Báo cáo khoa học: All Other Topics pdf

All Other Topics F1-1 Effects of atorvastatin on oxidative stress in L-NAME- treated rats R. Gelisgen 1 , D. Konukoglu 1 , H. Uzun 1 , R. Kalayci 2 , N. Arican 3 and M. Kaya 4 1 Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 2 Istanbul Medical Faculty Research Institute for Experimental Medicine, Istanbul University, Istanbul, TURKEY, 3 Istanbul Medical Faculty, Forensic Medicine, Istanbul University, Istanbul, TURKEY, 4 Department of Physiology, Istanbul Medical Faculty, Istanbul University, Istanbul, TURKEY Current evidence suggests that the beneficial effects of statins seem to be mediated not only by their lipid-lowering properties but also by improving vascular endothelial cell function. Chronic inhibition of endothelial nitric oxide synthesis by oral administration of N w-nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammation and subsequent arteriosclerosis. To test the relationship between statin (atorvastatin) therapy and oxidative stress, we determine the levels of plasma oxidative stress markers (protein carbonyl; PCO, lipid hydroperoxides; LPH, and oxidized LDL; ox-LDL) and plasma antioxidants (Paraoxonase -1; PON-1 and total thiol; T-SH) in L-NAME induced endothelial dysfunc- tion rat models. Adult male Wistar rats were divided into three groups, as saline (n: 8, controls), L-NAME (1 mg/ml of drinking water for three weeks, n: 8), and Atorvastatin plus L-NAME (4 mg/kg/day for 1 week during the third week of L-NAME treat- ment, n: 11). Plasma oxidative marker levels were higher and anti- oxidative marker levels were lower in L-NAME group than controls (for each, P < 0.01). Atorvastatin plus L-NAME group have lower plasma ox-LDL and LPH levels and higher PON-I activities than L-NAME group (for each, P<0.01). Therefore statins are likely to restore the L-NAME-induced inhibition of endothelial NO synthase activity by lowering oxidative stress. F1-2 Circulating oxidized LDL and antibodies against oxidized LDL in type 2 diabetes patients with and without coronary artery disease R. Gelisgen 1 , B. Cimen 1 , V. Yumuk 2 , M. Bolayirli 3 , M. Hacibekiroglu 3 and G. Burcak 1 1 Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 2 Department of Internal Medicine, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TUR- KEY, 3 Central Research Laboratory, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY We aimed in this study to investigate firstly whether ox-LDL and oLAB are increased in patients with type 2 diabetes mellitus com- pared with control subjects and secondly whether they differ between diabetic patients with and without coronary artery disease (CAD) .We studied type 2 diabetic patients (n=60) and nondia- betic control subjects (n=27). We divided the patients according to the presence of CAD. CAD was present in 29 diabetic patients. We assessed ox-LDL in plasma and oLAB, glucose, HbA1c, creati- nine, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total cholesterol, triglycerides, total protein and albumin in serum. ox-LDL and oLAB were measured with ELISA. In diabetic group ox-LDL,ox-LDL/LDL-C and oLAB were significantly higher than the control group.(P<0.001, P<0.001, P<0.05).Significantly higher val- ues for ox-LDL(P<0.001, P<0.001)and ox-LDL/LDL-C (P<0.001, P<0.001)were observed in both of the diabetic groups with and without CAD in comparison to the control group. oLAB level was found significantly higher only in the diabetic group with CAD than the control(P<0.01).ox-LDL concentra- tions did not display any significant difference between diabetic groups with and without CAD, but oLAB and ox-LDL/LDL-C were higher(P<0.05, P<0.05) in the diabetic group with CAD. F1-3 Decrease in lipid peroxidation as a result of black tea antioxidant action. W. Luczaj, E. Zapora and E. Skrzydlewska Medical University Of Bialystok, Bialystok, POLAND It has been widely postulated that reactive oxygen species (ROS) play an essential role in the aging process. The increased genera- tion of ROS during aging results in oxidative stress which causes changes in structure and function of important cellular compo- nents, especially lipids. Black tea has been recently proved to be a source of water-soluble antioxidants that may enhance cellular antioxidant abilities. The present study has been designed to investigate the efficiency of preventive effect of black tea on liver lipids oxidative modifications in 2-, 12-, 24-month-old rats. Lipid peroxidation was estimated by measurement of lipid hydroperox- ides, malondialdehyde, 4-hydroxynonenal and 8-isoprostaglandin F 2a by high-performance liquid chromatography (HPLC) and by spectrophotometric determination of conjugated dienes as well as phospholipase A 2 . Aging process causes the significant increase in the level of lipid oxidative modification products (6–53%). Admin- istration of black tea remarkably prevented the age-related increase in concentrations of all measured lipid peroxidation markers. In comparison to 2-month-old rats, the preventive effect of black tea causes decrease (6–20%) in the examined markers in the liver of 12- and 24-month-old subjects. Our findings indicate that black tea protects lipids against age-related oxidative modification. F1-4 Oxidation of serum low density lipoprotein in experimental hyperthyroidism; role of paraoxonase G. Simsek 1 , R. Gelisgen 2 , R. Yucel 1 , N. Dariyerli 1 , H. Genc 2 , Y. Karter 3 , C. Simsek 4 and H. Uzun 2 1 Department of Physiology, Cerrahpasa Medicine Faculty, Istanbul, TURKEY, 2 Department of Biochemistry, Cerrahpasa Medicine Fac- ulty, Istanbul, TURKEY, 3 Department of Internal Medicine, Cer- rahpasa Medicine Faculty, Istanbul, TURKEY, 4 Department of Public Health, Cerrahpasa Medicine Faculty, Istanbul, TURKEY Increased oxidative stress has been reported in hyperthyroid patients. It is known that oxidative stress causes atherosclerosis. Atherogenesis may be induced hyperthyroidism due to free radical mediated damage, e.g., lipid peroxidation, because lipid peroxida- tion plays a prominent role in lipoprotein oxidation. While oxid- ized low density lipoprotein (LDL) has atherogenic effect, high density lipoprotein (HDL) – associated paraoxonase1(PON1) pre- vents oxidation of LDL. This study was undertaken to compare the LDL oxidation and PON1 activity in hyperthyroid and control rats. Hyperthyroidism was induced by administration of L-thyrox- ine, 0.4 mg/100 g food, for 5 weeks. Plasma T3, T4, TSH, mal- ondialdehyde (MDA), total cholesterol, LDL cholestrol, HDL cholestrol, apolipoprotein A-1, triglycerides, oxidized LDL levels and PON1 activity were measured in hyperthyroid (n=12) and control (n=12) rats. Significantly increased MDA (P<0.001), oxidized LDL levels (P<0.01), decreased HDL-cholestrol (P<0.01), apolipoprotein A-1 levels (P<0.05) and PON1 activ- ity ( P<0.01) were found in hyperthyroid rats in comparison to control rats. There was no significant change in total cholestrol, triglycerides and LDL cholesterol levels. Our data indicate increased lipoprotein oxidation and reduced PON1 activity, thereby atherogenesis may be induced in hyperthyroidism. ª 2007 The Authors Journal compilation ª 2007 FEBS 265 Abstracts F1-5 Effects of hyperbaric oxygen treatment on glutathione and superoxide dismutase levels indistracted rat muscle S. Civelek 1 , S. Toklu 2 , Z. Kasymova 3 , H. Uzun 1 and A. Kaynar 3 1 Department of Biochemistry, Cerrahpasa Medical Faculty, University of Istanbul, Istanbul, TURKEY, 2 Department of Underwater and Hyperbaric Medicine, Medical Faculty Istanbul, University of Istanbul, Istanbul, TURKEY, 3 Istanbul University, School of Dentistry, Istanbul, TURKEY The aim of the study was to evaluate the effects of HBOT therapy on the muscle tissues surrounding the distracted rat tibia via bio- chemical parameters. The distraction protocol was carried out in optimum (0.5 mm/day) and hyperphysiologic (1 mm/day) rates. A total of 46 age-matched male Wistar rats were randomly divided into 5 groups; Group 1 (n=10) was distracted at 0.5 mm/day, Group 2 (n=10) was distracted at 0.5 mm/day and received HBOT treat- ment, Group 3 (n=10) was distracted at 1 mm/day, Group 4 (n=10) was distracted at 1 mm/day and received HBO treatment, and Group 5 was the sham operated control. At the end of experi- mental period of 5th and 9th days for 0.5 mm/day and 1 mm/day respectively, GSH levels and SOD activity were spectrophotometri- caly determined. Both GSH levels and SOD activity decreased with osteodistraction in muscle tissues. GSH levels and SOD activity in the in distracted rat muscle were higher in the HBO treated groups (Groups 2 and 4) than those in the untreated groups (Groups 1 and 3). Our preliminary data suggests that HBO may augment antioxid- ant levels in distracted rat muscle at both optimum and hyperphysio- logic rates. The implication of these findings is not yet completely known and therefore we are currently studying the effects of HBO therapy on bone healing and inflammatory cell recruitment in ani- mals, which underwent similar distraction conditions. Our findings may also elucidate the role of HBO therapy on soft and hard tissue healing in clinical applications of distraction osteogenesis. F1-6 Advanced glycation endproducts (AGEs) and antioxidant status in STZ induced diabetic rats: effects of copper supplementation S. Civelek, R. Gelis¸ gen, G. Andican, A. Seven, S. Ku ¨ cu ¨ k, M. Ozdogan and G. Burcak Department of Biochemistry, Cerrahpasa Medicine Faculty, Istanbul University, Istanbul, TURKEY We aimed to investigate the effects of Cu(II) supplementation on glycemic parameters, advanced glycation end products (AGEs), antioxidant status (GSH and total antioxidant capacity, TAOC) and lipid peroxidative damage (TBARS) in streptozotocin (STZ) induced diabetic rats and controls. Wistar albino rats were grouped as ‘control’, ‘STZ administered’, ‘Cu(II)supplemented’ ‘STZ admin- istered and Cu(II)supplemented’. STZ was administered (ip) at a single dose of 65 mg/kg and Cu II, 4 mg/kg (sc) every two days for 60 days. At the end of 60 days, glucose, Cu II, TBARS, TAOC were measured in plasma, GSH in erythrocytes and GHb in blood. Utilizing a flow system with two dedectors connected on line AGEs were measured spectrofluorometrically (k ex = 247 nm, k m = 440 nm) and low molecular peptides spectrophotometrically (k = 280 nm). In ‘STZ administered’ group plasma glucose, (P<0.01) GHb (P<0.05) and AGE(P<0.01) levels were higher than the control group. ‘Cu II supplemented’ group had lower plasma glucose (P<0.05) higher GHb (P<0.001), TAOC (P<0.05) and TBARS levels (P<0.001). In ‘STZ administered and Cu (II) supplemented’ group TAOC level was higher than the ‘STZ administered ‘group (P<0.01). Plasma AGE levels did not display any significant differences between Cu II supplemented and unsupplemented groups. The contribution of Cu II supplementa- tion to oxidative stress and AGE formation in diabetes remains to be elucidated. F1-7 Characterization of antioxidant capacities of 13 medicinal plant extracts F. X. Malcata 1 , A. Pereira 1 , I. Leita ˜ o 1 , M. Gia ˜ o 1 , J. Fernandes 1 , M. Pintado 1 , L. Belo 2 and A. Santos-Silva 2 1 College of Biotechnology, Porto, PORTUGAL, 2 Department Bioquı ´ mica, Faculdade de Farma ´ cia-Inst. de Biologia Molecular e Celular, UP, Porto, PORTUGAL Cardiovascular diseases (CVD) – mainly caused by atherogenesis, are the major cause of mortality and morbidity in the Western world. Infusions prepared with plant leaves are particularly rich in antioxidants, which seem to play a crucial protection role therein. Our aim was to evaluate the antioxidant features of 13 medicinal plant extracts: Agrimony, Avocato, Eucalyptus, Heath, Myrtle, Raspberry, Sage, Savory, Spanish wood Marjoram, Sweet amber, Thyme, Walnut-tree and Yarrow. Said features were determined by the ability to protect normal human red blood cells (RBC) against hydrogen peroxide – mediated oxidative damage in vitro. Our results showed that oxidative hemolysis of RBCs was sup- pressed by pre-treatment with all tested extracts (0.005%, w/v). In general, the protective effect increased in a dose-dependent manner (0.005–0.1%, w/v) – except for Heath, which exhibited a pro- hemolytic effect at 0.1% (w/v). The plant extracts with the highest protective effect, at the concentrations tested, were: Savory, Sage, Agrimony, Myrtle, Yarrow and Walnut-tree. These six extracts are thus potential nutraceutical ingredients towards CVD prevention. F1-8 On the dual nature and code of free radicals in biological systems S. Volovyk 1 and J. Siedow 2 1 Duke University Medical Center, Durham, NC, USA, 2 Duke University, Durham, NC, USA Free radicals, primordial ‘sea’ for the life origin and existence, induced by terrestrial and extraterrestrial radiation, are evolutional- ly archetypal, ubiquitous, and omnipotent in physiological-patho- physiological dichotomy in living systems. Delicate borderline norm-pathology with continuity-discontinuity dichotomy is a func- tion of immanent dual electronic nature and physiological func- tional ambivalence and complementarity of free radicals based on their dynamic charge transfer (CT)/redox ambivalence, correspond- ing code of chemical reactivity and selectivity and dynamic free- radical homeostasis. Subtle perturbations in radicals CT spatiotem- poral homeodynamics, in responsive signaling/controlling net- works, concomitant alterations in genes expression, transcription, and apoptosis, CT telomere/telomerase balance, DNA CT, hemi- spheric biochemical dominance/accentuation, including alteration of nitric oxide-superoxide complementarity, neurotransmission pat- tern, synaptic circuitry, etc have more fundamental impact on all organism systems functioning and deterioration than simple radi- cals inflicted oxidative cellular damage. This conceptualization of free-radical paradigm constitutes new dimension in deciphering molecular mechanisms of organism systems disorder – CT(redox)o- mics, that penetrates and links all ‘omics’. Acknowledgment: This material is based upon work supported by DOE under Award Number DE-FC01–06EH06028. 266 ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts F1-9 Ethanol-induced replication of hepatitis B virus requires IL-6 and JAK2 signaling pathway B. Kim, J. Kim and Y. Park Department of Biochemistry and Division of Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Seoul, REPUBLIC OF KOREA In patients infected with hepatitis B virus (HBV), alcohol intake may exacerbate clinical course of acute or chronic HBV infection and make effects on the susceptibility to HBV-related disease, such as cirrhosis and hepatocellular carcinoma. In this study, we studied the effect of ethanol on HBV replication and the signaling pathway involved in the event. In HepG2.2.15 cells, which are known to produce hepatitis B virus particles, the level of HBV transcripts and HBV DNA by ethanol treatment was increased in a dose- and time-dependent manner. In contrast, acetaldehyde as a product of the ethanol metabolism occurred in hepatocyte had no effect on the synthesis of HBV mRNA and ethanol-induced synthesis of HBV mRNA was not affected by pretreatment with 4-Methylpy- razole hydrochloride, alcohol dehydrogenase inhibitor. The synthe- sis of IL-6 mRNA was induced by ethanol treatment. Also, ethanol exposure induced the phosphorylation of JAK2, STAT3 and STAT5. Ethanol-induced phosphorylation of JAK2 was inhib- ited by pretreatment with IL-6 neutralizing antibody. Most import- antly, pretreatment with IL-6 neutralizing antibody reduced the ethanol-induced synthesis of HBV mRNA. Increase of HBV pro- moter activity by ethanol was abolished by pretreatment with the inhibitor of JAK2 (AG490) in HepG2 cells. Furthermore, ethanol- stimulated synthesis of HBV mRNA was completely inhibited by pretreatment with AG490 and by transfection of dominant negat- ive JAK2 plasmid. The level of HBV DNA enhanced by ethanol treatment was also blocked by pretreatment with AG490 in HepG2.2.15 cells. These results suggest that ethanol-enhanced rep- lication of HBV requires production of IL-6 and activation of JAK2. F1-10 Biochemical analysis of HCV E1E2 proteins containing entire and truncated ectodomains J. D. Tello,J.Go ´ mez-Gutie ´ rrez, B. Ye ´ lamos, M. J. Feito and F. Gavilanes Facultad de Ciencias Quı ´ micas. Universidad Complutense de Madrid, Madrid, SPAIN The hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which are components of the virus envelope. Their N-terminal ectodomains (residues 192–340 for E1 and 383–661 for E2) are thought to be responsible for the interaction between the virus and its receptor as well as for the fusion of the viral and cellular mem- branes. Using a baculovirus expression system we showed that, whereas E2 ectodomain could be successfully obtained with native- like properties, the efficient production of E1 was only achieved as a fusion protein with E2, either at the N-terminal (E1E2) or at the C-terminal (E2E1) end. Moreover, we analyzed several E1E2 chi- meras having the E2 ectodomain truncated at different positions in order to define the requirements for the expression of properly folded E1. Thus, we observed that the deletion of the C-terminal portion of E2 (residues 494–661) was dispensable, since protein E1E2–493 was correctly processed; however, the additional removal of the E2 473–493 fragment resulted in a missfolded poly- peptide, indicating that this region of E2 is necessary for the acqui- sition of the correct conformation of E1. The availability of different chimeras containing either the entire or truncated ectodo- mains has allowed us to develop comparative studies of their fuso- genic properties, by determining their ability to interact and destabilize phospholipid vesicles, and their interaction with hepatic cells. F1-11 Regulation of proteolytic processing of non-structural polyprotein in alphaviruses A. Lulla 1 , A. Golubtsov 2 , T. Ahola 2 and A. Merits 1 1 University of Tartu, Tartu, ESTONIA, 2 Institute of Biotechnology, Helsinki, FINLAND Semliki Forest virus (SFV) replication strategy relies on the pro- duction of replicase proteins in the form of non-structural polypro- tein precursor. Processing of the ns-polyprotein is performed by viral non-structural protease. During the course of infection poly- protein processing leads to rearrangements of the replication com- plex so that its RNA template preference is changed in favor of minus-strands over plus-strands. Previous results indicate that the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The determinants of cleavage efficiency are located in the region preceding the cleavage site and the protease recognizes at least residues P4 to P1’. Ran- dom mutagenesis analysis revealed that amino acid residues P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much vari- ation in contrast to residue P5, also mutation of residues P4 and P1’ had a significant effect on cleavage efficiency. However, it was found that only two cleavage sites are recognized by their short amino acid sequence, whereas processing of 2/3 site requires domain of nsP3 which is used for precise positioning of recognition sequence at the catalytic center of protease. Interestingly, the direct comparison of the requirements for the processing of cleavage sites in two prototype alphaviruses, SFV and Sindbis virus, indicates that in alphaviruses at least two different mechanisms of regula- tion, which lead to the similar outcome in the regulation of viral RNA synthesis. The results of current research significantly improve our understanding of the regulation of alphaviral replica- tion and reveal the principally new role of alphaviral nsP3 protein in the virus life cycle. F1-12 Effects of the Semliki Forest virus temperature sensitive mutations on the functions of virus-encoded proteins V. Lulla 1 , T. Ahola 2 and A. Merits 1 1 Institute of Molecular and Cell Biology, Tartu, ESTONIA, 2 Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND We have sequenced the nonstructural protein coding region of Sem- liki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. Several assays were used at dif- ferent temperatures to verify the phenotypic effects of the revealed mutations: titration of virus stocks, analysis of viral RNA synthesis, labeled polyprotein processing and other enzymatic assays. All mutations mapping to the protease domain of non-structural pro- tein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins. Two mutations mapping to nsP1 caused defects in RNA synthesis and did not affect the methyltransferase and guanylyl- transferase activities of nsP1 in vitro. ª 2007 The Authors Journal compilation ª 2007 FEBS 267 Abstracts F1-13 Changes of Ca 2+ affinity to membrane proteins of the sarcoplasmatic reticulum at experimental crash syndrome G. A. Kevorkian, L. H. Melkonyan, H. L. Hayrapetyan, A. G. Guevorkian, H. F. Khachatryan and L. N. Arakelyan H.Buniatian Inst. of Biochemistry, Yerevan, ARMENIA The experimental crash syndrome induced by crash of the hip soft muscle depends on two periods of the pathogenesis development: compression period (2 and 5 h) and decompression period (2, 4, 24, 48 h). Compression period was taken as control, and respectively, the decompression period was studied as experimental one. Our observations indicate that at decompression there take place a gen- eral intoxication of the organism and an infarction of myocardium. The experimental data coincide with clinical results obtained in the period of the 1988 earthquake in Armenia. By our results the devel- opment of the infarction of myocardium depended on toxic peptides released to blood from the randomized muscles and injured kidneys. In 2 h after decompression the membrane proteins of the sarcoplas- matic reticulum began to change Ca 2+ affinity, data of which were plotted in a Scatchard coordinate system. In 24 h after decompres- sion 5 acidic proteins and calsequestrin affinity almost wholly loose Ca 2+ affinity. Simultaneously the membrane protein with 32 kDa molecular weight, which does not possess Ca 2+ affinity, begins to bind Ca 2+ . It is shown that Ca 2+ is bound in 2 points: with high and low affinity. Hence, in 48 h after decompression the concentra- tion of Ca 2+ in high affinity part (Bmax and Kd) does not change, while Bmax and Kd of the low affinity part increase. Absolutely identical changes took place with the affinity to membrane proteins of the sarcoplasmatic reticulum at adrenal necroses, at injuries of myocardium at experimental pancreatitis, as well as at pancreatitis and crash syndrome. The problem of general mechanism of myocar- dium injury at pathologies with different etiology is represented. F1-14 A model for the influence of the polarizability on the ions solvation in the lipid membrane D. Ionescu 1 and R. A. Ionescu 2 1 University of Medicine and Pharmacy, Bucharest, ROMANIA, 2 National Institute for Physics and Nuclear Engineering ‘Horia Hulubei’, Bucharest, ROMANIA We discuss about conductance mechanisms at the level of lipid artificial membranes, taking into consideration the influence of the properties of the ions, emphasizing on their polarizability. One of the properties of the lipid membrane which is changed due to the presence of the ions in the bordering aqueous phase is the mem- brane dipole potential and this can be macroscopically seen by the amplitude of the diffusion currents through the lipid membrane given by differently charged ions. Our model takes a step deeper in the structure of the membrane and we intend to explain the man- ner in which the polarizability of the ions influences their solvation in the lipid membrane, supposing that they diffuse through the lipid phase surrounded by water molecules. Our calculations con- firm that the total energy of an ion into the lipid phase, which is given by the electrostatic and surface contributions, has a mini- mum at a radius of a few Angstrom. This means that it is energet- ically favorable for an ion to diffuse through the membrane together with water molecules. Because the polarizability of the ion plays an important role at the interface, we investigate the role of the hydrated ion total polarizability on the ion-lipid-water parti- tion. F1-15 Elongation and desaturation of fatty acids are critical in lipid metabolism, growth, and ontogeny of Caenorhabditis elegans K. Sakamoto, M. Horikawa, T. Hashimoto and T. Nomura University of Tsukuba, Tsukuba, JAPAN Obesity is a main causative factor in human lifestyle-related disease. Recently, it was reported that a deficit in the mouse stea- royl-CoA desaturase 1 (Scd1) gene decreases biosynthesis and accumulation of fatty acid and revitalizes the b-oxidation of fatty acid. To examine the physiological role of fatty acid desaturase (fat) and elongase (elo) in ontogeny, fatty acid accumulation, and individual lifespan, we performed bacteria-mediated RNA interfer- ence (RNAi) in the nematode Caenorhabditis elegans. Suppression of the expression of elo-2 gene mRNA caused a drastic decrease in the amount of body fat, miniaturization of the body, defects in egg-laying, and increased lifespan. The amount of body fat was markedly decreased, and body size reduced, by downregulation of fat-6 and fat-7—a functional homolog of the mouse Scd1 gene—- whereas lifespan was drastically reduced. RNAi of fat-6 decreased the mRNA levels of the genes involved in fatty acid synthesis, and RNAi of fat-7 increased the mRNA levels of b-oxidation-related genes. Additionally, RNAi of the elo-2 gene caused a remarkable decrease in fatty acid biosynthesis-related gene expression. These results indicated that the elongation and desaturation of fatty acids are integral to various phenomena such as ontogeny and lifespan and play important roles in fatty acid accumulation and consump- tion. F1-16 Anions modulate the interaction of hypericin/ BSA or amitriptyline complex with lipid membrane D. Ionescu, M. Dragusin, M. Dima, A. Popescu and C. Ganea Universtity of Medicine and Pharmacy ‘Carol Davila’, Bucharest, ROMANIA By the means of an electrophysiological method, Black Lipid Membrane (BLM), we studied the manner in which the electrical capacitance and conductance of the lipid membrane vary in the presence of some lyotropic anions, which are lately frequently encountered in various foods as additives, when the bovine serum albumin (BSA) – Hypericin complex was added. We previously reported (Ionescu and Ganea 2005) the increase in the conductance and the capacitance of the membrane in the presence of lyotropic anions when hypericin only is added and our recent results show that in the presence of perchlorate, acetate and chloride the electri- cal properties of the lipid membrane vary also when BSA – Hypericin complex is added, but with a different profile: the aug- mentation of the electrical conductance and capacitance of the lipid membrane when hypericin is added is steeper than the one obtained when adding BSA – Hypericin complex, suggesting a slower incorporation of the complex in the membrane. On the other hand, an increasing concentration of tricyclic antidepressant amitriptyline also modifies the insertion kinetics of hypericin in the lipid membrane in the presence of lyotropic anions showing a sat- uration of the binding process. Reference 1. Ionescu D, Ganea C. The Hofmeister effect of anions on the insertion of hypericin in lipid bilayers. Eur. Biophys. Journal 2005; 34: 699. 268 ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts F1-17 Effect of alpha-lipoic acid on myeloperoxidase activity and lipid peroxidation level in carrageenan-injected rats F. Odabasoglu 1 , H. Aygun 2 , Z. Halici 3 , Y. Bayir 1 , A. Cakir 4 , E. Cadirci 5 and F. Atalay 1 1 Department of Biochemistry, Faculty of Pharmacy, Ataturk University, Erzurum, TURKEY, 2 Department of Orthopedics and Traumatology, Medical Faculty, Ataturk University, Erzurum, TUR- KEY, 3 Department of Pharmacology, Medical Faculty, Atatu ¨ rk Uni- versity, Erzurum, TURKEY, 4 Department of Chemistry, Education Faculty, Atatu ¨ rk University, Erzurum, TURKEY, 5 Department of Pharmacology, Faculty of Pharmacy, Ataturk University, Erzurum, TURKEY Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mito- chondria as the coenzyme for pyruvate dehydrogenase and alpha-ke- toglutarate dehydrogenase. We have investigated alterations in the activity of myeloperoxidase (MPx) and level of lipid peroxidation (LPO) following oral administration of ALA, indomethacine (IND) and diclofenac (DIC) in rats with carrageenan-induced paw edema. Fortytwo Dawley rats divided into seven groups, of which paws of rats in six groups were injected with carrageenan (CAR). Then, they received ALA (50, 100 and 200 mg/kg), IND (25 mg/kg) and DIC (25 mg/kg) orally. Rats from the other group served as control. Fol- lowing euthanasia, paw tissues were scrapped and then ground within liquid nitrogen for activities of enzymes and GSH level. In the present study, we found that (i) ALA reduced the development of CAR-induced paw edema, at a smaller magnitude for ALA than for IND and DIC; (ii) All doses of ALA and IND have significantly decreasing effect on amplification in MPx activity resulting from induced paw edema; and (iii) All doses of ALA, IND and DIC ameli- orated amplifications in the LPO level caused by CAR injection. These results suggest that the anti-inflammatory effect of ALA on CAR-induced acute inflammation can be attributed to its amelior- ating effect on lipid peroxidation level and myeloperoxidase activity. F1-18 A Chinese hamster ovarian cell line imports cholesterol by high density lipoprotein degradation H. Stangl 1 , T. Pagler 1 , S. Golsabahi 1 , H. Laggner 1 , A. Lohininger 1 , S. Rhode 2 , G. J. Schu ¨ tz 2 , M. Pavelka 1 , C. Wadsack 3 and W. Strobl 1 1 Medical University of Vienna, Vienna, AUSTRIA, 2 Institute of Bio- physics, Johannes Kepler University Linz, Linz, AUSTRIA, 3 Clinic of Obstetrics and Gynecology, Medical University of Graz, Graz, AUSTRIA Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotec- tive role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol and transports its lipid load back to the liver. Thereby HDL is not dismantled but transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein deficient media degrades HDL and internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esteri- fication after HDL treatment. This HDL degradation pathway is distinct from the LDL receptor pathway, but also degrades LDL. 25-OH cholesterol, a potent inhibitor of the LDL receptor path- way, down-regulated LDL degradation in CHO7 cells only in part, and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycanes and lipases was tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, these CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. HDL degradation with similar prop- erties was also observed in a human placental cell line. F1-19 Structural modeling and site-directed mutagenesis of Zea mays phosphomethylpyrimidine kinase/thiamine phosphate synthase M. Rapala-Kozik 1 , M. Olczak 2 , A. Starosta 1 , J. Soroka 1 , K. Ostrowska 3 and A. Kozik 1 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, POLAND, 2 University of Wroclaw, Faculty of Biotechnology, Wroclaw, POLAND, 3 Jagiellonian University, Faculty of Chemistry, Krakow, POLAND Vitamin B1 (thiamine) can be synthesized by bacteria, fungi and plants from two precursors: 4-amino-5-hydroxymethyl-2-methylpy- rimidine phosphate (HMP-P) and 4-methyl-5-(2-hydroxyethyl) thi- azole phosphate (HET-P). HMP-P is then phosphorylated to HMP-PP which condenses with HET-P into thiamine phosphate (TMP). To perform two last steps, the higher plants, unlike other thiamine synthesizing organisms, use bifunctional enzymes with both HMP-P kinase and TMP synthase activities. These unique plant enzymes had not been structurally or kinetically character- ized until our recent isolation of recombinant Zea mays protein. In this work we present structural models of that protein. The pres- ence of separate HMP-kinase- and TMP-synthase domains was deduced from sequence comparisons with other enzymes with these activities. Based on the sequence similarity of N- and C-terminal domains to two bacterial enzymes with known three-dimensional structures, Salmonella typhimurium HMP-P kinase and Bacillus subtilis TMP synthase, respectively, we modeled their overall struc- ture and arrangement of active site residues, using the SWISS- MODEL server. Like the bacterial prototypes, the plant HMP-P kinase domain had a ribokinase fold and TMP-synthase form a typical TIM barrel. Site-directed mutagenesis was used to experi- mentally verify the model-predicted active site residues including G65, A72, D77, T96, Q98, M134, V161 and C268 at the HMP-P binding region and R375, K377, N405, D406, S444, T470, K473, G500, V523, S524 at TMP synthase center. F1-20 Structural and functional characterization of CheY from Helicobacter pylori K. H. Lam 1 , T. K. W. Ling 2 and S. W. N. Au 1 1 Department of Biochemistry, Centre for Protein Science and Crys- tallography, Faculty of Science, The Chinese University of Hong Kong, Hong Kong SAR, HONG KONG, 2 Department of Microbio- logy, The Chinese University of Hong Kong, Prince of Wales Hospi- tal, Hong Kong SAR, HONG KONG Helicobacter pylori is the human pathogen that causes gastritis, duodenal ulcer and gastric cancer. About 50% of the world popu- lation has been infected with H. pylori. Motility is an important virulence trait for H. pylori infection. From animal model in previ- ous studies, cheA, cheW and cheY mutants are non-chemotactic and show attenuated phenotype, suggesting that chemotaxis is essential in colonization. CheY belongs to response regulator superfamily that controls the clockwise or counter-clockwise move- ment of flagella. The process is determined by phosphorylation and dephosphorylation of CheY. Phosphorylation increases the affinity of CheY to FliM, a component of switch protein complex, altering the flagella rotation to opposite direction. To study the underlying mechanism of chemotactic regulation in H. pylori,we have expressed and purified recombinant CheY. Here, we report a 1.8 o A crystal structure of CheY. Structural comparison between CheY in H. pylori and other species will be discussed. The in vitro interaction between CheY and FliM will be investigated. ª 2007 The Authors Journal compilation ª 2007 FEBS 269 Abstracts F1-21 Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose K. Kim, W. Jung and S. Rhee Seoul National University, Seoul, REPUBLIC OF KOREA D-Psicose, a rare sugar produced by the enzymatic reaction of D- tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently character- ized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefac- iens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C3 position, with marked efficiency for D-psicose. The crystal struc- tures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enedio- late. Subsequently, Trp112 and Pro113 in the b4-a4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic res- idues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C3 position. F1-22 Crystallization and structure solution of major grass pollen allergen Phl p 3 V. Devanaboyina 1 , W. Keller 1 , A. Nandy 2 and H. Fiebig 2 1 University of Graz, Graz, AUSTRIA, 2 Allergopharma J.Ganzer KG, Reinbek, GERMANY Ig-E mediated allergies represent a major health problem in the industrialized world as they affect almost 25% of the population. For the precise analysis of the surface exposed IgE epitopes, the three dimensional structure of allergens are the most important source of information. The detailed knowledge derived from the three dimensional structure together with immunological data allows for the rational development of strategies to convert aller- gen molecule in to hypoallergenic derivatives through destruction or reorientation of IgE epitopes, such allergen derivatives may act as potent hypoallergenic vaccines for immunotherapy. The allergens from grass pollen are amongst the most potent and frequent elicitors of allergic disease. Phl p 3 is a timothy grass pol- len allergen with a molecular weight of 10 959 kDa. Phl p 3 crys- tallizes in two different forms at pH of 5.5 with PEG as precipitant. The Phl p3 sequence has a 57% sequence identity with Phl p 2, whose X-ray structure has been solved (PDB: 1WHO). A data set was collected to 2.3A ˚ at DESY, Hamburg. Structure is solved by Molecular replacement method. Phl p 3 has a FN-type III fold and it is beta sheet protein. Acknolledgement: This work is generously supported by Aus- trian science Foundation. FWF-SFB F018 F1-23 Reconstitution of azurocidin proteolytic activity by site-directed mutagenesis M. Olczak and T. Olczak University of Wroclaw, Wroclaw, POLAND Azurocidin (HBP or CAP37) is a 37-kDa polypeptide produced mostly in human neutrophils. The protein is proteolytically proc- essed during maturation by removal of 19-aa signal peptide and then two short 5-aa and 2-aa peptides from the N-terminus and 3-aa peptide from the C-terminus. Azurocidin belongs to serine proteinase family, closely related to neutrophil elastase, cathepsin G, proteinase 3, chymase and granzymes. However, the proteolytic activity of azurocidin was lost due to a substitution of His41 and Ser175 residues from catalytic triad by Ser and Gly residues, respectively. Using site-directed mutagenesis and non-lytic insect cell expression system we designed and expressed a double azuroci- din mutant with reconstituted catalytic triad and single mutants with Ser41 replaced by His or Gly175 substituted by Ser. All pro- teins contained respective protease cleavage site, inserted down- stream of a signal peptide sequence, allowing production of mature azurocidin after activation with specific protease (enterokinase, fac- tor Xa, or protease TEV). We showed that azurocidin with recon- stituted catalytic triad exhibited proteolytic activity when incubated with fluorescently labeled casein. Preliminary results also suggest autocatalytic removal of a tripeptide at the C-teminus of the protein. In contrast, single mutations of azurocidin has no effect on recovery of proteolytic activity. Our results confirms that the proteolytic mechanism of pro-peptide removal and azurocidin activation is preserved in the double mutant. F1-24 Study of operation of the molecular hinges in human PGK using site-directed mutagen esis B. Flachner, J. Szabo ´ , A. Varga, P. Za ´ vodszky and M. Vas Institute of Enzymology HAS, Budapest, HUNGARY The glycolytic enzyme 3-phosphoglycerate kinase (PGK) contains two structural domains. The domains each bind one of the two substrates: 1.3-BPG and MgADP or 3-PG and MgATP in the for- ward or reverse reaction. The transfer of the phospho group between the two substrates requires closure of the two domains. This motion brings the substrates into optimal distances for the reaction. Molecular graphical comparison of the open and closed crystal structures suggested the location of the main hinge region in b strand L. It may also regulate the other hinges at the ends of helix 7. In order to identify the side chains responsible domain clo- sure we constructed the following mutants of human PGK: S392A and T393A (bL), S398A (a14), N336A (bJ) as well as E192A (a7). The mutants were characterised by biophysical tests (CD, DSC) and by enzyme kinetic and substrate binding experiments. T393 is important in receiving the transmitted effect of 3-PG from the N- domain. The side-chain of N336 is located in the nucleotide-bind- ing site. It has an essential role in the enzyme activity and mediates the effect of the nucleotide substrate towards the main hinge. The side-chain of E192 connects a7tobL and has a role in the con- formational stability on the whole molecule. It also mediates the effect of 3-PG towards the bL. Based on these results a compre- hensive picture about operation of the main molecular hinge of PGK is presented. 270 ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts F1-25 The role of metal ion on active site in Pseudomonas stutzeri L-rhamnose isomerase H. Yoshida, M. Yamaji, M. Yamada, G. Takada, K. Izumori, T. Ishii and S. Kamitori Kagawa University, Kagawa, JAPAN L-rhamnose isomerase from Pseudomonas stutzeri (P. stutzeri L-RhI) is homo tetrameric enzyme and can catalyze the reversible isomerization between various aldoses and ketoses in the presence of appropriate metal ions. Each monomer contains two metal ions, ‘structural’ and ‘catalytic’ metals. L-RhI shows metal dependency and has the highest catalytic activity in the presence of Mn 2+ . The relative activity for Cu 2+ ,Co 2+ ,Zn 2+ compared to that of Mn 2+ is ca. 50, 40, 10%, respectively. To investigate the role of metal ion on active site in P. stutzeri L-RhI, we determined the crystal structures of P. stutzeri L-RhI in the presence of Zn 2+ (L-RhI_Zn), Mn 2+ (L-RhI_Mn), and in complex with a substrate, D-psicose (L-RhI_Mn/D-psi). In the electron density map of L-RhI_Mn, the ‘catalytic’ metal could be located at two positions, implying that Mn 2+ possibly moves between the two positions in each monomer, whereas the electron density maps of L-RhI_Zn and L-RhI_Mn/D-psi showed obvi- ously one position for ‘catalytic’ metal in each monomer. The movement of Mn 2+ as a catalytic metal might be related to cata- lytic activity of P. stutzeri L-RhI. F1-26 Gene expression profiling in the bone tissue of osteoporotic mice I. Orlic, F. Borovecki, P. Simic and S. Vukicevic Medical School, Zagreb, CROATIA Osteoporosis is a major public health problem that is characterized by microarchitectural deterioration, low bone mass and increased risk of fractures. Although widely studied, complex etiology of osteoporosis has still not been fully clarified. Ovaricetomized (OVX) mice represent an optimal animal model to investigate bone loss in osteoporosis. To further elucidate the underlying mecha- nisms of decreased bone formation and increased bone resorption following ovariectomy, we conducted gene expression profiling experiments using bone samples of OVX C57BL/6J mice. At 21 days following OVX we observed deregulation of genes involved in bone resorption, suggesting that this time point is very close to the peak of osteoclastic activity. Following OVX, genes involved in immune response, cell cycle regulation, growth, apop- tosis and bone resorption were upregulated, while genes that are important for mitosis, metabolism of carbohydrates, extracellular matrix structure, angiogenesis, skeletal development and morpho- genesis were downregulated. Among bone specific genes we observed upregulation of interleukin 7 (IL-7), IL-7 receptor and matrix metallopeptidase 8, while TGFb-3, procollagen type I and VI exhibited marked decrease in expression. We also discovered downregulation of two genes, parathyroid hormone receptor 1 and WD repeat domain 5, that are involved in skeletal development but were not previously reported to be altered in osteoporosis. In conclusion, OVX greatly influences expression of various genes involved in diverse biological processes confirming the notion that numerous pathways play an important role in pathophysiology of osteoporosis. F1-27 Effect of alloxan on urea, creatine and bilirubin levels in serum of rats A. Cebi, S. Yasar, G. Oto and H. Demir Yuzuncu Yil University, Van, TURKEY This study was carried out to investigate whether alloxan could affect biochemical parameters (urea, creatine, total bilirubin and direct bilirubin) in serum of rats or not. Twelve Sprague-Dawley albino rats were divided into two experimental groups; control and study groups. A single dose (100 mg/kg) of alloxan was injected in- traperitonealy to the study group rats. Same amount of physiologi- cal saline was injected to the control group rats. Various biochemical constituents were measured first, third and sixth hour after the alloxan injection. All biochemical parameters were meas- ured using an autoanalyzer (BNN/Hitachi-911) and the corres- ponding kit (DPC, Diagnostic Products Corporation, USA). One way ANOVA test was performed for statistical analysis. After all- oxan injection, urea levels increased significantly in all the meas- urements. Total bilirubin and direct bilirubin levels decreased as regard to control group in all the measurements after the alloxan injection whereas creatine levels decreased only in the measurement of first hour. It is concluded from this study that alloxan differ- ently effect urea, creatine and bilirubin levels in rats. F1-28 Differential regulation of homocysteine transport in vascular endothelial and smooth muscle cells X. Jiang 1 , F. Yang F 1 , E. Brailoiu 1 , H. Jakubowski 2 , N. Dun N 1 , A. I. Schafer 3 , X. Yang 1 , W. Durante 4 and H. Wang 1 1 Temple University School of Medicine, Philadelphia, PA, USA, 2 UMDNJ-New Jersey Medical School, Newark, NJ, USA, 3 University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 4 University of Missouri School of Medicine, Columbia, MO, USA We previously reported that homocysteine (Hcy) inhibits endothel- ial cell (EC) growth and promotes vascular smooth muscle cell (VSMC) proliferation. To begin to elucidate the underlying bio- chemical basis for these disparate effects, this study compared Hcy transport in cultured human aortic EC (HAEC) and smooth mus- cle cells (HASMC). L-Hcy (10 lM) was transported into both cell types in a time dependent fashion but was approximately 4-fold greater in HASMC. Hcy transport in HAEC had a Michaelis con- stant (Km) of 39 lM and a maximal transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, Hcy transport in HASMC had a lower affinity (Km=106 lM) but a higher transport capacity (Vmax = 4192 pmol/mg protein/min). Competition studies revealed that the small neutral amino acids tyrosine, cysteine, gly- cine, serine, alanine, methionine and leucine inhibited Hcy uptake in both cell types but the inhibition was greater for tyrosine, serine, glycine and alanine in HAEC. Sodium-depletion reduced Hcy transport to 34% in HAEC and 63% in HASMC. Increases in pH from 6.5 to 8.2 or lysosomal inhibition blocked Hcy uptake only in HAEC. In addition, Hcy shares carrier systems with cysteine, in a preferable order of ASC > XAG = L in HAEC and ASC > L > XAG in HASMC. The sodium-dependent system ASC is responsible for approximately 90% of Hcy uptake in HAEC and 65% in HASMC. The predominant role of system ASC and the lysosomal regulated feature of Hcy transport in EC may contribute to its cell specific proatherogenic effect and cardio- vascular disease. ª 2007 The Authors Journal compilation ª 2007 FEBS 271 Abstracts F1-29 Ciliated epithelial- and regional-spe cific expression and regulation of the estrogen receptor b 2 in rat fallopian tubes R. Shao, Sr. and H. Billig Neuroscience And Physiology, Go ¨ teborg University, Go ¨ teborg, SWEDEN In the present study, we have investigated the intracellular localiza- tion and regulatory patterns for ERb isoforms in rat fallopian tubes. Western blot analysis reveals that two ERb isoforms corres- ponding to ERb1 and ERb2 are expressed in rat fallopian tubes. However, ERb2 is the predominant form of ERb in this tissue. High-resolution confocal imaging and immunohistochemical analy- sis provide ample evidences that ERb expression is limited almost exclusively to the ciliated epithelial cells in contrast to ERa, which is widely distributed. Furthermore, within the ciliated epithelial cells, ERb is colocalized with b-tubulin IV at stem portion of the cilia. We show that ERb2 protein expression is tightly regulated by 17b-estradiol (E2) or diarylpropionitrile (DPN) in a time-depend- ent manner without changes in ERb1 expression. These estrogenic effects are inhibited by an ER antagonist ICI 182.780. In addition, significant alteration of ERb immunoreactivity is only detected his- tologically in the ampullary region. As the cilia are considered an essential determinant of tubal transport, we further demonstrate that E2- or DPN-induced ERb2 activation is associated with alter- ations in tubal protein expression crucial for the regulation of cal- cium-dependent ciliary beating. Given the coordinated regulation and interaction of ER and progesterone receptor in the cilia, we hypothesize that tubal ERb2 may facilitate the estrogen-mediated transport process by processing protein-protein interaction under physiological and/or pathological conditions. This study shows for the first time that a previously unrecognized localization of ERb isoform in rat fallopian tubes. F1-30 Recombinant starmaker T. M. Kapon 1 , G. Rymarczyk 1 , M. Nocula-Ługowska 1 , M. Jako ´ b 1 , Z. Szewczuk 2 and A. O _ zyhar 1 1 Department of Biochemistry, Wroclaw University of Technology, Wrocaw, POLAND, 2 Group of Chemistry and Stereochemistry of Peptides and Proteins, Faculty of Chemistry, University of Wroclaw, Wrocaw, POLAND Otoliths are essential elements in sensory system of the zebrafish (Danio rerio). These structures enable fishes to sense gravitational forces and linear accelerations. The biomineralization of the oto- liths is controlled by Starmaker (Stm) protein, which determines their morphology and even their crystal lattice structure. To facili- tate understanding of the molecular role Stm protein plays in bio- mineralization, it is necessary to obtain large amounts of pure Stm for in vitro studies. In this report, we describe bacterial expression system that allows to obtain about 1 mg of protein from 1 l of cul- ture. We have also elaborated purification procedure based on salt- ing out, as well as size exclusion and hydroxyapatite chromatography. Consequently, we have purified Stm devoid of any peptide tag, and the identity of the protein was confirmed by ESI MS. Subsequent gel filtration revealed extended conformation of Stm in solution, showing Stokes radius of 78.6 A ˚ , which is much higher than expected for a globular protein of the molecular mass of Stm (64.5 kDa). Such extended conformation, which is characteristic for intrinsically unstructured proteins, might result from high mean net charge of Stm combined with its low mean hy- drophobicity. Acknowledgement: Supported by a grant from the State Com- mittee for Scientific Research. F1-31 Prenatal cocaine exposure alters coronary artery reactivity in adult offspring L. Zhang 1 , D. Xiao 1 and S. Yang 2 1 Loma Linda University, Loma Linda, CA, 2 California State University, San Bernardino, CA, USA The present study tested the hypothesis that prenatal cocaine expo- sure alters myogenic reactivity of the coronary artery in adult off- spring. Pregnant rats received cocaine (30 mg/kg/day) or saline from days 15–21 of gestational age. Coronary arteries were isolated from 3 month-old offspring, and pressure-dependent myogenic tone was measured simultaneously with vessel wall intracellular Ca 2+ concentrations ([Ca 2+ ] i ). Prenatal cocaine exposure signifi- cantly decreased coronary artery myogenic responses without affecting [Ca 2+ ] i in adult male rats, resulting in a decrease in the ratio of changes in diameter to changes in [Ca 2+ ] i . In contrast, cocaine treatment caused a significant increase in pressure-induced myogenic tone and the ratio of changes in diameter to changes in [Ca 2+ ] i in coronary arteries of female offspring. Inhibiton of eNOS with L-NNA did not alter coronary artery myogenic responses in either saline or cocaine-treated offspring. The results suggest that prenatal cocaine exposure reprograms coronary artery function in a gender specific manner, resulting in a downregulation of pres- sure-dependent myogenic tone in male adult offspring, but an up- regulation in female offspring, which is caused by changes in the Ca 2+ sensitivity of myogenic mechanism. Acknowledgement: Supported in part by USA NIH grants HL82779 and S06GM073842. F1-32 Depolarization-evoked increase of cytosolic Ca 2+ : a new fluorescent assay in rat cortical neurons S. Francisconi, P. Salvati and C. Caccia Newron Pharmaceuticals SpA, Bresso (MI), ITALY Most of the studies in the literature on depolarization-induced Ca 2+ increase have been performed using synaptosomes and Fura2 as Ca 2+ indicator while only a few studies have been carried out in cultured neurons. In contrast to synaptosomes, which allow pharmacological studies only of presynaptic effects, neuronal cul- tures show intact neural circuits and synaptic integrity (pre-and postsynaptic cross-talk). Aim of the present study was to develop a fluorescence assay using a new and stable Ca 2+ dye (Fluo4) to measure the depolarization-induced Ca 2+ influx in cultured rat cortical neurons. In our experimental conditions, high K + depolar- ization induced a very fast increase in [Ca 2+ ] i , mainly due to Ca 2+ entry through voltage-dependent calcium channels (no increase in absence of Ca 2+ ions in the medium) and partly to the depletion of intracellular Ca 2+ storages (ryanodine receptor activation). Using specific toxins able to block different calcium channel sub- types, it was confirmed that R-, L- and P-type calcium channels played the major role in KCl-mediated Ca 2+ influx. The rise in Ca 2+ evoked by veratridine has to be ascribed only to Ca 2+ influx through voltage-dependent calcium channels, as it was completely abolished in absence of extracellular Ca 2+ . Our rat cortical neuron preparations respond to pharmacological characterization with ref- erence calcium and sodium channel blockers and therefore it could be a suitable model for screening novel compounds with therapeu- tic potential for channelopathies. 272 ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts F1-33 AGEs in experimental diabetic nephropathy: are pyridoxal phosphate and thiamine pyrophosphate beneficial ? G. Burcak, M. Ozdogan and S. Ku ¨ c¸ u ¨ k Department of Biochemistry, Cerrahpas¸ a Medical Faculty, _ Istanbul University, _ Istanbul, _ Istanbul, TURKEY Increased advanced glycation end product (AGE) formation is the major mechanism implicated in diabetic nephropathy (DN). In this study we questioned the benefit of pyridoxal phosphate (PLP) and thiamine pyrophosphate (TPP) in DN. In Wistar albino rats grouped as ‘Diabetic’,’Diabetic+PLP’,’Diabetic+TPP’, ‘Dia- betic+Insulin’ and ‘Control’, glucose HbA1c, AGE-peptides (plasma and kidney), aldose reductase (AR) activity (kidney), 8-epi PGF 2a (urine), creatinine clearance, microalbuminuria and b 2- microglobulinuria were measured. Our data revealed the estab- lishment of DN. AGE-peptides increased both in the kidney and in the plasma of diabetic rats (P<0.05, P<0.01) and were found to be correlated (P<0.01). AR acitivity did not display any significant difference between the groups. 8-epi PGF 2a was higher in diabetic rats than in control (P<0.001). Insulin treat- ment caused significant decreases in all parameters except renal hypertrophy and plasma AGE-peptide levels. PLP supplementation caused reduction in microalbuminuria and 8-epi PGF 2a lev- els.(P<0.01,P<0.05). TPP treatment appeared to have no effect on the measured parameters. To conclude, our results sug- gest that AGE-peptides in plasma reflect the AGE content in kid- ney. PLP treatment slowed the progression of DN and decreased glomerular injury. Considering the inhibitory effects of TPP and PLP on advanced glycation and oxidative stress, further studies addressing their role in DN are needed. F1-34 The relationship of bone metabolism with nitric oxide and cytokines in chronically ethanol treated rats S. Kaya 1 , I. Guner 2 , I. Birincioglu 3 , V. Sozer 4 , H. Uzun 1 , S. Aydin 1 , Y. Karter 5 , C. Simsek 6 , G. Yigit 2 and G. Simsek 2 1 Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 2 Department of Physiology, Cer- rahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 3 The Republic of Turkey Ministry of Justice Council of Forensic Medicine, Istanbul, TURKEY, 4 Yıldız Technical University Arts and Science Faculty Department of Chemistry, Istanbul, TURKEY, 5 Department of Internal Medicine, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 6 Department of Public Healty, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY We investigated the effects of NO, IL-lb, IL-6 and TNF-a on bone metabolism in chronically ethanol treated rats. Chronic ethanol intake was produced in 6 months old rats by gradual substitution (within 3 weeks) of tap water in diet for 5, 10, 15 and finally 20% of ethanol after which the animals were maintained under these conditions for 4 months. After 4 months of ethanol administration N w -nitro-L-arginine methyl ester (L-NAME) which is a NOS inhib- itor was given for three weeks along with ethanol to the same rats. Blood samples takes from the tail vein of rats were analysed for serum IL-lb, IL-6, TNFa, NO, Ca, P, PTH, 25(OH) D 3 , B-ALP. Ethanol treatment increased both cytokine and NO levels. Ca decreased. P, PTH and 25(OH) D 3 did not change. While the b- ALP decreased there was no change in T-ALP. ALT, AST and GGT increased. Simultaneous administration of ethanol and L- NAME in the same rats increased IL-6 and TNF-a levels whereas IL-lb were unchanged. Ca and P increased while PTH and 25(OH)D 3 decreased. T-ALP, b-ALP, ALT, AST and GGT increased. In this study we observed that ethanol suppressed bone turnover. Antiremodeling effect of ethanol may be mediated by NO F1-35 Transgenic rabbit as a source of recombinant human TNAP for pharmaceutical application L. Bodrogi 1 , R. Brands 2 , W. Raaben 3 , W. Seinen 2 , M. Baranyi 1 , D. Fichter 3 and Z. Bo} sze 1 1 Agricultural Biotechnology Center, Godollo, HUNGARY, 2 IRAS, University of Utrecht, Utrecht, NETHERLANDS ANTILLES, 3 AM-Pharma B.V., Bunnik, NETHERLANDS ANTILLES Transgenic rabbits are excellent bioreactors for the production of recombinant proteins in their milk both on an experimental and commercial scale. Alkaline phosphatases (AP) are promising thera- peutic agents in the Gram negative bacterial lipopolysaccharide (LPS) mediated diseases. LPS is dephosphorylated and thereby detoxified by placental AP at physiological pH. In our earlier experiments calf intestinal AP prevented 80% of mice from lethal Escherichia coli infection and attenuated LPS toxicity in piglets. Purified tissue non-specific AP (TNAP) is not available in large quantities from tissue sources which would enable to analyse its efficacy in animal sepsis models. We created two transgenic rabbit lines by pronuclear microinjection with the whey acidic protein promoter- humanTNAP minigene. Lactating females of both lines produced enzymatically active human TNAP which was two orders of magnitude higher compared to normal human serum levels and the molecular weight of recombinant protein was compliant with the authentic human form. After purification of the recombinant human TNAP from rabbit milk its effectiveness will be tested in animal sepsis models and it can be a valuable option with import- ant implication in attenuating LPS mediated inflammatory responses. Acknowledgement: This work was supported by the grants NWO-OTKA N37293 and GVOP-AKF 71/2004. F1-36 Expression of osteonectin correlates with levels of fin regeneration in zebrafish (Danio rerio) A. Brito, L. Cancela and P. Gavaia CCmar, University of Algarve, Faro, PORTUGAL Mammals have the ability to regenerate some tissues such as blood and liver, but the majority of organs fail to regenerate. In contrast, zebrafish is capable of regenerating complex organs/tissues such as optic nerve, scales, heart and fins, and is presently one of the most used metazoan in regeneration research. Zebrafish fin is composed of multiple fin rays with bony parts (lepidotrichia) originated by intramembraneous ossification. Fin regeneration is an epimorphic process dependent on formation of a specialized structure (blas- tema), consisting of mesenchymal-like cells located between stump tissues and the wounded epidermis, This mass of proliferative, plu- ripotent progenitor cells is the key intermediate for strict growth control and cell reprogramming leading to faithful restoration of lost parts. Osteonectin, a glycoprotein that complexes with colla- gen fibres and hydroxyapatite, was suggested to be involved in ini- tiation of active mineralization, found in regenerated tissues and its levels appeared to increase in tissues undergoing remodelling. Our main goal was to determine the pattern of osteonectin expres- sion during the first 96 h post amputation in zebrafish fin through real-time PCR and whole-mount in situ hybridization. The results showed a clear correlation between osteonectin expression and bone formation. Acknowledgements: This work was funded by FCT/POCI/ MAR/60883/2004 (XenoFish). AB Brito and PJ Gavaia are recipi- ents of FCT fellowships CCMAR/BTI/0041/2006 and SFRH/BPD/ 14528/2003. ª 2007 The Authors Journal compilation ª 2007 FEBS 273 Abstracts F1-37 Antihypertensive effect of a novel ACE-I inhibitory peptide from bigeye tuna dark muscle in spontaneously hypertensive rats S. Kim, Z. Qian, M. Kim and S. Lee Pukyong National University, Busan, REPUBLIC OF KOREA To produce bioactive peptides of fish processing by-products, big- eye tuna dark muscle was hydrolyzed using various enzymes (Alca- lase, a-chymotrypsin, Neutrase, papain, pepsin, and trypsin) for extraction of ACE I inhibitory peptide. Pepsin-proteolytic hydroly- sates exhibited the highest ACE I inhibitory activity among the others tested. An ACE I inhibitory peptide was purified by con- secutive chromatographic methods using a Hiprep 16/10 DEAE FF anion exchange column and an octadecylsilane (ODS) C18 reversed phase column. In the result of N-terminal amino acid sequencing analysis, the peptide purified from pepsin-digests of bigeye tuna muscle protein (BTMP-Pepsin) is a dodeca-oligopep- tide with rich trypsin in c-terminus (WPEAAELMMEVDP; 1.5 kDa of molecular weight). The ACE I inhibitory peptide, BTMP-Pepsin, exhibited potent inhibitory activity with 21.6 lMof ACE IC 50 value, and it was evaluated as a non-competitive inhib- itor against ACE I in the assay for inhibitory pattern by Lineweav- er-Burk plotting. Antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of BTMP-Pepsin can decrease systolic blood pressure significantly (P<0.05). In addition, MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5). The result of this study suggests that ACE inhibitory peptides derived from BTMP could be potential candidates to develop nutraceuticals and pharmaceuticals. F1-38 Genetic variation in rocky mouse, Apodemus mystacinus (Danford and Alston, 1877) (Mammalia: Rodentia) in Turkey R. C¸ olak 1 , G. Olgun 1 , I. Kandemir 2 ,E.C¸ olak 1 and N. Yig ˘ it 1 1 Ankara University, Faculty of Science, Department of Biology, Ankara, TURKEY, 2 Karaelmas University Faculty of Science & Art, Department of Biology, Ankara, TURKEY Apodemus mystacinus is widely distributed from Balkans to Middle East and Caucasus. Three subspecies of A. mystacinus are distribu- ted in Turkey. Despite of several previous studies, the taxonomic status of A. mystacinus in Turkey is problematic. The aim of the this study was to survey genetic structure based on DNA markers and to contribute to the taxonomy and population genetics of A. mystacinus in Turkey. A total of 22 specimens were collected from 11 locations in Turkey. To explore the extent of genetic var- iation in A. mystacinus populations, a Randomly Amplified Poly- morphic DNA marker system was used. The estimates of NEI’s standart genetic identity and genetic distance were calculated to show the genetic relationships between studied populations. All estimations were calculated with the POPGENE software. With the 60 RAPD markers tested, 14 of them yielded 147 polymorphic DNA bands. The estimated genic diversity for A. mystacinus popu- lations was ranged from H = 0.0227 (P=4.55) in Trabzon to 0.2045 (P = 40.91%) in Mugla population. The total gene diver- sity was calculated as H T = 0.3087 in A. mystacinus populations. G ST value calculated was high (0.7438) indicating that genetic dif- ferentiation among the studied populations was substantial. Dend- ogram constructed with genetic distance data contained 3 clusters. The groupings in the A. mystacinus cluster were consistent with the previously assigned subspecific categories. The implication of the results with respect to the genetic variation of subspecies was also discussed. F1-39 Immunohistochemical distribution of the novel peptide alarin in the adult rat brain N. Eberhard 1 , S. M. Schmidhuber 1 , I. Rauch 1 , G. Sperk 2 and B. Kofler 1 1 Department of Pediatrics, Paracelsus Private Medical School, Salz- burg, AUSTRIA, 2 Department of Pharmacology, University of Inns- bruck, Innsbruck, AUSTRIA Recently, the expression of a splice variant of the Galanin-like pep- tide (GALP) gene, named Alarin, was observed in gangliocytes of human neuroblastic tumors and different neuronal tissues. Given that GALP has potent species-specific and time-dependent effects in the central nervous system and has been suggested to constitute a link between metabolism and reproduction, we aimed to deter- mine the distribution of Alarin in the rat brain. For immunohisto- chemical studies, an affinity purified polyclonal antibody directed against synthetic murine Alarin peptide was used. Alarin-like-im- munoreactivity (Alarin-LI) was observed mainly in the basal gan- glia of the rodent brain, which was further confirmed by RT-PCR. Additional immunostained areas were observed in the amygdala, in the pirifom cortex, and in the CA1, stratum lacunosum molecul- are of the hippocampus. Relatively dense staining was also noted in the tuberomammillary nucleus (TM), a cluster of magnocellular cells in the posterior hypothalamus, which is the main source of neuronal histamine in the brain. The expression of Alarin in cells of neuronal origin, mainly in the basal ganglia and the TM indi- cates possible association in motor control, emotion, cognition and learning. Acknowledgement: Supported by the Paracelsus Private Medical University F1-40 Response of gingival mast cells in streptozotocin-induced type 2 diabetic rats N. Ozsoy Department of Biology, Faculty of Science, Ankara University, Ankara, TURKEY Diabetes is a complex disease, which triggers various complications including tissue destruction mechanisms. One of these complica- tions is gingivitis. Occurrence of gingival tissue damage in diabetes is well known, but its mechanism has not yet been adequately explained at cellular and molecular levels. Although type 2 diabetes comprises 80% of all diabetics, type 1 diabetic cases are more fre- quently investigated. Cytological studies in especially type 2 diabet- ics and their conclusions are far less than desired. Experimental diabetes should be a logical method for investigating oral changes due to diabetes. In this study, diabetes has been induced in neona- tal male rats by intraperitoneal injection of 90 mg/kg streptozoto- cin (STZ). The effects of experimental diabetes on gingival mast cell responses were investigated ultrastructurally. The ultrastructur- al study of the mast cell demonstrated mast cells had evidence of secretion. Gingival biopsies obtain from rats with type 2 diabetes mellitus showed more mast cell secretion than control biopsies. Mast cell exhibited an atypical degranulation, which included cyto- plasmic granules filled by partially or fully dissolved granular mat- rices. It was found that the integrity of the membrane of some granules deteriorated and a common membrane surrounded some of them. There was pycnosis in the nuclei of some mast cells, and granules having less dense dissolved granule matrix material filling were noted around them. Some mast cells presented a number of cytoplasmic secretory granules containing less dense, irregular threads. These ultrastructural features suggest that diabetes causes abnormal mast cell degranulation which in turn triggers gingival tissue breakdown associated with diabetes mellitus. 274 ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts [...]... heliotherapy, cold mud ointment and swimming into the salted water of the lake, in addition to electrotherapy, kinetotherapy and massage The studied group included 25 patients (6 female and 19 male), 13 of them with AS (following ACR criteria for diagnosis) and the other 12 with osteoarthritis 20 patients underwent cold mud ointment and 5 heated mud packing The patients were clinically and paraclinically... single-strand conformation polymorphism (SSCP) analysis was used All samples showing aberrant SSCP patterns and adequate number of randomly chosen ones with normal pattern were additionally sequenced No mutations R141H and F119L neither allele IVS5+22A have been detected The estimated frequency of IVS5+19T allele was 0.958 and IVS5+19C allele was 0.042, while the estimated incidence of heterozygotes... ELISA and the chemically synthesized P1 could bind to VEGFR-3 specifically in a dose dependent manner What’s more the flow cytometry assay and immunoflorenscence showed that the FITC labeled P1 could bind to VEGFR-3 positive carcinoma cells with specificity In the competition assay, the phages displayed P1 could specifically inhibit the binding of P1 and VEGFR-3 and the P1 could specifically inhibit the FITC-P1... can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains ª 2007 The Authors Journal compilation ª 2007 FEBS H Hung1, K Chang1 and G Liu2 1 National Chung-Hsing University, Taichung, TAIWAN, 2 Chung-Shan Medical University, Taichung, TAIWAN Human mitochondrial NAD(P)+-dependent malic enzyme is a regulatory enzyme allosterically activated by fumarate Structural studies... spectrofotometrically using Gly-Pro p-nitroanilide as substrate Results: A statistically significant decrease in serum DPP IV was found in patients with rheumatoid arthritis (30.45 ± 3.04 U/l) compared to the control group (48.37 ± 1.10 U/l) (P < 0.001) In contrast, serum DPP IV activities in patients with psoriatic arthritis (49.96 ± 3.80 U/l) and osteoporosis (43.11 ± 3.9 U/l) were not statistically significantly... dysfunction, in normal and preeclamptic pregnants Nitrite was measured via an electrochemical method and other parameters were detected via HPLC method Hypoxanthine, xanthine, uric acid, allantoin and xanthine oxidase activity has been found with higher levels in preeclampsia than normal pregnants levels Other parameters are not changed in both groups According to our results, we concluded increased xanthine... in all 3 cell lines with the highest in GaMG (3 fold) On protein level, OPN, CA9, EPO and HIF-1a were expressed in GBM at a higher rate than in LGA OPN was 3-fold overexpressed in GBM compared to LGA In vitro, CAIX and HIF-1a showed a clear hypoxia regulated protein overexpression pattern EPO was less induced than CAIX in all three cell lines OPN and CA9 mRNA were clearly upregulated at 0.1% O2 in all. .. group In these subjects early breast milk was taken (usually 5th day after childbirth) In all human milk samples levels of selected antioxidants (e.g tocopherols, ascorbate, carotenoids – RP-HPLC-MS), total antioxidant status (TRAP), fatty acid levels (GC-MS) and protein profiles (PAGE-SDS, microfluidic electrophoresis) were measured Monthly changes of all parameters were evaluated and average values were... protein profiles were very similar among all subjects and relatively independent on diet of mothers The main protein fractions were lactalbumine and caseins On contrary, lipid composition of breast milk was more variable and depended on dietary intake While levels of saturated and monounsaturated fatty acids as well as arachidonic acid were quite similar among all subjects and among average monthly values,... color smooth texture and other side black color Toluene was injected to C3H and C57 for conditioning place preferences test at day and night time points Toluene was injected every two days as a 800 mg/kg and then placed to the white side of shuttle box for 30 min Other days, which was not given toluene, olive oil was injected and then placed to the black side of box for 30 min Totally, injections was eight . reorientation of IgE epitopes, such allergen derivatives may act as potent hypoallergenic vaccines for immunotherapy. The allergens from grass pollen are amongst. together with immunological data allows for the rational development of strategies to convert aller- gen molecule in to hypoallergenic derivatives through