitta.Eklund@biokemi.uu.se The thiopurine derivatives trans-acetylvinylthioguanine (tAVTG) and cis-acetylvinylthiopurine (cAVTP) are glutathione-activated prodrugs of 6-thioguanine (6-TG) and 6-mercaptopurine have (6-MP) The activation has been investigated kinetically with human glutathione transferases (GSTs) and glutathione (GSH) GSTs have broad substrate specificities and are involved in several cellular processes, but their main function is to detoxify xenobiotic molecules by conjugating them with GSH In contrast, the rationale behind the prodrugs studied is toxification The design of the prodrugs make them more tissue specific chemotherapeutic agents, since they will preferentially exert their cytotoxicity in tumour cells that have increased levels of GSH The reactions between GSH and the prodrugs are presumed to be GST-catalyzed, but activities of human GSTs have not previously been determined Cytosolic human GSTs investigated were obtained by heterologous expression in Escherichia coli and included members of the Alpha class: A1-1, A2-2, A3-3, A4-4; the Mu class: M1-1, M2-2, M3-3; Omega class: O1-1; Pi class: P1-1; and Theta class: T1-1 The results establish that all of the Alpha and the Muclass GSTs catalyze the reaction between GSH and the thiopurine produgs With tAVTG GST A4-4 and GST M1-1 are the most active enzymes With cAVTP GST M1-1 and GST M2-2 have the highest specific activities with all Alpha class members have specific activities lower by an order of magnitude GST A4-4, which has the highest activity with tAVTG, has markedly lower activity with cAVTP V1-026P Synthesis of phosphorodithoate compounds and study of their inhibitory effects on acetylcholinesterase (AChE) enzyme S Emadi, B Kaboudin and D Elhamifar Organic Lab 2, Department of Chemistry, Institute for Advanced Studies in Basic Sciences, Zanjan, Iran E-mail: emadi@iasbs.ac.ir Phosphorodithioates are among thiolic derivatives of phosphoric acid which show different degrees of toxicity and have found many application in pharmacology and pesticide research Their toxicity is primarily due to binding to the enzyme acetylcholinesterase which play pivotal role in neurotransmission process In this study the anticholinesterase activities of five synthesized compounds were investigated on acetylcholinesterase from electric eel, human erythrocytes by the spectrophotometric method of Ellman and his coworkers This study showed that pre-incubation of the enzymes with the synthesized compounds caused AChEs to show lower activities (10–30% lower than control sample) in compare with the enzyme mixture containing no synthetic phosphorodithioate compounds The work is in progress, and with special attention to the mode of interaction of these compounds with AChE, we are at the process of examining other synthesized phosphorodithioate compounds with presumably greater inhibitory effects 546 Stannous chloride (SnCl2) is widely used in daily human life, for example, to conserve soft drinks, in food manufacturing and biocidal preparations It has genotoxicity, immunotoxicity, neurotoxicity and generate free radicals Therefore, the present experiment was carried out to determine the effectiveness of L-ascorbic acid (AA) in alleviating the toxicity of SnCl2 on lipid peroxidation and some enzyme activities of male New-Zealand white rabbits Six rabbits per group were assigned to one of four treatment groups: mg AA and mg SnCl2/kg BW (control); 40 mg AA/kg BW; 20 mg SnCl2/kg BW (1/500 LD50); 20 mg SnCl2 plus 40 mg AA/kg BW Rabbits were orally administered their respective doses every other day for 12 weeks Liver and Kidney specimens were processed for histopathologic studies Results obtained showed that SnCl2 significantly (P < 0.05) induced free radicals in rabbit liver, testes, kidney, lung, brain and heart While, the activity of glutathione S-transferase (GST) and the levels of sulfhydryl groups (SH-groups) were decreased (P < 0.05) in all tested organs except brain and heart Aspartate aminotransferase (AST) activity was increased (P < 0.05) in liver and decreased in testes, but alanine aminotransferase (ALT) did not change The activities of alkaline phosphatase (AlP) and acid phosphatase (AcP) were decreased (P < 0.05) in liver, testes, kidney and lung Also, the activity of acetylcholinesterase (AChE) was significantly decreased in brain and plasma of rabbits treated with SnCl2 compared to control group Histopathologic studies showed marked changes in hepatocytes as well as proliferation of duct epithelium, dilatation and congestion of blood vessels as well as mononuclear inflammatory infiltrate The kidneys were also severely affected by SnCl2 the Bowman’s space was increased, with infiltration of renal parenchyma by mononuclear inflammatory infiltrate and changes in cells lining convoluted tubule Ascorbic acid alone significantly decreased the levels of free radicals, and increased the activity of GST and the levels of SH groups in tested organs except brain and heart While, the rest of the tested parameters were not affected Results showed that AA alleviated the harmful effects of SnCl2 This was proved histpathologically by the great improvement in liver and kidney histology were hepatocytes retained normal architecture with mild dilatation and congestion of blood vessels Bowman’s space of kidneys were almost normal, with normal lining of proximal and distal convoluted tubules In conclusion AA could be effective in the protection against stannous chloride toxicity V1-028P Evaluation of optimal conditions for arginase activity in streptozotocin-induced diabetic rats ´ ´ M Erytyr1, E Ercel2, S Yilmaz1 and S T Ozan1 ¸ Department of Biochemistry, Firat University, Elazig, Turkey, Province Veterinary Control Laboratory, Elazig, Turkey E-mail: mineerisir@yahoo.com The assay conditions needed to achieve maximal activity of liver and kidney arginase in diabetic and non-diabetic rats were inves- Abstracts tigated and compared The physicochemical and kinetic properties of liver arginase in diabetic and control rats were very similar, those of kidney arginase were significantly different It was found that pre-incubation temperature (68 °C), pre-incubation period (20 min), optimal pH (10.1) of liver arginase and Km (3.2) to its substrate, l-arginine, did not change in diabetic and nondiabetic rats As a consequence of diabetes, the optimum Mn2+ concentration for liver arginase only changed from to mm Although pre-incubation temperature and period for activation of kidney arginase in control rats was unnecessary, they were found to be 56 °C and 12 in diabetic rats pH profile of arginase in kidney of diabetic rats was different than that of control rats The Km value (6.7) of arginase for l-arginine in kidney is unchanged in diabetes, whereas a marked decrease in Vmax was found Optimum Mn2+ concentration (2 mm) for kidney arginase unchanged in diabetes The activity of arginase in liver of diabetic animals was higher 1.5- to 1.7-fold than that of controls Diabetes caused about 53% decrease of arginase activity in kidney of female rats, 26% in that of male These findings may suggest the idea that encoded arginases by separate gene loci may be differently affected by pathological and hormonal status V1-029P Characterization of Pur3, a monophosphatase from the puromycin biosynthetic pathway of Streptomyces alboniger ´ ´ ´ P Barrado, M B Sanchez, A Jimenez and M Fernandez Lobato ´noma of Madrid, Centro de Biologı´a Molecular, Universidad Auto Madrid, Spain E-mail: mfernandez@cbm.uam.es Pur3 is the product of a gene (pur3) from the puromycin biosynthetic pur cluster of Streptomyces alboniger A comparative analysis of its deduced amino acid sequence with those from data banks indicated that Pur3 is a phosphatase enzyme These comparisons detected in Pur3 a Mg++ binding motif and other motifs that are supposed to be implicated in the catalytic activity of a variety of phosphatases A structural modeling of Pur3, based on the SWISS-MODEL program, indicated that Pur3 contains these motifs located in a central core of 155 amino acids In contrast to other phosphtases, the G201, A226, G227 and G228 residues of Pur3 are located opposite to the active center at the external face of the molecule These findings might suggest an implication in regulatory function(s) The pur3 gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag and then was purified to apparent homogeneity by filtration through a TALON niquel column In accordance to the above referred to high similarity this recombinant protein showed monophosphatase activity It was able to dephosphorylate a wide variety of substrates amongst which lowest Km values were obtained for the putative intermediates of the puromycin ´ biosynthesis pathway 3’-amino-3-dAMP (Km = 1.37 mm) and puromycin aminonucleoside (Km = 1.40 mm) The identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway V1-030P Application of dotblotting for detecting the expression of p5cs gene in transgenic olive plantlets M Farzaneh, F Rastegar Jazi and N Motamed Biochemistry, Department of Biology, University of Tehran, Tehran, Enghelab, Iran E-mail: mfarzaneh57@yahoo.com Olive is an ever-green tree of the Oleaceae family and of the genus Olea, grown under a wide range of climates and soils all over the world in Iran, 50% of all growing areas encountered by high-salt concentration conditions and drought Water deficit caused by drought and high salinity has been an important factor limiting crop productivity To cope with these environmental stresses, application of molecular plant breeding is necessary In response to osmotic stress elicited by condition of high salt, the expression of P5CS gene altered in olive plants for this, constructs (S,E,X) were prepared by introducing P5CS sequence in to PBI121 Constructs cloned in binary vector were transformed to the disarmed Agrobactrium tumefaciens Olive embryo were transformed with A tumefaciens harboring the various gene constructs and salt-tolerance plants were regenerated in media containing adequate concentration of salt The total protein was extracted from fresh leaves of both control and transformed regen To probe the effect of p5cs overexpression on salinity stress tolerance, poly clonal antibody developed in rabbit against p5cs protein The result of immunological methods for specific detection of p5cs gene, strongly indicated P5CS up-regulation in stressed transformed plants vs non-transformed plants, erated plants V1-031P Repression of SOX6 transcriptional activity by SUMO modification R Fernandez-Lloris1,2, N Osses1, E Jaffray2, L Shen2, O A Vaughan2, D Girdwood2, R Bartrons1, J L Rosa1, R T Hay2 and F Ventura1 4171, Ciencies Fisiologiques II, Universitat de Barcelona, Hospitalet De Llobregat, Spain, 22.08, Biomolecular Sciences, University of St Andrews, St Andrews, UK E-mail: rakylloris@yahoo.es SOX6, a member of the SOX family of Sry-type HMG transcription factors, plays key functions in several developmental processes, including neurogenesis, sex determination and skeleton formation In this report, we show that SOX6 is covalently modified in vitro and in vivo by SUMO1, and on two consensus sites (IK364NE and VK377DE) Mutation of both lysines to arginine abolished SOX6 sumoylation and increased SOX6 transcriptional activity as well as SOX6/SOX9 synergistic activation of the Col2a1 enhancer SUMO dependent repression of SOX6 transcription was demonstrated by Uubc9 overexpression whereas siRNA to Ubc9, cotransfection of a catalytically inactive UBC9 or a SUMO specific protease increased SOX6 transcriptional activity Immunofluorescence analysis showed a predominant diffused nuclear localization of SOX6 when expressed alone Coexpression of SOX6 with SUMO1 and/or SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with PML PML body localization of SOX6 was abolished by mutations in SOX6 sumoylation sites Thus, SUMO modification of SOX6 alters its subnuclear localization and leads to transcriptional repression V1-032P Usage of the mannose-binding protein (lectin) Con A and the fucose-binding lectins UEA-I and PA-IIL for the study of glycoproteins from human milks and other body fluids N Gilboa-Garber, B Lerrer and E Lesman-Movshovich Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel E-mail: garben@mail.biu.ac.il Human milk, saliva, and seminal fluid are rich in glycoconjugates that act as decoys to which pathogens are attracted instead of binding to the host cells for infection initiation Such compounds function in the front line against infections in adults, and are especially 547 Abstracts important for the newborn protection The fucose-binding Ulex europaeus lectin (UEA-I), which is selective for the H type epitope, detects only the glycans of the ’secretors’ who contain H epitope in their secretions, The ‘‘non-secretor’’ body fluids, that not express the same H epitope, contain other fucosylated glycoforms, which are not detected by UEA-I but might be detected by other fucose-specific lectins The present communication describes the usage of fucose-binding lectin of Pseudomonas aeruginosa (PA-IIL), which may contribute to this pathogen adhesion, as a probe for fucosylated anti-adhesive glycans in the human body fluids PA-IIL was found to display highest sensitivity to milk glycans, followed by those of seminal fluids and salivas, while UEA-I was only inhibited by ‘‘secretor’’ body fluids, most strongly by seminal fluids, followed by milks and salivas Conclusions: Body fluids are rich in fucosylated glycans, detectable by UEA-I and the P aeruginosa lectin PA-IIL These lectins are useful as probes for identification of glycoprotein glycans that might efficiently block fucose-dependent adhesion of that bacterium and of other pathogens Acknowledgments: These results are parts of the PhD theses of B.L and E.L.-M V1-033P NMR Conformational studies of the interaction between the V3 loop of HIV-1 coat glycoprotein gp120 and chemokine receptor CCR5, at peptide level P A Galanakis1, N Kandias1, G A Spyroulias1, M Sioumpara2, D Morikis3, A Rizos4 and E Krambovitis2 Department of Pharmacy, University of Patras, Patras, Greece, Department of Applied Immunology & Biochemistry, Institute of Molecular Biology & Biotechnology, FORTH, Heraklion, Greece, Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, CA, USA, 4Department of Chemistry, University of Crete, Heraklion, Greece E-mail: pgalanak@upatras.gr Recent convincing evidence indicates that the majority of the cells that die due to HIV-1 are not actually infected by the virus Instead, HIV-1 or its components lead these cells to programmed cell death by altering their physiological function (Zafiropoulos A et al BBRC 2001; 284: 875–879] after the activation of apoptotic mechanisms Ionic interactions between the variable V3 domain of the HIV-1 coat glycoprotein gp120 and the amino terminal of the chemokine receptor CCR5 (Baritaki S et al BBRC 2002; 298: 574–580] play a prominent role in this process Standard multidimensional and multinuclear NMR spectroscopy was applied to probe the structural and physicochemical determinants of three representative peptides from V3 domain of the HIV-1 and a 22-residue peptide, representing the amino terminal of the chemokine receptor CCR5, in their free or interacting state Titration of CCR5 peptide with V3-peptides was performed in NMR tube, at 286K 1D 1H NMR spectra and 1H-15N HSQC were recorded after each addition of V3 peptides Analysis of the NOESY maps, acquired for free and interacting peptides at 278K, suggests that the free CCR5 construct is structured, giving rise to numerous NOEs Data analysis of HSQC and NOESY spectra verifies the interaction of the V3-CCR5 peptide constructs and suggests a remarkable role for the (i) 7-residue CCR5 N-terminal domain and particularly for Tyr3, which is the first among the four N-terminal tyrosines of CCR5, and (ii) for the 7/9-residue V3 N-terminal peptide fragment, which especially in V3 LAI peptide is rich in basic residues 548 V1-034P Construction of a double copy (DC) retroviral vector for efficient expression of small hairpin RNAs (shRNAs) in avian cells P Gooz and S Hoffman Department of Medicine, Rheu, Medical University of South Carolina, Charleston, SC, USA E-mail: goozp@musc.edu RNA interference (RNAi) has recently become a powerful tool for studying gene functions in vitro and in vivo The human H1 promoter is widely used for expression of shRNAs within the cells These shRNAs are processed into double stranded siRNA molecules that lead to specific degradation of their target mRNAs We wanted to validate the use of the H1 promoter in avian systems, which are useful models for studying angiogenesis and embryonic development We directly compared the green fluorescence protein (GFP) silencing efficiency of the human H1 promoter in the quail QCE-6 and the human HEK 293 cell lines, which were previously transfected with GFP We found that in the avian cell line the silencing effect developed slower (6 days vs days) and the silencing efficiency was lower (~60% vs >95%), compared to the 293 cells On Northern blot we were not able to detect the shRNAs transcribed from the H1 promoter in the QCE-6 cells while the 293 cells produced high amount of the fully processed siRNA product Thus we hypothesized that the human H1 promoter cannot drive sufficiently high level transcription of shRNA products in avian cells To increase the level of transcripts in QCE-6 cells beside the single copy (SC) vector we constructed a double copy (DC) retroviral vector by cloning the human H1 promoter into the U3 region of the 3’LTR of the pLEGFP-N1 retroviral vector (Clontech, Palo Alto) This vector expresses eGFP that we used to assess infection efficiency and to obtain pure populations of cells that express the shRNA by Fluorescence Activated Cell Sorting Then we designed several shRNAs targeting the 75 kDa gelatinase that is expressed endogenously by the QCE-6 cells and cloned them into both SC and DC vectors Northern blot analysis showed a significant increase in the expression of siRNA molecules when the cells were infected with the DC retrovirus as compared to the single copy vector Moreover one of these constructs very effectively (>98%) inhibited the expression of 75 kDa gelatinase when expressed as a DC design The maximum inhibition developed about days after infection and the silencing effect was stable for more then 10 passages (~2 month) of the cells We concluded, that we constructed a retroviral vector that can be used for highly efficient gene silencing in avian cells V1-035P Changes in antioxidant enzyme activities, glutathione and lipid peroxidation levels in rat erythrocytes with age S Gumuslu and O Ozturk Department of Biochemistry, Akdeniz University, Faculty of Medicine, Antalya, Turkey E-mail: sgumuslu@akdeniz.edu.tr We have studied the activities of enzymes [glucose-6-phosphate dehydrogenase (G-6-PD), copper,zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST)], and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and thiobarbituric acid-reactive substances (TBARS) in rat erythrocytes and estimated the ratio of GSH/GSSG and the redox index Male Wistar rats at ages of 1, and 12 months were used The activities of G-6-PD and Cu,Zn-SOD, the levels of GSSG and TBARS were increased, while the activity of Se-GSHPx and the level of GSH were decreased with age GSH/GSSG ratio was significantly decreased with age We found a positive Abstracts profile of Per1 mRNA at P10 but not at P3 At P10, the daily profile of PER1 production was also slightly modulated by the photoperiod The data demonstrate rhythmic production of PER1 in the SCN at P3 and gradual maturation of the rhythm between P3 and P10 The sensitivity of Per1 expression and PER1 production to photoperiod appears between P3 and P10 At P10, the modulation of the rhythmicity by photoperiod is still only partial one as compared with adult animals Acknowledgments: This work was supported by grant Nos 309050350 and 30902D093 V1-055P Expression of actinomycin synthetase genes in Streptomyces chrysomallus is controlled by a promoter with homology to sigma70consensus of E coli M Lang, I Ortel, J Vater and U Keller Laboratory of Dr Keller, Institut fuăr Chemie, Fachgruppe Biochemie und Molekulare Biologie, Technical University of Berlin, Berlin, Germany E-mail: julianmanuellang@web.de It was shown previously that actinomycin synthesis in actinomycin-producing Streptomyces antibioticus is repressed by glucose In order to analyze the regulation of expression of actinomycin synthetase genes in the actinomycin producer S chrysomallus, we have mapped the transcriptional start points in the intercistronic region between the oppositely arranged actinomycin synthetase genes The results indicate divergently arranged promoter regions directing expression of the genes acmA and acmB encoding actinomycin synthetases I and II, respectively For acmA two promoters and for acmB one promoter were deduced with -10 and -35 boxes showing high similarity to the sigma 70-dependent promoters from streptomycetes and E coli In particular, promoters showed sequence similarity to promoters of streptomycete genes known to be repressible by glucose such as of amylases or of malE, the maltotriose uptake system from Streptomyces coelicolor Several directed and inverted repeats in the acmA/B promoter region were reminiscent of operators found in the promoter regions of these genes indicating binding sites for regulators of acm synthesis Heterologous expression of acmA in Streptomyces lividans under the control of its own promoter in a high copy plasmid was repressible by glucose and stimulated by maltose By contrast, expression of acmA in strain JN241, a S lividans strain lacking Reg1, the S lividans ortholog of MalR repressor, was derepressed in the presence of glucose Surprisingly, in Streptomyces chrysomallus expression of the actinomycin synthetase genes showed maximum activity during growth phase with sharp decline after transition in the stationary phase regardless whether glucose or maltose served as carbon source This indicates constitutive expression of the actinomycin synthetase genes in S chrysomallus V1-056P Expression of proinflammatory cytokines IL-23 in mice infected with Leishmania major M Lamsal1, F T Cottier2 and P Launois2 Department of Biochemistry, BP Koirala Institute of Health Sciences, Dharan, Sunsari Nepal, 2WHO/IRTC, Department of Biochemistry, University of Lausanne, Lausanne, Switzerland E-mail: madhablamsal@yahoo.co.uk Introduction: IL-23 is a heterodimeric proinflammatory cytokine which shares the P40 subunit of IL-12 along with another novel chain p-19 The IL-12Rb1 subunit of the IL-12 receptor is also shared by IL-23, in addition to its unique receptor subunit IL-23 R Involvement of IL-12 in protective immunity through Th-1 response might in part or wholly be contributed by IL-23 The present study was undertaken to explore the roles of IL-23 in the murine models infected with Leishmania major Aims and objectives: To detect the expression of IL-23 in murine models infected with Leishmania major Materials and methods: C57BL/6 and BALB/C mice were infected by footpad inoculation of million parasites of L major (LV39) Animals were sacrificed after h, h, 24 h, days, 10 days and 40 days after inoculation Controls without Leishmania infection were also included Lymph nodes & spleen were collected, homogenized and RNA was prepared The cDNA, amplification of HPRT, p-19, p-35, p-40, p-28, EBI3 & IFN-b was done by PCR, products detected on agarose gels and recorded by KODAK Scanner Quantification of the PCR product was done using Image J program Results and discussion: IL-23 in BALB/C was expressed in the initial hour of inoculation but the level fell down after days as compared to the C57BL/6 Critical phase of IL-23 transition was observed between h and day during which the IL-4 upsurge also occurs IL-23 and IFN-b may counter balance the effect of IL-4 thereby leading to Th1 modulation which may be necessary for protective immunity in C57BL/6 as compared to the BALB/C Conclusion: This study shows the involvement of IL-23 in the protective immunity against Leishmania major Acknowledgment: This work was supported by MCBN-UNESCO V1-057P Hydrolysis catalyzed by human prostatic acid phosphatase: cooperativity in substrate binding and cooperative inhibition by tartrate E Luchter-Wasylewska, M Chrzaszcz, W Halaburda, S Gaweda and P Kolodziejczyk Institute of Medical Biochemistry, Jagiellonian University, Krakow, Poland E-mail: mbwasyle@cyf-kr.edu.pl Human prostatic acid phosphatase (PAP) exhibits positive cooperativity in substrate binding [1, 2] Degree of cooperativity grows with increased enzyme concentration Ligand-induced concentration-dependent dissociation-association of active PAP oligomeric forms (monomer–dimmer–tetramer), suggested from kinetics, was confirmed in physicochemical studies [3] Further studies on kinetics were performed using o-carboxyphenyl phosphate as substrate Positive cooperativity was found for this substrate Degree of cooperativity and half saturation constant both grow, but catalytic constant remains constant, when enzyme concentration increases L(+)-tartrate anion, a transition state analogue, is a stereospecific inhibitor of PAP Inhibition studies of hydrolysis of 1-naphthyl phosphate and phenyl phosphate have been performed, after the cooperativity in substrate binding of PAP was stated Kinetic constants were calculated by fitting the experimental results to Hill equation Tartrate was found not as a simple competitive inhibitor of PAP, as it was earlier stated, but as the non-linear competitive inhibitor Half-saturation constant grows, Hill cooperation coefficient drops but the catalytic constant remains constant in the presence of inhibitor (I) The shape of nonlinear Dixon plots (1/vo vs [I]) depends on both: substrate and enzyme concentrations References Luchter-Wasylewska E Biochim Biophys Acta 2001; 1548: 257–264 Luchter-Wasylewska E, Iciek M J Coll Interf Sci 2004; 273: 632–637 Luchter-Wasylewska E, Wasylewski M, Rohm KH J Prot ă Chem 2003; 22: 243–247 555 Abstracts V1-058P Laser hemotherapy influence on structural and functional properties of blood proteins in brain ischemia J I Musienko Biochemical Laboratory, 5th Clinical Hospital, University of Neurology, Neurosurgery and Physiotherapy, Minsk, Belarus E-mail: musienko2002@mail.ru Brain ischemia/reperfusion is characterized by disturbances of redox and acid-base metabolism Intravenous laser irradiation of blood (ILIB) is expedient pathogenetically in cerebral ischemia This work deals with study of mechanism of interaction between low power laser radiation and blood proteins with estimation of acid-base status (ABS) and blood oxygen transport (BOT) in ILIB after experimental local ischemia of brain (LIB) LIB was made by bilateral h occlusion of common carotid arteries under thiopental anesthesia He-Ne laser radiation power of light guide inserted in otic vein was 2.5 mW Rabbits with LIB underwent by 10-min exposure for days Optical spectroscopy method is used to determine proteins reaction on LPLR with analysis of absorption spectrum of hemoglobin and plasma proteins in infra-red (IR) and ultra-violet (UV) spectra Parameters of ABS and BOT [actual acidity (pH), bases excess (BE), oxyhemoglobin dissociation curve (ODC) and hemoglobin saturation] were measured in jugular vein by gas analyzer The hemoglobin-oxygen affinity (HOA) was assessed by P50(blood pO2 under its 50% saturation by O2) Postischemic period was characterized by metabolic acidosis with worsening of BOT Laser application after LIB modeling contributed for normalizing of ABS findings and tendency to BOT improving compared to controls, what showed ODC shift leftwards and increasing hemoglobin affinity to oxygen by 10% Laser hemotherapy causes to marked changes of spectral characteristics of hemoglobin, porphyrin and plasma proteins both in IR and in UV diapasons ILIB decreases luminescence intensity of plasmatic proteins It was established that improvement of oxygen transport under ILIB influence in brain ischemia is connected to changes of blood protein structure including hemoglobin V1-059P Purification and characterization of alpha amylase inhibitor from Schinopsis brasiliensis seeds (SbAlpha-AI) actives against insect pests V L Silva1, F R Brenardo1, D V Duarte1, R B Gomes1, A C Treitler1, C B Bloch-Junior2, M V Prates2, ´ M F Grossi-de-Sa2 and F R Melo1 Laboratory of Quı´mica e Bioquı´mica, Department of Agronomia e Zootecnia, University Unia Pioneira de Integraca Social, Brası´¸ ˜o ˜o lia, Distrito Federal Brazil, 2Laboratory of Bioquı´mica e Controle de Pragas da Agricultura, Biotecnologia, Centro Nacional de Recursos Gene´ticos e Biotecnologia – EMBRAPA, Brası´lia, Distrito Federal Brazil E-mail: etemelo@cenargen.embrapa.br Several studies have been suggested that amylases inhibitors could be a seed protective agent against insect attack These proteins hold great promise for crop protection using biotechnological technical In this work, we searched for amylases inhibitors actives against insect pests, using Schinopsis brasiliensis seeds as study source This Brazilian savannah tree represent an important source of commercially available wood, nevertheless, few biochemical studies about proteins composition were done Thus, a crude extract was prepared using seeds flours and 0.1 m NaCl, 0.01 m HCl solution with continuous stirring The material was centrifuged at 10 000 ·g, °C for 20 and the supernatant was used on enzymatic assays against amylases from Acanthoscelides obtectus (common bean weevil), Spodoptera fugiperda (fall 556 armyworm) and Anthonomus grandis (cotton boll weevil) larvae, according to Bernfeld (1955) The results shown, for the first time, inhibitory activities of 98, 35 and 15% for the three insects mentioned above, respectively Then, this extract was applied in a chromatography column, Red-Sepharose and the retained peak was dialyzed, lyophilized and applied in a reverse-phase HPLC column (Vydac C-18) using a linear acetonitrile gradient (0– 100%) to elute the protein peaks The major peak was applied in a reverse-phase HPLC column Vydac C-12, using different successive acetonitrile gradient concentration until to obtain the purified inhibitor, which was named SbAlpha-AI This protein was visualized by SDS-PAGE and the molecular mass was determined by mass spectrometry (MALDI TOF/TOF) at 12 773 Da Now, this inhibitor is being sequenced in a PPSQ-23 sequencer from Shimadzu Co and future in vivo assays could to confirm the potential insecticide of this new inhibitor V1-060P Nuclear proteins of a cytoplasmic-replicating avian reovirus J Martinez-Costas, L Vazquez-Iglesias, C Costas-Iglesias and J Benavente Martı´ nez Laboratorio de Virologia Molecular, Departamento de Bioquimica y Biologia Molecular Facultad Farmacia, University of Santiago de Compostela, Santiago de Compostela, Spain E-mail: bnjmm@usc.es Reoviruses are non-enveloped, icosahedric viruses, with a segmented, double-stranded RNA (dsRNA) genome, which is enclosed within a double-protein capsid shell These viruses replicate and assemble within dense cytoplasmic globular inclusions termed viral factories, which are initially formed by the non-structural protein muNS Fluorescence microscopy analysis of reovirusinfected chicken embryo fibroblasts (CEF) revealed that both the inner shell protein sigmaA and the nonstructural protein p17 are present within the nucleus of the infected host Protein p17, which is a small (17 kDa) non-structural viral protein encoded by the second cistron of the S1 gene, has been found to be a transcription-dependent, nucleocytoplasmic shuttling protein, that exits the nucleus through a CRM1-independent pathway On the other hand, the dsRNA-binding protein sigmaA was detected in both the nucleolus and cytoplasm of infected cells The fact that the two proteins are also detected within the nucleus of transiently transfected cells indicates that no other viral factors are required for these proteins to be transported into the nucleus Finally, mutagenic analysis allowed us to identify specific nuclear localization signals in the primary amino acid sequence of the two proteins V1-061P Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa by the disulfiram metabolite methyl diethylthiocarbamoyl sulfoxide ˜ R A Munoz-Clares1, V J Zaldı´ var-Machorro1, R Velasco-Garcı´ a2, P Demare3, M R Ramı´ rez3 and I Regla3 Department of Biochemistry, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, D.F Mexico, Laboratory of Osmoregulation, FES Iztacala, National Autonomous University of Mexico, Mexico City, D.F Mexico, 3Laboratory of Drugs Synthesis, FES Zaragoza, National Autonomous University of Mexico, Mexico City, D.F Mexico E-mail: clares@servidor.unam.mx Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) catalyses the NAD(P)+-dependent oxidation of betaine aldehyde to glycine Abstracts betaine, an efficient osmoprotectant In the human opportunistic pathogen Pseudomonas aeruginosa this reaction participates in the catabolism of choline or choline precursors, which are abundant in the infected tissues As osmotic stress is frequently present in these tissues, glycine betaine may play the dual role of an osmoprotectant and a metabolic intermediate in these bacteria BADH inhibition would block choline catabolism, seriously affect the ability of the bacteria to withstand osmotic stress, and lead to the build-up of the toxic betaine aldehyde Thus, BADH might be a potential drug target, which is much needed given the high prevalence of antibiotic resistant P aeruginosa strains We have previously shown that disulfiram, a drug used for years in treatment of alcoholism, inactivated this enzyme in a time- and dose-dependent manner by forming a mixed disulfide with the catalytic cysteine residue Since disulfiram is readily metabolized in vivo to methyl diethylthiocarbamoyl sulfoxide (MeDTC-SO) we have now tested in vitro this compound as a BADH inhibitor and found it to be about 100-fold more potent than disulfiram At neutral pH, inactivation cannot be reverted by a physiological concentration of glutathione and takes place even in the presence of the reductant Moreover, NAD(P)+ increases the inactivation rate These results suggest that MeDTC-SO might inhibit P aeruginosa BADH in vivo by forming stable carbomyl-cysteine adducts Acknowledgments: This work was supported by CONACYT grant 37820 and DGAPA-UNAM grant IN206505 V1-062P The survey of changes in complete blood count (CBC) and RBC indices at different temperatures up to 48 h M Mahmoodi1, G Asadikaram1, M Khaksari2, N Seyedi1, A Rahnema3, M Hajizadeh1, M Mirzae1 and A Sayadi4 Department of Biochemistry and Biophysics, Faculty of Medicine, Rafsanjan Medical University, Rafsanjan, Iran, 2Department of Physiology, Faculty of Medicine, Rafsanjan Medical University, Kerman, Iran, 3Department of Pathology, Faculty of Medicine, Rafsanjan Medical University, Rafsanjan, Iran, 4Department of Psychology, Faculty of Medicine, Rafsanjan Medical University, Rafsanjan, Iran E-mail: mahmoodies@yahoo.com Background and objectives: CBC is one of the most common and conventional blood test that physicians usually request The result of this test is effective in physician’s decision to diagnosis and treatment However the results of this test are affected by different factors such as, the temperature and duration of incubation, duration between sampling and doing the test, patient situation, therefore the aim of this survey was to evaluate the effect of temperature and time of incubation on CBC, RBC indices and white blood cells (WBC) differential count in a crosssectional study Materials and methods: Blood samples were taken from 30 healthy medical students of Rafsanjan University (15 males and 15 females) The samples divided into three parts; CBC were done on the samples up to 48 h incubation at temperature of 25, 30, and 37 °C at the time of sampling, and after 2, 8, 24 and 48 h Data were statistically analyzed and the following results were obtained Results: RBC count, hematocrit, MCH, percent of monocytes and eosinophils were constant in different temperatures, WBC count, MCHC, hemoglobin, platelets count, the percent of lymphocytes and neutrophils were constant up to 24 h and then tend to increase with increasing temperature except lymphocytes percent that tend to decrease MCV decreased with increasing temperature up to h and then increased WBC, hematocrit, MCV, platelets count, and neutrophils’ percent tend to increase by the time of incubation, but RBC count, MCHC, lymphocytes’ percent decreased Hemoglobin, MCH, and the percent of monocytes and eosinophils were constant Conclusion: The finding of this survey showed that some of CBC parameters can be changed with the incubation, therefore it is better to the CBC test after blood taking as soon as possible V1-063P Alteration of the expression of heterotrimeric G protein b subunits and the activities of effectors by stress in rat brain C.-S Myung1, H B Nguyen1, S I Yang2 and S Y Lee2 Department of Pharmacology, Chungnam National University College of Pharmacy, Daejeon, South Korea, 2Department of Pharmacology, Sung Kyun Kwan University College of Pharmacy, Suwon, South Korea E-mail: cm8r@cnu.ac.kr Various heterotrimeric G protein bc subunits (Gbc) are regionspecifically expressed in brain where associated with ‘‘stressaxis’’ and these specific Gbc may play an important role in regulating stress This study was designed to examine the changes of Gbc expression and Gbc-mediated effector activity in rat brain by stress Experimental stress was induced by immobilization (2 h/day for d) and the level of mRNAs and proteins for Gbs (Gb1–5) was measured by using reverse transciptase-polymerase chain reaction (RT-PCR), immunohistochemistry and western hybridization in five different regions of rat brain including frontal cortex, striatum, hypothalamus, hippocampus, and cerebellum The ability of brain tissue to activate effectors such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein (MAP) kinase was measured and compared in between non-stressed and stressed rats Immobilization caused the stress to experimental animals by increasing in the level of plasma corticosterone, and corticotropic-releasing factor (CRF) and preopiomelanocortin (POMC) in hypothalamus Although there was difference to some extent among Gbs, all Gbs of both mRNA and proteins expressed all five different regions of brain tested The expression of Gb1–5 was increased by stress, especially in hypothalamus and hippocampus, which induced the activation of effectors such as PI3K/ Akt and Erk phosphorylation These results suggest that stress activate the Gbc-mediated PI3K/Akt and MAP kinase signaling pathway V1-064P Functional proteomics of MAPK-regulated cytoskeleton rearrangements in yeast M V Metodiev Biological Sciences, University of Essex, Colchester, Essex, UK E-mail: mmetod@essex.ac.uk Signal-induced rearrangements of the cytoskeleton were found to correlate with protein phosphorylation controlled by pheromone inducible MAPK in Saccharomyces cerevisiae A set of cytoskeleton associated MAPK target proteins was identified by phosphoaffinity capture and nano-LC/MS/MS analysis Some of these phosphoproteins localize in an asymmetric fashion during the activation of the pathway, which is likely to contribute to the establishing and maintenance of the axis of polarity The possible molecular mechanisms responsible for this asymmetry are discussed 557 Abstracts V1-065P HPV DNA testing in Cyprus: epidemiology and identification of novel HPV types P I Neophytou1, V Tanos1, A Chrysanthou1,2, S Lerios1,2, K E Tsitsanou1 and D Philippidou1, S K Ulucay1, S Spyrou1, G Maos1, M Phylactou1, I Andreou1, S Demetriou1, S Ellinas1, J Papageorgiou1 and A A Albayrak1 Mendel Center for Biomedical Sciences, Egkomi, Cyprus, Institute of Reproductive Medicine, Nicosia, Cyprus E-mail: pav@mendelcenter.org Human papilloma virus (HPV) infection is the cause of cervical cancer We developed an HPV DNA test that includes high-resolution typing and semi-quantitative measurement of viral load A higher viral load was associated with an increased risk for cervical intraepithelial neoplasia (CIN) We conducted a prospective, cross-sectional epidemiological study of Cypriot women and identified the most frequent HPV types in Greek-Cypriot women (6, 16, 66, 53, 31) and in Turkish-Cypriot women (58, 66, 11) Turkish-Cypriot women had a much higher prevalence of HPV infection (27%) than Greek-Cypriot women (15%), which is explained by poor population screening in the north part of Cyprus The main epidemiological factors of increased risk in Greek-Cypriot women were smoking and multiple sexual partners These two factors were strongly associated with each other and with HPV-positivity in Greek-Cypriot women, but not in Turkish-Cypriot women DNA sequencing of putative HPV’s displaying novel RFLP’s resulted in the identification of two novel types, four subtypes, 10 variants and four human loci amplified by HPV-specific primers MY09 and MY11 Identification of the latter allows for an increased specificity of our HPV DNA test by elimination of false positives In conclusion, we developed an HPV DNA test with high specificity and used it to conduct an epidemiological study in Cyprus We identified the prevalent types in our population, the main factors of risk and a number of novel HPV types These results will allow the design and implementation of more effective cervical cancer prevention programs V1-066P Methionine sulfoxide reductase B: catalysis and structural specificity F Neiers, A Olry, S Boschi-Muller and G Brnalant Laboratoire MAEM UMR 7567 CNRS UHP, Faculte´ des Sciences, Univerite´ Henri Poincare´, Nancy, France E-mail: fabrice.neiers@maem.uhp-nancy.fr Methionine oxidation into methionine sulfoxide is known to be involved in the etiology of a panoply of pathologies and to exert regulatory effects on the function of the modified proteins The oxidation process can be reversed by enzymes named methionine sulfoxide reductases (Msrs) Msrs belong to two structural-unrelated classes, MsrA and MsrB which display opposite stereoselectivity towards the R and S sulfoxide function They share a similar three-step chemical mechanism including, (i) formation of a sulfenic acid intermediate with concomitant release of one mol of Met per mol of Msr, (ii) formation of an intramonomeric disulfide bond, the rate of which has been shown to be limited by that of the sulfenic acid intermediate and (iii) reduction of the disulfide bond by thioredoxin This latter step has been shown to be rate-limiting in the overall process Biochemical and kinetic data will be presented which illustrate the role of the aminoacids which are involved in the catalysis of the reductase step and in the substrate specificity of MsrB from Neisseria meningitidis 558 V1-067P Metabolic control and treatment of malignant growth: tumor, regularities of amino acid imbalance formation and anti-cancer drug ‘‘Ukrain’’Ò L I Nefyodov1, Y M Doroshenko1, V Y Smirnov1, A V Karavay2 and J W Nowicky3 Department of Biochemistry, Yanka Kupala State University, Grodno, Belarus, 2Oncology Department, Medical University, Grodno, Belarus, 3Ukrainian Anti-Cancer Institute, Vienna, Austria E-mail: l.nefyodov@grsu.by Aim: The aim of the work was elucidation of the mechanisms of the anticancer effects of preparation Ukrain Methods before and after the treatment with Ukrain in experimental (W-256, SM-1 and PC-1 tumors) and clinical trials (cancers of the mammary gland, urinary bladder and prostate) were comparing the processes of formation of the pool of free amino acids and their derivatives in the blood plasma and biopsy materials of the tumor and unchanged tissues, as well as processes of protein biosynthesis, gluconeogenesis and energy production in tumor cells, accumulation of Ukrain in tumor tissue and elimination the malignant growth-induced metabolic imbalance Results: In experimental and clinical studies Ukrain was proven to be safe in use and highly effective against cancer It inhibits protein synthesis in tumor cells, selectively gets accumulated in tumor tissue after i.v administration, eliminates the malignant growth-induced metabolic imbalance As an anti-cancerous drug Ukrain acts destructively on the metabolic processes of the tumors, manifested in disturbances of the metabolism, involves a control of the transport and intermediate metabolism of amino acids governing the activities of malignant growth processes Its cancerostatic action realized by controlling the amino acid pool formation in the host organism and in the tumor and prevented active transport of these compounds into the tumor tissue, causing inhibition of the activities of some essential metabolic processes Conclusion: On the basis of the literature data and the results of our studies we suggest that the biological activity of Ukrain in malignant growth is realized according to several relatively independent mechanisms: the cancerostatic effect of the agent mediated by a decrease in activities of tumor growth limiting metabolic reactions, angiogenesis inhibition and collagen production enhancement to form a connective tissue capsule, that prevents growth and spreading, around the primary tumor as well as activation of proteolysis and degradation of cancerocyte structural elements; the immunomodulating and immunocorrecting effects of the substance and local immunity activation; alleviation of the metabolic imbalance (the ratios in the rates of endogenous compound chemical conversions along different pathways to form and expend energy in normal cells) by affecting the regulatory links in metabolic flows in the tumor-bearing organism (metabolic correction and therapy) V1-068P Is H2O2 an activator or an inhibitor of NF-kappaB activation induced by TNF-alpha? V Oliveira-Marques1, L Cyrne1, H S Marinho1 and F Antunes1,2 Centro de Quı´mica e Bioquı´mica, Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias da Universidade de Lisboa, Lisbon, Portugal, 2Instituto de Investigaca Cientı´fica Bento da ¸ ˜o Rocha Cabral, Lisbon, Portugal E-mail: vmmarques@fc.ul.pt At high concentrations reactive oxygen species are responsible for oxidative damage, but at moderate/low concentrations they Abstracts are involved in redox regulation [1] H2O2 is commonly used to create oxidative conditions in cells Usually, cells are exposed to a bolus addition of H2O2 i.e., a high dose of H2O2 is added at the beginning of the experiment High concentrations of H2O2 are required to compensate its natural consumption by cells and still induce any possible oxidative effect When exposing cells to a steady state (s.s.) of H2O2 lower concentrations can be used, which is a better approach to mimic the conditions in vivo [2] We studied the effect of H2O2 and TNF-a on the NF-jB activation pathway in MCF-7 cells In the bolus approach, H2O2 acts as a negative modulator in p65 subunit translocation to the nucleus induced by TNF-a On the contrary, when using the s.s approach, the stimulatory effects of H2O2 and TNF-a add up, or there is even a slight synergism When cells are pre-incubated with H2O2 in s.s before TNF-a exposure, a negative modulation occurs, similar to that observed after the bolus addition These differences justify some contradictory results found in the literature about NF-jB activation, which are probably related with the conditions used to subject cells to H2O2 References Droge W Physiol Rev 2002; 82: 4795 ă Antunes F, Cardenes E Free Radic Biol Med 2001; 30: 1008– 1018 V1-069P Biosynthesis of D-lysergylpeptides in the ergot fungus Claviceps purpurea I Ortel and U Keller Laboratory of Dr Keller, Institut fuăr Chemie, Fachgruppe Biochemie und Molekulare Biologie, Technical University of Berlin, Berlin, Germany E-mail: i.ortel@freenet.de Ergot fungi such as Claviceps purpurea produce a variety of pharmacologically active compounds containing d-lysergic acid In these compounds, the d-lysergic acid moiety is attached in amide-like fashion to tripeptide chains as in the ergopeptines or to a simple amino alcohol (alaninol) as in ergometrine Previously we have isolated and characterized the non-ribosomal peptide synthetases LPS and LPS 2, catalyzing assembly of the d-lysergic acid tripeptide backbone of ergopeptines LPS (144 kDa) was shown to consist of a simple module catalyzing recruitment of d-lysergic acid, whereas LPS (355 kDa) harbors the three modules for assembly of the three amino acids of the tripeptide chain Enzymatic analysis revealed that assembly proceeds via d-lysergylmono-, di-, and tripeptide intermediates on the surface of LPS Here we show, that besides LPS and LPS 2, strains of C purpurea selected for ergometrine high production contain a 180 kDa NRPS which exclusively activates alanine as thioester Presently performed characterizations of this enzyme in conjunction with LPS indicates that it may interact with LPS in the catalysis of d-lysergylmonopeptide formation such as of dlysergylalanine which is the immediate precursor of ergometrine The data suggest a dual role of the d-lysergic acid module of LPS participating in the formation of different alkaloid classes i.e the ergopeptines and the simple D-lysergic acid amides V1-070P Incorporation and photodynamic activity of methylene blue in HeLa cells C S Oliveira, A J Kowaltowski and M S Baptista Departamento de Bioquı´mica, Universidade de Sa Paulo, ˜o Sao Paulo, Brazil E-mail: carlaoli@usp.br Sensitizers used in Photodynamic Therapy (PDT) generate a series of reactive species upon excitation, which can result in cell death We investigated incorporation of Methylene Blue (MB) in HeLa cell cultures Cellular viability with MB and in dark was determined Only MB at concentrations higher than 100 mm decreased cellular growth MB incorporation was carried out at increasing MB concentrations and time Absorption spectra in the buffer and cellular fraction served to determine incorporation The uptake is high, about 70% Furthermore, the maximum incorporation was obtained after h of incubation We characterized the relationship between incorporation and mitochondrial electrochemical potential Cells were incubated with MB and drugs that alter the potential Incorporation decreased with low potential and increased with high potential The data indicate the MB incorporation is dependent on mitochondrial membrane potential and suggest MB is in mitochondria MB can induce type I (radicals) and type II (singlet oxygen) photochemical reactions depending on its aggregation state We characterized the dimer/monomer ratio in cells Surface absorption spectra indicated dimer formation was favored at increasing MB concentrations Singlet oxygen is believed to be the main cause of apoptotic cell death induced by PDT Singlet oxygen formation was verified with an near-infra red emission apparatus Singlet oxygen formation increased at increasing MB concentrations and mitochondrial membrane potentials After incubation with MB, cells were irradiated with laser and cell death was verified with flow cytometry The data indicate MB induced apoptosis We are studying a series of structurally related photoactive drugs to establish a structure–activity relationship in PDT treatments V1-071P Sulfhydryl group reactivity of meldola blue I Ozer Department of Biochemistry, Hacettepe University, School of Pharmacy, Ankara, Turkey E-mail: iozer@hacettepe.edu.tr The reactivity of meldola blue (CI 51175; MB), a phenoxazine dye employed in biosensors, towards reduced glutathione (GSH) and protein-SH groups was studied at 25 °C in 50 mm MOPS buffer, pH 9.0, containing 25–80 lm MB and (a) GSH (0.25– mm), (b) human serum albumin (HSA, 100 lm) or (c) sorbitol dehydrogenase (SDH, 40 mU/ml) The reactions were monitored spectrophotometrically tagging changes in dye structure at 570 nm (a and b) or enzyme activity at 340 nm (c) GSH caused a triphasic change in the spectral properties of MB, pointing to rapid formation of an adduct (Kd = 1.1 ± 0.02 mm), followed by two GSH-dependent (pseudofirst-order) processes with rate constants in the /min and /h range The faster of the two kinetic components was found to be associated with a spectral shift; the slower component reflected the formation of a leuko form of the dye The reaction of MB with HSA exhibited a single protein-related kinetic component (k = 0.06 ± 0.02/min at 25 lm MB) The reaction with SDH (carried out in the presence of 300 mm fructose and 0.2 mm NADH as substrate and cofactor) was much faster (k ‡ 3.5/min at 80 lm MB) and resulted in total inactivation of the enzyme The susceptibility of MB to SH groups may pose a significant problem in dye-mediated analysis of real samples V1-072P Catalase and cytosolic carbonic anhydrase activities in carcinoma patients O Ozensoy1, S Isik1, O Arslan1, F Kockar2 and M Alkan3 Biochemistry, Chemistry, Balikesir, Balikesir, Turkey, 2Molecular Biology, Biology, Balikesir, Balikesir, Turkey, 3Physical Chemistry, Chemistry, Balikesir, Balikesir, Turkey E-mail: ozensoy@balikesir.edu.tr The effects of the cytosolic carbonic anhydrase I and II (HCA-I and HCA-II, E.C.4.2.1.1) and catalase activity (CAT, EC 559 Abstracts 1.11.1.6) have been studied Hydrogen peroxide is involved in physiological phenomena and in the pathogenesis of various diseases The enzyme catalase has the ability to decompose hydrogen peroxide into oxygen and water: This enzyme plays a central role in controlling the hydrogen peroxide concentration in human cells The aim of this study was to determine the erythrocytes CAT activity and erythrocyte CA activity in normal subjects with different age categories, and patients whom suffer from various type of cancer In 31 healthy individuals the mean and S.D values of blood catalase activity were 0.003123 ± 0.000747 with no significant correlation between male and female subjects Total CA activity of control groups was determines as 157.7883871 ± 34.61446404 In total 108 samples of the bloods from patients with different types of cancer were taken and total CA and catalase activity was 204.1362593 ± 91.32110608 and 0.003464815 ± 0.00146678620, respectively The results suggest the presence of high CA activity in carcinoma patients but it was not observed a meaningful value for the CAT activities in the samples V1-073P Analysis of the reaction mechanism of aldoxime dehydratase containing heme as the active center K.-I Oinuma1, H Kumita2, T Ohta3, K Konishi1, K Ishida1, Y Hashimoto1, H Higashibata1, T Kitagawa3, Y Shiro2 and M Kobayashi1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki Japan, 2RIKEN Harima Institute/ SPring-8, Sayo, Hyogo Japan, 3Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi Japan E-mail: oioinuma@yahoo.co.jp Background: Aldoxime dehydratase (OxdA) is a unique hemecontaining enzyme which catalyzes the dehydration of aldoxime [R-CH¼N-OH] into nitrile [R-CBN] [1]; the reaction is surprising, as previous with extra particularly in the formation of the carbon-nitrogen triple bond and the efficient dehydration of the substrate in the presence of water In the previous study, we characterized the enzymological and physicochemical properties of OxdA and found that OxdA is active when the heme is in the 5-coordinate high-spin ferrous state, but not in the 6-coordinate low-spin ferric state [2] Nevertheless, the catalytic cycle is yet unclear In this study, we examined the structure of the active center, and analyzed the reaction of OxdA with the substrate on the millisecond scale Results: The combination of site-directed mutagenesis of OxdA, enzyme assay on the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the distal histidine (His320), which would play a crucial role in the catalytic cycle On stopped-flow analysis of OxdA, a spectrum very similar to those of bis-histidine ligated hemes was detected on mixing ferrous OxdA with butyraldoxime; it gradually changed into that of ferrous OxdA with an isosbestic point at 421 nm On the other hand, the spectral change on the addition of butyraldoxime to the ferrous H320A mutant showed the formation of a substratecoordinated mutant, the absorption spectrum of which closely resembled that of the above intermediate These findings demonstrate that the reaction intermediate is the substrate-coordinated form, in which the substrate binds to the ferrous heme via its nitrogen atom References Oinuma K-I, Hashimoto Y, Konishi K et al J Biol Chem 2003; 278: 29600–29608 Oinuma K-I, Ohta T, Konishi K et al FEBS Lett 2004; 568: 44–48 560 V1-074P Plasmodium berghei produce neuraminidase K I Ononiwu1 and N M Useh2 Laboratory of the Leather Research Institute, Biochemistry, Ahmadu Bello University, Zaria, Kaduna Nigeria, 2Microbiology and Pathology Laboratory Veterinary, Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna Nigeria E-mail: ononiwu_kingsley@yahoo.com A study was conducted to investigate the production of neuraminidase (sialidase, EC 3.2.1.18) by Plasmodium berghei and we detected neuraminidase activity in specific pathogen free balb c albino mice (in vivo) experimentally infected with P berghei Infected animals were treated with chloroquine phosphate (5 mg/ kg, i.m.) and a plant rauwolfia vomitoria (50, 100 and 150 mg/ kg i.m.) chloroquine (5 mg/kg i.m.) and rauwolfia plant (50 mg/ kg i.m.) produced the best therapeutic results The packed cell volume (PCV) of infected mice were much lower than those of the controls and the difference was statistically significant (P < 0.001) During infection and treatment, some of the experimental animals had higher PCV than the pre-infection values and this was attributed to dehydration There was leukopenia in infected animals, compared to pre-infection values and controls and the decrease in leukocyte counts was statistically significant (P < 0.001) During infection, parasitemia was characterized by pyrexia, anorexia and dullness Neuraminidase activity increased both during infection and treatment and the increase compared to the pre-infection values and controls was statistically significant (P < 0.001) Neuraminidase activity followed a specific pattern, as it increased both during infection and treatment, but decreased post-treatment The difference between neuraminidase activity during infection, during treatment and post-treatment was statistically significant (P < 0.001) There was a negative correlation between neuraminidase activity and leukocyte counts in all the infected groups Erythrocyte surface sialic acid (ESA) decreased during infection and treatment, but increased posttreatment and the decrease in the former, when compared to the pre-infection values and controls was statistically significant (P < 0.001), suggesting the regeneration of sailic acid on the erythrocyte surface Free plasma sialic acid levels increased during infection and during treatment, but decrease post-treatment The increase in plasma sialic acid (PSA) levels during infection and during treatment compared to the pre-infection values and controls was statistically significant (P < 0.001) The significance of leukopenia decreased ESA and elevated PSA levels during infection is discussed The therapeutic potential of the rauwolfia plant used to treat P berghei infection in the mice used for the study is discussed It was concluded that further work should be conducted to ascertain the role of neuraminidase in the pathogenesis of P berghei infection It is suggested based on our findings in this study that the therapeutic efficacy and advantage of the rauwolfia plant in ameliorating P berghei infection should be exploited as resistance to quinoline antimalarial is on the increase globally V1-075P Expression profile of glutathione S-transferase in renal cell carcinoma M Pljesa-Ercegovac, J Mimic-Oka, A Savic-Radojevic, M Opacic and T Simic Institute of Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade, Serbia and Montenegro E-mail: mpljesa@eunet.yu Cytosolic glutathione S-transferases (GST) are a superfamily of enzymes composed of four different classes, that protect normal Abstracts cells by catalyzing conjugation of electrophylic compounds of exogenous and endogenous origin with glutathione In solid tumors increased expression of GST Pi and Mi has been linked to drug resistance and malignant phenotype The aim of this study was to elucidate the expression profile of GSTs in renal cell carcinoma (RCC), one of the most chemotherapy-resistant tumors Expression of different classes of GST was determined in cytosolic fractions of tumor and non-tumor tissue of 15 patients suffering from RCC Isoenzyme classes were identified with specific antibodies against classes Alpha, Mu and Pi by using Western blot In non-tumor tissue of patients with RCC all of three examined classes were detected, with dominant expression of GST Alpha In tumor tissue significant downregulation of GST Alpha subtype was observed in all examined patients GST Mu class expression was decreased (5/15), absent (4/15) or increased (6/15) Expression of GST Pi, a well established tumor marker, was found to be unchanged (7/15), increased (4/15) or decreased (4/15) in RCC Based on our data we conclude that renal cell carcinogenesis is associated with significant changes in the profile of expressed GST isoenzymes The results obtained are consistent with the principle that GST Pi and Mu influence drug resistance and malignant phenotype of RCC V1-076P Detection and characterization of rat proteins recognizing DNA damaged by N-acetoxyacetyloaminofluorene and cis-platinum M Pietrowska, M Kalinowska and P Widlak Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland E-mail: m_pietrowska@io.gliwice.pl Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in extracts from rat tissues, using a damaged 36 bp oligonucleotide as a substrate and electrophoretic mobility shift assay Two major complexes binding DNA damaged by AAAF were detected that possess different electrophoretic mobility The low mobility complex contained loosely attached protein extracted with 0.2 m NaCl from isolated nuclei The amount of this protein was estimated as about 104 copies per cell The affinity of this protein to AAAF-damaged DNA was ~10-fold higher than to undamaged DNA and its probable size was about 42 kDa, as detected by Southwestern blotting assay Analogous AAAF-DDB complexes were also detected in extracts from brain, testis and kidney The second higher mobility complex contained protein extracted with 0.4 m NaCl The amount of this protein was estimated as about 105 copies per cell, and its probable size was about 26 kDa, as detected by the Southwestern blotting assay The affinity of this protein to DNA damaged by AAAF was ~70-fold higher as compared to undamaged DNA The protein was termed AAAF/ DDP-DDP because it also bound with high affinity to DNA damaged by cis-platinum The AAAF/DDP-DDB protein was detected also in brain, spleen and kidney, but not in testis The levels of both damaged DNA-binding proteins detected in rat tissues were not affected in animals treated with the carcinogen 2-acetylaminofluorene Acknowledgment: This work was supported by the Polish State Committee for Scientific Research (KBN) Grant 3PO5A10524 V1-077P Glyconectin to glyconectin recognition – a primordial self-assembly and non-self discrimination pathway leading to multicellularity O Popescu1 and G N Misevic2 Molecular Biology Center, Institute for Interdisciplinary Experimental Research, Babes-Bolyai University, Cluj-Napoca, Romania, Laboratoire des Processus Inte´gratifs Cellulaires, UMR 6037 CNRS, Faculte´ des Sciences et Techniques de Rouen, Mont St Aignan, France E-mail: opopescu@biolog.ubbcluj.ro The emergence of multicellularity has been tightly joined to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self Large cell surface glycoconjugate molecules, such as glyconectins (GNs), are the key candidates for the primordial recognition and adhesion functions in biological self-assembly systems Atomic force microscopy investigations demonstrated that the binding strength between a single pair of Microciona prolifera cell surface GN1 glycoconjugates can hold the weight of 1600 cells, proving their adhesion functions Here, measurement of molecular selfrecognition of GN1, -2 and -3 purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination Physicochemical and biochemical characterizations of these three GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans In functional and blotting assays, GNs displayed species-specific recognition and adhesion Monoclonal and affinity-purified monospecific polyclonal antibodies prepared against GN glycans selectively inhibited cell adhesion in the respective sponge species The molecular mechanism of glycan-mediated homophilic GN interactions in Porifera is based on highly species-specific and Ca2+-dependent associations, and approaches the degree of selectivity of the evolutionarily advanced heterophilic immunoglobulin superfamily recognition system Our study establishes a new paradigm for the essential function of primordial GN glycans in the self-assembly and non-self discrimination pathway of cellular adhesion prior to the appearance and evolution of multicellularity V1-078P Expression of the granulocyte macrophagecolony stimulating factor receptor (GM-CSFR) on human myocardiocytes from end-stage heart failure patients L Postiglione1, S Montagnani2, P Ladogana1, C Castaldo2, G Di Spigna1, E M Bruno1, L De Santo3 and G Rossi1 Department of Cellular and Molecular Biology and Pathology L Califano, University of Federico II, Naples, Italy, 2Department of Bio-morphological and functional Sciences, University of Federico II, Naples, Italy, 3Department of Cardiothoracic and Respiratory Sciences, Second University, Naples, Italy E-mail: lorposti@unina.it In remodeling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeletal filament of myocardiocytes, associated with fibrosis, cellular damage and death, which lead to alterations in heart geometry, size and mechanical properties Recently several observations have demonstrated the role of some cytokines and haemopoietic growth factors in the damage and regeneration mechanisms of cardiac tissue during acute myocardial infarction 561 Abstracts After heart damage, the development of scarred tissue has been considered as the only reaction, since myocytes are thought to be terminally differentiated cells and thus unable to proliferate On the contrary, recent studies in animal models and adult human hearts have shown that myocytes can proliferate before reaching their final size and that their proliferation may be stimulated and modulated by several factors Against this background, we have investigated the expression of the receptor for GM-CSF, an haemopoietic growth factor, in healthy and end-stage human cardiac tissues, to clarify the possible role of GM-CSF and its receptor in the regenerative process of cardiac tissue in chronic cardiomyopathy We have studied GM-CSFR expression on human cardiac tissue from explanted hearts of chronic patients with heart failure (ischemic and dilatative cardiomyopathy) and on cardiac biopsies from normal human hearts, by immunohistochemistry, cellular and molecular biology assays Our results demonstrated the presence of GM-CSFRb in cardiac tissue; in particular GM-CSFRb positive cells were a constant finding in pathological cardiac tissues and significantly increased as compared to normal control tissues At the moment we can only hypothesize that GM-CSF, like other growth factors, plays a role in apoptotic and/or ECM deposition processes as well as in the cytoskeleton modification in myocardium V1-079P Proteomic analysis of clusterin in colorectal cancer ´ ´ A M Rodrı´ guez-Pineiro1, D Ayude1, A Lopez-Saco2, ˜ ´ F J Rodrı´ guez-Berrocal1 and M Paez de la Cadena1 Laboratory of Biochemistry and Molecular Biology, Department of Biochemistry, Genetics and Immunology, University of Vigo, Vigo, Spain, 2C.H Xeral-Cı´es, Vigo, Spain E-mail: mpaez@uvigo.es Proteomic techniques have been previously employed by our group to seek for proteins altered in serum of colorectal cancer (CRC) patients One of the proteins found differentially expressed was identified as clusterin This is a heterodimeric highly conserved glycoprotein, expressed in a wide variety of tissues and present in all human body fluids It has been implicated in numerous processes, but its most striking functions affect cellular death, being described both as a pro- and anti-apoptotic protein These opposed roles might be related to specific proteomic profiles (either the overall pattern of clusterin isoforms or the ratio between them), due to different cellular stimuli In CRC, clusterin was reported overexpressed in the cytoplasm, and a translocation of a nuclear form (proapoptotic) to the cytoplasm (where it exerts cytoprotective functions) was described Sera were processed through a Concanavalin A column, obtaining a fraction (FI) enriched in O- or non-glycosylated proteins, and another one (FII) enriched in N-glycoproteins and free of albumin The latter fractions were separated by two-dimensional electrophoresis (2-DE), and clusterin N-glycoforms were compared, finding different levels of expression in some isoforms between healthy individuals and patients On the other hand, the whole serum and both fractions were analysed by native- and SDSPAGE, performing immunodetection with anti-clusterin antibodies after Western blot We detected differences in serum and FII, corresponding with those found by 2-DE Surprisingly enough, we also detected a 40-kDa form eluted in FI from patients, although clusterin has been described as only N-glycosylated This new finding will be further discussed 562 V1-080P Influence of anticancer drugs on interactions of tumor suppressor protein p53 with DNA H Pivonkova, K Nemcova, M Brazdova, J Kasparkova, V Brabec and M Fojta Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, Brno, Czech Republic E-mail: hapi@ibp.cz Protein p53 is a tumor suppressor whose functions are dependent on its interactions with DNA In addition to the sequence-specific binding to response elements (p53CON) in promoters of downstream genes, p53 can bind DNA in a structure-selective mode Action of many anticancer drugs involves their covalent or noncovalent interactions to DNA We investigated effects of various cytostatics on the p53-DNA binding DNA not containing the p53CON modified with cisplatin was bound by p53 with a higher affinity than the same but unmodified DNA This ability was remarkably more pronounced in the latent (post-translationally unmodified) p53 than in the active one [1] Protein deletion studies showed that the p53 C-terminal domain is primarily responsible for the damaged DNA recognition DNA adducts with other platinum complexes were not recognized by p53 [2] On the other hand, DNA modification with some platinum complexes reduced the binding of p53 to p53CON Sensitivity of p53 interactions with different p53CONs was dependent on their particular nucleotide sequences Anticancer agents interacting with DNA primarily non-covalently (such as doxorubicin) affected p53 binding to p53CON and to supercoiled DNA in agreement with changes in DNA conformation caused by intercalation of the drugs Acknowledgment: This work was supported by a grant No NC/7574 – from the IGA MH CR References Fojta M, Pivonkova H, Brazdova M, Kovarova L, Palecek E, Pospisilova S, Vojtesek B, Kasparkova J, Brabec V Biochemical Pharmacology 2003; 65: 1305–1316 Kasparkova J, Fojta M, Farrell N, Brabec V Nucleic Acid Research 2004; 32: 5546–5552 V1-081P Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells ´ ´ ´ R Peracaula1, G Tabares1, A Lopez-Ferrer2, S Barrabes1, ` L Pages1, R Brossmer3, C de Bolos2 and R de Llorens1 Unit of Biochemistry, Department of Biology, University of Girona, Girona, Spain, 2Molecular and Celular Biology Unit, Institut Municipal d’Investigacio´ Me`dica (IMIM), University Pompeu Fabra, Barcelona, Spain, 3Biochemie-Zentrum, University of Heidelberg, Heidelberg, Germany E-mail: rosa.peracaula@udg.es Sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumour cells and related to their metastatic potential While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been performed regarding the activity and expression of the a2,3-sialyltransferases in pancreatic tumour cells We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplasic differentiation stages and in normal human pancreatic tissues Total ST activity measured on asialofetuin varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression Human pancreatic tissues also showed ST expression and activity However, it presented a much higher Abstracts expression of neutral fucosylated structures compared to sialylated structures In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialylLewis antigens, in addition to fucosyltransferase activities V1-082P Characterization of RNase glycans from endothelial cells by 2D electrophoresis and lectins ` ´ ´ L Pages1, S Barrabes1, G Tabares1, M Moenner2, R De Llorens1 and R Peracaula1 Biochemistry and Molecular Biology, Department of Biology, University of Girona, Girona, Spain, 2Me´canismes Mole´culaires de l’Angioge´nesis, Department of Biology, University of Bordeaux, Talence, France E-mail: lpages@ozu.es Pancreatic adenocarcinoma is one of the neoplasias with worse prognosis mainly due to the fact that at moment of its detection the tumor has already developed metastasis Previous studies show that RNase 1, a serum protein secreted mainly by pancreas, could be useful as a tumor marker of this neoplasia because it shows glycosylation differences between normal and tumor states However, serum RNase could be originated from other sources, like endothelial cells For this reason it is necessary the glycan characterization of the RNase from endothelial cells in order to determine whether it could interfere in the detection of serum pancreatic RNase We have developed a method to purificate the RNase from endothelial origin (from conditioned media of EA.hy926, HUVEC, HULEC and HUMMEC cell lines) The process of purification has consisted of concentration of the media and three chromatographic steps: Cibacron Blue affinity chromatography, MONO-S cationic exchange chromatography and VYDAC-C4 reverse phase chromatography With the purified endothelial RNase we have characterized the glycans using lectins In addition we have studied the pattern of glycosylation of the protein using 2D electrophoresis combined with glycosidase digestions The results show that RNase secreted by the endothelial cell lines has sialic acid, which can be found in serum RNasa 1, in addition to the pancreatic RNase isoforms that have been characterized as neutral ones V1-083P Capsid-targeted viral inactivation for dengue, the Trojan horse approach C Qin and E Qin Laboratory of Virology, Institute of Microbiology and Epidemiology, Beijing, PR China E-mail: virelery@gmail.com Dengue infections pose a serious public health problem in most tropic and subtropic areas Considering the current lack of antivirals and vaccines, much effort focus on new antivirals against dengue One potential protein-based antiviral strategy is called ‘‘capsid-targeted viral inactivation’’ (CTVI), in which a viral capsid protein is attached to a deleterious enzyme, e.g., a nuclease, via genetic engineering During virus infection, the capsid protein moitey targets the fusion protein to the assembling virus particles, and the antiviral moiety de novo sepcifically inactivates the newly produced virions from within just like the heroes within the Torjan horse CTVI represented a unique and potent antiviral strategy for retroviruses, or potentially for other viruses Here we successfully demonstrated utilizing CTVI to a sigle-strand sense RNA virus, dengue virus We constructed plasmids expressing fusion protein consisting of dengue virus capsid protein (D2C) and straphylococcal nuclease (SN), and then developed experimental system to evaluate the antiviral effects in a prophylactic and therapeutic model We established a BHK cell line stably expressing the fusion protein D2C-SN and subsequently challenged with wild dengue virus And the virion infectivity from D2C-SN expressing cultures sharply decreased up to 104-fold compared with that from control cultures And in dengue-infected cultures, D2C-SN as a therapeutic agent caused a reduction up to 60-fold in infectious titers Biochemical analysis for the progeny virions suggested D2C-SN were incorporated within, where D2C-SN effectively destroyed the viral RNA genome These results strongly indicated dengue virus therefore should be particularly vulnerable to CTVI approach V1-084P Cooperative binding of pheophorbide a to poly-l-Lysine O A Ryazanova, V A Karachevtsev, O B Nesterov, I M Voloshin and V N Zozulya Department of Molecular Biophysics, B Verkin Institute of Low Temperature Physics and Engineering of NAS of Ukraine, Kharkov, Ukraine E-mail: ryazanova@ilt.kharkov.ua The pheophorbide a (Pheo) is anionic porphyrin derivative It is widely used as a photosensitizer in photodynamical therapy of tumors because of its high photosensitizing activity in vitro and in vivo The cooperative binding of Pheo to model polycationic polypeptide matrix, poly-l-Lysine (pLL), in buffered (pH6.9) aqueous/ethanol solutions (volume ratio 19:1) of different ionic strength has been investigated by means of absorption and polarized fluorescence spectroscopy The measurements were carried out in a wide range of molar phosphate-to-dye ratios, P/D, at different ionic strengths The absorption and fluorescent properties of aggregates formed by Pheo chromophores stacking at the cationic poly-l-Lysine were established Such parameters of cooperative binding as the number of binding sites per monomer unity of pLL, cooperativity parameter and the cooperative binding constant were estimated by Schwarz’s method V1-085P Protein tyrosine phosphatase SHP-1 and Akt/ PI3K pathway regulate cellular adhesion and spreading in human prostatic PC3 cancer cells F J Rodriguez-Ubreva1, M C Almaraz1, J Angulo2, R M Martin-Orozco1, M Lopez-Ruiz1 and B Colas1 ´ Department of Biochemistry, University of Alcala, Alcala De Henares, Madrid Spain, 2Department of Urology, Hospital of Getafe, Getafe, Madrid Spain E-mail: begona.colas@uah.es Prostatic cancer mortality and morbidity result, mainly, from tumour spread beyond the prostate Therefore, cellular adhesion and spreading are crucial processes in metastatic events These processes are controlled, partly, by protein tyrosine phosphorylation and dephosphorylation that are under the control of the tyrosine kinases and tyrosine phosphatases, respectively Recently, we have demonstrated that the tyrosine phosphatase SHP-1 is expressed in normal prostate, benign prostatic hyperplasia and well-differentiated adenocarcinoma, but not in poorly differentiated prostate cancer In addition, our group has described that overexpression of SHP-1 in human prostatic PC3 cancer cells reduces cellular proliferation and increases cellular adhesion and spreading on fibronectin In this sense, previous studies have shown that PC3 cell adhesion on extracellular matrix (ECM) components, such as osteopontin, vitronectin and fibronectin, induces the activation of the Akt/PI3K pathway [1] Thus, we analyzed the role of the Akt/PI3K pathway and tyrosine phosphatase SHP-1 in regulation of cellular adhesion on collagen type I, the major bone ECM component Our results show that collagen type I induces Akt and focal adhesion kinase (FAK) 563 Abstracts phosphorylation in PC3 cells Furthermore, FAK associates with SHP-1 and the p85 subunit of PI3K The PI3K inhibitor LY294002 reduces both cellular adhesion and spreading of PC3 cells on collagen type I At the same time, when SHP-1 phosphatase activity was inhibited, we also observed a significant decrease in cellular adhesion on this ECM component These results suggest that the tyrosine phosphatase SHP-1 and Akt/PI3K pathway play a very important role in prostatic cancer progression Acknowledgments: Source of Funding: Fondo de Investigaciones Sanitarias (FIS) P1020964 and Consejeria de Sanidad de Castilla-La Mancha (01035) Reference Zheng DQ, Woodward AS, Tallini G, Languino LR J Biol Chem 2000; 11: 24565–24574 V1-086P Antiviral activity of lactoferrin against bovine viral diarrhea virus A M Roseanu1, A Raducanu1, C Stoica1, M Icriverzi1, C Lazar2, A Deliu2 and N Nichita2 Department of Ligand-Receptor Interaction, Institute of Biochemistry, Bucharest, Romania, 2Department of Molecular Glycobiology, Institute of Biochemistry, Bucharest, Romania E-mail: roseanu@biochim.ro Lactoferrin (Lf) is known to present antiviral activity towards a wide range of human and animal viruses This paper describes the antiviral property of Lf using for the first time bovine viral diarrhea virus (BVDV), a model for HCV We found that bovine Lf (bLf) and human Lf (hLf) are potent inhibitors of BVDV-infection in Madin–Darby bovine kidney (MDBK) cells only when pre-incubated with virus for h at 37 °C before infection The effect was shown both on the level of viral infectivity and viral protein expression The anti-BVDV action was found to be specific and not influenced by iron-content of Lf bLf hydrolysate, a pool of pepsin digested fragments, was less efficient than native protein suggesting that the size or conformation are important for the antiviral action Our findings revealed the possibility to use Lf as antiviral agent able to remove circulating viruses V1-087P Influence of LY 300164 (Talampanel) – a novel antiepileptic drug, on the anticonvulsant activity of newer antiepileptics: an isobolographic analysis M J Swiader1, M Wielosz1 and S J Czuczwar2 Department of Pharmacology and Toxicology, Skubiszewski Medical University, Lublin, Poland, 2Department of Pathophysiology, Skubiszewski Medical University, Lublin, Poland E-mail: swiaderm@eskulap.am.lublin.pl LY 300164 (Talampanel) is a newer antiepileptic drug, which acts through the inhibition of AMPA/kainite receptors In initial clinical studies, it demonstrates broad spectrum of anticonvulsant action The objective of this study was to isobolographically evaluate the interactions between Talampanel and a number of antiepileptic drugs against maximal electroshock (MES)-induced convulsions in mice Electroconvulsions were produced by means of an alternating current (ear-clip electrodes, 0.2-s stimulus duration, tonic hindlimb extension taken as the endpoint) The isobolographic analysis distinguishes three most important types of interactions, among them the most accepted are: pure additivity, supra-additivity and sub-additivity The protective activity of two-drug mixture, applied in three fixed dose ratio combinations, was estimated and expressed as the ED50 values (dose of antiepileptic drug protecting 50% of animals) of these drugs against 564 MES-induced seizures in mice Moreover, the adverse effect was determined in the chimney test in mice Interactions between LY 300164 and topiramate, felbamate and lamotrigine caused pure additivity, in both MES and chimney tests Also, pharmacokinetic interactions could be excluded, because there were no changes in brain or plasma concentrations of LY 300164 as well as topiramate Finally, the isobolographic analysis revealed that LY 300164 combined with some newer antiepileptics might result in positive (additive) interactions, in clinical practice V1-088P High redox state in transitional cell carcinoma is mediated by increased antioxidant enzyme activities A Savic-Radojevic, M Opacic, M Pljesa-Ercegovac, J Mimic-Oka and T Simic Institute of Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade, Serbia and Montenegro E-mail: rrade@tehnicom.com High intracellular redox state in tumors is often associated with high proliferation rate and resistance to drugs It is believed, that oxidant/antioxidant balance in transitional cell carcinoma (TCC) of urinary bladder, is in favor of reduced state, based on increased glutathione (GSH) levels However, the data on the activity of GSH-replenishing and antioxidant enzymes in TCC are lacking We examined spectrophotometrically the specific activities of GSH-replenishing enzymes involved in GSH synthesis (gammaglutamylcysteine synthetase, gamma-GCS), GSH regeneration (glutathione reductase, GR) as well as antioxidant protection (glutathione peroxidase, GPX and superoxide dismutase, SOD) in the cytosolic fraction of tumors and the surrounding normal tissue of 28 TCC patients The results obtained have shown a significant increase in the activity of both GSH-replenishing and antioxidant enzymes in tumors in comparison with adjacent normal uroepithelium Mean gamma-GCS and GR activities in tumors were about 2-fold higher than in corresponding normal tissue (11 ± 0.35 vs 4.2 ± 0.35 U/mg and 59 ± 5.5 vs 25 ± 2.5 U/ mg, P < 0.001, respectively) Similarly, GPX and SOD activities in TCC were markedly increased (28 ± 5.1 vs.7.0 ± 0.8 U/mg and 3.0 ± 0.29 vs 1.8 ± 0.23 U/mg, P < 0.001, respectively) We conclude that enhanced GSH-replenishing pathways account for increased GSH levels in TCC Upregulated GPX and SOD, also contribute to high redox potential in TCC V1-089P Glutathione S-transferase isoenzyme profile in urothelial carcinoma T Simic, M Opacic, M Pljesa-Ercegovac, J Mimic-Oka and A Savic-Radojevic Institute of Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade, Serbia and Montenegro E-mail: tsimic@eunet.yu In this study, we featured a systematic functional investigation of different GST classes in transitional cell carcinoma (TCC) of urinary bladder and the surrounding normal uroepithelium of the same individuals Tumor samples and surrounding normal uroepithelium were obtained from 24 patients with TCC of urinary bladder The following substrates with differential specificities were used: 1-chloro-2, 4-dinitrobenzene for overall GST activity; 7chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST Alpha; 1,2-dichloro-4-nitro-benzene for GST Mu; 4-vinylpyridine for GST P1-1; and 1,2-epoxy-3-(p-nitrophenoxy)propane for GSTT1-1 GSTP1-1 and GSTT1-1 activities were demonstrated in all uroepithelial and TCC samples, while GST Mu activities were detectable in 11 of 24 patients In tumors significant upregulation of all expressed GST Abstracts subtypes was observed The mean GSTP1-1 and GSTT1-1 levels in TCC were increased twofold and 3.6-fold, respectively, compared with mean levels in normal uroepithelium (P < 0.001) Tumor GSTT1-1 activities correlated statistically significant with the stage of the tumor (P < 0.05) We conclude that in tumors and adjacent normal uroepithelium of patients with TCC three major cytosolic GST classes, Mu, Pi and Theta, are expressed Although GST isoenzyme pattern in TCC was similar to that of corresponding normal uroepithelium, during cancer progression there is a clear tendency towards an increase in all the GST subtypes expressed For the first time, distinct GSTT1-1 activity levels were demonstrated in human uroepithelium, followed by its pronounced upregulation in the TCC V1-090P Dietary protein intake and nitrogen emissions from dairy sheep 1 1 A Sevi , M Albenzio , M Caroprese , R Marino , A Muscio and L Taibi2 Department Pr.I.M.E., University of Foggia, Foggia, Italy, Istituto Sperimentale per la Zootecnia, Segezia-Foggia, Italy E-mail: a.sevi@unifg.it The experiment involved 48 Comisana ewes, divided in two groups of 24 each Ewes received a low (LP) or a moderate (MP) Crude Protein dietary level (13% and 16% CP of dry matter, respectively) Milk yield was recorded daily Individual milk samples were analyzed weekly for composition, and fortnightly for bacteriological characteristics After the last milk sampling (day 49 of the study period), eight animals from each group were placed in a metabolic box and their individual feces and urine were collected for three consecutive days The amounts of urine and feces excreted, and urinary and fecal N outputs were measured No differences emerged in milk yield among groups The MP animals required more dietary nitrogen to synthesize milk protein than the LP ewes Significant effects of CP level of diet were found for total water excretion and for total and fecal N emission, with the MP group displaying the higher values for all those parameters The low CP level resulted in smaller volumes of less wet feces and led to higher ammonia release in the urine compared to the moderate CP level Results suggest that the choice of proper dietary CP levels can be critical for sustainable management of intensive dairy sheep production systems Under the conditions of the present study, the ewes receiving the 13% CP ration displayed an enhanced efficiency of dietary N utilization, which results in milk yields comparable to the ewes fed the 16% CP ration and in a marked reduction of N output to the environment V1-091P A novel self-cleavage assay for measuring DNA binding of restriction endonucleases N Y Sidorova, S Muradymov and D C Rau LPSB, NICHD, National Institutes of Health, Bethesda, MD, USA E-mail: sidorova@mail.nih.gov DNA-protein interactions can be analyzed by methods based on physical separation of complexes from free DNA fragments (gel mobility shift and filter binding assays) or by techniques probing DNA-protein equilibrium directly in a solution (e.g fluorescence and calorimetry) These later techniques require larger amounts of materials and not provide enough sensitivity for measuring equilibrium association constants in the range 109–1012 M-1 Both gel mobility shift and filter binding assays that can measure these large binding constants, however, have been criticized for the danger of disrupting equilibrium by physically separating DNA-protein complexes from free species We have developed a novel self- cleavage or self-footprinting assay that uses the nuclease activity of restriction endonucleases to measure sensitively their specific binding to DNA At sufficiently high concentrations of neutral osmolytes cleavage reaction can be triggered at only those DNA fragments with bound enzyme Under these conditions the fraction of specific DNA fragment cleaved reliably reflects the fraction of DNA initially bound to the enzyme The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level We compare here the self-cleavage and gel mobility shift assays applied to the dissociation kinetics and binding equilibrium of two restriction endonucleases, EcoRI and BamHI The technique can be used, in principle, for any protein possessing DNA cleavage activity if its interactions with DNA are sensitive to osmotic stress V1-092P Inhibition of hexosaminidase secretion in Aspergillus oryzae causes changes in its glycosylation J Sklenar1, K Hoffbauerova1, P Pompach1 and K Bezouska1,2 Laboratory of Protein Architecture, Department Immunology, Institute of Microbiology, Prague, Czech Republic, 2Department of Biochemistry, Charles University, Prague, Czech Republic E-mail: sklenar@biomed.cas.cz Aspergillus oryzae cultivated in GlcNAc (N-acetyl-b-D-glucosamine) based medium produces considerable level of b-hexosaminidase While GlcNAc serves as a sole source of energy, only small fraction of ManNAc (N-acetyl-b-D-mannosamine) is incorporated in the medium containing a mixture of both sugars Under these conditions we measured hexosaminidase turnover by pulsechase experiment The speed of intracellular production was the same irrespective of the saccharide used On the contrary, the speed of secretion was highest in GlcNAc based medium, and decreased proportionally with the rise of ManNAc concentration Also, the total protein concentrations in mycelium increased in media with higher concentrations of ManNAc These observations suggest that ManNAc activates an unknown mechanism causing slowdown of hexosaminidase secretion To confirm these observations, we decided to analyze hexosaminidase glycosylation Assuming the slowdown of protein secretion may lead to a change in the intracellular flow through the secretory compartments, we hypothesized this change might influence protein glycosylation Analysis of N-linked glycans of hexosaminidase (by liquid chromatography and mass spectrometry) secreted under different conditions revealed small, yet significant, changes in ratios of distinct high-mannose oligosaccharides, which correlated with increasing ManNAc concentration Thus glycosylation pattern sensitively indicates the intracellular fate of a protein after translation Presented work provides the basis for delineation of a mechanism that modulates the protein secretion, mechanism that could be important for heterologous expression in filamentous fungi Acknowledgements: This work was supported by: Czech Grant Agency GACR 203/04/1045, GACR 203/05/0172, and AVCR No 50200510 V1-093P Active site mutants of thymidine kinases from human and Caenorhabditis elegans T Skovgaard, M Thunø and B Munch-Petersen Department of Life Sciences and Chemistry, Roskilde University, Roskilde, Denmark E-mail: scubamedown@svanenet.dk The DNA-precursors, the deoxynucleotides, are synthesized from small inorganic molecules via de novo pathway or from deoxynu- 565 Abstracts cleosides (dNs) via the less energy demanding salvage pathway, where the key step is phosphorylation of the dNs to deoxynucleoside monophosphates (dNMP), catalyzed by deoxynucleoside kinases (dNKs) The dNMPs are subsequently di- and triphosphorylated by other kinases and thereafter incorporated into DNA Apart from their natural substrates, dNKs also phosphorylate nucleoside analogs such as acyclovir (ACV) and azidothymidine (AZT) In their triphosphorylated forms, the analogs inhibit the DNA synthetic machinery or induce apoptosis In anti-cancer gene therapy a dNK from herpes simplex virus type (HSV1-TK) is used to phosphorylate nucleoside analogs (ACV) in cancer cells and thereby prohibit cell growth Mammalia have four different dNKs with overlapping substrate specificities None of them can phosphorylate all four natural deoxynucleosides, but the most restricted kinase is thymidine kinase (TK1), which only phosphorylates thymidine (dT) and deoxyuridine (dU) A TK1-like enzyme (Ce-TK1) has been found in the nematode C elegans and appears to be the only dNK in this worm In spite of the sequence similarity between the two TK1-enzymes, Ce-TK1 phosphorylates deoxyguanosine in addition to dT and dU The recently solved structure of human TK1 helps to clarify which amino acids are likely to influence the substrate specificity In order to understand the broader substrate specificity of Ce-TK1 relative to Hu-TK1 and to create a better kinase for gene therapy, we have created a number of point mutations in the active sites of both Hu-TK1 and Ce-TK1 and examined the effect on specificity and kinetics V1-094P Antimicrobial activity of ketoconazole & fluconazole against metronidazole resistance strains of Helicobacter pylori F Siavoshi1, F Safari1, S Latifi-Navid2, R Malekzadeh3 and S Agah4 Microbiology, Tehran University, Tehran, Iran, 2Microbiology, Ardabil Azad, Rdabil, Iran, 3Digestive Diseases Research Center, Tehran University of Medical Sciences, Tehran, Iran, 4Hazrate Rasoul Hospital, Tehran, Iran E-mail: safarif2003@yahoo.com Ketoconazole and fluconazole as potent antifungal agents are practically inactive against bacteria and most protozoa, in order to search for the more efficacious drugs against H pylori the antibacterial effects of fluconazole and ketoconazole were assessed against H pylori isolates, which exhibited either resistance or susceptibility to metronidazole 35 isolates of H pylori from patients with digestive disorders were recruited, among them were 11 strains resistant to metronidazole MICs of ketoconazole and fluconazole for H pylori isolates were determined by disk diffusion procedure To examine the effect of ketoconazole and fluconazole on resistance of H pylori to metronidazole, blood agar plates containing ketoconazole or fluconazole with concentrations below MIC (1/2 MIC), were surface inoculated with bacterial suspension Blank discs were then deposited on the agar and inoculated with 10 ll of metronidazole (4, 8, and 32 lg/ml) Plates were incubated microaerobically, and examined for the visualization of inhibition zones All the 35 H pylori strains were inhibited by ketoconazole and fluconazole MICs of ketoconazole for and 31 out of 35 strains were determined as lg/ml and lg/ml respectively All strains were inhibited by fluconazole with the MICs ranging from lg/ml to lg/ml All the 11 strains which were resistant to metronidazole turned susceptible when treated with ketoconazole and fluconazole in the concentration below MIC Ketoconazole and floconazole, showed an excellent in vitro activity against the H pylori isolates Both antifungals at concentration below MIC (1/2 MIC) converted metronidazole resistant strains to susceptible 566 V1-095P NMR models of the Angiotensin-I Converting Enzyme Zn(II) catalytic sites: the basis for a structural study on the enzyme-substrate interaction G A Spyroulias1, A S Galanis1, G Pairas1, E Manessi-Zoupa2, I P Gerothanassis3 and P Cordopatis1 Department of Pharmacy, University of Patras, Patras, Greece, Department of Chemistry, University of Patras, Patras, Greece, Deparment of Chemistry, University of Ioannina, Ioannina, Greece E-mail: G.A.Spyroulias@upatras.gr The Angiotensin-I Converting Enzyme (ACE) is a gluzincin Zinc Metallopeptidase and constitutes one of the major partners of the Renin-Angiotensin System This system possesses a pivotal role in blood pressure regulation ACE catalyzes the hydrolysis of the Angiotensin-I (AI) carboxy-terminal dipeptide His-Leu after Renin-mediated AI liberation to the blood from the Angiotensinogen ACE transforms AI to the vassopressor octapeptide Angiotensin-II (AII) and it is encountered in two distinct forms in humans, the somatic and the testis form These differ from the structural point of view, mainly in size (1306 and 732 amino acids for somatic and testis isoform, respectively) bearing and Zn(II) catalytic sites Only recently the X-ray structure of testis ACE has been determined [1] The reconstitution of the catalytic sites of ACE, has been performed using synthetic peptides and followed by Zn(II) addition The solution structures of these constructs have been studied using high-resolution multinuclear NMR spectroscopy Data analysis has led to the: (i) identification of the amino acid Zn(II) ligands, (ii) identification of the fold and the elements of secondary structure which are typical for gluzincin Zn(II) Metallopeptidase [2] and (b) 3D solution structure determination The NMR models of two ACE catalytic sites set the basis in order to understand the different substrate binding specificity and study their interaction with physiological substrate, such as Angiotensin I and Bradykin, as well as with various inhibitors References Natesh R et al Nature 2003; 421: 551–554 Spyroulias GA et al Curr Topics Med Chem 2004; 4: 403–429 V1-096P Genetic immunization of schistosomiasis using DNA plasmid encoding for key glycolytic enzyme M A Saber and E El Dabaa Biochemistry, Theodor Bilharz Research Institute, Cairo, Egypt E-mail: maasaber@yahoo.com Schistosomiasis is a major health problem in tropical and subtropical regions Available drug for treatment, has no prophylactic effect, therefore, vaccination is an essential requirement The gene encoding S mansoni fructose–1,6-bisphosphate aldolase (SMALDO) (a key glycolytic enzyme) was isolated from cercarial cDNA expression library using protective sera of rabbits vaccinated with irradiated cercariae Introns study indicated that this isozyme is encoded by one exone The SMALDO was cloned into the eukaryotic expression vector cDNAI/Amp and used as DNA vaccine, by intramuscular immunization of out bread mice (100 lg/mouse) Mice have been boosted once (100 lg/mice ) two weeks later Five weeks after the 1st booster, the mice have been challenged with cercariae and perfused 10 weeks later Vaccinated mice sera IgG recognized specifically both native (40 kDa) and recombinant SMALDO enzyme and has no cross reactivity with human and mice aldolases DNA vaccination induced both cellular and humeral immune response and achieved significant protection (21.3%) against challenge infection with S mansoni Abstracts cercariae Histopathology of the liver of vaccinated animals, revealed significant reduction (31.2%) of granuloma size accompanied by significant increase of lymphocytes (228%) and macrophages (108%) and highly significant decrease of eosinophils (51.3%), indicating an increased Th1 and decreased, but not abrogated, Th2 cellular response (which have a good impact on the pathology of the disease) In addition, no auto immune reaction has been developed in vaccinated animals in the site of injection In conclusion; SMALDO DNA immunization, has stimulated specific humeral and cellular immunity with no cross reactivity with host ALDO, having ant pathological and moderately protective effects, and could be a promising candidate genetic vaccine for schistosomiasis V1-097P The inhibitory effect of alcohols on alkaline phosphatase activity R Sariri1, A Ghanadzadeh2, E Laiali1 and R Hasan Sajedi1 Biochemistry, Department of Biology, The University of Guilan, Rasht, Gilan Iran, 2Physical chemistry, Department of Chemistry, The University of Guilan, Rasht, Gilan, Iran E-mail: sariri@guilan.ac.ir Alkaline phosphatase (EC 3.1.3.1) is a dimeric protein containing Zn and Mg ions with a molecular weight of 140 KD Unlike many enzymes that origin from cytoplasm, alkaline phosphatase is located in the cellular membrane by PGI anchor and, therefore, is not easily released from the cell wall Alkaline phosphatase plays four major and quite important catalytic activities namely, hydrolase, phosphotransferase, protein phosphatase and pyrophosphatase Various isozymes of alkaline phosphatase are elevated in plasma during different physiological disorders such as bone and kidney carcinoma Therefore, assessment of this enzyme in a clinical laboratory could be a reliable diagnostic tool Alkaline phosphatase is used in a variety of analytical technologies such as ELISA and recombinant DNA technology There are some drugs that affect the plasma level of alkaline phosphatase, an aspect which could be used for the design of new drugs and study their mechanism of action In this research, the effect of some alcohols on the activity of calf alkaline phosphatase upon one of its substrates, para-nitrophenyl phosphate (pNPP) was studied The results showed that although the inhibitory effect of ethanol was much higher than higher alcohols, i.e propanol and butanol and also bifunctional alcohols such as ethylene glycol, but none of the alcohols could inhibit 50% of enzyme activity This is a low inhibition rate when compared with inhibition of the same enzyme by gluthatione From the Lineweaver burk plots of the enzyme in the presence of inhibitors, it was also found that all alcohols were reversible non-competitive inhibitors V1-098P The effect of malachite green and methyl green on human plasma cholinesterase T Z Tuylu Kucukkilinc and I Ozer Faculty of Pharmacy Department of Biochemistry, Hacettepe University, Ankara, Turkey E-mail: ttuylu@hacettepe.edu.tr Butyrylcholinesterase (BChE, EC 3.1.1.8) is a plasma esterase, long considered to have a detoxifying function in vivo Recent studies suggest that it may, moreover, be involved in a number of developmental processes and in overall brain function This emerging role has added to the interest in cholinesterase inhibitors as therapeutic agents, especially in connection with neurodegenerative diseases such as Alzheimer’s disease The present study aimed to evaluate cationic triarylmethane dyes (TAM+s) as potential anticholinesteratic agents The inhibitory effects of malachite green (MG+) and methyl green (MeG+) on human plasma cholinesterase were studied in 100 mM MOPS, pH 8.0, at 25 °C Both dyes acted as competitive inhibitors, with Ki, MG = 0.28 lM and Ki, MeG = 0.15 lM The inhibitory potency of TAM+s was higher than most of the inhibitors studied to date (Representative Ki values for ChE inhibitors in clinical use are: tacrine, 0.007 lM; donepezil, 2.3 lM; huperzine, 76 lM) The competitive nature of the inhibition was a further feature, which distinguished TAM+s from the majority of ChE inhibitors reported to act as mixed-type inhibitors The results suggest that inhibitory mechanism and potency may relate to affinity towards the central and peripheral binding sites on the enzyme V1-099P Differential regulation of hypoxia inducible factor 1a (HIF-1a) expression and HeLa cell survival and proliferation by hypoxia, desferrioxamine, cobalt and quercetin: the role of iron A Triantafyllou, P Liakos, A Tsakalof, E Georgatsou, G Simos and S Bonanou Laboratory of Biochemistry, Department of Medicine, University of Thessaly, Larissa, Greece E-mail: atriand@med.uth.gr Low concentrations of oxygen increase the expression of the hypoxia-inducible factor 1a (HIF-1a), by interfering with O2 and Fe2+ – dependent proline hydroxylation steps, which target it for proteolysis Working with HeLa cells, we investigated the characteristics of induction of HIF-1a by hypoxia or its chemical mimetics, desferrioxamine, cobalt chloride or quercetin and examined the effect of adding excess Fe3+ on HIF-1a expression and cell survival and proliferation We found that addition of Fe3+ abolished the induction of HIF-1a by desferrioxamine or quercetin, but not by hypoxia or cobalt, whereas addition of the antioxidant N-acetyl-cysteine reduced the expression of HIF-1a only in the cobalt-treated cells These results suggest that quercetin, like desferrioxamine, acts by chelating the iron required by the proline hydroxylases, whereas the action of cobalt involves reactive oxygen species HeLa cell viability and incorporation of [3H]-thymidine into DNA were not influenced by hypoxia up to 48 h, but were reduced to less than 50% of the control by desferrioxamine and to less than 25% of the control by cobalt or quercetin Addition of Fe3+ to the desferrioxamine-, cobalt- or quercetin-treated cells, restored cell numbers and DNA synthesis to near control levels Under all conditions of culture, however, there was no correlation between DNA synthesis and HIF-1a protein levels In conclusion, our data highlight the ways iron affects the expression of HIF-1a during true or chemical hypoxia, elucidate the mechanism of action of quercetin and cobalt and demonstrate that in HeLa cells it is iron and not HIF-1a that is essential for survival and proliferation V1-100P NAD+ metabolism in breast cancer L Turker Sener1, L Yalcintepe1, D Tiryaki1, A Sener2 and E Bermek1 Department of Biophysics, Istanbul University, Istanbul Faculty Of Medicine, Istanbul, Turkey, 2Department of General Surgery, Hospital of Oltu, Erzurum, Turkey E-mail: leylaturker@hotmail.com The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation Human CD38 is 43-kDa transmembrane protein that acts as a bifunctional ectoenzyme, catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of NAD to ADP-ribose Total 14 patients breast tissues were collec- 567 Abstracts ted during surgical operation of women diagnosed with breast carcinoma according to pathological results NADase and ADPribosyl cyclase activities were measured in the tissue samples both malign lesion and normal region from the same breast and detected to have significantly higher NADase and ADP-ribosyl cyclase activities than normal SDS-PAGE analysis of tissue samples followed by Western blotting revealed the presence of a 43 kDa band that immunoreactive in breast cancer compared normal region with human CD38-specific monoclonal antibody OKT10 Immunohistochemical data indicated that the increase of CD38 expression was due to plasma cell of cancer tissue The results suggest that the relative abundance of proteins immunologically related to CD38 may account for the elevated levels of glycohydrolase and cyclase activities in breast cancer patients this study we show that recombinational telomere elongation in a telomerase deletion mutant of Kluyveromyces lactis occurs via amplification of sequence originating from a single telomere Furthermore, the presence of a single elongated telomere partially suppressed cellular senescence and led to the highly preferential spreading of its sequence to all other telomeres Strikingly, the transition between a state resistant to recombination and a state capable of recombination is abrupt, typically occurring when telomeres are ~3–4 repeats in length Finally, mutant repeats defective at regulating telomerase are shown to also be defective at regulating telomere length during recombinational telomere maintenance Acknowledgement: This work was supported by NIH grant GM61645-01 V1-101P Effects of cyclosporine A on deformability and oxidative stress in the peripheral lymphocytes of rats S Tamer1, E Ademoglu2, I Albeniz3 and C Gokkusu2 Department of Physiology, University of Istanbul, Istanbul, Turkey, 2Deaprtment of Biochemistry, University of Istanbul, Istanbul, Turkey, 3Department of Biophysics, University of Istanbul, Istanbul, Turkey E-mail: tamers@istanbul.edu.tr Cyclosporin A (CsA) is a principle immunosuppressive agent used in transplant surgery and offers an effective treatment for autoimmune disorders Unfortunately, the therapeutic benefits of CsA are often limited by the occurrence of side effects such as nephrotoxicity, hepatotoxicty and cardiotoxicity Although the importance of rheological properties of lymphocytes in the pathophysiology of various diseases has been recognized, little information is available about the effects of CsA on rheological properties and oxidant-antioxidant system in peripheral lymphocytes The objective of this study is to investigate the effects of CsA on lymphocyte deformability and oxidant-antioxidant system in peripheral lymphocytes in rats Twenty, female WistarAlbino rats (n = 10) were injected 10 mg/kg SandimmunÒ i.p., every day for 30 days The control rats (n = 10) were injected 0.9% NaCl CsA Administration caused a significant reduction in the deformability of lymphocytes (P < 0.001) and produced a significant increase in peripheral lymphocytes lipid peroxide level (P < 0.001) and a significant decrease in nitric oxide (NO) level (P < 0.01) The activity of superoxide dismutase (SOD) in peripheral lymphocytes did not show significant differences between CsA administrated and control rats However, the total thiol (t-SH) content of lymphocytes was significantly lower in CsA administrated rats (P < 0.01) The present data demonstrate that CsA administration increased lipid peroxidation and decreased the NO levels and t-SH content in the peripheral lymphocytes of rats This effect was accompanied by a decrease in lymphocytes deformability These results indicate that disturbances in lymphocytes rheology may be an additional risk factor in the pathogenesis of side effects associated with CsA V1-102P A single source for recombinationally elongated telomeres in K lactis Z Topcu1, K Nickles2, C Davis2 and M J McEachern2 Department of Pharmaceutical Biotechnology, University of Ege Faculty of Pharmacy, Izmir, Turkey, 2Department of Genetics, University of Georgia Fred Davison Life Sciences Complex, Athens, GA, 30602, USA E-mail: ztopcu@yahoo.com Eukaryotic cells lacking telomerase, including some human cancers, can sometimes maintain telomeres using recombination In 568 V1-103P Kunitz type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies A P U Araujo1, D Hansen2, D F Vieira1, C Oliveira2, L A Santana2, L M Beltramini2, C M Sampaio2, M U Sampaio2 and M L V Oliva2 Centro de Biotecnologia Molecular Estrutural, Instituto de Fı´sica de Sa Carlos – USP, Sao Carlos, SP Brazil, 2Departamento de ˜o Bioquı´mica, Universidade Federal de Sa Paulo – Escola Paulista ˜o de Medicina, Sao Paulo, SP, Brazil E-mail: anapaula@if.sc.usp.br Bauhinia bauhinoides Cruzipain Inhibitor (BbCI) and Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) respectively are cysteine and serine proteinase inhibitors homologous to plant Kunitz-type inhibitors, but lacking disulfide bridges A potent inhibitor of tissue kallikreins, BbKI may aid in the design of synthetic inhibitors for kallikreins BbCI effectiveness in cruzipain inhibition should likewise be considered in the design of potential drugs against Trypanosoma cruzi infection Additionally, as BbCI inhibits elastase, it warrants consideration for therapeutics aimed at physiopathological processes in which elastase is involved Based on cDNA sequences we found that BbKI and BbCI are initially synthesized as a prepropeptide with the following structure: N-signal peptide (19 residues), mature protein (164 residues), C-terminal-peptide (10 residues) The heterologous expression in E coli of both mature proteins containing an N-terminal His-tag allowing a high purity level in the inhibitors production Activity assays showed that both proteins are biologically active Circular dichroism spectra deconvolution of BbCI and BbKI were in agreement with the structural pattern b-trefoil fold described for Kunitz inhibitors Finally, the development of a method for the production of large amounts of correctly folded recombinant inhibitor of high purify will facilitate structural studies, as spectroscopics, crystallization, as well studies of their function in physiological processes such as inflammation, coagulation, diabetes, cancer and insect development, currently under investigation Acknowledgment: This work was supported by Brazilian Agency FAPESP Abstracts V1-104P Late induced plant regeneration from protoplast-derived callus of Helianthus annuus L cv Turbo, by subsequent changes of media and culture conditions S M Vesa1, L Rakosy-Tican1 and A Gilbert2 Plant Genetic Manipulation Group, Department of Biology, Babes-Bolyai University, Cluj-Napoca, Romania, 2Laboratoire Biotechnologie et Ame´lioration de Plantes, Ecole Nationale Superieure Agronomique, Institut National Politechnique, Toulouse, France E-mail: simona_vesa@yahoo.com Cultivated sunflower (Helianthus annuus L.), together with soybean, oil seed rape and peanuts is one of the four most important annual species for edible oil production Regeneration of fertile plants from sunflower protoplasts has met with considerable difficulties In contrast with the production of callus, which is rather easy to achieve, the successful regeneration of shoots and production of entire plants have been reported in only few cases Moreover, all these reports are referring to plant regeneration within 3–4 months old cultures, after this period the regeneration potential is considered to be lost Our objective was to induce late plant regeneration from protoplast derived calli by subsequent changes in the culture media and hormonal balance Protoplasts were isolated from 7-days old hypocotyls of sunflower cv Turbo, embedded in agarose droplets at a final density of 106 protoplasts/ml The culture protocol was according to Krasniansky and Menczel (1993) up to callus stage After two months, the small emerging calli were transferred to the regeneration medium R501 (Burrus et al., 1991) supplemented with 0.5 mg/l ANA, mg/l BAP and 0.1 mg/l GA3 The cultures were maintained on this medium for nine months The white calli presenting green spots were transferred to weekly refreshed MA1 liquid media (Escalant et al., 1994), supplemented with ANA mg/l and kinetin, 0.2 mg/l and maintained on a shaker at 125 rpm Developing shoots were observed and plants started to develop after two months in culture and were transferred to solid culture media without hormones The present data show that by changing culture media and hormone balance late plant regeneration from protoplast-derived callus can be induce and encourages further investigations on sunflower protoplast regeneration with special reference to highly responsive genotypes V1-105P Quantitative simulation analysis of functional contribution of human alcohol dehydrogenase family to ethanol metabolism S J Yin1 and S L Lee2 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan ROC, 2Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Taiwan ROC E-mail: yinsj@ndmc.idv.tw Human alcohol dehydrogenase (ADH) constitutes a complex family The family members have been grouped into five distinct classes, designated ADH1-ADH5, primarily on the basis of the structural characteristics Interclass and intraclass positional identities of the amino acid/nucleotide sequences are approximately 60% and 90%, respectively To elucidate kinetic mechanism of human ADH family functioning as ethanol dehydrogenase, initial velocity, product inhibition, and dead-end inhibition experiments were performed with the recombinant ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH2, and ADH4 The mechanism for class I, II, and IV ADHs deduced from steady state kinetics is best described as an ordered sequential mechanism for both eth- anol oxidation and acetaldehyde reduction Steady state and kinetic isotope effect studies suggest rate limiting at the coenzyme release Computer simulations using the mechanism-based complete steady-state rate equations in conjunction with the experimentally determined kinetic parameters indicate that high-Km forms ADH2 and ADH1B3 in liver as well as high-Km ADH4 in stomach may contribute significantly to first-pass metabolism of ethanol, that low-Km AHD1B1 appears to be most sensitive to acetaldehyde product inhibition, and that ADH family members may be inhibited to varied extents by increased [NADH]/[NAD] redox ratio in the cell during ethanol metabolism V1-106P Role of vitamin E in ameliorating the malathion-induced changes in oxidative stress, hemato-biochemical parameters of male rabbits M I Yousef Hormon Lab, Environmental Studies, University of Alexandria, Institute of Graduate Studies and Research, Alexandria, Egypt E-mail: mokhtar_yousef@yahoo.com The present investigation deals with determining the efficacy of vitamin E (100 mg/kg BW) in combating the effects of sublethal dose of malathion (164 mg/kg BW; 1/25 LD50) on lipid peroxidation and enzyme activities in male New-Zealand white rabbits Results obtained showed that malathion significantly (P < 0.05) induced free radicals in plasma, liver, testes, brain and kidney While, vitamin E alone significantly decreased the levels of free radicals, and alleviated the negative effect of malathion The activity of glutathione S-transferase (GST) and sulfhydryl groups (SH-groups) were significantly increased in liver, testes, kidney and plasma of animals treated with malathion and/or vitamin E alone Liver and testes aminotransferases, phosphatases, and liver, testes and brain lactate dehydrogenase and phosphorylase activities were significantly decreased due to malathion administration While, the activities of these enzymes were significantly increased in plasma The activity of acetylcholinesterase was significantly decreased in brain and plasma Vitamin E alone did not cause any significant change in the activities of these enzymes, while minimized the toxic effect of malathion The present results suggest that vitamin E seems to have a beneficial effect in alleviating the effects of malathion V1-107P Antibody production in plants (Plantibody) A Zebarjadi and M Jalali Javaran Plant Breeding, Tarbiat Modarres University, Tehran, Iran E-mail: zebarjad@nrcgeb.ac.ir Recently, various strategies have been developed to exploit plants as bioreactors for the production of pharmaceutical antibodies, to engineer antibody mediated pathogen resistance or to alter the plant phenotype by immunomodulation Antibodies are bioactive molecules that, owing to their individual and specific binding properties, allow a large diversity of potential applications These include medical diagnosis and therapy, the sensitive detection and removal of environmental contaminants, control of pathogens Plants can not potential produced antibodies, but production of full length antibody and antibody fragments with using animal genes and expression of them in transgenic plants have attracted more researchers for using this technology in molecular biology and plant breeding This paper discusses about antibody that produced in plants (Plantibody) 569 ... was observed when COS cells were co-cultured with macrophages and this effect was reduced by NOS inhibitors The cause of the induction of NOS II is not clear: the levels of some cytokines as interferongamma... Bickle2 Laboratory of Dr Ettrich, Institute of Physical Biology of USB and Institute of Landscape Ecology of AS CR, Nove Hrady, Czech Republic, 2Laboratory of Dr Bickle, Department of Microbiology,... characterized by disturbances of redox and acid-base metabolism Intravenous laser irradiation of blood (ILIB) is expedient pathogenetically in cerebral ischemia This work deals with study of mechanism of