L7.1

Molecular investigations in medical practiceAnna Tylki-Szymańska

The Children’s Memorial Heath Institute, Warsaw, Polande-mail: Anna Tylki Szymanska <atylki@op.pl>

The ability to perform molecular tests for diagnostic and prognostic purposes in medical practice dates back to the eighties of the last century. Molecular tests have proved crucial in the diagnosis of certain diseases and particularly important in identification of the carrier state in pre-symp-tomatic patients, in the family and even population wide screening tests.Initially, the molecular research was associated with hope of finding a simple relationship between genotype and phenotype. But along with the data gathering and experi-ence gaining it was proved that the molecular tests only bring some information about the potential of the disease phenotype. They cannot form the basis for accurate pre-diction of the clinical course of illness. Association of the defect type in the gene with the dysfunction of its product, e.g. enzyme protein, allowed for a better understanding of biochemical processes in many metabolic disorders. Sur-prisingly, the molecular studies have brought a lot of inter-esting and surprising information about the founder effects and migration in the area of Europe.

Session 7: Molecular Basis of Diseases

LecturesL7.2

Molecular basis of diabetes mellitusKrzysztof Zabłocki, Dorota Dymkowska, Katarzyna Wierzbicka, Beata Drabarek, Sylwia Wojciechowska

Nencki Institute of Experimental Biology, Department of Biochemistry, Warsaw, Polande-mail: Krzysztof Zablocki <k.zablocki@nencki.gov.pl>

Diabetes mellitus (DM) is a serious incurable metabolic disease which affects approximately 1% o the global hu-man population and the increase in incidence of diabetes is expected to double in a generation. DM is subdivided into two major classes: type 1 diabetes (T1D) accounts for 10% of all diabetes and type 2 (T2D) accounts for 80-90% of all diabetic patients. While an etiology of T1D is rela-tively clear and attributed to the autoimmune damage to the beta cells, pathophysiology of T2D is poorly under-stood. It is thought that changes of life style, particularly western-type diet, leading to obesity and hypertension are an important cause of rapid increase of diabetes preva-lence. Together with environmental and behavioral factors a genetic background seems to have an important but not well understood impact on susceptibility of individuals to insulin resistance and diabetes. Although the pathophysiol-ogy of T2D is very complex and heterogeneous, in some cases diabetes may result from well defined mutations or be secondary to specific genetic disorders. Among a number of concepts concerning mechanism of insulin resistance and T2D, the “mitochondrial” hypothesis is broadly discussed and accepted. There is a growing body of evidence indicating that an apparently slight mitochon-drial dysfunction might increase (probably together with other factors) the probability of insulin resistance and T2D development.The focus of this lecture is to present and discuss mod-ern opinions on diabetes mellitus with special emphasis on T2D.

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L7.3

Hepatitis C virus: epidemiology, molecular biology and antiviral therapyAnna Boguszewska-Chachulska1Genomed, Ponczowa 12, 02-971 Warsaw, Poland2Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664 Warsaw, Polande-mail: Anna Boguszewska Chachulska <annab-ch@genomed.pl>

About 180 million people worldwide are chronically infect-ed with Hepatitis C virus (HCV), an agent responsible for 50–76% of all cases of liver cancer, and for two thirds of all liver transplants in the developed world (WHO, 2007). HCV remains one of the most important viral threats to human health in spite of advanced studies on its life cy-cle and encouraging results of studies on the development of new drugs. Mechanisms of viral entry, replication and virion formation are dissected in view of obtaining new anti-HCV drugs. The present treatment for HCV infec-tion, however, is still limited to the pegylated interferon-α (IFN-α) in combination with ribavirin, with response rates between 40 and 50% for genotype 1, the most prevalent genotype in Europe and the United States and up to 80% for genotypes 2 and 3. The development of antiviral agents directly targeting the viral life cycle seems to be the most promising therapeutic strategy, as it should block HCV replication and thus spread of infection. This goal could be achieved by direct inhibition of viral enzymes involved in the replication process, such as the NS3/4A protease protein, exerting both serine protease and RNA helicase/NTPase activities, or the NS5B protein - the RNA-depend-ent RNA polymerase. A few drugs targeting the NS3/4A protease, or the NS5B polymerase, are already in advanced clinical trials and are expected to be approved for the HCV treatment in a few years’ time. However, even the introduc-tion of new directly acting antivirals targeted against the vi-ral protease and polymerase will not solve one of the major problems encountered when treating HCV infections - that of viral breakthroughs due to the emergence of HCV mu-tants resistant to the treatment applied, observed also dur-ing the clinical trials with a protease inhibitor, telaprevir, a prospective new drug. Still, as an enzyme indispensable for HCV replication, that unwinds double-stranded forms of RNA and allows viral replication and translation to occur, the NS3 helicase/NTPase represents a tempting target for specific anti-HCV drug design. Another advantage is that NS3 helicase does not possess close homologs among hu-man cellular enzymes. The NS3 helicase inhibitors will be discussed as potential components of a multidrug therapy against HCV.

L7.4

Dysregulation of cellular calcium homeostasis — a cause or a result of neurotoxicityLudmila Zylinska

Department of Molecular Neurochemistry, Medical University, Lodz, Polande-mail: Ludmila Zylinska <luska@csk.umed.lodz.pl>

Calcium ions belong to a group of the most effective sign-aling molecules that regulate physiological events in every type of the cells. To keep the intracellular free Ca2+ concen-tration at safe, nanomolar resting level, the cells developed a dynamic homeostatic system that equilibrates a release from intracellular calcium stores, controls fluxes across plasma membranes and buffering capacity of cellular pro-teins. Dysregulation of calcium homeostasis and concomi-tant cellular derangement are the important mediators of neurotoxicity. Altered membrane excitability, overload or deficiency of neurotransmitters may result in substantial changes in expression of calcium-regulated genes, thereby either trigger some adaptive mechanisms or lead to cell death. Among transporters engaged in calcium flux, the key role is played by channels, exchangers and pumps; however only the last group is energy-consuming and use one mol-ecule of ATP for transport of one or two calcium ions. Disturbances in cytosolic calcium levels by deregulation of different homeostatic control mechanisms have been ob-served in a number of neuropathological conditions includ-ing brain ischemia, asphyxia, and several neurodegenerative disorders. Triggering of the specific pathway activated by Ca2+ depends on spatio-temporal cooperation of a variety of components in the plasma membrane, endoplasmic re-ticulum and mitochondria, which serve to amplify the Ca2+ signals. Calcium overload disrupts the mitochondrial func-tion by decreasing ATP synthesis, increasing the produc-tion of reactive oxygen/nitrogen species and finally, leads to enhancement of apoptosis or necrosis. Acknowledgements:Supported by the grants 502-16-809, 502-16-810 and 503-6086-2 from the Medical University of Lodz.

Abstracts114

L7.5

Cell cycle dysregulation in Alzheimer’s diseaseUrszula Wojda

Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Polande-mail: Urszula Wojda <ulawojda@iimcb.gov.pl>

Alzheimer’s disease (AD) is the most common dementia in the elderly characterized by progressive neuronal loss. Ma-jority of AD cases occur sporadically and have unknown etiology (SAD). The Abeta peptide deposition in the neu-ritic plaques is one of the major AD hallmarks. However, the role of Abeta as a primary driver in AD progression arouses controversy. Mounting evidence indicates that AD pathogenesis is triggered by genomic instability and reentry into the cell cycle of differentiated, postmitotic neurons. In SAD neurons, activation of cell cycle proteins accompa-nied by partial or complete DNA replication without signs of mitosis was demonstrated. It is supposed that at the early stages of SAD, activation of cell cycle proteins may represent a protective response to stress factors, allowing neurons survive due to additional alleles of genes produced by DNA replication. The price for survival, however, is the appearance of pathological changes characteristic of neu-rodegeneration and higher susceptibility of these neurons to death triggered by stress factors later in the SAD proc-ess. Our recent data suggest that some of the cell cycle chang-es can be also observed in human peripheral cells such as lymphocytes. Our results from PCR arrays involving 90 cell cycle genes and from immunobloting experiments in the EBV-immortalized lymphocytes from SAD patients showed most significant changes in the expression of genes and proteins engaged in the control of G1/S cell cycle phases. The most striking difference occurred in p21 protein level, which was significantly elevated in SAD com-paring to control lymphocytes. Alterations in the expres-sion of cell cycle genes and p21 increase observed for SAD lymphocytes were accompanied by prolongation of G1 phase with simultaneous shortening of S phase, as demon-strated by flow cytometry and BrdU-labeling.These results emphasize that cell cycle dysregulation is found not only in AD neurons, but also in peripheral lym-phocytes. Thus, easily accessible human lymphocytes have a potential diagnostic value in AD.

L7.6

Mechanisms of delayed postischaemic neuronal death and regenerationBarbara Zabłocka, Małgorzata Beręsewicz, Joanna E. Kowalczyk

Molecular Biology Unit, Mossakowski Medical Research Centre, Pawińskiego 5, 02-106 Warsaw, Polande-mail: Barbara Zablocka <zablocka@cmdik.pan.pl>

Ischaemic stroke is a common and devastating disease and its frequency is on the increase in aging populations. It oc-curs when the blood supply to the brain is disturbed (due to cerebral thrombosis, embolism or cardiac arrest) starving neurones of oxygen and rapidly causing some cells to die and leaving others damaged. Unfortunately, no treatment is available while the human and the economic costs of this disease are enormous. The major problem in adult ischae-mic stroke is that the pathological events quickly become self-sustaining and, within hours, any treatment becomes ineffective.Neuronal injury after transient ischaemia is induced by a combination of hypoxia, hypoglycemia, glutamate-de-pendent excitotoxicity as well as free radical damage. An extracellular glutamate accumulation leads to overstimu-lation of postsynaptic glutamate receptors with intracel-lular Ca2+ overload, reorganization of protein complexes at excitatory synapses forming postsynaptic density (PSD), changes in the activity of protein kinases and cytochrome c release from mitochondria. A biphasic increase in cytosolic cytochrome c in the hippocampi following transient glo-bal ischaemia in gerbils was demonstrated. It takes part in ischemic signal propagation via endoplasmic reticulum and amplification of apoptotic cascade. But, transient ischae-mic insult stimulate complex changes which play a role in neurodegeneration as well as in tissue remodeling and re-pair. It was shown that brain ischaemia induces changes in endogenous expression of the specific alternatively spliced IGF-1 variant (called MGF or IGF-1Ec). In the post-ichae-mic hippocampus the levels of both MGF mRNA and the peptide were increased selectively in neurones resistant to the ischaemic insult. It suggests that MGF may form a part of a novel neuroprotective mechanism in the brain. In ad-dition, the endogenous production of MGF by stressed neurones suggests that strategies to specifically up-regulate expression of this isoform or gene therapy targeting MGF to neurones could be viable approaches.

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Oral Presentations

O7.1

Laminopathies and molecular background of disease phenotypes — are we ready for a development of universal stem cell and gene therapy?Magdalena Zaremba-Czogalla, Katarzyna Kozioł, Magda Dubińska-Magiera, Maria Żeleźnik, Janusz Wiśniewski, Ryszard Rzepecki

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Wrocław, Polande-mail: Magdalena Zaremba CZogalla <rzepecki@ibmb.uni.wroc.pl>

The term laminopathies defines a group of human degener-ative disorders which are associated with mutations in genes coding for nuclear lamina (LMNA, LMNB1, LMNB2) and nuclear envelope proteins (EMD, LAP2, LBR) and which include systemic disorders and tissue restricted diseases. To date, more than 340 unique mutations have been reported from more than 1000 patients. Laminopathies arise mainly from missense and frameshift mutations of lamin genes or defective posttranslational processing of pre-lamin A. It is still unclear yet how specific mutations result in a particular tissue specific laminopathy phenotype considering the fact that A and B type lamins are expressed in nearly all differ-entiated cell types. Many different mutations can give rise to the same clinical conditions. On the other hand the same single mutation can result in different phenotypes probably depending on genetic background of particular patient. It might be explained by the two hypothesis: structural and gene expression hypothesis. There is also possibility that in some cases, effects of the mutated LMNA gene are altered by mutations in one or more other genes. Considering the gene therapy approach for the laminopathies we face sever-al problems. They can be roughly divided into two groups. The first set of problems and difficulties is associated with not fully known molecular mechanisms of diseases and interactions between proteins of interest and other pro-teins. The second set of problems concerns the enormous difficulties associated with the design of the more or less universal and efficient gene therapy protocol for possible treatment. We consider two main strategies for a develop-ment of therapy of laminopathies. One may involve the ex vivo gene therapy of hematopoietic stem cells or mesoan-gioblasts of the patient using replication deficient retrovi-rus vector and transdifferentiation into muscle stem cells followed by systemic delivery. The second may be the in vivo gene therapy directed specifically to muscle stem cells or myotubes using fully “gutted”, specifically pseudotyped retrowirus vectors.Acknowledgements:The research was supported by Wroclaw Research Centre EIT+ under the project „Biotechnologies and advanced medical technologies” - BioMed (POIG.01.01.02-02-003/08) financed from the European Regional De-velopment Fund (Operational Programme Innovative Economy, 1.1.2).

O7.2

Plasma proteome changes in chronic kidney disease-related arteriosclerosis. The analysis of low-abundant proteinsMagdalena Łuczak1, Dorota Formanowicz2, Marek Figlerowicz1

1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland; 2Department of Clinical Biochemistry, Poznań University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Polande-mail: Magdalena Łuczak <magdalu@ibch.poznan.pl>

Chronic kidney disease (CKD) is a progressive loss of renal function. CKD can be divided into five stages (CKD1-5) based on glomerular filtration rate (GFR). The latter de-scribes the flow rate of filtered fluid through the kidney. Arteriosclerosis is one of the most serious and frequent complications occurring in patients suffering from CKD. It was also found to be the major cause of mortality in patients with mild to moderate and end-stage renal disease. Moreover, the risk of cardiovascular events increases with the reduction of renal function. On the other hand, renal dysfunction is one of the most serious complications of cardiac surgery, and often cardiovascular disease (CVD) pa-tients have a kidney function decline. Thus, the close con-nection between kidney dysfunction and arteriosclerosis is undeniable. However, some of the symptoms are different or even opposite for CKD-related arteriosclerosis (CKDA) and for classical CVD. To increase our knowledge on the mechanisms underlying CKDA development we attempted to identify the proteins specifically accumulating in plasma of patients in different stages of the disease. In addition, the results of our analysis were confronted with analogous data obtained for patients with CVD and normal renal function. The studies involved 60 patients: 15 patients in the initial stage of CKD with the first CKDA symptoms (CKD 1-2), 15 pre-dialyzed pa-tients (CKD 3-4), 15 patients with end-stage renal disease and full CKDA symptoms (CKD 5) and 15 patients with CVD and normal renal function and 20 healthy volunteers as controls. Earlier, it was suggested that in case of human blood plas-ma the disease-specific biomarkers usually occur among proteins of low abundance. Accordingly, our studies were focused on this fraction of molecules. After the depletion of high-abundant proteins with commercially available MARS-Hu7 columns plasma samples were subjected to a standard proteomic analysis (2DE and MALDI-ToF-MS). The undertaken experiments revealed quantitative differ-ences in the accumulation of several proteins. As a result, biomarkers characteristic for the particular stages of CKD and differentiating between CKDA and CVD were identi-fied.

Abstracts116

O7.3

The -675_-674insG polymorphism of the PAI- I gene in determining the predisposition to ischaemic stroke in childrenI. Żak1, T. Iwanicki1, A. Balcerzyk1, E. Emich-Widera2, I. Kopyta2, E. Pilarska3, K. Pienczk-Ręcławowicz3, M. Nowak1

1Department of Biochemistry and Medical Genetics, Medyków 18, 40-750, Katowice, Poland; 2Department of Neuropediatrics Medical University of Silesia, Katowice, Poland; 3Department of Neurology, Developmental Neurology Clinic, Medical University of Gdańsk, Gdańsk, Polande-mail: Tomasz Iwanicki <t_iwanicki@wp.pl>

Background: Stroke in children is relatively rare in com-parison to adults. Incidence of this disease entity are ap-proximately six cases in every 100 000 children per year. The most often stroke impairs are neurological functions and children`s social abilities. The cause of the one- third of cases remains unknown but as a multifactorial disease stroke has a number of possible risk factor. Hypercholeste-rolemia, oxidative stress or disorders in coagulation system can increase a risk of ischaemic stroke presence. In recent years, epidemiological studies have shown that abnormali-ties in some hemostatic parameters may help to predict the risk for ischemic events. Plasminogen Activator Inhibitor I (PAI-I) is a glycoprotein attending an important role in fibrinolysis. Its main function is to inhibit a synthesis of plasmin- an enzyme dissolving fibrine. High levels of PAI-I seems to influence processes of smooth muscle cell pro-liferation, plaque, and matrix remodeling in the direction of promoting atherothrombosis. The -675_-674insG is a insertion/ deletion polymorphism located in the promoter of PAI- I gene. 4G allele produces up to 6 times mRNA in vitro and is associated with high plasma PAI-I activity in vivo. The aim of this study was to evaluate possible associa-tion between PAI-I gene polymorphism and the childhood stroke.Material and methods: The study population included 70 affected children and 133 healthy children as a control group. Stroke children were also analyzed with their biolog-ical patients (n=140). The age of patients in acute phase of ischaemic stroke was 8.65± 5.58. The -675_-674insG poly-morphism was genotyped with PCR- RFLP method. The Transmission/ Disequilibrium Test (TDT) was used for the analysis of specific allele transmission from heterozygous patients to their affected children. The statistical analysis was performed with Statistica 7.1 and EpiInfo software.Results: There were 57 informative trios for the -675_-674insG polymorphism of PAI- I gene. Test TDT did not show a preferential transmission of any alleles from het-erozygous parents to their affected children (χ2= 0.00, p= 1.00). There was no statistical significance in the genotypes and alleles frequencies between stroke children and control group (4G/ 5G: χ2= 0.00, p= 0.99).Conclusion: The obtained results did not reveal any as-sociation between -675_-674insG polymorphism of PAI-I gene and ischaemic stroke in children.

O7.4

Expression of vascular endothelial growth factor and its receptors in human neointimaAndrzej Małkowski1, Radosław Kowalewski2, Marek Gacko2, Krzysztof Sobolewski1

Medical University of Białystok, 1Department of Medical Biochemistry, Poland; 2Department of Vascular Surgery and Transplantology, Białystok, Polande-mail: Andrzej Małkowski <amalkow@umwb.edu.pl>

Endothelial dysfunction is a key factor underlying many vascular pathologies. Invasive surgical procedures, such as angioplasty or peripheral vascular by-pass grafting, result in mechanical damage of the arterial wall, which initiates nu-merous cellular and molecular mechanisms. Disturbed ves-sel wall homeostasis leads to proliferation and migration of smooth muscle cells, which often exceeds the repair proc-ess and results in extensive neointima formation and arte-rial stenosis. Vascular endothelial growth factor (VEGF) is one of the main factors that stimulate angio- and vasculo-genesis. It stimulates cell division, their migration, and in-creases the permeability of capillaries. Furthermore VEGF rouses cells to increased extracellular matrix metabolism. In experimental models it was observed that exogenous VEGF, intensifying the proliferation of endothelial cells, reduces neointima formation. However increased VEGF expression in the neointima was found in various animal models. The aim of the study was to evaluate VEGF-A and its two receptors (VEGFR1 and VEGFR2) in the neointi-ma, when compared to normal aortas. The studied mate-rial consisted of 12 neointima samples, that were obtained from patients with chronic limb ischaemia in the age range 45–60 years, after aorto-femoral by-pass reconstructions. The tissue samples were collected from distal anastomoses during secondary operations performed due to graft oc-clusion 6 to 24 months after primary procedure. Normal aortas without macroscopically visible lesions, that were harvested from age-matched organ donors were the con-trol material. Expression and content of VEGF-A, VEG-FR1 and VEGFR2 were evaluated with RT-PCR, Western blot and ELISA methods respectively. We have observed in human model, that artery injury followed by neointima formation shows increase in expression of VEGF-A and its receptors type 1 and 2. It has confirmed data obtained from in vitro and animal experiments that VEGF is one of the key agents involved in neointima formation. The over-expressed VEGF-A in the neointima influences many gene expression via VEGFRs, including these involved in cell proliferation, extracellular matrix metabolism and artery wall remodeling.

45th Annual Meeting of the Polish Biochemical Society 117

Posters

P7.1

Multi-sumoylation of Usp protein - heterodimerization partner of EcR from Drosophila melanogasterKatarzyna Bielska1, Justyna Seliga1, Elżbieta Wieczorek1, Rainer Niedenthal2, Andrzej Ożyhar1

1Department of Biochemistry, Wroclaw University of Technology, Wroclaw, Poland; 2Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Hannover, Germanye-mail: Katarzyna Bielska <katarzyna.bielska@pwr.wroc.pl>

Reversible posttranslational modification is a rapid and ef-ficient system to control the activity of proteins. Modifi-ers range from small chemical moieties to proteins. Small ubiquitin-like modifier (SUMO) belongs to a family of a proteins that have been found to be covalently attached to lysine residues of specific target proteins. Sumoylation modulates important functional properties, depending on the protein substrate. The ultraspiracle protein (Usp), which together with the ecdysone receptor (EcR) forms a functional EcR/Usp complex in Drosophila melanogaster, provides an ideal model system for construction of gene switches in humans. However, there is no information about sumoylation of Usp. To investigate this modification of Usp we used Ubc9 fusion-directed sumoylation (UFDS) system (Jakobs et al., 2007) which is based on fusion of conjugating enzyme Ubc9 with substrate protein. We pre-pared constructs coding fusion proteins where Ubc9 was attached to the N- or C-terminus of Usp (Ubc9-Usp, Usp-Ubc9), expressed them in human embryonic kidney cells (HEK293) and analyzed the protein extracts by western blotting with an Ubc9 antibody. The results revealed that both Ubc9-Usp and Usp-Ubc9 are sumoylated by SUMO1 and SUMO3, however Usp-Ubc9 less efficiently. We used bioinformatic tools (SUMOplot, SUMOsp and PCI-SUMO) to predict and score potential sumoylation sites in Usp. Using this data, we prepared some point mutants. To determine region of the modification, we also prepared fragments of Usp including A/B region, DNA binding do-main (DBD) and ligand binding domain (LBD) fused to Ubc9. For A/B region and LBD we showed that there are potential residues that can be sumoylated. Identification of Usp sumoylation sites is currently underway.References:Jakobs A, Himstedt F, Funk M, Korn B, Gaestel M, Niedenthal R (2007) Nucleic Acids Res 35: e109.Acknowledgements:This work was supported by the Wroclaw University of Technology.

P7.2

Effect of high-carbohydrates diet on the level of mRNA expression for selected elongases and desaturases in liver tissue of ratsJagoda Drąg, Anna Goździalska, Anna Gawędzka, Jerzy Jaśkiewicz

Department of Analytical Biochemistry, Faculty of Pharmacy, Collegium Medicum, Jagiellonian University, Kraków, Polande-mail: Jagoda Drąg <jagoda.drag@gmail.com>

Hepatic lipids contain fatty acid derived from diet or from endogenous synthesis. Both sources provide saturated and n-9 unsaturated fatty acid. In addition, diet is the only source of essential polyunsaturated n-3 and n-6 fatty acids. In human body the structure of fatty acids is modified by fatty acid elongases and desaturases. The activity of these enzymes is regulated by different factors including diet, hormones and transcription factors. Fatty acid profile of the body is determined by the activity of elongases and desaturases. Qualitative and quantitative changes of fatty acids affect structure and function of cell’s membrane. De-velopment of knowledge on enzymes modifying the com-position of fatty acid molecules may be helpful in elucidat-ing the pathomechanism of different diseases.The aim of our study was determine the influence of the high-carbohydrate diet on hepatic gene expression for Elovl 2, Elovl 5, Δ5 desaturase, Δ6 desaturase, Scd1 and Scd2. We demonstrated that mRNA expression level of Elovl 5, Δ5 desaturase, Δ6 desaturase and Scd2 in liver of rats fed high-carbohydrate diet was similar to the levels of mRNA for the same enzymes in liver of animals fed stand-ard diet. Statistically significant difference was shown be-tween mRNA expression of Elovl2 and Scd1.High carbohydrate diet increases the level of glucose and triggers insulin synthesis and release which in turn cause the activation of transcription factor called SREBP. It is also known that ChREBP is required for the carbohydrate-induced transcriptional activation of several additional enzymes involved in fatty acid synthesis, most notably SCD-1. High carbohydrate diet augments the synthesis of monounsaturated fatty acids which are major components of triglycerides that are transported by blood and stored in adipose tissue. Obtained results suggest that the analysis of SCD1 mRNA expression together with other biochemical markers can be added to diagnostic panel of diabetes and obesity.References:1. Wang Y et al. (2005) J Lipid Res 46: 706–715.2. Popeijus et al. (2008) Int J Obes 32: 1076–1082.3. Shanmugam M et al. (2009) Nutr Metab 6: 27.4. Iizuka K et al. (2008) Endocr J 55: 617–624.5. Moon YA et al. (2009) J Lipid Res 50: 412–423.6. Weickert MO et al. (2006) Diabetologia 49: 1732–1741.7. Dentin R et al. (2006) J Nutr 136: 1145–1149.8. Dentin R et al. (2005) Biochimie 87: 81–86.

Abstracts118

P7.3

The effect of physical effort with incremental load on the lipid profile in the blood serum of non-athletesWioleta Dudzinska1, Anna Lubkowska1,2

1Faculty of Natural Sciences of Szczecin University, Department of Physiology, Szczecin, Poland; 2Pomeranian Medical University, Department of Biochemistry and Medical Chemistry, Szczecin, Polande-mail: Wioleta Dudzi?ska <wiola@univ.szczecin.pl>

The aim of this study was to assess the influence of physi-cal activity with increasing load on the serum lipid profile in non-athletes.The study was carried out on 22 healthy men, aged 21.9±2.33 years. The subjects underwent a single test on an ergometer (Kettler X-7, Germany), with progressively increasing load. The test began with resistance of 70W, and the participants were asked to maintain 70 revolutions per minute. The effort was continued with increasing load (by 20W every 3 minutes) until refusal, that is until the partici-pant was not possible to continue at the required rate of revolutions. A Polar S610 heart rate monitor (Polar, Fin-land) was used to record a resting heart rate and its changes during exercise. In order to assess the uptake of oxygen during exercise, an Oxycon gas analyzer was used (Jaeger, Germany).Blood samples were taken from the cubital vein three times: before the test, during 3 minutes after the completion of the exercise, and in the 30th minute after the completion of the exercise. The determinations of the studied lipid pa-rameters (triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol) were performed in blood serum us-ing enzymatic methods.In the studied group, an exercise with incremental load, continued until refusal, lead to a significant increase in trig-lycerides in the 30th minute of rest, a significant increase in lipoprotein HDL and HDL/LDL ratio, and a decrease in cholesterol/HDL ratio.A single physical exercise favorably modified the concen-tration of anti-atheroscerotic lipoproteins.

P7.4

The Membrane-Type Matrix Metalloproteinases (MT1-MMP and MT2-MMP) of the umbilical cord in preeclampsiaZofia Galewska1, Lech Romanowicz1, Stefan Jaworski2, Edward Bańkowski1

1Departments of Medical Biochemistry and 2Gynecology, Medical University of Białystok, Białystok, Polande-mail: Zofia Galewska <zofia.galewska@umwb.edu.pl>

The vascular system of mother and placenta plays an im-portant role in the intrauterine development of the fetus. Preeclampsia is a conditions characterized by systemic vas-cular endothelial dysfunction. Our previous research dem-onstrated that preeclampsia - associated accumulation of collagen and proteoglycans in the umbilical cord tissues is a result of increased biosynthesis and decreased degrada-tion of these components. Metalloproteinases are enzymes engaged in degradation of collagen and protein cores of proteoglycans, including those which bind peptide growth factors. The Membrane-Type Matrix Metalloproteinases (MT-MMPs), with the exception of MT4-MMP, are clas-sical extracellular matrix (ECM)-degrading endopeptidas-es, and such they exhibit a broad spectrum of substrate specificity degrading one or more ECM components. We used Western Immunoblot method and immunoenzy-matic assay (ELISA) for detection of metalloproteinases. We found that the human umbilical cord arteries, vein and Wharton’s jelly of control and preeclamptic newborns con-tained MT1-MMP (MMP-14) and MT2-MMP (MMP-15). Free latent form of both investigated MT-MMPs was not detected in all investigated tissue extracts. The indicated molecular weight of them (about 54 kDa) corresponds to free active form of respective MMP. The umbilical cord tissues of control and preeclamptic subjects contain both enzymes in a form of complexes with other extracellular matrix components. Furthermore, we observed a distinct increase in the amount of MT1-MMP in preeclamptic um-bilical cord vessel walls and significant decrease in preec-lamptic Wharton’s jelly in comparison to control materials. It may be one of the mechanism of extracellular matrix re-modelling in the umbilical cord of preeclamptic newborns.

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P7.5

Characterization of the activation mechanism of human HtrA2 proteaseDorota Żurawa-Janicka, Mirosław Jarząb, Agnieszka Lubomska, Przemysław Glaza, Barbara Lipińska

Department of Biochemistry, University of Gdansk, Gdansk, Polande-mail: Mirosław Jarząb <biomja@ug.edu.pl>

Human HtrA2 protein belongs to the HtrA family of stress-induced serine proteases, characterised by the pres-ence of a conserved trypsin-like protease domain and at least one PDZ domain at the C-terminal end. Under physi-ological conditions HtrA2 promotes cell survival by the maintenance of mitochondrial homeostasis. In stressful conditions HtrA2 functions as a proapoptotic factor, trig-gering cell death. Disturbancesin its function contribute to the development of Parkinson’s disorder and cancer.Analysis of crystal structure of HtrA2 trimers was a basis of a model of the HtrA2 regulation wherein C-terminal fragments of PDZ domains interact with protease domains through van der Waals contacts and block the access to the protease active sites.The aim of the study was to characterize the mechanism of thermal activation of HtrA2 and find out which residues are involved in stabilization of the active site architecture. We generated several point mutation-containing HtrA2 mu-tants using site-directed mutagenesis. Hydrophobic amino acid residues theoretically involved in the stabilization of active site architecture have been substituted by hydrophilic amino acids:V226K, F331Y, and I329N within the protease domain, and V325D in the PDZ domain. The wtHtrA2 and mutated variants of HtrA2 were overproduced in bacterial cells and purified. Their proteolytic activity was character-ized in the range from 20 to 55oC.While proteolytic activity of the wtHtrA2 increased gradu-ally with temperature, mutated HtrA2s: F331Y, I329N, V325D were unable to degrade the substrate. The proteo-lytic activity of HtrA2V226K was higher than the activity of wtHtrA at all temperatures tested. It suggests that these residues play an important role in regulation of the HtrA2 proteolytic activity. Their hydrophobic features are prob-ably indispensable for proper architecture of the HtrA2 active site. Understanding of the molecular basis of HtrA2 regulation will be helpful for pathogenesis studies and generation of new therapeutic strategies for diseases connected to HtrA2 dysfunction.

P7.6

Lipid droplet-like structure formation in stressed endothelial and adipose stromal vascular fraction (SVF) cells in presence of TNFα and fatty acidsBeata Kiec-Wilk1, Anna Knapp1, Magdalena Korczynska1, Urszula Czech1, Joanna Goralska1, Agnieszka Sliwa1, Anna Gruca1, Horst Robenek2, Gerd Schmitz3, Aldona Dembinska-Kiec1

1Departament of Clinical Biochemistry Collegium Medicum, Jagiellonian University; Cracow, Poland; 2Leibniz Institute for Arteriosclerosis Research, University of Muenster; Muenster, Germany; 3Department of Clinical Biochemistry, University Hospital Regensburg, Germanye-mail: Beata Kiec <mbkiec@gmail.com>

In 20–40% adipose tissue consists of the mesenchymal stromal vascular fraction (SVF) containing preadipocytes, and in 10–20% of preendothelial cells. The di-/dedifferen-tiation of SVF is poorly understood. Free fatty acids(FFA) and insulin resistance are responsible for lipotoxicity and li-pid droplets (LD) formation. Nutritional overload induces endoplasmic reticulum (ER)-stress, changes in mitochon-drial membrane potential, LD formation accompanied by autophagy important for survival and protection from ap-optosis. Study is aimed to find the sequence of the gene ex-pression/protein biosynthesis and the lipid content chang-es characteristic for the LD formation and disappearance in preadipocytes and HUVEC does not accumulating lipids.Methods: HUVEC and SVF were cultured in EBM with PA, AA, EPA (30 μM), VEGF, or L-Arginine (SIGMA) for 24 hours (SVF), when with PA or TNFα (5 ng/ml) for 1, 4 and 24 h (HUVEC). Changes in gene expression was analyzed by microarray. Metabolic activity of mitochondria were analyzed by ATP production and oxygen requirement (Oxygraf 2-K Oroboros). The changes DY, PAT protein and selected ER chaperones were followed by the fluores-cence microscopy imaging (Bioimager BD) and by freeze-fracture technique combined with replica immunolabeling for high-resolution imaging.Results: VEGF, as well EPA and AA inhibited, when PA promoted differentiation SVF to adipocytes. SVF me-tabolism (oxygen consumption and ATP) was higher than HUVEC. FFA did not significantly change oxygen con-sumption, but ATP generation was decreased by PA and OA. TNF induced expression of adipophyllin HUVEC. Microarray analysis revealed an induction of intracellu-lar substrate transporters, metabolism and angiogenesis genes, as well as induction of ER-shock chaperones. The electron-microscopic technique of freeze-fracture replica immunolabeling illustrates the localization of the proteins p61ER, GRP78, and GRP94 on the E-face of the ER membrane, the E-face of the inner nuclear membrane and in the lumen of the ER.Conclussion. The ER shock associates formation of the LD-like structures in lipid not accumulating HUVEC.Acknowledgements:Supported by EU F7 HEALTH-2007-2.1.1-6 :LIPIDOMICNET.

Abstracts120

P7.7

Modulation of PPAR-gamma in mice influences EPC functions.Jerzy Kotlinowski1, Anna Grochot-Przęczek1, Magdalena Kozakowska1, Ewa Zuba-Surma1, Jakub Zimoch1, Rafał Derlacz2,3, Laszlo Nagy4, Józef Dulak1, Alicja Józkowicz1

1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Cracow, Poland; 2Adamed Ltd, R&D Department, Pieńków, Poland; 3University of Warsaw, Faculty of Biology, Department of Metabolic Regulation, Warsaw, Poland; 4University of Debrecen, Medical and Health Science Centre, Department of Biochemistry and Molecular Biology, Debrecen, Hungarye-mail: Jerzy Kotlinowski <alicja.jozkowicz@uj.edu.pl>

PPARγ nuclear receptor is a target for thiazolidinediones (TZD), the commonly used insulin sensitizers. TZD can also improve some functions of endothelial progenitor cells (EPC) in diabetic patients, but the role of PPARγ in this pathway has not been proven. Our aim was to check how modulation of PPARγ activity affects EPC functions in healthy and diabetic mice.Experiments were performed on the 12-week old mice with different expression of PPARγ (wild type PPARγWT, heterozygous PPARγHT, and heterozygous with knock-out in monocytes PPARγHT/Mo-) or on 10-week old wild type and diabetic db/db mice. EPC were characterized as the KDR, Sca-1 and lectin positive cells. We found that re-duced expression of PPARγ had no effect on percentage of EPC in the bone marrow: in all animals tested the KDR, Sca-1 and lectin positive progenitors constituted approxi-mately 0.014% of cells. Furthermore, PPARγ expression did not affect the EPC fraction in the blood of healthy mice. However, we observed significantly reduced mobili-zation of EPC 24 h after hind limb ischemia in PPARγHT and PPARγHT/Mo- animals: the number of EPC in the blood of wild type individuals raised by 6.8-fold, while in mice with reduced PPARγ levels this increase ranged from 2.2 to 2.8-fold. Analysis of the gene expression pattern in EPC suggested that this effect may be associated with down-regulation of receptors for VEGF, the major fac-tor responsible for EPC mobilization. What is more, EPC from PPARγHT/Mo- mice produced in vitro significantly less SDF-1a, whereas expression of VEGF did not depended on PPARγ level.Importantly, percentage of EPC was significantly reduced in the bone marrow of diabetic mice (to 0.009%). Reduc-tion related to blood glucose levels suggested that diabetes influences the number of EPC according to its severity. These defect was fully reversed by an oral application of TZD (rosiglitazone or pioglitazone, 10 mg/kg, 28 days), however loss of the correlation between blood glucose and EPC at the end of the study indicated that TZD did not act directly by lowering glucose levels. In in vitro assays, EPC isolated from db/db mice displayed impaired migra-tory and angiogenic potential, also restored by rosiglitazone (10 μmol/L, 24 h) in a PPARγ-dependent manner. Thus, PPARγ is an important regulator of EPC mobilization and its activation may be used to improve the EPC functions.

P7.8

Nuclear lamina and nuclear envelope proteins — multiple roles in mitotic entry and exit, new implications for a development of laminopathyKatarzyna Kozioł, Magdalena Zaremba-Czogalla, Magda Dubińska-Magiera, Ryszard Rzepecki

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Polande-mail: Katarzyna Kozioł <rzepecki@ibmb.uni.wroc.pl>

The cell nucleus is separated from the rest of the cell by nuclear envelope (NE). It includes two lipids bilayers (outer and inner nuclear membrane — ONM, INM respectively), nuclear pores and the nuclear lamina (NL). ONM is func-tionally similar to ER membranes, INM has specific com-position and functions. NL is composed of lamins and is located between INM and the peripheral chromatin. Lam-ins together with nuclear envelope proteins plays impor-tant role in maintenance of the nuclear shape, spacing of nuclear pore complexes, organization of chromatin, DNA replication, regulation of transcription factors, epigenetics, DNA repair, transcription, cell cycle regulation, cell devel-opment and differentiation, nuclear migration and apop-tosis. Recent studies have provided evidences in support of nuclear proteins function in virus infection, tumorogen-esis, mitosis and for linking the nucleoplasm to all major cytoskeletal networks. Mutations in nuclear proteins genes may cause a wide range of heritable human diseases col-lectively called laminopathies/envelopathies.During mitosis entire structure of cell nucleus is remod-eled, NE and NL are depolymerized and mitotic spindle forms allowing separation of chromosomes into daughter cells. Then the NE and NL reassembles around separated chromatids. We analyzed the localization, distribution and dynamics of selected NE and NL proteins. In Xenopus we analyzed XLAP2 proteins, lamins, BAF, membranes and tubulin. In HeLa cells we analyzed distribution of emer-in, LAP2, lamins, dynein, TPX2, pericentrin, tubulin and membrane fractions. We find that emerin, LAP2, XLAP2 and lamins together with membranes of NE transiently as-sociate with mitotic spindle microtubules and mitotic spin-dle matrix both in Xenopus and mammalian cells. Knock-down of such proteins is either lethal, as in the case of XLAP protein or affects mitosis resulting in abnormal phenotypes (emerin, LAP2). Similarly overexpression of protein domains necessary for interaction with mitotic ap-paratus results in abnormal mitosis. We suggest that ab-normal mitosis may be the additional feature of cells with laminopathy and may contribute to manifestation of dis-ease phenotype in patients.

45th Annual Meeting of the Polish Biochemical Society 121

P7.9

Lysenin, a toxin which specifically recognizes sphingomyelin in rafts and affects signaling of immunoreceptor FcγIIAMagdalena Kulma, Katarzyna Kwiatkowska, Andrzej Sobota

Nencki Institute of Experimental Biology, Laboratory of Plasma Membrane Receptors, Warsaw, Polande-mail: Magdalena Kulma <mkulma@nencki.gov.pl>

Sphingolipids and cholesterol of the plasma membrane are organized into microdomains, named rafts. Within rafts dis-tinct tyrosine kinases and scaffolding proteins are anchored allowing formation of signaling platforms of immunore-ceptors, including FcγRIIA. To analyze the dynamics and functions of rafts in FcγRIIA signaling we developed a probe, GST-lysenin, a toxin which selectively recognized membrane sphingomyelin, one of the major constituent of lipid rafts. Interaction of GST-lysenin with membranes of various composition was examined by measurements of surface plasmon resonance. This analysis revealed that GST-lysenin bound 2-fold stronger to SM/DOPC than to SM/DPPC liposomes. Incorporation of cholesterol to the liposomes facilitated formation of sphingomyelin-rich do-mains and improved the binding of GST-lysenin. Binding of GST-lysenin to sphingomyelin membranes was corre-lated with protein oligomerization and pore assembly. To analyze the effect of lipid membrane composition on the pore formation by lysenin, we measured efflux of carboxy-fluorescein from liposomes of various compositions. We found that GST-lysenin released carboxyfluorescein 2.5-fold more effectively from SM/DOPC/cholesterol lipo-somes than from SM/DPPC. Liposomes without sphin-gomyelin were insensitive to lysenin. The data suggest that GST-lysenin binds preferentially to membranes of lipid composition corresponding to that of rafts. To analyze in-teraction of lysenin with the plasma membrane, U937 cells pretreated with lysenin were solubilized in Triton X-100 and fractionated by gradient centrifugation. GST-lysenin formed oligomers (hexamers) upon binding to cell which were detected exclusively in isolated raft fraction. The size exclusion chromatography of lysates of cells pretreated with GST-lysenin followed by FcγRIIA activation showed that the toxin induced formation of large complexes of activated FcγIIA. Binding of the toxin to intact U937 cells affected FcγRIIA signaling as indicated by enhancement of phosphorylation of FcγRIIA and proteins of signal-ing cascade. The data indicate that incorporation of GST-lysenin to the sphingomyelin-rich microdomains induces the formation of large signaling platforms in the plane of the plasma membrane and facilitates signaling of FcγRIIA receptor. The data indicate that GST-lysenin can be an useful tool to study organization of sphingomyelin in the plasma membrane and the role of the lipid in the receptor signaling.

P7.10

Expression of gene encoding TGF-β and genes encoding its receptors in amelanotic melanoma cell cultures C-32 exposed to Photodynamic Therapy (PDT) with PhotolonDariusz Kuśmierz, Małgorzata Latocha, Aleksandra Zielińska, Monika Rybarz, Elektra Sliupkas-Dyrda

Department of Cell Biology, Medical University of Silesia, Jedności 8, 41-200 Sosnowiec, Polande-mail: Dariusz Kuśmierz <dkusmierz@sum.edu.pl>

The transforming growth factor beta (TGF-β) family of proteins are a signaling molecules with unique and potent immunoregulatory properties. TGF-β is produced mainly by macrophages, neutrophiles, thrombocytes and acti-vated T and B lymphocytes. Its immunosupressive prop-erties are i.a consequence of its ability to inhibit: T and B lymphocytes and NK cells proliferation, MHC class II expression, Tc lymphocytes development and IgM and IgG antybodies production. Frequently, effect of TGF-β on immune response is more efficient than in case of im-munosupressive drugs. TGF-β may exert tumor promoter activities at later stages of carcinogenesis. Most human tu-mors secrete large amounts of TGF-β, which directly influ-ences the microenvironment and promotes tumor growth, invasiveness and capacity to form metastases. Elevated lev-els of the TGF-β in plasma of cancer patients contribute to systemic immunosupression promoting tumorgenesis. Increased expression and secretion of different TGF-β isoforms in melanoma cell lines was observed in several studies. TGF-β1 is secreted by normal melanocytes and melanomas at various stage, while TGF-β2 and TGF-β3 are secreted only heterogenously in nevi and melanoma.The aim of the study was to investigate the expression of gene encoding transforming growth factor β (TGF-β) and genes encoding its receptors in amelanotic melanoma cell cultures C-32 exposed to PDT with a Photolon as a pho-tosensitiser. The photosensitizer was applied to the melanoma cell cul-tures at a concentration of 0.01 mg/ml. After 1h of incuba-tion, cell cultures were irradiated with laser light (l=662nm) at three doses: 5, 10, 20 J/cm2. Next, after incubation time of 3h, total RNA was extracted with TRIzol reagent (In-vitrogen). Spectrophotometry was performed to analyse the extracted total RNA for concentration and purity. To analyze the relative changes in examined genes expression the real-time PCR was performed. PCR products were de-tected in 8% polyacrylamide gel electrophoresis. The results obtained in our study show a very low levels of expression of TGF-β and its receptors TGF-β RI, TGF-β RII i TGF-β RIII in amelanotic melanoma cells C-32. In melanoma cells previously subjected to PDT-Photolon a statistically significant decrease of mRNA copy number of TGF-β and all its receptors were observed.

Abstracts122

P7.11

Endocytosis as a component of LPS signaling in macrophagesMaciej Czerkies1, Galyna Kleveta1,2, Andrzej Sobota1, Katarzyna Kwiatkowska1

1Nencki Institute of Experimental Biology, Lab. Plasma Membrane Receptors, Warsaw, Poland, 2Ivan Franco Lviv National University, Department of Biochemistry, Ukrainee-mail: Katarzyna Kwiatkowska <k.kwiatkowska@nencki.gov.pl>

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. Upon bacterial infection LPS activates macrophages triggering produc-tion of cytokines and other mediators of inflammatory responses. If excessive host reactions towards LPS take place, deadly systemic inflammation named sepsis devel-ops. Stimulation of macrophages with LPS engages CD14 and TLR4 receptors and triggers two signaling pathways with one originating from the plasma membrane and the second one linked presumably to endocytosis. In order to reveal whether endocytosis is a component of LPS signal-ing, CD14 and TLR4 distribution in LPS-stimulated J774 cells was examined. At 1–1000 ng/ml LPS did not induce changes of the CD14 cell surface level nor affected the total pool of CD14, as indicated by flow and laser scan cytometry. On the contrary, the amount of the exofacial radio-labeled TLR4 was transiently diminishing by about 50% in LPS-treated cells. Under the influence of dynasore, an inhibitor of GTPase dynamine and hence clathrin-dependent endocytosis, the reduction of the cell-surface TLR4 level was augmented and degradation of TLR4 took place. Simultaneously, LPS-induced signaling exemplified by NFκB and Erk1/2 phosphorylation and cytokine pro-duction was inhibited. Confocal microscopy revealed that during LPS stimulation macrophages polarized. Co-locali-zation of CD14 and TLR4 was limited to the leading edge of stimulated cells and the Pearson’s correlation of CD14 and TLR4 co-labeling reached only 0.41 in relation to 1 as the maximal value. The data indicate that in LPS induces transient co-localization of CD14 and TLR4 followed by TLR4 internalization. However, TLR4 was dispensable for an uptake of 1 μg/ml LPS-Alexa Fluor 488 while function blocking anti-CD14 antibody abolished LPS internaliza-tion. Similarly, dextran sulfate, a ligand of scavenger recep-tors, inhibited internalization of 1 μg/ml LPS. Contrary to TLR4 endocytosis, the uptake of LPS was up-regulated by dynasore but required participation of the actin cytoskel-eton and was negatively regulated by ceramide. We hypoth-esize that TLR4 undergoes clathrin-dependent endocytosis together with a small signaling pool of LPS while CD14 remains on the cell surface and can transfer higher amounts LPS to scavenger receptors which mediate actin-dependent uptake of LPS leading to its detoxification.

P7.12

Role of heme oxygenase-1 in ochratoxin A-induced kidney damageAnna Stachurska1, Aleksandra Sierpniowska1, Christine Boesch-Saadatmandi2, Gerald Rimbach2, Alicja Jozkowicz1, Jozef Dulak1, Agnieszka Loboda1

1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Kraków, Poland; 2Christian Albrechts University Kiel, Institute of Human Nutrition and Food Science, Kiel, Germanye-mail: Agnieszka Loboda <agnieszka.loboda@uj.edu.pl>

The main target organ of ochratoxin A (OTA) is kidney. It is also hepatotoxic in rats and teratogenic in a number of species including mice and potentially human. Heme oxygenase-1 (HO-1), an enzyme involved in heme degradation, possessing anti-apoptotic, immunomodulato-ry and anti-oxidant properties, has been recently suggested to play an important role in kidney pathology. To assess the role of HO-1 in OTA-induced toxicity, HO-1+/+, HO-1+/– and HO-1–/– mice were injected with 2.5 mg/kg/bw OTA i.p., every other day for 20 days. 38% of mortality was observed only in OTA-injected HO-1–/– animals, however after OTA delivery all mice lost their weights. Moreover, HO1–/– mice had larger kidneys com-pared to other mice and OTA potently reduced the size of kidneys in HO1–/– mice. The expression of several genes important for kidney func-tioning was changed after OTA treatment. Vascular en-dothelial growth factor (VEGF) level was downregulated in OTA-injected animals. Expression of p21, a protein which mediates cell cycle G1 phase arrest in response to stress, as well as genes encoding proteins involved in apoptosis (e.g. Bax, PUMA) was potently elevated by OTA. Level of mRNA for MDM2, negative regulator of p53 suppressor protein, was increased in HO-1+/+ and HO-1+/– but not in HO-1–/– animals. Interestingly, OTA induced expression of NF-E2-related factor-2 (Nrf2), the major factor regulating the antioxidant response, and its target gene HO-1. However, expression of catalase and superoxide dismutases (SOD-1, 2) was diminished by OTA delivery. Additionally, expression of genes encoding enzymes involved in glutathione metabo-lism, like glutathione reductase (GSR) and glutathione S-transferase (GSTA) was attenuated and elevated after OTA, respectively. OTA may also affect the expression of profibrotic genes. We observed induction of expression of tissue inhibitor of metalloproteinases-1, 2 (TIMP-1, 2) as well as transform-ing growth factors β (TGFβ1 and TGFβ2), with the fold of induction the highest in HO-1–/– mice. OTA also di-minishes expression of markers of epithelial-mesenchymal transition (EMT) — cadherin 1 (CDH1) in HO-1+/+ and CDH2 in HO-1+/– animals.Taken together, toxic effect evoked by OTA in kidneys is reflected in alterations in various pathways. Lack of HO-1 enhances susceptibility to OTA but the mechanism needs further investigation.Acknewledgements:Supported by the grant No N N401 297835 from the Polish Ministry of Science and Higher Education.

45th Annual Meeting of the Polish Biochemical Society 123

P7.13

Disruption of sphingomyelin metabolism in plasma membrane inhibits receptor FcγIIA signalingAnna Łukasik, Katarzyna Kwiatkowska, Andrzej Sobota

Nencki Institute of Experimental Biology, Departament of Cell Biology, Warsaw, Polande-mail: Anna Łukasik <a.lukasik@nencki.gov.pl>

Sphingomyelin (SM) is a structural component of the plasma membrane, asymmetrically distributed in the bilayer but being concentrated in the extracellular leaflet of the membrane. There, together with cholesterol forms tight-ly packed microdomains termed lipid rafts. The rafts are considered as a signaling platforms as long as they are col-lecting elements necessary for signal transduction, like acti-vated receptors and protein tyrosine kinases.Previous studies in our laboratory show that stimulated re-ceptor FcγIIA associates with sphingolipid/cholesterol do-mains. At the onset of the receptor activation, SM hydroly-sis in the extracellular part of the plasma membrane takes place leading to formation of ceramide. The hydrolysis of SM is mediated by acid sphingomyelinase (ASMase), en-zyme being extruded onto the cell surface during FcγRIIA stimulation and increasing ceramide level. On the other hand, in the plasma membrane sphingomyelin synthase 2 (SMSase 2) is also located which use ceramide as a substrate to rebuild SM pool.In this study we modulated activities of these two en-zymes in U937 cells diminishing their gene expression by RNA interference technique. Down-regulation of ASMase caused significant decrease in level of protein phosphoryla-tion triggered by FcγRIIA activation. Similar disturbances in phosphorylation cascade appeared in cells deficient in SMSase 2 activity. We measured the quantity of SM in the plasma membrane to examine changes ensuing from per-turbationes at the metabolism of the lipid. Surprisingly, si-lencing of SMSase 2 as well as ASMase gene expression correlated with decrement in SM level. At this conditions, the receptor association with rafts was examined. We as-sessed accumulation of the activated receptor in detergent resistant membrane fractions containing raft-derived mem-branes. We found that down-regulation of ASMase and SMSase 2 facilitated association of activated FcγRIIA with rafts. Furthermore, both ASMase and SMSase 2 depletion favored formation of bigger complexes containing stimu-lated receptor, as shown by size exclusion chromatography of detergent cells lysates.All together, the results indicate that disturbances in sphin-gomyelin metabolism influence negatively phosphorylation cascade triggered by FcγRIIA activation, but do not per-turb association of the receptor with rafts.

P7.14

Role of -1607 1G/2G MMP-1 and 372 C/T TIMP-1 gene polymorphisms in pathogenesis of open angle glaucomaŁukasz Markiewicz1, Karolina Przybyłowska1, Anna K. Kurowska2, Anna Kamińska2, Jerzy Szaflik2, Ireneusz Majsterek1

1Department of Clinical Chemistry and Biochemistry, Medical University in Lodz, Lodz, Poland; 2Department of Ophthalmology and Eye Clinic Of 2nd Medical Faculty in Warsaw, Medical University of Warsaw, Warsaw, Polande-mail: Łukasz Markiewicz <markiewicz77@gmail.com>

Extracellular matrix remodeling process mediated by matrix metalloproteinases (MMPs) is an important determinant factor for the development of glaucoma. Well-balanced activity of MMPs and tissue inhibitors of matrix metalo-proteinases (TIMP’s) seems to be crucial for maintaining proper outflow of humor aquosus from eye anterior cham-ber. The main aim of this study was an evaluation of the -1607 1G/2G MMP-1 and 372 C/T TIMP-1 gene polymor-phisms association with the development of primary open angle glaucoma (POAG) in Polish patients. DNA obtained from blood samples of 99 adult patients with POAG and 107 control subjects were enrolled in our study. The poly-morphism analyses were performed using the polymerase chain reaction, restriction fragment length polymorphism (RFLP-PCR). An association between genetic polymor-phisms and the risk of POAG were estimated by χ2 test and logistic regression. Performed research of the -1607 1G/2G MMP-1 gene polymorphism showed increased chance of POAG occurrence in case of 2G2G genotype (OR 2.81, 95% CI 1.33 – 5.95, p<0.01) as well as 2G allele (OR 2.31, 95% CI 1.53 – 3.47, p<0.01). Analysis of 372 C/T TIMP-1 showed increased chance of POAG occur-rence in case of C allele (OR 1.70, 95% CI 1.14 – 2.54, p<0.01). In conclusion we suggested that -1607 1G/2G MMP-1 and 372 C/T TIMP-1 gene polymorphisms might be associated with the risk of primary open angle glaucoma in Polish population.Acknowledgements:This work was supported by grant N N402 248936 from Polish Ministry of Science and Higher Education.

Abstracts124

P7.15

Analysis of consequently changed signal — a novel method for analyzing microarray dataJakub Mieczkowski, Karolina Swiatek-Machado, Bozena Kaminska

The Nencki Institute of Experimental Biology, Laboratory of Transcription Regulation, Department of Cell Biology, Warsaw, Polande-mail: Jakub Mieczkowski <j.mieczkowski@nencki.gov.pl>

A common microarray analysis focuses on a long list of differentially expressed genes. Extracting biological infor-mation from such list is a major challenge. Moreover widely used single gene-based statistics has some limitations, e.g it neglects interactions between genes or cannot detect multivariate changes. Additionally, frequently two lists of differentially expressed genes obtained by two different groups studying the same biological condition may contain a small number of overlapping genes. On the other hand, it is known that deregulation in a group of functionally re-lated genes, such as signaling pathway, plays an essential role in many diseases. A microarray technology allows to shift attention from gene-based analysis to analysis of gene categories, in particular signaling pathways. Since most of the existing methods of pathway analysis treat pathways as an ordinary set of genes, neglecting genes interactions, novel pathway-specific approaches are desired. We propose a novel analysis of consequently changed sig-nal (ACCS) for investigation of signaling pathways. Our method combines microarray gene-specific data with es-tablished knowledge of gene interactions. The ACCS al-lows to pinpoint pathways within which signal is impor-tantly changed under specific conditions. We illustrate our method using expression datastes previously published in biologically oriented papers. This guarantees an unbiased control for paths pointed by our method. In our work we used signaling pathways from KEGG (Kanehisa et al., 2010, Nucleic Acids Res 238: D355–D360 ). By combining expression data and pathway information, ACCS may fa-cilitate analysis of microarray data.

P7.16

Amino trasferase activity in the cerebral cortex of rats with intermittent alcohol intoxicationM. N. Kurbat, V. V. Lelevich

Grodno State Medical University, Department of Biochemistry, Grodno, Belaruse-mail: Kurbat Mikhal <vwmisha@mail.ru>

Not the last role in the mechanisms of ethanol dependence syndrome is a violation of amino acid metabolism in the CNS, therefore, would seem appropriate fully integrated study of the level of metabolism in different models of ex-perimental alcoholism. The purpose of this study — to de-termine the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cerebral cortex of rat brain in a state of intermittent alcohol intoxication. Ex-periments were carried out on white male rats, with a mass at the beginning of the experiment 200–220 g, contained on a standard diet vivarium with free access to water. Etha-nol (25% solution) was administered intragastrically twice a day at a dose of 3.5 g/kg b.w., period of alcohol addiction was 4 days, and cancel — 3 days. Cycles of alcoholism/can-cellation repeated 2 and 4 times. Decapitation of animals was carried out on 14 and 28 days of the experiment, which allowed the dynamics to study the development of this form of alcohol intoxication. Determination of transami-nase activity in brain cortex tissue was performed by kinetic method. The experimental results showed that in all experi-mental groups the activity of key enzymes of transamina-tion of amino acids (ALT and AST) differ from those in intact animals. The degree of change depends on the dura-tion of receipt of alcohol and number of cycles of alcohol addiction and correlated with published data on enhancing the activity of cytoplasmic fractions of ALT and AST and mitochondrial AST during prolonged alcohol abuse among animals. Change of studied enzymes may also be related to the disruption of metabolic adaptation, modification of enzyme kinetics and related disorders with vitamins (espe-cially B6) under the influence of chronic ethanol proceeds into the organism. Undoubtedly, the destabilization of ami-notransferase activity in the CNS may be one reason of metabolism disturbances in the brain in case of intermit-tent alcohol consumption, because studied enzymes are the link between carbohydrate, protein, amino acid and energy metabolism.

45th Annual Meeting of the Polish Biochemical Society 125

P7.17

Synergistic effect of CYBA gene A640G polymorphism and hypercholesterolemia increases the risk of coronary artery diseasePaweł Niemiec1, Iwona Żak1, Tomasz Nowak1, Jolanta Krauze2

1The Medical University of Silesia, School of Health Care, Department of Biochemistry and Medical Genetics, Medyków 18, 40-752 Katowice, Poland; 2The First Department and Clinic of Cardiology, Ziołowa 45/47, 40-635 Katowice, Polande-mail: Tomasz Nowak <tn1984@o2.pl>

Background: Oxidative stress is implicated in the patho-genesis of coronary artery disease (CAD). NAD(P)H oxi-dases are the main source of superoxide in the vasculature. The p22phox peptide encoded by the CYBA gene is a criti-cal component of NAD(P)H oxidases and a functionally relevant A640G and C242T polymorphisms of the CYBA gene modulate generation of reactive oxygen species. This report focuses on the interactions between A640G CYBA polymorphism and traditional risk factors of CAD like hy-percholesterolemia and cigarette smoking.Aims: Aim of the study was to analyze whether A640G CYBA and C242T polymorphisms and the CYBA haplo-types are associated with CAD and its traditional risk fac-tors.Methods: The study population of white Polish Cauca-sians included: 93 patients and 78 controls. The CYBA gene polymorphisms were genotyped using PCR-RFLP method. Statistical analyses were performed using Statis-tica 6.0 software. Gene-traditional risk factors interactions were analyzed using 4x2 tables, with the Rothman’s synergy index (SI) as a determinant of the power of interaction.Results: Case-control study did not reveal any statistically significant differences in genotypes and alleles frequencies of A640G polymorphism between analyzed groups. How-ever, there is a tendency to the prevalence of 242T allele and TT242 genotype among patients with CAD. The differences of haplotypes frequencies of both CYBA gene polymor-phisms did not show statistical significance, although there is a tendency to the prevalence of G640+T242 haplotype in the patients group (39.1 vs 31.6). The hypercholesterolemic carriers of 640G allele were more frequent in the patients group than in the controls (43% vs 15.4%, p<0.0001). The risk of CAD associated with the presence of hypercholes-terolemia was about eight-fold higher in 640G allele carriers than in AA640 homozygous subjects (SI=7.71).Conclusion: The A640G polymorphism can not be con-sidered as an independent risk factor of CAD, but it modu-lates the risk of CAD associated with hypercholesterolemia.

P7.18

Effect of tau and MAP2 on PrP-induced oligomerization of tubulinKatarzyna M. Osiecka, Hanna Nieznanska, Krzysztof Nieznanski

Nencki Institute of Experimental Biology, Department of Biochemistry, Pasteur 3, 02-093 Warsaw, Polande-mail: Katarzyna Osiecka <k.osiecka@nencki.gov.pl>

Microtubules (MT) are cytoskeletal structures composed of tubulin which are involved in many cellular processes such as chromosome segregation, cell division, intracellu-lar transport, secretion, cell motility and determination of cell morphology. Dynamic instability of MT that is cycling between assembly and disassembly is crucial to fulfill their function in the cell, hence it is precisely regulated by nu-merous MT-associated proteins (MAPs). Dysregulation of the balance between assembly and disassembly may result in cell death as it takes place in Alzheimer disease in which hyperphosphorylation of tau abolishes its ability to stabi-lize MT. In previous studies we have demonstrated that prion protein (PrP) - a key molecule engaged in fatal neu-rological disorders called prion diseases interacts with MT/tubulin which leads to oligomerization of tubulin and con-sequent inhibition of MT assembly. Molecular mechanism of PrP neurotoxicity may include this phenomenon. In this report we present an effect of MAPs on PrP-induced oli-gomerization of tubulin. Taking advantage of turbidimetry we have shown that MAPs fraction isolated from pig brain inhibits PrP-induced oligomerization of tubulin. Further studies with purified tau and MAP2 - major constituents of MAPs fraction have demonstrated that both proteins affect formation of the oligomers. Moreover, we have found that tau phosphorylation modulates the effect of tau on PrP-induced oligomerization of tubulin. Based on the above results we propose that hyperphosphorylation of tau - a key event observed in Alzheimer disease promotes PrP-induced oligomerization of tubulin which exacerbates dis-ruption of microtubular cytoskeleton.

Abstracts126

P7.19

Enolase epitopes as plasmonogen-binding receptors in parasites — in silico analysisMichał Piast

University of Arts and Sciences, Department of Public Health and Environmental Sciences, Kielce, Polande-mail: Michał Piast <piast80@gmail.com>

Enolase [EC 4.2.1.11] is a glycolytic pathway enzyme, cata-lyzing 2-phospho-glycerate dehydratation. Enolase per-forms several functions, ie. an eye tau-crystallin protein, sur-face receptor for the binding of plasminogen, Myc-binding protein, etc. Enolase ability to act as a plasminogen-binding protein in bacteria and parasites has been recently studied.In this research, selected enolase amino acid sequences from bacterial and parasites organisms were chosen for analysis together with human alpha enolase to check for possible antibody binding sites.Data analysis involved ClustalX sequence alignment, MEGA4 evolutionary analysis and SIFT analysis to find the most conserved parts of molecules.According to previous work on alpha-enolase-like proteins, they share common sequence DLDFKSPDDPSRYISP, lo-cated on the external part of molecules, binding to mono-clonal antibody.Our research indicate strong sequence similarity between human alpha enolase and four analysed parasites — F. hepatica, S. japonicum, P. falciparum and T. gondii (average of 79.75% similarity). These four molecules share identical string 257-YDLDFK-262 with human alpha enolase. Fur-thermore, latter part of string, after position 262, consists of amino acids predicted as tolerated substitutions. Se-quences from E. histolytica, M. balamuthi and analysed bacte-ria show strong difference in indicated region. Theoretical model of F. hepatica enolase was conducted in SWISS-MODEL using existing human alpha enolase as template. Model quality was checked with anolea, verify3d and Ramachandran plot. Previously indicated string, possi-bly involved in plasminogen binding is located on external part of molecule.Results shown might have implication in understanding process of enolase-plasminogen interaction and might prove useful in treating parasite induced diseases.References:1. Arza et al. (1997) Thromb Haemost 78: 1097–1103.2. Chumchua et al. (2008) Bioinformation 3: 18–23.

P7.20

Short-term pre- or post-treatments with TNF-α, IFNα and IFNγ do not affect insulin-dependent expression of MRF and MyHC in C2C12 myotubesBarbara Pijet1, Maja Pijet1, Anna Litwiniuk1, Arkadiusz Orzechowski1,2

1Warsaw University of Life Sciences (SGGW), Faculty of Veterinary Medicine, Department of Physiological Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; 2Polish Academy of Sciences, Mossakowski Medical Research Center, Department of Cell Ultrastructure, Pawinskiego 5, 02-106 Warsaw, Polande-mail: Barbara Natalia Pijet <basia.pijet@wp.pl>

Skeletal muscle cachexia is associated with muscle loss. NF-κB activated by TNF-α may lead to proteolysis of muscle proteins. Furthermore, STAT kinases could play a significant role in alteration of TNF-α action by IFNs. The main symptom of accelerated protein catabolism in muscle seems to be the reduced expression and/or degra-dation of skeletal, fast myosin heavy chain (MyHC). The aim of the present study was to examine the effect of in-sulin and/or IFNs on the TNF-α-dependent activity of NF-κB, STAT kinases, and protein levels of MyHC protein in C2C12 myotubes analyzed by immunoblotting. TransAm multiplate test was used to examine transcriptional activ-ity. IFNs increased TNF-α-stimulated degradation of IκB protein, the NF-κB translocation to nuclei, and it’s DNA-binding capacity. Surprisingly, long-term (48 h) exposure of 6-days C2C12 myotubes to TNF-α and IFNγ alone, or in combination did not alter expression of MyHC. IFNα alone and together with TNF-α elevated expression of MyHC in 8-days myotubes. The protein level of myogenin in 8-days old myotubes was decreased merely in the long-term treatment with TNF-α. MyoD expression level re-mained unchanged. 24-hours pretreatment of 5-days old C2C12 myotubes with insulin strongly enhanced expres-sion of MyHC in the 8th day of differentiation. This effect was not altered by 48-hours exposure to cytokines (TNF-α, IFNγ alone or with TNF-α). Myogenin and MyoD expres-sion did not change, either. Cytokines in 24-hours pretreat-ment prior insulin administration also had no impact on MyHC and selected MRF expression levels in 8-days myo-tubes. Cytokines used in short-term application after long-term exposure to insulin did not affect insulin-dependent expression of MyHC. Insulin action was apparently domi-nant. These results indicate that insulin participates in myo-genesis from C2C12 cells by contributing to the elevated expression of MyHC and modulation of NF-κB activity.Acknowledgements:This work was supported by the grants No N 401 033 32/0759 and No N 308 111 738 from the Ministry of Science and Higher Education, Poland.

45th Annual Meeting of the Polish Biochemical Society 127

P7.21

Down-regulation of CDK4 and CDK6 genes by RNA interference as therapeutic approach for Alzheimer’s diseaseDanuta Piotrzkowska, Małgorzata Sierant, Milena Sobczak, Barbara Nawrot

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Polande-mail: Danuta Piotrzkowska <danka@cbmm.lodz.pl>

Alzheimer’s disease (AD) is an incurable, neurodegenera-tive brain disease that destroys memory and thinking skills. Several concepts were proposed to explain pathogenesis of AD, among them the most widespread the β-amyloid cas-cade hypothesis (reviewed in [1]). Some investigators link the neuronal cells death in AD and reactivation of the cell cycle. They related abnormal activation of the cell-cycle components such as the cyclin-dependent kinases (CDK4, CDK6, CDC2), cyclins (B1, D) and cdk inhibitors of the INK4 family in pathologically affected neurons in the AD brain [2]. Arendt et al. suggested that aberrant activation of the Ras-MAPK signaling pathway triggers reactivation of the cell cycle. Usually, in postmitotic and terminally dif-ferentiated neurons, the cell-cycle activity is arrested by the enrichment of cdk inhibitors, and neurons are “locked” in the G0 phase. At the beginning of neuropathogenesis, neurons leave the G0 phase and progress until the S phase. Very likely that neurons die at the G1-M transition. The aim of our studies was to down-regulate the expres-sion of two kinases CDK4 and CDK6 in neural cells through RNA interference-based gene silencing. Thus, sixteen siRNAs of sequences complementary to human, mouse and rat transcripts of CDK4 and CDK6 were de-signed and synthesized in house. Their silencing activity was screened in human (HeLa, SH-SY5Y), mouse (Neu-ro2a, RAW 264.7) and rat (PC12) cell lines. The mRNA and target protein levels in cells transfected with screened siRNAs were determined by quantitative RT-PCR (Ro-che) and by Western blot analyses, respectively. The cells transfected with non-silencing siRNA (100% expression of target gene) were used as a control. For CDK4 protein an average 60–90% lowering of mRNA level and 60-80% lowering of protein were observed, while for CDK6 corre-sponding values of 50–80% and 60–85% were found. The sequences of the most powerful siRNA silencers were used to design shRNA and micro RNA inserts, which were sub-sequently cloned into pSilencer 2.0-U6 (Ambion) plasmid downstream of the U6 promoter and into pEGFP-C1 (BD Biosciences) plasmid downstream of the CMV promoter. These constructs down-regulated the CDK4 and CDK6 genes to 30–70% and 30–60% (mRNA) and 21–95% and 20–65% (protein), respectively. References:1. Nawrot B, Sierant M, Paduszynska A (2009) RSC Drug Discovery 3: 230–270.2. Yang Y, Mufson EJ, Herrup K (2003) J Neurosci 23: 2557.

P7.22

The effect of different nitric oxide availability on high fat diet induced metabolic changes in miceUrszula Razny1, Beata Kiec-Wilk1,3, Anna Polus1, Grzegorz Dyduch2, Bogdan Solnica1, Lukasz Wator 1, Maciej Malecki3, Romana Tomaszewska2, John P. Cooke4, Aldona Dembinska-Kiec1

Jagiellonian University Medical College, 1Department of Clinical Biochemistry, Kopernika 15a, 31-501 Cracow, Poland; 2Department of Pathomorphology, Grzegorzecka 16, 31-531 Cracow, Poland; 3Department of Metabolic Diseases, Kopernika 15, 31-501 Cracow, Poland; 4Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5406, USAe-mail: Urszula Razny <urazny@cm-uj.krakow.pl>

Background: High fat diet impairs NO bioavability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. Accordingly, the purpose of this study was to investigate the effect of high fat diet on metabolism in transgenic mice with decreased (eNOS–/– mice ) and in-creased (DDAH overexpressing mice) NO bioavailability. Materials and Methods: Transgene eNOS–/–, DDAH, and WT mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and in-sulin were monitored. The 3D matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis. Gene expression in adipose tissues was analyzed by micro-array and Real Time PCR.Results: eNOS–/– mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose lev-el was observed. Fasting insulin and cholesterol levels re-mains unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-reg-ulated, whereas in eNOS–/– mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were up-regulated.Conclusions: Our results indicate that response to a high fat diet is modulated by NO availability. Increased NO availability attenuates some HFD-induced alterations in metabolism and gene expression associated with insulin resistance. Acknowledgements:This study was supported by: K/PBW/000048, K/ZDS/000623, NuGO FP6-506360.

Abstracts128

P7.23

Altered sphingolipid composition in human Wharton’s jelly of preeclamptic newbornsLech Romanowicz, Edward Bańkowski

Department of Medical Biochemistry, Medical University of Białystok, Białystok, Polande-mail: Lech Romanowicz <lroman@umwb.edu.pl>

Wharton’s jelly is a myxomatous substance surrounding the umbilical cord vessels to protect them against exten-sion, bending, twisting and compression. Preeclampsia (hypertension, edema, proteinuria) is the most common pregnancy-associated pathological syndrome. It is ac-companied by significant alterations in the umbilical cord composition. Here we describe the sphingolipids of Whar-ton’s jelly from 10 newborns delivered by healthy moth-ers and from 10 babies of mothers with preeclampsia. Thin layer chromatography, solid phase extraction and high-performance liquid chromatography were employed. Control tissues were abundant in sphingomyelins and ce-ramides, whereas the amounts of sphingoid bases were distinctly lower. Preeclampsia is associated with a signifi-cant increase in sphingomyelins, ceramides, sphingosine, sphinganine, 4-OH-sphinganine, sphingosine 1-phosphate and glycosylated sphingosine. Furthermore, a decrease in sphinganine 1-phosphate was found. Stearate (C18:0) is the dominating fatty acid in sphingomyelins and cera-mides of control tissue. In contrast to that preeclamptic material contained the highest amount of laurate (C12:0) in sphingomyelins and myristoleate (C14:1) in ceramides. Sphingolipids and some sphingoid bases are bioactive mol-ecules which contribute to regulation of signal transduc-tion pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of Wharton’s jelly re-sulting in remodeling of its composition.

P7.24

The cytotoxic and mutagenic effect of methylmethane sulfonate in M13 phage replicated in Escherichia coli wild and alkB strainsKarol P. Ruszel1, Paweł Kowalczyk2, Beata Sokołowska1, Agnieszka M. Maciejewska1, Jarosław T. Kuśmierek1

1Department of Molecular Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland, 2Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Warsaw, Polande-mail: Karol Ruszel <kruszel@ibb.waw.pl>

We have evaluated the role of AlkB dioxygenase in repair of DNA alkylation damage in E. coli cells. Using M13mp18 phage lacZ system, we compared the survival and muta-genesis of MMS treated phage DNA in E. coli wild-type or alkB mutant. Single stranded DNA of M13mp18 was modified in vitro by MMS and subsequently DNA samples were transfected into E. coli strains JM105 wild type (as a control) and JM105 alkB (adapted or not by MMS). The obtained DNA of mutants was isolated and sequenced us-ing the Sanger method with primer BT3 for lacZ gene. We have found that MMS treated M13mp18 DNA had lower survival and higher mutation frequency in alkB cells than in the wild type. Preliminary results of sequencing of mutants showed that the most frequent mutations are simultaneous big deletions of 93 (DM15 deletion of F’lacZ) and 54 (M13 polylinker) nucleotide fragments of phage DNA. These de-letions are more frequent when phage is replicated in non adapted alkB or in adapted alkB than in adapted wt strain. The most frequent base substitution in non adapted wild type were the G=>A transitions followed by C=>A and T=>A transversions. Among the frameshifts, insertions of a single C or single A and deletions of a single A or G in runs of GA, were detected. In non adapted alkB strain, among the frameshifts, additions of a single A, single C, single T were observed. The detected base substitutions were frequent G=>A transitions in runs of GA (as in wild type), C=>A, A=>C, G=>C transversions and A=>G transition. In adapted alkB we observed mainly frameshifts such as: additions of single A and single C, deletion of sin-gle G. Among the base substitutions: G=>A transitions and G=>C transversions were also observed. The effect was more prominent than in the adapted wild type.

45th Annual Meeting of the Polish Biochemical Society 129

P7.25

Recombinant form of EBA-140 a novel ligand of Plasmodium falciparum —expression and purificationJoanna Rydzak, Ewa Jaskiewicz

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Immunochemistry, Wrocław, Polande-mail: Joanna Rydzak <joanna.rydzak@iitd.pan.wroc.pl>

Malaria causes considerable morbidity in some tropical ar-eas what results in upwards of half a billion episodes of clinical malaria and the deaths of more than a million chil-dren each year in sub-Saharan Africa alone [1]. Plasmodium falciparum, malaria parasite, causes the most severe form of malaria in humans. That parasite can use multiple host receptors to invade human red blood cells (rbc) [2]. Two main invasion pathways has been identified: the sialic acid dependent and the sialic acid independent. Most studies in-dicate that rbc glycophorins serve as host receptors for in-vasion via the sialic acid-dependent pathway [3]. The EBA-140 is a member of DBL protein family of P. falciparum that has been shown to be a novel ligand for human eryth-rocyte glycophorin C. It shares structural homology with EBA-175, another P. falciparum DBL ligand. The conserved binding domain of each member of DBL protein family, called Region II, consists of two subdomains F1 and F2. The F2 domain seems to be more important for receptor binding [4]. We have cloned Region II of EBA-140 ligand from genomic DNA of P. falciparum clone Dd2. Expres-sion of the recombinant form of EBA-140 RII was carried out in two bacterial expression systems using the following vectors: pET28a+ (Novagen), pMALp2x and pMALc2x ( New England BioLabs). The recombinant form of EBA-140 RII was purified from cytoplasm, periplasm and inclu-sion bodies on Nti-Agarose for the pET28a+ system or by affinity chromathography on amylose for pMAL system, respectively. The purified recombinant form of EBA-140 RII will be used in successive studies on parasite ligand and erythrocyte receptor interaction. References:1. Cowman AF, Crabb BS (2006) Cell 124: 755–766.2. Li X (2008) Biochem Biophys Res Commun 376: 489–493.3. Thompson JK et al. (2001) Mol Biol 41: 47–58.4. Pandey KC et al. (2002) Mol Biochem Parasitol 123: 23–33.

P7.26

Acid sphingomyelinase is translocated onto the cell surface upon activation of immunoreceptor FcγRIIAAgata Samonek, Andrzej Sobota

Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Polande-mail: Agata Samonek <asamonek@nencki.gov.pl>

Fcγ receptor IIA (FcγRIIA) is expressed exclusively in human cells of the immunodefense system and mediates phagocytosis of pathogens coated with IgG. Upon activa-tion the receptor is phosphorylated which triggers down-stream signaling pathways. Our previous results showed that activated immunoreceptor FcγRII in U937 cells un-dergoes clustering and is recruited from glycerophosphol-ipid-rich environment of the plasma membrane to sphin-gomyelin/cholesterol enriched microdomains, called lipid rafts. The interaction between activated receptors and rafts seems to be facilitated by ceramide, a product of sphingo-myelinase activity. In this study we demonstrated that during FcγRII acti-vation acid sphingomyelinase (ASMase) is translocated onto the cell surface. This was concluded on the basis of measurement of activity of ASMase detected at the cell extracellular leaflet of the plasma membrane. We observed elevation of ASMase activity (~1.5-fold) during FcγRII ac-tivation. The profile of ASMase activity in the course of FcγRII activation was correlated with generation of the cell surface ceramide and decreasing level of sphingomyelin in crude plasma membrane fraction.To examine if translocation of ASMase is a specific re-sponse to FcγRII activation, the activity of this enzyme was estimated in U937 cells after cross-linking of transferrin receptor, CD55 and glycoproteins and glycolipids by con-canavalin A. These types of cell stimulation did not induce translocation of ASMase onto the cell surface.Electron microscopy performed on isolated membrane sheets showed translocation of ASMase onto the cell sur-face during FcγRII cross-linking. This enzyme was accu-mulated in electron-dense structures in the plane of the plasma membrane in which FcγRII and NTAL (lipid raft marker) were also concentrated. These structures seem to be assembled by coalescence of lipid rafts.Taken together, the data indicate that activation of immu-noreceptor FcγRII induces translocation of acid sphingo-myelinase onto the cell surface where ceramide is generated and this process seems to be specific for activation of im-munoreceptor FcγRII in U937 cells.

Abstracts130

P7.27

Effect of sodium azide and sodium nitrite on rat liver aldehyde dehydrogenasesValentina I. Satanovskaya

Institute of Pharmacology and Biochemistry of the NAS of Belarus, Grodno, Belaruse-mail: Valentina Satanovskaya <visatanovskaya@gmail.com>

Purpose: Hypoxia induced by various factors is accompa-nied by appearance of reactive oxygen species, free radi-cals and activation of lipid peroxidation (LPO). The toxic aldehydes produced (malondialdehyde, 4-oxynonenal, etc.) are metabolized by the system of aldehyde dehydrogenases (ALDH) occurring as a variety of isoforms.Methods: Male Wistar rats weighing 180–220g were used in the experiment. The hemic hypoxia was induced by administration of sodium nitrite (50 mg/kg, i.p., 4 days), whereas tissue hypoxia — by injections of the cytochrome oxidase inhibitor, sodium azide (10 mg/kg, i.p., 4 days). Liver tissue was assayed for the activities of NAD-and NADP-dependent ALDH with various Km for aldehydes.Results: Both the substances did not affect markedly the NAD-dependent with high and low Km ALDH forms but activated NADP-dependent ALDH with high Km for acetaldehyde: control — 1.9+0.1 mU, sodium azide - 2.4+0.2, p<0.02; sodium nitrite — 2.7+0.2, p<0.01. In us-ing aromatic aldehyde (benzaldehyde) as substrate, activa-tion by sodium nitrite was found: 2.8±0.05 mU — control; 2.8±0.1 — sodium azide; 3.4±0.2, p<0.01, — sodium ni-trite. This enzyme form (NADP-dependent with high and low Km to aliphatic and aromatic aldehydes) belongs to the Class ALDH-3 which is expressed during tumor growth. Some authors (Reisdorph R et al., 1998) show that expres-sion of ALDH-3 genes is regulated by a receptor of poly-cyclic hydrocarbons (cancerogenes), and by dimer of this receptor with a hypoxia-inducible factor 1 alpha (HIF-1-alpha), or by LPO products.Conclusions: Our data suggest that systemic intoxication by nitrates and azide can induce neoplastic changes in the liver. References:Reisdorph R, Lindhal R et al. (1998) Biochem Biophys Res Commun 249: 709–712.

P7.28

Sumoylation of the Drosophila 20-hydroxyecdysone receptorJustyna Seliga1, Katarzyna Bielska1, Elzbieta Wieczorek1, Rainer Niedenthal2, Andrzej Ozyhar1

1Department of Biochemistry, Wroclaw University of Technology, Wroclaw, Poland; 2Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Germanye-mail: Justyna Seliga <justyna.seliga@pwr.wroc.pl>

The 20-hydroxyecdysone (20E) coordinates crucial biologi-cal processes in insects such as development and reproduc-tion. It acts through a specific nuclear receptor complex, 20-hydroxyecdysone receptor (EcR) and ultraspiracle (USP). EcR and its ligands are absent in vertebrates and thereby used in commercial ecdysone-inducible expression systems. They are also regarded as building elements of potential gene switches with future therapeutic usage. But EcR action is still poorly understood. Now its activity regu-lation sets the stage for a new research field, especially as far as posttranslational modifications are concerned. Till now there has been no study about EcR sumoylation - attach-ment of the small ubiquitin-like modifier protein (SUMO) that plays a vital role in regulating functions of wide range of cellular proteins. First we performed bioinformatic anal-ysis of sumoylation sites of Drosophila melanogaster EcR with four predictors: PCI-SUMO, SUMOplotTM, SUMOpre, SUMOsp 2.0. Then to confirm SUMO conjugation to EcR we used the Ubc9 fusion-directed SUMOylation (UFDS) system. We prepared a set of constructs coding all three isoforms of EcR from Drosophila melanogaster — EcR-A, EcR-B1 and EcR-B2 fused with sumoylation E2 enzyme — Ubc9 on N- or C-termini. After expression in HEK293 line, cell extracts were fractionated by SDS-PAGE, fol-lowed by immunobloting with anti-Ubc9 antibodies. Elec-trophoretic mobility changes for all proteins were observed suggesting modification by SUMO, both endogenous and coexpressed. Sumoylaion pattern is different depending on location of Ubc9 on N- or C-termini and specific for each isoform but common for SUMO1 and SUMO3. It is also preserved while EcR is coexpressed with its dimerization partner — Usp. In order to identify sumoylation sites we prepared another series of constructs with cDNA of do-mains and fragments of Drosophila melanogaster EcR which revealed regions associated with modification by SUMO protein.Acknowledgements:This work has been supported by the Polish Ministry of Science and Higher Education Grant N302 035 32/2827 and The Wroclaw University of Technology.

45th Annual Meeting of the Polish Biochemical Society 131

P7.29

Oxidative RNA damage and its potential role in cellular responses to radiationM. Skonieczna1, S. Student1, A. Cieślar-Pobuda1, R. Herok2, R. Jaksik1, J. Rzeszowska-Wolny1,2

1Institute of Automatic Control, Silesian Uiversity of Technology, Gliwice, Poland; 2Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Branch Gliwice, Polande-mail: Magdalena Skonieczna <magdalena.skonieczna@polsl.pl>

mRNA stability is an element of the regulation of gene expression, and its modulation is important for proper responses to inter- and intra-cellular signaling and cellu-lar reactions to environmental changes. The structure of transcripts and the presence of nucleotide sequence motifs that are specifically targeted by proteins or miRNAs are crucial for this modulation. Damage introduced in RNA by different factors can influence the specific interactions be-tween RNA and modulators of its stability. One such fac-tor could be oxidative damage to cellular macromolecules by exposure to ionizing radiation. Accumulation of 8-oxo-7,8-dihydroguanosine (8-oxoG) in RNA can be measured experimentally by high pressure liquid chromatography with electrochemical detection (HPLC-EC). In the present work we followed the changes in 8-oxoG levels in RNA of colon cancer HCT 116 p53+/+ and p53–/– cells exposed to ionizing radiation (IR). Cells grown in DMEM-F12 medi-um supplemented with 10% FBS, under standard CO2, hu-midity and temperature were irradiated by 4 Gy of X-rays from a Clinac GMV system, and at different times after irradiation RNA was isolated using a Total RNA Isolation Kit (A&A Biotechnology). The level of 8-oxoG in RNA from irradiated cells changed with time, reaching the high-est value a short time (1 h) after irradiation. Transcript pro-files measured in parallel by the microarray method showed high numbers of up-regulated mRNAs that were enriched in miRNA-targeted sites and ARE sequence motifs. Our results are consistent with the hypothesis that after irradia-tion, this changed abundance of many transcripts enriched in miRNA-targeted and protein binding AU-rich motifs re-flects modulation of mRNA-miRNA and mRNA-protein interactions due to radiation-induced damage to RNA by reactive oxygen species. This mechanism could be impli-cated more generally in cellular responses to stresses where ROS levels increase.Acknowledgements:This work was supported by grant No. N N514 411936 from the Polish Ministry of Education and Science and No. PBU-48/Rau1/2009 from the Silesian University of Technology.

P7.30

Tumor necrosis factor in varicose veins and varicose veins complicated by thrombophlebitisRadosław Kowalewski1, Andrzej Małkowski2, Krzysztof Sobolewski2, Marek Gacko1

1Medical University of Białystok, Department of Vascular Surgery and Transplantology, Białystok, Poland; 2Medical University of Białystok, Department of Medical Biochemistry, Białystok, Polande-mail: Krzysztof Sobolewski <zdbioch@umwb.edu.pl>

Varicose veins are among the most common features of chronic venous insufficiency (CVI), which exerts a high impact on patient quality of life. The disease pathogenesis is still not clearly understood. Although valvular incompe-tence, alterations in smooth muscle cells (SMCs) arrange-ment, and changes in extracellular matrix (ECM) com-position are the most commonly noted factors affecting the development of varicose veins, the exact mechanisms through which they interact in disease pathogenesis remain unknown. Extensive extracellular matrix remodeling of the vein wall is involved in the pathogenesis of varicose veins. The process is controlled by numerous factors. Among them tumor necrosis factor α (TNF-α) is one of the most important cytokines involved in proliferation and growth of cells responsible for inflammatory and immunological response. Furthermore TNF-α influences synthesis many other factors, which can modulate the ECM metabolism. The aim of the study was to evaluate TNF-α and its two receptors (TNF-R1 and TNF-R2) in the wall of varicose and varicose veins complicated by thrombophlebitis, when compared to normal ones. Segments of normal, varicose and varicose veins complicated by thrombophlebitis were collected during varicose veins surgery in 17 patients (9 women and 8 men, 33 – 54 years age range). Expression and content of TNF-α, TNF-R1 and TNF-R2 were evalu-ated with RT-PCR, Western blot and ELISA methods re-spectively. Decreased TNF-α expression were observed in the wall of varicose veins. However in the varicose veins complicated by thrombophlebitis its expression was signifi-cantly higher in comparison with the wall of normal veins. The evaluation of TNF receptors type 1 and 2 showed slight differences in their expression, both in varicose and varicose veins complicated by thrombophlebitis walls. The overexpressed TNF-α in the wall of thrombophlebitis vari-cose veins via TNF receptors may influence gene expres-sion of enzymes involved in extracellular matrix metabo-lism and play a role in vein wall remodeling, as well as in the disease pathogenesis.

Abstracts132

P7.31

Polymorphisms of β-carotene 15,15’-monooxygenase I (BCMO1) gene and metabolic syndromeTeresa Staszel1, Iwona Wybranska1, Beata Kieć-Wilk1,2, Małgorzata Malczewska-Malec1, Katarzyna Kosno1, Maciej Małecki2, Aldona Dembinska-Kiec1

Jagiellonian University, Collegium Medicum, 1Departament of Clinical Biochemistry, 2Department of Metabolic Disorders, Cracow, Polande-mail: Teresa Staszel <teresa.staszel@cm-uj.krakow.pl>

Introduction: The β-carotene 15,15'-monooxygenase I (BCMO I) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A/retinoic acid (RA). BCMO1 is expressed in adipocytes as a peroxisome pro-liferator-activated receptor (PPAR) regulated gene. Recent studies in a BCMO1-deficient mouse model implicate that a tissue specific conversion of β-carotene may influence lipid metabolism. Therefore β-carotene claimed to be protective against metabolic syndrome complications in which adi-pose tissue accumulation (obesity) plays a key role. Recent data show that double BCMO1 mutant R267S and A379V demonstrate reduced catalytic activity by 57% (Leung WC et al., 2009, FASEB J 23: 1041–1053), affecting the cell Vit A and retinoic acid concentrations. The aim of this study was to investigate the possible link between the two BCMO I single nucleotide polymorphisms (R267S and A379V) and biochemical parameters in patients with metabolic syndrome. Methods: 60 subjects of patients with metabolic syndrome diagnosed according to WHO were genetic screened to iden-tification of common nonsynonymous single nucleotide polymorphisms (R267S:rs12934922; A379V:rs7501331) using denaturing HPLC method. At the same time follow-ing biochemical parameters as: blood lipids (TG and FFA), insulin, glucose, leptin were measured during oral glucose (OGTT) and lipid (OLTT) tolerance tests. Results and comments: Patients with BCMO I A376V vs CC genotype presented significantly higher insulin level and increase of FFA level after 6 hours in OLTT in comparison with TC genotype. There were no significant differences in biochemical parameters between TT and CC genotypes of A376V polymorphism (what may result from low frequen-cy of this genotypes in our study). Different genotypes in R267S did not differ in biochemical parameters. Variant allele frequencies R267S and A379V was 55.2% and 28% respectivelly, and were similar to that published by Leung et al. 2009. In our current study the number of double mutants was too small to find significant differences in biochemical parameters. Acknowledgements:Sponsored by European Network of Excellence NUGO.

P7.32

Quantitative characteristics of endogenous retroviruses in porcine kidney cell line PK-15Barbara Strzałka-Mrozik, Joanna Gola, Jolanta Adamska, Urszula Mazurek

Department of Molecular Biology, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Polande-mail: Barbara Strzałka Mrozik <emusialik@sum.edu.pl>

The presence of Porcine Endogenous Retroviruses (PERV) state a potential infectious risk to a recipient when porcine tissues and organs are used for xenotransplantation.In vitro studies on human cell cultures allow to assess the risk of infection and influence of PERV on human cells.Aim: Quantitative detection of Porcine Endogenous Ret-roviruses genomes in PK-15 pig cells and the asessment of the possibility of their transmission to human cells.Materials and methods: Porcine kidney samples and PK-15 cell line derived from porcine kidney were used in this study. DNA extraction was performed with the use of DNA Genomic Mini Kit. RNA was extracted with the use of phenol-chlorophorm method. The number of PERV A, PERV B i PERV C genomes was determined by QPCR i QRT-PCR techniques. The quantitative analysis was carried out with the use of OpticonTM DNA Engine Continuous Fluorescence Detector. Sequence specificity of amplimers was proved by analysis of their melting temperatures and by the separation on polyacrylamide gel with the visualiza-tion by silver staining.Results: PERV A i PERV B genomes were detected in all tissue samples. PERV C genome was detected in 70% of tissue samples. In PK-15 cells PERV A i PERV B genom-es were detected. The number of PERV copies in PK15 cells were as follows: 4093814 ± 3779658 DNA PERV A/μg DNA, 10356962 ± 2356943 RNA PERV A/μg RNA, 953223 ± 685398 DNA PERV B/μg DNA oraz 1411619 ± 956151 RNA PERV B/ μg RNA. PERV C genome was not detected in studied cell line. Conclusions: PK-15 cells derived from porcine kidney are characterised by highest number of PERV A genomes than PERV B. Taking into account the possibility of recombina-tion to more pathogenic PERVA/C, the absence of PERV C in this cell line is advantageous factor in xenotransplanta-tions.

45th Annual Meeting of the Polish Biochemical Society 133

P7.33

The heat shock regulates Mdm1 gene expression in spermatocytesManuela Mikolajec1, Aleksandra Wojtas1, Oksana Shtapenko1, Joanna Korfanty1, Małgorzata Kus-Liskiewicz2, Natalia Vydra1, Agnieszka Toma1, Wieslawa Widlak1

1Department of Tumor Biology, M. Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, Gliwice, Poland; 2Branch Campus of the Faculty of Biotechnology, Rzeszów University, Sokołowska 26, Kolbuszowa 36-100, Polande-mail: Wiesława Widłak <wwidlak@io.gliwice.pl>

The mouse Mdm1 (transformed mouse 3T3 cell double minute 1), similarly to Mdm2 (transformed mouse 3T3 cell double minute 2 ), was described as a gene amplified 25-30-fold in transformed mouse cells containing numerous double minute chromatin particles. However, in contrast to Mdm2, very little is known about functions of Mdm1. Mdm1 is expressed at high level in testes and retina, and at low level at some other mouse tissues. The global gene expression analysis by Affymetrix microarrays revealed el-evated expression of Mdm1 in isolated spermatocytes sub-jected to the heat shock. Here we aimed to analyze further the Mdm1 gene expression in mouse testes and somatic tis-sues subjected to hyperthermia.The level of Mdm1 transcript assessed by RT-PCR in iso-lated spermatocytes was elevated up to 24h after one hour treatment at 38 or 43oC (compared to its expression at physiological temperature 33oC). However, in whole testes subjected to the heat shock in vivo this effect was not visible. Noteworthy, in liver the low level of Mdm1 expression was further decreased after the heat shock. In a search for puta-tive sequences that bind heat shock transcription factors (Hsf1 or Hsf2) in Mdm1 promoter we found HSE (heat shock element)-like sequence located 627-612 bp upstream of ATG codon. Importantly, using the chromatin immu-noprecipitation (ChIP) the binding of Hsf2 (but not Hsf1) to the Mdm1 promoter in spermatocytes was found after the heat shock. Expression of Mdm1 protein in testes was analyzed by im-munohistochemistry. The protein was detected in all cells of control testes excluding myoid cells and spermatozoa. Very strong punctuate staining of Mdm1 protein was ob-served in cytoplasm of metaphase to telophase spermato-cytes. In pachytene spermatocytes the heat shock induced redistribution of Mdm1 from a cytoplasmic to paranuclear localization, which started 2 hours after the treatment at 42oC. It has been shown that in spermatocytes the first features of the hyperthermia-induced apoptosis are de-tected within 2 hours of the treatment. Importantly, the hyperthermia-induced relocalization of Mdm1 was similar to redistribution of mitochondria and Bax observed in ap-optotic germ cells. Noteworthy, in liver cells, which do not induce apoptosis after the heat shock, only a weak stain-ing of Mdm1 was observed in nuclei 2–6 hours after the heat shock. Our observation suggests the involvement of Mdm1 in the hyperthermia-induced apoptosis of sperma-tocytes, which leads to infertility.

P7.34

Insulin-like growth factor I in salivaMałgorzata Wolańska, Ewelina Taudul

Department of Medical Biochemistry, Medical University of Białystok, Mickiewicza 2c, 15-089 Białystok, Polande-mail: Małgorzata Wolańska <ma.wolanska@interia.pl>

Introduction: There are many proteins, including peptide growth factors, present in saliva. Among them probably the insulin- like growth factors can be found. IGF-I and IGF-II are polypeptides of the molecular mass of 7.5 kDa. They circulate as complexes with binding proteins in plasma. The main IGF binding protein is BP-3 in plasma. Complexes of those growth factors with binding proteins (IGFBP-3) reg-ulate their bioavailability and modify effects of their action.The aim of the study: The aim of the study was to detect of IGF and IGF binding proteins in saliva of healthy per-sons of different age.Material and methods: Material for the study was non-stimulated mixed saliva taken with the spiting out method from generally healthy volunteers. The 70 persons aged 5–75 were divided into 7 groups taking into account age and sex. For determination of IGF-I and IGFBP-3 elec-trophoretic method and Western immunoblot technique were used.Results: The electrophoretic pattern indicates that pro-teins with molecular mass above 54 kDa are present in the saliva. They are probably albumins. There are not sig-nificant differences in electrophoregrams of investigated groups. Western immunoblot results for IGFBP-3 show bands of the molecular mass of about 35 kDa. They could correspond to the complex of binding protein BP-3 with IGF-I. Free form of IGF-I is not observed.Conclusions: Our preliminary results indicate that IGF-I is present in the saliva in a form of complexes with binding proteins. Its expression does not depend on sex and age.

Abstracts134

P7.35

Physical-chemical properties of stacking complexes of heterocyclic aromatic amines with caffeine and other methylxanthinesAnna Woziwodzka1, Anna Gwizdek-Wiśniewska2, Jacek Piosik1

Intercollegiate Faculty of Biotechnology University of Gdańsk and Medical University of Gdańsk (IFB UG-MUG), 1Department of Molecular and Cellular Biology, 2Department of Biotechnology, Kładki 24, 80-822 Gdańsk, Polande-mail: Anna Woziwodzka <anna.woziwodzka@biotech.ug.gda.pl>

Methylxanthines (MTX), particularly caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many statistical data indicate protective effects of CAF in-take against several types of cancer. This protective action of CAF may be explained by its ability to form non-cova-lent stacking (π-π) complexes with aromatic molecules of many mutagens and carcinogens.Heterocyclic aromatic amines (HCAs) are a group of mu-tagenic and carcinogenic compounds produced during the cooking of meat. Their mutagenic activity in the Ames test is higher than other food-borne mutagen, aflatoxin B1. Due to their aromatic character, HCAs are likely to interact di-rectly with MTX by formation of stacking complexes.In order to investigate interactions between HCAs and three MTX: CAF, pentoxifylline (PTX) and theophylline (TH), UV-Vis spectroscopy was used. Significant spectral changes were observed in absorption spectra of HCAs ti-trated with MTX, indicating mixed complexes formation between analysed compounds. The use of the statistical-thermodynamical model of mixed aggregation enabled us to calculate the parameters of the interactions, such as con-centrations of all mixture components and neighbourhood association constants (KAC), which were estimated at about 102 M-1.In order to determine the heat effects of MTX-HCA com-plexes formation, the series of microcalorimetric titrations was performed. The favourable enthalpies (about -30 kJ/mol) of MTX-HCA complexes formation were observed for all analysed compounds. The values of obtained en-thalpies are characteristic for stacking interactions, indi-cating that HCA-MTX complex formation is based on interactions between π-π electron systems from polycyclic aromatic rings present in both HCAs and MTX molecules.Molecular modeling studies confirmed mixed hetero-asso-ciation between MTX and HCAs, and allowed us to de-termine the most favourable geometry of such aggregates.The results of this study suggest that MTX interact directly with HCAs by formation of stacking complexes, which may contribute to decreased biological activity of HCAs in the presence of MTX.Acknowledgements:The authors thank Karolina Jagiełło, Anita Dopierała and Professor Jan Mazerski from Gdansk University of Technology for the disclosure of microcalorimeter and excellent technical support during microcalorimet-ric measurements.This work was supported by Polish Ministry of Science and Higher Edu-cation Grant N N301 029834.

P7.36

Novel mammalian homocysteine thiolactone hydrolase. Purification and characterizationJaroslaw Zimny, Ewa Bretes, Andrzej Guranowski

Poznań University of Life Sciences, Department of Biochemistry and Biotechnology, Poznań, Polande-mail: Jarosław Zimny <zimny@up.poznan.pl>

Objective: Homocysteine thiolactone (HcyTL) is a reac-tive form of homocysteine. It spontaneously homocystei-nylates proteins impairing their functions. Till now, two mammalian homocysteine thiolactonases (HcyTL-ases) were discovered; first one (paraoxonase) in the serum and second one (bleomycin hydrolase) in the cells. We have found another HcyTL-ase activity assaying extracts from different organs of transgenic mice with knock-outs of genes encoding aforementioned enzymes. The highest ac-tivity of this novel HcyTL-ase was found in the kidney ex-tracts. Therefore we decided to purify and characterize the enzyme from kidneys.Methods: HcyTL-ase activity was purified from 18 rat kidneys using classical biochemical techniques, such as anion- and cation exchange chromatography, gel filtration and SDS/PAGE. The enzyme activity was assayed by the use of TLC which allowed to monitor the disappearance of HcyTL. Mass spectrometry analysis was performed on LTQ-FT apparatus combined with nanoACQUITY chro-matographic system.Results: The purified 27 kDa-protein was identified as a biphenyl hydrolase-like protein. Its gene is conserved in hu-man, other mammals, chicken, zebrafish, fruit fly, mosqui-to, and C.elegans. In humans the biphenyl hydrolase gene is on 6th chromosome (location 6p25), and cDNA is present in many organs. However, endogenous substrate of biphe-nyl hydrolase remained unknown. We are collecting basic kinetic data on the reactions catalyzed by this proteinConclusion: We have purified and identified a novel protein that exhibits HcyTl-ase activity. LC-MS analysis revealed that this is well known biphenyl hydrolase. Discovery of serum and cellular thiolactonases increase our knowledge on the mechanisms that minimize homocysteine toxicity. Novel, strongly conserved and widely distributed thiolac-tonase will be another element to be considered in studies on the metabolism of homocysteine and diseases caused by its excess.

45th Annual Meeting of the Polish Biochemical Society 135

P7.37

Correlation between expression of HINT1 gene, family history of gastric cancer and H. pylori infection in gastric mucosa of dyspeptic patientsKarolina Zuk1, Lukasz Peczek1, Krystyna Stec-Michalska2, Blazej Michalski1,3, Barbara Nawrot1

1Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland; 2Medical University of Lodz, Department of Gastroenterology and Internal Diseases, Lodz, Poland; Medical University of Lodz, 35th Clinical Hospital, Lodz, Polande-mail: Karolina Żuk <kzuk@cbmm.lodz.pl>

Introduction: The histidine triad nucleotide binding pro-tein 1 (HINT1) is a member of the evolutionary highly conserved HIT protein super family. HINT1 acts as an adenosine phosphoramidase. HINT1 is a novel tumor sup-pressor which is ubiquitously expressed in mammalian tis-sues. It is involved in the regulation of apoptotic pathways by inducing an up-regulation of p53 and the proapoptotic factor Bax expression and a concomitant down-regulation of the apoptosis inhibitor Bcl-2. Its transcriptional regula-tion occurs by interacting with MITF or CDK7.Aims and methods: While the decreased level of expres-sion of HINT1 was observed in cancer cells, we examined if there any decrease in expression of this gene in gastric mucosa of patients with family history of gastric cancer which may face increased risk of developing of that cancer and whether the H.pylori infection has any influence on the expression of HINT1. The study comprised 43 ethnically homogenous patients with dyspeptic symptoms, selected in four groups: groups I and II consist of H. pylori negative patients without and with family history of gastric cancer (16 and 8 individuals, respectively), and groups III and IV consist of H. pylori positive patients without and with fam-ily history of gastric cancer (9 and 10 individuals, respec-tively). qRT-PCR amplification was used to determine the level of mRNA of HINT1 protein. The results were nor-malized to the level of mRNA of a housekeeping GAPDH gene in the biopsy specimen taken from the stomach an-trum and corpus.Results: Family history of gastric cancer correlates with decrease of the level of expression of HINT1 in antrum mucosa only (p < 0.05) which is observed in the H. pylori negative patients as well as in the H. pylori positive group. We have also noticed that H. pylori infection up-regulates HINT1 in patients with family history of gastric cancer but in corpus tissue only and the level of expression of this gene is higher when compared to the antrum (p < 0.05).Conclusions: The level of expression of HINT1, a novel tumor suppressor gene, is decreased in patients with family history of gastric cancer, therefore, we can presume that these patients might be in higher risk of development of gastric cancer.The H. pylori infection up-regulates HINT1 in patients with family history of gastric cancer but the mechanism of this phenomenon is unclear.