Lipids and carbohydrates biochemistry


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Lipids and carbohydrates biochemistry

  1. 1. Session I. Lipids and Carbohydrates Biochemistry Lectures L9.2 L9.1 The role of long-chain fatty acids in the pathogenesis of the Metabolic Syndrome Lipid compounds of the umbilical cord artery Lech Romanowicz, Edward Bańkowski Department of Medical Biochemistry, Medical University of Białystok, Białystok, Poland e-mail: Edward Bańkowski <> The lipid composition of arterial walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of extracellular matrix both in the umbilical cord vessels and in the surrounding Wharton’s jelly. The lipids of the umbilical cord were not studied till now, therefore it was decided to evaluate the specific features of lipid composition of the umbilical cord artery (UCA) and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that UCA wall, as most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature of UCA wall is the presence of high amount of long chain polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with a deep decrease in most free fatty acids and acylglycerols. The total amount of long chain PUFA: C18:2, C:18:3, C20:4, C20:5 and C22:6 in these lipid fractions decreased by half. In the same time an increase in free cholesterol and its esters was observed. The role of these phenomena in pathobiology of UCA and foetal circulation is discussed. The shortage of PUFA may reduce prostaglandin synthesis in arterial wall and impair blood flow in foetal vascular system. Agnieszka Dobrzyń Laboratory of Cell Signaling and Metabolic Disorders, Nencki Institute of Experimental Biology PAS, Warszawa, Poland e-mail: Agnieszka Dobrzyń <> The incidents of obesity has increased dramatically in recent years, making it one of the most pressing public health concerns worldwide. Obesity is commonly associated with comorbid conditions, most notably insulin resistance, diabetes, heart disease, and hypertension, and the coexistence of these diseases has been termed the Metabolic Syndrome. The precise etiology of the many abnormalities that occur in the obese people is still unknown; however an increasing body of evidence indicates that several manifestations of the Metabolic Syndrome and type 2 diabetes mellitus are associated with alterations in intracellular lipid metabolism. Obese humans and animals not only accumulate significant amounts of triglyceride in adipose tissue, but also in liver, muscle and other peripheral tissues. Storage of even modest caloric surplus in lean tissues leads to lipid-induced dysfunction in those tissues. If non-adipose tissues are exposed to excess of long-chain fatty acids, unless leptin action increases their oxidation sufficiently, unoxidized fatty acids enter damaging pathways of nonoxidative metabolism, such as ceramide synthesis. Free fatty acids and ceramides are probably the most toxic lipids and are a cause of lipoapotosis of pancreatic β cells and myocardiocytes leading to diabetes and lipotoxic cardiomyopathy. In the skeletal muscle, the resulting functional impairment causes insulin resistance. These facts demonstrate that reduction in intracellular fatty acid accumulation in non-adipose tissue may be a very promising therapeutic strategy in the prevention of diabetes and other components of the Metabolic Syndrome.
  2. 2. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 L9.3 L9.4 Nitric oxide and cPLA2 regulation in Parkinson and Alzheimer diseases 205 Role of fatty acids and carnitine in the heart Małgorzata Chalimoniuk1, Anna Stolecka1,2, Jozef Langfort2, 3, Joanna B. Strosznajder1 1Department of Cellular Signaling, Medical Research Centre PAS, Warszawa, Poland; 2Department of Physiology, Academy of Physical Education, Katowice, Poland; 3Department of Experimental Pharmacology, Medical Research Centre PAS, Warszawa, Poland e-mail: Malgorzata Chalimoniuk <> Cytosolic phospholipase A2 (cPLA2) appears to play an important role in the central nervous system. It belongs to a family of enzymes which catalyze the hydrolysis of the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of these reactions may exist as second messenger themselves, or be metabolized to eicosanoids and lysophospholipids. In Parkinson’s and Alzheimer’s diseases, many studies have observed increased expression and activity of cPLA2, accompanied with increased release of AA, induction of COX-2 enzyme and higher production of prostaglandins. We investigated if the cGMP/cCGP-dependent protein kinase (PKG) signaling pathway was involved in cPLA2 activation in Parkinson and Alzheimer diseases. We found increased levels of total and phosphorylated cPLA2 and increased AA release in the nigrostriatal system of MPTPinduced parkinsonism mice and in experimental model of AD [in PC12 cells with the Swedish double mutation in amyloid beta precursor protein (APPsw) and in transfected with human APP (APPwt)]. We used cPLA2-specific inhibitors and Ca2+-independent PLA2 (iPLA2), and we found that cPLA2 released more AA after stimulation with MPTP/MPP+ than iPLA2 and that there was a time-dependent delay of AA release by iPLA2 compared to cPLA2. Moreover, our results indicated that the direct relationship between Ab concentration, NOS activity and cPLA2 level and AA release in experimental model of AD. The PKG inhibitor KT5823 decreased MPTP-induced AA release in the nigrostriatal pathway. KT5823, in addition to PKC and ERK1/2 inhibitors, decreased cPLA2 activity as well as total and phosphorlyated cPLA2 protein levels in the midbrain and striatum of MPTP-induced parkinsonism mice.. Inhibition occurred within 30 minutes and persisted for up to 24 hours. Similar results were also observed in MPP+-treated PC12 cells. Dual treatment with PKG and PKC inhibitors had the same effect on cPLA2 activity and protein levels. PKG is involved in the enhancement of cPLA2 phosphorylation at Serine-505 and in AA release in PC12 cells exposed to MPP+. In PC12 cells, inhibitors of cPLA2 and PKG increased viability and prevented MPP+-induced apoptosis. Our results indicate that the nNOS/cGMP/PKG pathway stimulates cPLA2 phosphorylation at Ser-505 by activation of PKC or ERK1/2. Our results also suggest that upregulation of the nNOS/ cGMP pathway observed in experimental models of PD and AD may mediate neuron degeneration and death through activation of cPLA2. Ryszard T. Smolenski Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Ryszard Smolenski <> Fatty acids are predominant energy substrates for cardiomyocytes in the healthy heart, but it is now well appreciated that it is also dangerous fuel for cardiac metabolism, particularly in the ischemic or failing heart. Both basic research and clinical evidence highlight deleterious effects of excess of fatty acids on cardiac cell viability and function. Cardiomyocytes maintain one of the highest levels of fatty acid binding protein among all cells in the body that indicates importance of sequestration of free fatty acid molecules inside the cell. Number of mechanisms could contribute to impairment of cardiomyocyte function by fatty acids. These could be a consequence of biophysical or chemical properties of fatty acids such as detergent effect or specific effects on enzymes or membrane transport. There is strong clinical evidence that correlates deterioration of heart function in patents suffering from heart failure with elevated blood free fatty acid concentration. Reduction of fatty acid entry into cardiac cells and replacement of its metabolic role with carbohydrate substrates is therefore an important target for treatment of heart failure and ischemic heart disease. Carnitine is vital for cardiomyocyte fatty acid transport across mitochondrial inner membrane but its role under pathological conditions extends into additional pathway to sequester fatty acids inside cardiac cells. Carnitine supplementation is therefore believed to provide therapeutic benefits in heart disease. Our studies on carnitine metabolism in the failing heart provided further support for this concept. First, contrary to common view that carnitine is supplied exogenously to cardiac cells we have shown that key enzyme of this pathway arginine:glycine amidinotransferase (AGAT) is present and active in the heart. Furthermore, we have shown that expression and activity of AGAT is increased several times in heart failure, possibly indicating activation of compensatory mechanism under conditions of increased demand for carnitine. Optimizing fatty acid metabolism in the heart seems to be important in heart failure and ischemic heart disease and this pathway could be target for new effective drugs.
  3. 3. The Congress of Biochemistry and Cell Biology — Abstracts 206 L9.5 L9.6 Regulation of the activity of glycolytic enzymes upon their binding to lipid and membrane structures 2008 Surface plasmon resonance (SPR) — applications in carbohydrate analysis Jan Gutowicz Department of Physical Chemistry of Microorganisms, Institute of Genetics and Microbiology, University of Wroclaw, Poland e-mail: Jan Gutowicz <> Interaction between most of glycolytic enzymes and intracellular membranous or protein insoluble structures in vitro as well as their co-localization in situ have been evidenced in a wide range of plant, animal and microbial cells. In this report, the representative recent studies (own studies of the author and his coworkers included) on the interaction, its mechanism, and its consequences for the molecular and functional properties of some glycolytic enzymes are reviewed and discussed. The interaction results in reversible binding of the enzymes to the structures. Since in almost all studied cases the binding is highly sensitive for the change of environmental conditions like pH and ionic strength influencing ionic bonds the multi-elelectrostatic interaction between the molecules of the enzymes and the charged surface of the cellular substructure binding sites has been postulated to be the initial and main forces in the binding mechanism. The implications of the association with negatively charged surface for the activity have been studied using phospholipid bilayer model structures. The electrostatic interaction mechanism has been supported by the studies. In many of the studied enzymes more specific substances like metabolites, substrates, cofactors, some drugs etc. have been shown to alter the binding. The binding of the enzymes to both: natural membrane or phospholipid bilayer structures induces modification of their catalytic properties and the effect differs among the izozyme forms. For aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase the contribution of conformational changes in the activity modification upon the binding with anionic phospholipid bilayers has been evidenced. All these findings support the concept that the equilibrium between bound and unbound forms of the enzymes in vivo is sensitive for metabolic state of a cell. The role of the capacities of the enzymes for such dynamic association is discussed in terms of the regulation of the enzyme activity and of the pathway. Hubert Krotkiewski1, Bożena Krotkiewska2 1Ludwik Hirszfeld Institute of Immunology & Experimental Therapy, PAS, Wrocław, Poland; 2Department of Biochemistry, Medical University, Wrocław, Poland e-mail: Hubert Krotkiewski <> BIAcore optical biosensor, constructed in 1990 (Biacore AB, Uppsala, Sweden) was designed to measure, in a real time, an interaction between two substances (biological interaction analysis, BIA), for example antigen and antibody, lectin and glycoprotein, etc. As a detection method in the instrument the surface plasmon resonance (SPR) technique was used; it is an optical phenomenon which arises during the total inner light reflection when light illuminates thin conducting films under specific conditions, originally discovered by Turbadar (1959). During BIAcore operation the following methodology is used: one of two interacting substances, a ligand, is covalently bound to a working channel of a sensor chip; the second interacting substance, an analyte, is introduced into the working channel as a solution of known concentration, under controlled flow rate in the range of 1–100 µl/min. A detector, based on the surface plasmon resonance, detects the mass increase in the channel, which is a direct consequence of the ligand-analyte complex formation; a signal generated by the detector is given in the relative resonance units (RU); it is proportional to an increase of mass in the working channel (1 RU = 1 pg/mm2 for protein). In the course of investigating the carbohydrate structures, using BIAcore, the following sets of experiments were performed: analysis of the level of desialylation of human glycophorin A (use of Psathyrella velutina, Triticum vulgaris and Sambucus nigra lectins), analysis of reaction of Triticum vulgaris (wheat germ agglutinin, WGA) with glycophorin A, isolated from erythrocytes of blood group A, B and O (looking for fine differences in glycophorin glycosylation), analysis of the level of agalactosylation of IgG, isolated from the patients with rheumatoid arthritis (RA) before and after pharmacological treatment, using Ricinus communis and Griffonia simplicifolia II lectins. The method with BIAcore use showed up as a procedure sensitive, without any need of chemical derivatization of the reagents and repeatable. In all experiments the lectins served as the ligands and the proteins were used as the analytes. Reference: Turbadar T (1959) Proc Phys Soc (London) 73: 40–44.
  4. 4. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 L9.7 Oral Presentations The role of hexokinase in sugar sensing and signaling in plant cells 207 O9.1 Iwona Ciereszko Institute of Biology, University of Białystok, Białystok, Poland e-mail: Iwona Ciereszko <> Sugars affect germination, plant growth and metabolic processes via the expression of numerous genes. Glucose, sucrose and other sugars might serve as elicitors of sugar signaling. Plants developed mechanisms of sensing and transduction of sugar signals. Hexokinase (EC, sugar transporters and specific sugar receptors have been proposed as components of sugar sensing machinery. However, only hexokinase1 (HXK1) is the relatively well characterized sugar receptor until today. HXK1 play a dual role in glucose signaling and hexose phosphorylation (enzyme of the glycolytic pathway), both in plants and other organisms (yeast, animals). Genetic and biochemical analyses of Arabidopsis mutants, gin2, have shown that diverse glucose responses can be mediated by the HXK1 mutations with little or no catalytic activity and provided evidence for uncoupling of glucose-signaling functions from metabolic activities. Hexokinases are encoded by a relatively large gene family (e.g. 6 genes in Arabidopsis or 10 genes in rice). Different subcellular localizations were determined for HXK protein family members. HXK proteins can be soluble in the cytosol or associated with organelles like mitochondrium, chloroplast, Golgi and nucleus. Recent studies have provided evidence for the nuclear localization of hexokinase1 and showed that this nuclear HXK1 forms a signaling complex with VHA-B1 (vacuolar H+-ATPase B1) and RPT5B (19S regulatory particle of proteasome). Such complex directly modulates gene transcription, independently of glucose metabolism. The role of actin cytoskeleton in HXK-dependent glucose signaling during plant growth has recently also been suggested. Raffinose family of oligosaccharides and galactosyl cyclitols in legume seeds Lesław Lahuta, Ryszard Górecki University of Warmia and Mazury, Department of Plant Physiology and Biotechnology, Olsztyn, Poland e-mail: Lesław Lahuta <> One of the major reasons for limited consumption of grain legume seeds is the presence of relatively high quantities of the raffinose family of oligosaccharides (RFOs) that result in flatulence. Therefore, legume plant breeders try to reduce or eliminate the flatulence-producing oligosaccharides from seeds. RFOs are ubiquitous in legume seeds but their compositions vary among species. The seeds of some species contain also α-D-galactosides of myo-inositol isomers or methylated ethers (galactosyl cyclitols). The presence of galactosyl cyclitols in seeds is correlated to the presence of appropriate cyclitols in vegetative tissues. Accumulation of both types of oligosaccharides starts during seed filling and accelerates during seed maturation drying. Changes in the activity of enzymes of the RFO pathway throughout seed development and maturation and catalytic properties of purified or recombinant enzymes indicate that in the biosynthesis of RFOs and galactosyl cyclitols the same set of enzymes is engaged. Therefore, it is possible to improve nutritional quality of legume seeds by the replacement of RFOs by defined α-D-galactosides of cyclitols (with lower flatulence potential and heath promoting property). Before this effort proceeds to far, seed physiologists need to determine the physiological effects of such modification of oligosaccharides in seeds. Feeding free cyclitols into isolated developing seeds or via the cut stem explants was introduced as an experimental model to obtain seeds of Pisum sativum L., Phaseolus vulgaris L., Lathyrus odorathus L. and Vicia sp. with modified α-D-galactosides composition. The amount and composition of newly formed galactosyl cyclitols was closely related to genotype, seed developmental stage and cyclitols type and their concentration used in experiments. The formation of galactosyl cyclitols inhibited the accumulation of higher homologues of raffinose (especially verbascose) and decreased the total content of RFOs in seeds. The accumulation of new cyclitols and galactosyl cyclitols does not affect seed physiological quality (germinability and desiccation tolerance). Acknowledgments: This work was partially supported by grant N303 125 32/4015 obtained from Ministry of Sciences and Education in Poland.
  5. 5. The Congress of Biochemistry and Cell Biology — Abstracts 208 O9.2 O9.3 N-oligosaccharides of α3β1 and αvβ3 integrins in metastatic melanoma WM9 and WM239 cell lines Underappreciated catabolism of glycoconjugates in alcohol abuse Małgorzata Przybyło1, Marcelina E. Kremser1, Dorota Hoja-Łukowicz1, Ewa Pocheć1, Angela Amoresano2, Andrea Carpentieri2, Monika Bubka1, Anna Lityńska1 2008 Napoleon Waszkiewicz1, Sławomir D. Szajda2, Anna Jankowska3, Agata Szulc1, Beata Konarzewska1, Krzysztof Zwierz2 1Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Kraków, Poland; 2Dipartimento di Chimica e Biochimica, Complesso Universitario Monte S’Angelo, Napoli, Italy e-mail: Małgorzata Przybyło <malgorzata.przybylo@> It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched tri- and tetraantennary complex type N-glycans, the presence of poly-Nacetyllactosamine structures, and high sialylation of cellsurface glycoproteins. Carbohydrates have been shown to interact with proteins during recognition events, so any changes in the structure of the sugar component may influence the glycoprotein’s ligand binding ability. Integrins, a large family of cell membrane glycosylated receptors, are involved in important processes such as cell-cell and cellECM adhesion. Their altered expression is associated with tissue invasion and metastasis in many types of cancer. In melanoma, α3β1 and αvβ3 integrins are recognised as specific markers of tumour progression, and αvβ3 in particular is used as a marker to distinguish the radial growth phase (RGP) from the vertical growth phase (VGP). The aim of this study was to analyse glycan pools of α3β1 integrin purified from human melanoma cell line WM239 (skin metastasis), with the use of MALDI MS, and to compare them with previously analysed glycosylation patterns for integrin α3β1 from WM9 cells and αvβ3 from both cell lines. Integrin α3β1 was purified from cell extracts by affinity chromatography. The collected material was separated by SDS/PAGE under non-reducing condition, and protein bands corresponding to a3 and b1 subunits were excised from PVDF sheet. After protein alkylation, in situ digestion, sugar extraction and microcolumn clean-up of sugars, MALDI MS was performed and oligosaccharides were detected as [M + Na+] or [M + H+] ions in the positive ion mass spectrum. Integrins α3β1 and αvβ3 purified from two melanoma cell lines derived from particular metastasis sites differ in their glycosylation profiles. Both subunits of α3β1 integrin in WM239 cells showed less diverse glycan types than did WM9 cells. Our previous results concerning αvβ3 integrin glycosylation have shown reverse correlation: the glycan pool from WM239 cells was much more diverse than in WM9 cells. Nevertheless, both integrins showed the presence of tumour-associated glycans regardless of the differences in glycosylation profiles between these integrins in the two cell lines. Acknowledgements: This work was supported by the Polish State Committee for Scientific Research (PB/0939/P05/2004/26). 1Department of Psychiatry, 2Department of Pharmaceutical Biochemistry, 3Department of Paedodontics, Medical University of Białystok, Białystok, Poland; e-mail: Napoleon Waszkiewicz <> Glycoproteins, glycolipids, and proteoglycans, are referred to as glycoconjugates or complex carbohydrates [1, 4]. Glycoconjugates are important for maintaining cellular and extracellular homeostasis. The biological roles of the oligosaccharide units of individual classes of glycoconjugates include: maintaining conformation and stability of proteins, being target for microorganisms and masking of such a target structures, control of the half-life of proteins and cells, modulation of protein functions, mediating of ligands binding, as well as cell-matrix and cell-cell interactions. Alcohol abuse is a widespread social and medical problem in a large section of populations all over the world [2, 3]. Ethanol and its metabolites induce a variety of pathogenic reactions, affecting almost every organ of human body, disrupting also developmental processes (e.g. alcoholic liver disease and fetal alcohol syndrome). Ethanol, acetaldehyde, reactive oxygen species, and other metabolites of alcohol (e.g. fatty acid ethyl esters), participate in destabilization of processes of glycoconjugate metabolism [2–4]. As the majority of changes in the cellular metabolism of glycoconjugates are ascribed to the alcohol-induced alterations in synthesis, transport, glycosylation and secretion of glycoconjugates [2, 3], the degradative and elimination processes, of which the former occurs also in extracellular matrix, seem to be underappreciated. We therefore reviewed all of these aforementioned crucial areas of the glycoconjugate metabolism and went into conclusion that further research, based on all metabolic steps of the glycoconjugate metabolism, including important catabolic process, need to bridge the transdisciplinary research in enhancing our understanding of alcohol- and glycoconjugate-related diseases. References: 1. Varki A (1993) Glycobiology 3: 97–130. 2. Tomas M et al. (2002) J Neurochem 83: 601–612. 3. Lands WE (1993) Glycobiology 3: 415–416. 4. Waszkiewicz N et al. (2008) Alcohol Alcohol doi: 10.1093/alcalc/ agn027.
  6. 6. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 Posters P9.2 P9.1 209 Evaluation of the influence of age on the activity of salivary lysosomal exoglycosidase in diabetes type 2 Changes in mammalian erythrocyte membrane permeability induced by amphotericin B, nystatin and verapamil Agnieszka Knopik-Skrocka, Józef Bielawski, Joanna Grochowska, Marta Mot Institute of Experimental Biology, Department of Cell Biology, Adam Mickiewicz University of Poznań, Poland; e-mail: Agnieszka Knopik-Skrocka <> Erythrocyte membrane lipids (cholesterol, phospholipids) are very important targets of some hemolytic agents. The membrane activity of amphotericin B (AmB) and nystatin (Nys), belonging to antimicrobial polyene antibiotics, is the effect of sterol-antibiotic complex formation [1, 2]. Similar to AmB and Nys, verapamil (Ver) known as a calcium channel blocker and P-gp inhibitor shows a high affinity to membrane lipids, mainly to anionic phospholipids [3]. The hemolytic activity of AmB and Nys is highly dependent on red blood cell (RBC) membrane specie`s and specimen’s features [4–6]. Its modification is caused by some changes in incubation conditions [2, 3]. In the present study, it is of interest how the changes in chemical composition and temperature of incubation medium influence the hemolysis induced by the polyenes in chosen mammalian erythrocytes. Ver interacts with RBC’s membranes inducing cell shape changes [7]. However, the kinetics of Ver-induced hemolysis and the effect of species features of RBC’s membranes are not known in detailes. In standard conditions (isoosmotic KCl, 37oC), the kinetics of hemolysis induced by Ver, measured with absorption spectrophotometric method (l =590 nm), was very similar to that of AmB and Nys. However, the resistance (C50) of the mammalian erythrocytes to Ver was about 3 orders of magnitude higher than to AmB and 2 orders of magnitude higher than to Nys. No statistic difference was found between C50 calculated for human and pig erythrocytes treated with Ver. The kinetics of AmB or Nys-induced hemolysis was modified when KCl was replaced by other isoosmotic media. However, smaller changes in C50 values were found for Nys. For both antibiotics, the effect of cations and anions tested is dependent on specie’s features of erythrocyte membrane. With the increase of temperature from 15oC to 37oC, the rates of hemolysis induced by the polyenes in human as well as in pig RBCs do not increase markedly. The results for AmB and Nys are similar. The values of the ratios C50 37oC/C50 15oC, calculated from the rates of hemolysis, were near 1.0. References: 1. Knopik-Skrocka A, Bielawski J (2002) Cell Mol Biol Lett 7: 31– 48. 2. Knopik-Skrocka A et al. (2003) Cell Mol Biol Lett 8: 439–454. 3. Knopik-Skrocka A, Buldańczyk A (2004) Biol Lett 41: 27–39. 4. Speelmans G et al. (1995) Biochim Biophys Acta 1238: 137–146. 5. Deuticke B (1968) Biochim Biophys Acta 163: 494–500. 6. Coutinho A, Prieto M (1995) J Biophys 69: 2541–2557. 7. Charbonneau C et al. (2001) Biophys Chem 91: 125–133. Slawomir D. Szajda1, Malgorzata Knas1, Katarzyna Knas-Karaszewska2, Jakub Karaszewski3, Malgorzata BorzymKluczyk1, Anna Stypulkowska1, Wiesław Zarzycki4, Krzysztof Zwierz1 1Department of Pharmaceutical Biochemistry, 3Department of Maxillofacial Surgery, 4Department of Endocrinology, Diabetology and Internal Diseases, Medical University of Białystok, Poland; 2Unpublic Institution of Health Care “Stomatology Dr. Knas”, Białystok, Poland e-mail: Slawomir Szajda <> The diabetes (DM — diabetes mellitus) is a systemic disease which has an influence on glucose level in the blood. Type 2 diabetes mellitus is a disease that affects a rapidly increasing number of patients. Most patients with Type 2 diabetes will develop vascular complications. This may be microvascular disease, such as nephropathy, retinopathy or polyneuropathy, and also macrovascular disease, such as coronary heart disease, stroke or peripheral artery disease. β-galactosidase is one of the lysosomal exoclycosidases which catalyzes removal of galactose residues from the non-reducing end of oligosaccharide chains of glycoconjugates. β-galactosidase also degrades lactose to galactose and glucose in small intestine. The aim of the study was evaluation of the influence of age on salivary lysosomal exoglycosidase (β-galactosidase) in diabetes type 2. Material and methods: 3–5 ml of unstimulated saliva was obtained one to three hours after last meal from the patients with diabetes type 2. 40 patients were divided into two groups: I — patients to 65 years old and II — patients after 65 years old. Unstimulated saliva was collected, by spitting method, into polyethylene tubes, kept on ice. βgalactosidase activity was determined by Chatteriee et al method in Zwierz et al. modyfication. Results: We showed significant decrease in activity (pKat/ kg protein) of β-galactosidase (p=0.008126) in group II (patients after 65 years old with type 2 diabetes) in comparison to group I (patients to 65 years old with type 2 diabetes). Conclusion: Age has influence on enzymatic activity of saliva in patients with type 2 diabetes.
  7. 7. The Congress of Biochemistry and Cell Biology — Abstracts 210 2008 P9.3 P9.4 Evaluation of the influence of diabetes type 1 on the activity of salivary lysosomal exoglycosidases Evaluation of the influence of diabetes type 1 on exoglycosidases activity in human saliva Krzysztof Zwierz1, Malgorzata Borzym-Kluczyk1, Malgorzata Knas1, Katarzyna Knas-Karaszewska2, Jakub Karaszewski3, Slawomir D. Szajda1, Anna Stypulkowska1, Wiesław Zarzycki4 Malgorzata Knas1, Katarzyna Knas-Karaszewska2, Jakub Karaszewski3, Malgorzata Borzym-Kluczyk1, Slawomir D. Szajda1, Anna Stypulkowska1, Wiesław Zarzycki4, Krzysztof Zwierz1 1Department of Pharmaceutical Biochemistry, 3Department of Maxillofacial Surgery, 4Department of Endocrinology, Diabetology and Internal Diseases, Medical University of Białystok, Białystok, Poland; 2Unpublic Institution of Health Care “Stomatology Dr. Knas”, Białystok, Poland e-mail: Krzysztof Zwierz <> 1Department Type 1 diabetes is usually diagnosed in children and young adults, and was previously known as juvenile diabetes. In type 1 diabetes, the body does not produce insulin. Insulin is a hormone that is needed to convert sugar (glucose), starches and other food into energy needed for daily life.Glucuronidase is located in lysosomes and plays an important role in recycling cellular components by cleaving glucuronide moieties from proteins. Glucuronidase exhibits both endo-glycosidase and exo-glycosidase activities, meaning that it can cleave monosaccharides from the middle of a chain or from the end. One of diagnosing method of periodontal disease in a patients is detecting elevated concentrations of β-glucuronidase in saliva. The aim of the study was evaluation of the influence of diabetes type 1 on the activity of salivary lysosomal exoglycosidase — β-glucuronidase. Material and methods: 3–5 ml of unstimulated saliva was obtained one to three hours after last meal from the patients with diabetes type 1. Unstimulated saliva was collected, by spitting method, into polyethylene tubes, kept on ice. β-glucuronidase activity was determined by Chatteriee et al. method in Marciniak et al. modyfication. Results: We showed significant increase in concentration of activity (nmol/ml/min) β-glucuronidase activity (p= 0.000000) in the saliva in type 1 diabetic patients in comparison to control group. Conclusion: The significant increase of the activity of β-glucuronidase probably is connected with increase in catabolism of glycoconjugates mostly related to inflammatory state of tissues. Diabetes mellitus is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycaemia) resulting from low levels of the hormone insulin with or without abnormal resistance to insulin’s effects. Diabetes type 1 is usually due to autoimmune destruction of the pancreatic beta cells. N-acetyl-β-hexosaminidase and β-galactosidase are lysosomal exoglycosidases that degrade oligosaccharide chains of glycoconjugates, such as glycoproteins and glycolipids that constitute cell membranes, and glycosaminoglycan chains of proteoglycans that constitute extracellular matrix. Nacetyl-β-hexosaminidase releases N-acetylglucosamine and N-acetylgalactosamine, and β-galactosidase releases galactose from non-reducing end of oligosaccharide chains of lycoconjugates. Changes in the levels of these enzymes could be associated with breakdown of the periodontal ligament or the oral mucosa.The aim of the study was evaluation of the influence of diabetes type 1 on the activity of salivary lysosomal exoglycosidases. Material and methods: 3–5 ml of unstimulated saliva was obtained one to three hours after last meal from the patients with diabetes type 1. Unstimulated saliva was collected, by spitting method, into polyethylene tubes, kept on ice. Lysosomal exoglycosidases (N-acetyl-β-Dhexosaminidase and β-galactosidase) activity was determined by Chatteriee et al. method in Zwierz et al. modyfication. Results: We showed significant increase in concentration of activity (nmol/ml/min) N-acetyl-β-D-hexosaminidase (p=0.000000) and β-galactosidase (p=0.000000) in the saliva in type 1 diabetic patients in comparison to control group. Conclusion: The significant increase of the activity of lysosomal exoglycosidases probably is connected with increase in catabolism of glycoconjugates mostly related to inflammatory state of tissues. of Pharmaceutical Biochemistry, 3Department of Maxillofacial Surgery, 4Department of Endocrinology, Diabetology and Internal Diseases, Medical University of Białystok, Białystok, Poland; 2Unpublic Institution of Health Care “Stomatology Dr. Knas”, Białystok, Poland e-mail: Małgorzata Knaś <>
  8. 8. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 P9.5 P9.6 Activity of a-fucosidase (a-Fuc) in rats liver after 5 days of hypoxia Enzymes of glycogen catabolism from Contracaecum rudolphii (Nematoda) Danuta Dudzik1, Małgorzata Knaś1, Róża Wisniewska2, Małgorzata BorzymKluczyk1, Krzysztof Zwierz1 211 Krystyna Żółtowska1, Elżbieta Łopieńska-Biernat1, Jerzy Rokicki2 1Department 2Department of Pharmaceutical Biochemistry, of Pharmacology, Medical University of Białystok, Białystok, Poland e-mail: Danuta Dudzik <> Background: Hypoxia is defined as a reduction in the amount of oxygen passing into the blood and it is a state of oxygen deficiency in the blood which is sufficient to cause an impairment of organism function. Hypoxia is caused by the reduction in partial pressure of oxygen, inadequate oxygen transport, or the inability of the tissues to use oxygen. α-fucosidase is one of the lysosomal exoglycosidases which catalyzes removal of fucose residues from the non-reducing ends of oligosaccharide chains of glycoconjugates. The aim of our work was determination of α-fucosidase activity in rats liver after 5 days of hypoxia. Materials and Methods: Hypoxia was induced by five days of anoxemia resulting from putting laboratory rats into gas mixture (2% oxygen and 98% nitrogen) with stable flow, and without pressure changes until first apnoea incident. Activity of α-fucosidase (pKat/kg of protein) in rats liver was determined by Chatteriee et al. method in the modification of Zwierz et al. Results: The mean activity of α-fucosidase in homogenates of rats liver were: in control group – 0.633 nKat/kg of protein and – 1.88 nKat/kg of proiein, after 5 days of hypoxia. We observed significant increase in the activity of α-fucosidase in liver tissue after 5 days hypoxia, in comparison to the control group (p=0.000000). Conclusions: It may be suggested that hypoxia causes increase in degradation of glycoconjugates containing fucose in liver tissue. 1Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Olsztyn, Poland, 2Invertebrate Zoology Division, Gdańsk University, Gdańsk, Poland e-mail: Krystyna Żółtowska <> The nematode Contracaecum rudolphii is a cosmopolitan parasite of piscevorous birds as cormorants, pelicans and see ducks. It can contribute to mortality in birds and causes economical losses to the fish industry. In the last decade this species is starting to close its development cycle also in Poland. Biochemistry of this parasite is particularly interesting because of the complexity of its life cycle. Only little information was found on the metabolism of C. rudolphii. Sugars are an important energetic source for parasitic nematodes. This study was aimed at carbohydrates and the enzymes of glycogen catabolism from C. rudolphii. Larvae third (L3) and fourth (L4) stage and the adult male and female C. rudolphii were isolated from stomachs (n = 10) of cormorants (Phalacrocorax carbo) from lake Bełdany (north-eastern Poland). In the extracts from them the contents of total sugars, glycogen, trehalose and glucose were estimated. The activities of glycogen phosphorylase and enzymes hydrolyzing glycogen as α-amylase, glucoamylase and disaccharidases: maltase, lactase and saccharose were studied. The concentration of total sugars was 6 - 8% of fresh matter. Female parasites accumulated significantly more sugars than male. The concentrations of studied sugars may be put in order: glycogen, glucose, trehalose. Glycogen was a main sugar of this parasite. Its content was higher in adult nematodes than in their larvae. Differently trehalose was more in larvae than in adult. The activity of glycogen phosphorylase was especially high in L3 (2.89 μmol/mg). Both amylases had high activities, and they were almost the same in all parasite stages. The disaccharidases activities were higher in larvae than in adults. Among the disaccharidases activities maltase was the most active (2.67 μmol/mg in L3). Our results indicate that at C. rudolphii age dependent differences in carbohydrate catabolism were appearing.
  9. 9. The Congress of Biochemistry and Cell Biology — Abstracts 212 2008 P9.7 P9.8 The properties of trehalose 6-phosphate synthase from the third larval stage of Anisakis simplex (Nematoda, Anisakidae) Increase of 11-beta-hydroxysteroid dehydrogenase type I mRNA level in rat white adipose tissue after starvation-refeeding Elżbieta Łopieńska-Biernat1, Marta Czubak1, Jerzy Rokicki2 Tomasz Śledziński 1Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Olsztyn, Poland; 2Invertebrate Zoology Division, University of Gdansk, Gdynia, Poland e-mail: Elżbieta Łopieńska-Biernat <ela.lopienska@uwm.> Anisakis simplex is a parasite gastrointestinal nematode with a complex life cycle; the definitive hosts — marine mammals – as well as in humans. As anisakiasis is a serious condition that be fatal to a patient and may cause allergic reaction in patients sensitive to A. simplex allergens, it necessary to intensify research on biochemisty of the parasite’s larvae. For example, little is known about its carbohydrates, in particular — synthesis of trehalose. This sugar is of special importance for parasites owing to its physical and chemical properties. Besides the function of energy reserve, it fulfills a protective role under stress conditions. There is non information available on synthesis of trehalose in the third larval stage of A. simplex that’s why in the present research we decided to mark determine the properties of enzyme participating in synthesis of trehalose of A. simplex. Activity of TPS (EC, was determined using the method by Giaever et al. (1988). The end of the product of reaction – trehalose was determined using HPLC. Protein content according to Bradford (1976).The optimum pH was 7.0. TPS showed optimal activity at 55oC. The activity of enzyme decreased rapidly at a temperature higher than 60oC. The activity of TPS was found to be unaffected by the 15-min. preincubation at temperatures up to 50oC. Temperatures higher than 65oC resulted in inactivation of enzyme. Trehalose showed an activator effect on the enzyme. At 300 mmol trehalose increased 24-fold of TPS activity. Fructose and sorbitol exhibited an activity effect at 100 mmol 2-fold and 3-fold respectively, higher concentrations up at 400 mmol had inhibitory effect on the enzyme. Glucose and proline had an inhibiting effect on the enzyme at 100 and 200 mmol concentration. Na+ had an activator effect on he enzyme, maximal effect was observed at 50 mmol, where it increased the enzyme activity by about 5-fold, higher concentration of NaCl to 400 mmol inhibited almost 27% control activity. K+ showed no significant effect on the enzyme. Mg2+ had an activator effect at higher concentrations (20, 25 mmol) about 30%. Co2+ and Cu2+ had an inhibitory effect with a maximum at 5 mmol. Reference: Bradford J (1976) Anal Biochem. 72: 248-254. Giaever H M et al. (1998) J Bacteriol 170: 2841-49. Acknowledgements: The study was supported by the Polish Ministry of Science and Higher Education grant No. P04C02428. Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Tomasz Śledziński <> 11-beta-hydroxysteroid dehydrogenase type I (11βHSD1) catalyses conversion of less active cortison (11-dehydrocorticosterone in rodents) to more active cortisol (corticosterone in rodents). 11βHSD1 gene is expressed in liver, white adipose tissue (WAT) and other tissues. Cortisol acts on adipose tissue promoting adipocytes differentiation. Mice overexpressing 11βHSD1 gene display enhanced adipocytes differentiation measured by increased fat cell size, and consequently higher adipose tissue mass. Increased activity of 11βHSD1 has been postulated to play important role in pathogenesis of obesity. Another important factor for the development of obesity is increased fatty acid biosynthesis. It has been shown that fastingrefeeding leads to increased lipogenesis. The aim of the study was to investigate the effect of fasting-refeeding on 11βHSD1 gene expression in WAT of rats. 11βHSD1 and lipogenic enzymes genes mRNA level were analysed by real-time PCR in perirenal WAT of 2 months old male rats fasted for 3 days and then fed for another 3 days ad libitum. The results were compared to results obtained in control rats. As expected fasting-refeeding resulted in increase of lipogenic enzymes genes expression (fatty acid synthase and malic enzyme) in WAT of rats. The new finding of this study is that fasting-refeeding leads also to increase of 11βHSD1 gene expression in WAT of rats.
  10. 10. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 P9.9 P9.10 Synthesis, uptake and biotransformation of [3H]-pantethine in brain structures after its intracerebroventricular administration Analysis of expression and tissue localization of phosphoglucan water dikinase (PWD) gene from Solanum tuberosum L. Valery Hurynovich1, Inna Katkovskaya1, Gennady Badun2, Zinaida Tyasto2, Natalya Gulyaeva3, Andrey Moiseenok1 213 Sławomir Orzechowski1, Joanna Simińska1, Agnieszka Grabowska1, Mirosław Sobczak2 1Institute of Pharmacology and Biochemistry NAS, Grodno, Belarus; 2M.V. Lomonosov Moscow State University, Moscow, Russian Federation; 3Institute for Higher Nervous Activity and Neurophysiology, RAS, Moscow, Russian Federation e-mail: Valery Hurynovich <> To obtain the labeled preparation, we used tritium thermal activation and D-pantethine substance (Sigma, USA). The biotransformation of [3H]-PT was studied after its cerebroventricular administration (4 μl, uni- or bilaterally, with specific radioactivity of 1.2 mCi/ml (1.6 Ci/mmol)) to Wistar male rats. After dithiothreitol treatment (10 mM), tissue perchlorate extracts were assayed for radionuclides using HPLC in the regimen of isocratic elution on a 10 μm, 250×4 mm μBondapak C18 column with a mobile phase of 50 mM potassium phosphate buffer-methanol in the ratio of 91.5:8.5 (v/v). [3H]PT uptake by brain structures was the following: hippocamp > large hemisphere cortex > frontal cortex > brain stem > cerebellum. Maximum level of [3H]PT and its metabolites was noted in the hippocamp and large hemisphere cortex after 10–20 min when the radionuclide content 5 to 10-fold exceeded the levels of labeled products in other brain structures. No differences were found in [3H]-PT biotransformation after its uni- and bilateral administration. The samples assayed showed PT as pantetheine (PN). After 10–20 min, the hippocamp and the large hemisphere cortex demonstrated formation of the following metabolites: phosphopantothenic acid (PPA) (6–10%), pantothenic acid (PA) (2–9%), phosphoPN (PPN) (52–57%), PN (26–31%). After 1 h following the administration of the preparation, the PPA and PA fractions were found to increase up to 23–30% while the PPN and PN fractions – to decrease (32–45% and 5–9%, respectively). Since that time [3H]-CoA fraction was seen in the hippocamp (trace amounts) and the large hemisphere cortex (1–3% after 1-10 h, and 11% after 24 h). After 3–24 h following the [3H]PT intracerebroventricular administration the amount of the labeled PPA and PA constituted the bulk of PA labeled metabolites, whereas the amount of PPN and PN fractions decreased down to 6–17 and 5– 11%, respectively. [3H]PT biotransformation in the frontal cortex, brain stem and cerebellum was distinguished by a rapid increase in the PPA fraction (27–40%), CoA (4%) being detected in the frontal cortex at this period. The results obtained indicate a possibility of direct PT transport (in the form of PN) into the CNS structures, biotransformation to phosphopantetheine and CoA, PN hydrolysis by pantetheine kinase from brain (Vecsei L, 1990) to give PA and its phosphorylation leading to considerable accumulation of phosphopantothenic acid. 1Department of Biochemistry, 2Department of Botany, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Warszawa, Poland e-mail: Sławomir Orzechowski <slawomir_> Starch is the most abundant storage carbohydrate produced in plants. All transient starch granules synthesised during the day undergo the breakdown during the night, providing sugars that become included in the metabolism of whole plant. Key meaning in the starch degradation is attributed to α-amylase, product of its activity β-maltose is transported to the cytosol where it is subjected farther conversions. There are same important elements affecting rate of starch decomposition: day durnal cycle, starch phosphorylation and posttranslational regulation of enzyme activities. We isolated from Solanum tuberosum L. tubers and partly described expression pattern of a novel protein implicated in starch metabolism — phosphoglucan, water dikinase (PWD, EC We cloned a part of PWD sequence from potato. The cDNA sequence designated as StPWD1 was submitted to the National Center for Biotechnology Information (accession no. EL595870). On the basis of this sequence we prepared specific primer pairs to localize expression of PWD gene in different potato tissues and organs using in-situ RT-PCR methods. Expression of PWD takes place both in heterotrophic and autotrophic tissues at potato. This is in agreement with our previous observations and implies a possible involvement of PWD in storage and transient starch metabolism. We also observed higher levels of PWD expression at the end of light period than during the rest of the day. Such diurnal changes in transcript abundancy are typical for genes involved in starch degradation in chloroplasts. Apparently, PWD plays a regulatory role in starch degradation in potato. Acknowledgements: This project was supported by Ministry of Science Grant no. N302061134.
  11. 11. The Congress of Biochemistry and Cell Biology — Abstracts 214 2008 P9.11 P9.12 The effect of JA-Me on the degradation of α-Dgalactosides in germinating yellow lupin seeds The influence of trensgenic BT corn pollen on chosen carbohydrates and protein level in worker honeybees (Apis mellifera carnica) Bartosz Nitkiewicz1, Lesław B. Lahuta2, Kazimierz Zalewski1 1Department of Biochemistry, 2Department of Plant Physiology an Biotechnology, University of Warmia and Mazury, Olsztyn, Poland e-mail: Bartosz Nitkiewicz <> The aim of the study was the determination of the effect of jasmonate metyl-ester (JA-Me) at different concentrations (10–3–10–6 M) on the degradation of α-D-galactosides during 48 h of yellow lupin (Lupinus luteus L. cv. Polo) seeds germination. The composition and amounts of soluble carbohydrates were analyzed by gas chromatography method in axes and cotyledons (separately) after 0, 24 and 48 h of seeds germination. In dry seeds raffinose family of oligosaccharides (RFOs) were the main fraction of soluble carbohydrates. Seeds contained also α-D-galactosides of D-pinitol (galactosyl pinitols) and α-D-galactosides of D-chiro-inositol (fagopyritols), but at several fold lower concentration than that of RFOs. During germination the degradation of RFOs (and other galactosides) started earlier in axis, than in cotyledons. The level of sucrose, fructose, glucose and free cyclitols in axis increased according to hydrolysis of oligosaccharides. In axis tissues JA-Me delayed degradation of RFOs, regardless of its concentration. The rate of hydrolysis of RFOs decreased according to increasing concentration of JA-Me, as expected. However, during the first 24 hours of germination JA-Me at 10–5 and 10–6 M induced disappearance of RFOs in cotyledons tissues, opposite to axis. Thus it can be concluded, that the effect of JA-Me on the hydrolysis of α-D-galactosides in germinating yellow lupin seeds is associated with changes in the susceptibility of both axis and cotyledons tissues to this growth regulator. Marek Farjan1, Zbigniew Lipiński1, Krystyna Żółtowska1, Benedikt Polaczek23 1Division of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Olsztyn, Poland; 2Institute of Zoology, Free University of Berlin, Berlin, Germany e-mail: Marek Farjan <> Honeybee is of paramount importance as a pollinator in the natural environment and agriculture. A recent increase of the genetically modified crops areas require researches about their thread to the beneficial insects. Carbohydrate metabolism is an essential marker of the physiological condition of insects. We have examined how GM corn pollen affects the levels of protein content as well as maltose and trehalose – sugars crucial in insects biochemistry. We have chosen the pollen of Limagrain LG 22.43 (without genetic modifications, experimental field near Olsztyn, Poland) and MON863 x MON810 corn (experimental field near Berlin, Germany), which is a hybrid of two lines containing cry3Bb1 and cry1Ab genes, from bt genes family, coding Bt crystal proteins, thus making plants resistant to coleopteran and lepidopteran pests. In order to assess potential impact of transgenic insect-resistant (MON 863 x MON 810) Bt corn-pollen consumption on hive honeybees, 2-, 3-, 4- and 5-day old workers were chosen. The bees were placed in queen shipment cages and fed during 5 days with a honey breed - mixture of honey and sugar (control), honey with non-transgenic Limagrain corn pollen (gr I) and honey with Bt corn pollen (gr II). The protein content, trehalose and maltose levels were estimated. The protein content was measured by Bradford method. HPLC was used to determine trehalose and maltose content. There were no significant differences in protein content tested among the groups of bees fed all of the tree diets (example for 7 days-old bees: control — 2.97±0.49, gr I — 2.68±0.57, gr II — 2.63±0.40 mg protein/g tissue). The sugars contents were singnificantly higher in the control (0.889±0.027 and 0.243±0.075 mg/g tissue) than in both of corn pollen consuming groups (gr I 0.483±0.001 and 0.176±0.031 mg/g tissue, and gr II 0.459±0.145 and 0.147±0.039 mg/g tissue) respectively for trehalose and maltose. Our data indicate that the transgenic pollen has no adverse impact on studied markers of young hive honeybees under our experimental conditions.
  12. 12. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 P9.13 P9.14 Purification and characterization of trehalose-6-phosphate synthase from muscle of Ascaris suum (Nematoda) 215 The effect of membrane cholesterol depletion on hemolytic activity of bile salts Małgorzata Dmitryjuk, Marta Dopieralska, Elżbieta Łopieńska-Biernat, Marek Farjan Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Olsztyn, Poland e-mail: Małgorzata Dmitryjuk <> The synthesis of trehalose in nematodes is catalysed by the action of two enzymes: trehalose-6 phosphate synthase (TPS; EC2.4.1.15) that catalyzes the condensation reaction of UDP-glucose and glucose-6-phosphate to give intermediate trehalose-6-phosphate (T6P), and trehalose6-phosphate phosphatase (TPP; EC3.1.3.12) that convert T6P to free trehalose and Pi. The properties of TPS in nematodes was only researched in Aphelenchus avenae (Loomis et al. 1980). The first information concerning the pathway of trehalose synthesis in reproductive tissues and muscle of A. suum presented Feist et al. (1965). The goal of these researches was purification of TPS from muscles of parasitic nematode A. suum and investigating its properties such as optimum pH and temperature, thermal stability, isoelectric point and molecular weight, Km, Vmax, substrate specificity and influence of potential activators and inhibitors on activity of the enzyme. Activity of TPS was determined by Giaever et al. (1988). The end product of reactions — trehalose was determined using HPLC. Protein concentration was determined by the method of Bradford (1976). Absorbance at 280 nm was used for monitoring protein in the column eluate. To determine the optimum pH was used 0.1 mol acetic acidammonia buffer in range of pH 3.0–8.6. Enzyme optimal temperature and thermal stability was studied measuring TPS activity at 20–90oC. Trehalose-6-phosphate synthase was isolated from muscle of A. suum 265-fold by fractionating with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on sepharose 6B. TPS was a monomer with molecular mass on native and on SDS/PAGE of 66 kDa. The enzyme has the optimum pH 3.8, pI 5.4, Km of 6.6 × 10–4 mol and Vmax = 3.5 nmol × min–1 × mg–1 for UDPG and Km of 1.8 × 10–3 mol and Vmax = 6.2 nmol × min–1 × mg–1 for G6P. Basides glucose-6-phosphate, it is use fructose-6-phosphate as acceptor of glucose and it does not act on glucose and fructose. TPS from A. suum muscles was activated by 10 mmol MgCl2, NaCl or CaCl2 and inhibited by EDTA, KCl, FeCl3 and ZnCl2. References: Bradford MM (1976) Anal Biochem 72: 248–254. Feist CF et al. (1965) J Parasitol 51: 76–78. Giaever HM et al. (1988) J Bacteriol 170: 2841–2849. Loomis SH et al. (1980) J Exp Zoology 211: 311–320. Acknowledgements: This work was supported by Grant: 2PO4C 124 29, from 2005 to 2008. Lucyna Mrówczyńska, Katarzyna Wawrzyniak, Józef Bielawski Department of Cell Biology, Institute of Experimental Biology, Poznań, Poland e-mail: Lucyna Mrówczyńska <> Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. The membrane fluidity (cholesterol/phospholipid ratio) of a cell determines the extent to which it is susceptible to compounds with detergent properties. The present study was undertaken to examine the effect of membrane cholesterol content on the kinetics of hemolysis induced by unconiugated bile salts differ in number of hydroxyl groups and hydrophobicity index (HIx) – two hydrophobic (monohydroxy litocholic LChol and dihydroxy deoxycholic — NaDChol) and one hydrophilic (trihydroxy cholic — NaChol). Using metylbeta-cyclodextrin we directly get the depletion of ~33% cholesterol from the erythrocyte membrane. Digitonin known to from complexes with cholesterol in membranes was used as positive control in our study. The kinetics of hydrophobic and hydrophilic bile salts-induced hemolysis of erythrocytes treated with methyl-β-cyclodextrin appeared to be similar to observe for the control cells and indicated the colloid osmotic type. Depletion of membrane cholesterol increased bile salts-induced hemolysis rate in the concentration under and above their CMC in the order monohydroxy < dihydroksy < trihydroxy. No significant decrease (p>0.05) the erythrocyte resistance to deoxycholic and cholic bile salts was observed after methyl-beta-cyclodextrin treatment. Cholesterol depletion did not influence the hemolytic activity of the most hydrophobic lithocholic acid. To compare, depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed digitonin-induced hemolysis and significantly increased the erythrocyte resistance to its action (p<0.05). Cholesterol depletion had no effect on the erythrocyte shape. The obtain results showed that the partial depletion of plasma membrane cholesterol is not essential for the significant decrease of erythrocyte resistance against unconiugated hydrophobic and hydrophilic bile salts. We conclude that bile salts are able to interact with membrane differently depending on their physicochemical properties.
  13. 13. The Congress of Biochemistry and Cell Biology — Abstracts 216 2008 P9.15 P9.16 Influence of JA-Me in biosynthesis of phospholipids in germinating yellow lupin (Lupinus luteus) seeds Sex-related differences in lipogenic enzymes and SREBP-1 genes expression in rat subcutaneous adipose tissue by progesterone treatment Kazimierz Zalewski, Bartosz Nitkiewicz Ewa Stelmanska1, Elzbieta Goyke1, Julian Swierczynski1 Department of Biochemistry, University of Warmia and Mazury, Olsztyn, Poland e-mail: Kazimierz Zalewski <> The study presents jasmonic acid methyl ester influence on biosynthesis and composition of phospholipid compounds in germinating Lupinus luteus var. Polo seeds. Fully matured lupin seeds chosen to experiment acquired form seeds production central (OLZNAS-CH Sp. z o. o.). Seeds were germinating for 48 h in 21oC in darkness. Phospholipid (TLC) isolation and separation was conducted according to Nichols et al. (1965). Phosphorus content was analyzed according to Ames (1966). 3H-sodium acetate (Polatom, Świerk) was used as a precursor to lipids synthesis. Samples radioactivity was indicated by scintilation counter LS-1801 (Beckman). Growth regulator was applied in four concentrations 10–3 M, 10–4 M, 10–5 M, 10–6 M. Seeds germinating in distilled water were used as control sample. Intensivity of different phospholipid synthesis varied and depended mainly on JA-Me concentration applied. Table 1. 3H-sodium acetate (185 kBq/cm3) incorporation to phospholipids of germinating lupin seeds (48 h, 21°C) (cpm/100 embryonic axes) Phospholipid combination PS PI PC PG PE PA H20 14708 15151 4131 16102 38210 11449 JA-Me 10-3 M 2380 8137 7396 21341 49280 2303 JA-Me 10-4 M 15658 13521 2003 15756 31731 4017 JA-Me 10-5 M 17517 10040 2063 12867 26548 2906 JA-Me 10-6 M 15790 11434 2923 22816 6443 3402 Table 2. 3H-sodium acetate (185 kBq/cm3) incorporation to phospholipids of germinating lupin seeds (48 h, 21°C) (cpm/100 cotyledons) Phospholipid combination PS PI PC PG PE PA H20 19875 23266 73435 27034 34478 11898 JA-Me 10-3 M 6768 25722 11568 39531 53723 11606 JA-Me 10-4 M 11383 19973 5264 59608 38199 15931 JA-Me 10-5 M 14538 18373 10660 32391 26168 21746 JA-Me 10-6 M 14488 17862 21697 20218 43002 11272 1Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Ewa Stelmanska <> Steroid hormones play an important role in the regulation of lipogenesis, however the effect of progesterone on lipogenesis is not clear. The aim of present study was to investigate the influence of pharmacological doses of progesterone on the lipogenic enzymes genes expression in subcutaneous adipose tissue of female and male Wistar rats. Two-month old rats received a single dose (100 mg) of progesterone as a subcutaneous implant. After 1 month of such treatment rats were killed, blood and adipose tissues were collected. Fatty acid synthase, ATP citrate-lyase, malic enzyme, and SREBP-1 genes expression in adipose tissue was measured. Serum progesterone concentration in progesterone-treated rats was higher than in control animals (76.8 ng/ml versus 31.3 ng/ml in female and 29.5 ng/ml versus 1.7 ng/ml in male). Progesterone treated female rats gained weight more rapidly than the control animals. In contrast no effect of progesterone on body weight gain in male rats was found. Lipogenic enzymes genes expression (measured as mRNA level and enzyme activity) was significantly higher in adipose tissue of female rats treated with progesterone as compared with the control rats. Moreover, SREBP-1c mRNA level in this group of rats was also significantly higher. No significant differences in lipogenic enzyme genes expression in subcutaneous adipose tissue of male rats treated with progesterone as compared with the control rats were observed. In the present study we demonstrated sex-related differences in lipogenic enzymes genes expression in subcutaneous adipose tissue by progesterone treatment. Moreover, our data suggested that SREBP-1c plays important role in progesterone-induced stimulation of lipogenic enzyme genes expression in female rats.
  14. 14. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 P9.17 P9.18 Effect of platelet-activating factor (PAF) on motility, capacitation and acrosome reaction of frozen-thawed boar spermatozoa 217 Regulation of hormone-sensitive lipase/cholesterol esterase (LIPE) gene expression by transcription factor C/ EBPα in the adrenal cortical cells Władysław Kordan, Marek Lecewicz, Jakub Tobolski Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury in Olsztyn, OlsztynKortowo, Poland e-mail: Władysław Kordan <wladyslaw.kordan@uwm.> Fertility in mammals involves a series of specific receptor-ligand interactions between different substances during sperm-oocyte fusion. Capacitation and subsequent acrosome reaction are important events in the process of sperm-egg fertilization. It has been demonstrated that platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero3phosphorylcholine, PAF) can influence the fertilization processes by modulating motility, capacitation and the acrosome reaction in spermatozoa. However, there is no information in the literature regarding the effect of PAF on sperm function following semen cryopreservation.The aims of this study were to 1) analyze sperm motility and viability and 2) determine if frozen-thawed boar spermatozoa could undergo capacitation and acrosome reaction following the supplementation of different concentrations of PAF to the thawing medium.Ejaculates, collected from four Large White race boars, were frozen using the Kortowo freezing protocol (Strzeżek et al., 1985). PAF used at concentrations ranging from 1 × 10–8 to 1 × 10–5M was supplemented to semen samples prior to and after freezing-thawing. Motility parameters were assessed using a CASA system. Sperm viability was assessed using the Live/DeadÒ Sperm viability kit (Molecular Probes). The antibiotic chlorotetracycline was used to monitor sperm capacitation status (Saling & Storey, 1979), whereas the Giemsa staining method was used to assess the sperm acrosomal status (Watson, 1975). It was confirmed that the supplementation of PAF at concentrations ranging from 1 × 10–5 to 1 × 10–6M had a beneficial effect on postthaw sperm quality characteristics. This phenomenon was manifested in enhanced motility and survivability of the frozen-thawed spermatozoa. Furthermore, PAF used at a concentration of 1 × 10–5M caused a marked increase in the percentage of membrane-intact frozen-thawed spermatozoa. In addition, the supplementation of 1 × 10–5M PAF to the thawing medium seemed to induce capacitation and acrosome reaction in frozen-thawed spermatozoa. It appeared that PAF used at lower concentrations did not have any significant effect on capacitation and the acrosomal status of frozen-thawed boar spermatozoa. The findings of this study emphasized the role of PAF in mammalian reproductive processes. References: Saling, Storey (1979) J Cell Biol 83: 544–555. Strzeżek J et al. (1985) Med Wet 6: 349–353. Watson PF (1975) Vet Rec 97: 12–15. Acknowledgements: This study was supported by funds from UWM in Olsztyn (0103.0206) Maciej Czajkowski, Katarzyna Kulcenty, Marcin Hołysz, Wiesław H. Trzeciak Department of Biochemistry and Molecular Biology, University of Medical Sciences in Poznań, Poznań, Poland e-mail: Marcin Hołysz <> Experiments on human and animal cell lines revealed that the steroidogenic process is regulated by corticotropin (ACTH). This hormone activates protein kinase A (PKA), which phosphorylates and thereby activates hormonesensitive lipase/cholesterol esterase (HSL) responsible for cholesterol supply for steroid hormone synthesis. We demonstrated that in the adrenal cortical cells Y-1 expression of LIPE gene, encoding hormone-sensitive lipase/cholesterol esterase, was induced by activation of the PKA signaling pathway. The analysis of the LIPE promoter sequence indicated that a number of transcription factors might bind to this region. One of them is C/EBPα, which binds to the proximal region of the promoter, between –46 and –59 bp. The overexpression of C/EBPα in these cells increased transcriptional activity of the LIPE promoter. The expression of the C/EBPα gene in these cells was confirmed by detection of the C/EBPα transcript by real-time qPCR and the presence of the C/EBPα protein by Western blotting. Transfection of Y-1 cells with the vector containing the LIPE promoter fragments, and co-transfection with the vector overexpressing C/EBPα confirmed that LIPE transcription was stimulated by C/EBPα and probably its coregulators GATA-2 and GATA-3, which bind to the proximal region of the promoter between –30 and –60 bp. To confirm that protein kinase A and C/EBPα took part in the stimulation of LIPE expression, the cells were transfected with the vector overexpressing C/EBPα, along with the vector containing the LIPE promoter fragments and simultaneously were incubated with the activator of adenylyl cyclase – forskolin. It is postulated that the cAMP-mediated activation of LIPE expression is independent of the stimulatory effect of C/ EBPα. However, the possibility that C/EBPα and GATA proteins may be phosphorylated by protein kinase  A can not be excluded.
  15. 15. The Congress of Biochemistry and Cell Biology — Abstracts 218 2008 P9.19 P9.20 Regulation of hormone-sensitive lipase/cholesterol esterase (LIPE) gene expression by steroidogenic factor 1 (SF-1) in the adrenal cortical cells The content of protein as well as fat, free fatty acids and phospholipids in cotyledons of developing seeds of Andean lupine (Lupinus mutabilis Sweet) growing in vitro in various conditions of carbon and nitrogen nutrition Katarzyna Kulcenty, Maciej Czajkowski, Marcin Hołysz, Wiesław H. Trzeciak Department of Biochemistry and Molecular Biology, University of Medical Sciences in Poznań, Poznań, Poland e-mail: Katarzyna Kulcenty <> Synthesis of steroid hormones in the adrenal cortex is regulated by corticotropin (ACTH). This hormone activates adenylyl cyclase, which results in increased synthesis of cAMP and activation of protein kinase A (PKA). A common transcription factor controlling gene expression via protein kinase A in the adrenal cortex is steroidogenic factor 1 (SF-1) also known as adrenal binding protein-4 (Ad4BP). It regulates the expression of several genes encoding cytochromes involved in steroid hormone synthesis as well as cholesterol transporters and the ACTH receptor. An important step in steroid hormone synthesis is free cholesterol supply by hormone-sensitive lipase/cholesterol esterase (HSL) which hydrolyses cholesterol esters stored in the lipid droplets. It is not known, however, whether SF-1 is involved in the expression of the LIPE gene, encoding HSL. The aim of our investigations was to elucidate the mechanism of regulation of LIPE gene expression by ACTH and to determine if SF-1 plays a role in this process. The investigations were conducted in adrenocortical Y-1 cells in culture. Specific mRNA levels were determines by real time PCR and the activities of SF-1 and LIPE promoters were estimated by means of luciferase reporter gene expression. To investigate the role of SF-1 in the regulation of LIPE expression, the cells were transfected with plasmids harbouring -343, -1150 and -2150 bp fragments of the LIPE promoter fused to the firefly luciferase reporter gene and the transfection efficiency was normalised to the Renilla luciferase. It was evidenced that overexpression of SF-1 significantly increased transcriptional activity of the LIPE promoter fragments of -1150 and -2150 bp, but not -343 bp. The analysis of LIPE promoter sequence revealed SF-1 binding sites at around -1400 bp. Electrophoretic mobility shift assay (EMSA) using specific probe confirmed that the interactions between SF-1 and the identified sequence of the LIPE promoter were direct. To reveal the importance of SF-1 in PKA-dependent LIPE gene expression, the SF-1 gene, was silenced using specific siRNA leading to a significant reduction in transcriptional activity of the LIPE gene. It was concluded that in the adrenocortical cells SF-1 plays an essential role in the protein kinase A -mediated regulation of LIPE gene transcription. Sławomir Borek1, Stanisława Pukacka2, Krzysztof Michalski3, Lech Ratajczak1 1Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland; 2Institute of Dendrology PAS, Kórnik, Poland; 3Plant Breeding and Acclimatization Institute, Poznań, Poland e-mail: Lech Ratajczak <> Accumulation of storage compounds in developing seeds is very important for the following germination. It is also important to human, for example as a source of high quality oil or animal fodder. Could the protein seeds of lupines be a good alternative to widely used soy? We know that the quantity and quality of storage compounds in seeds closely depend on cultivation conditions. In lupine seeds, five (I-V) developmental stages can be distinguished upon morphological and anatomical features. Total lipid level in whole developing seeds obtained from field cultivation in 2007 year was determined. Lipid content was higher in consecutive developmental stages. The maximum level was achieved in stage V and was about 12% of fresh weight. In stage III endosperm disappears and lipid content is about 50% of maximum level. In this stage cotyledons were isolated and cultivated in vitro for 96 hours on the Heller’s medium with 60 mM sucrose or without the sugar. The medium was additionally enriched in 35 mM asparagine (Asn) or 35 mM NaNO3. Light conditions were 75 mM light quantum × m–2 × s–1. Soluble protein, total lipid content, free fatty acids and phospholipids were determined. Protein content in cotyledons growing on medium without sucrose, Asn and NaNO3 was the same as in cotyledons used for the preparation of in vitro culture. Addition of sucrose to the medium caused an increase in protein content and addition of Asn (with and without sucrose) resulted in even greater increase in protein content. Similar effect was caused by NaNO3, but it was weaker than in the case of Asn. Addition of sucrose increased lipid level as well; however, Asn inhibited this process. This effect was more intensive in cotyledons growing on medium without the sugar. Phosphatidylinositol, phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine were detected. The highest concentration was found for phosphatidylcholine. The content of almost all phospholipids was smaller in cotyledons growing on the medium with Asn (with and without sucrose). Palmitic, stearic, oleic, linoleic, linolenoic and eicozic acid were determined. The highest concentration was found for linoleic and oleic acids. No erucic acid was found. Our results show that higher accumulation of protein and lipid in developing cotyledons can be achieved by good sucrose supply. However, in Andean lupine seeds there is negative correlation between protein and lipid accumulation. Acknowledgements: This work was supported by grant no. 2 P06A 004 29 from science fundings in years 2005–2008.
  16. 16. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 P9.21 P9.22 Sucrose regulates activity of acyl-CoA oxidase and PEP carboxykinase in germinating seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus albus L.), and Andean lupine (Lupinus mutabilis Sweet) growing in vitro 219 Characterisation of glycoforms of ascitic fluids by lectins Sławomir Borek, Lech Ratajczak Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland e-mail: Sławomir Borek <> Storage lipids, accumulated in developing seeds, are converted into glucose and sucrose during germination. Through catabolic and anabolic repression these sugars can regulate expression of hundreds of genes. We know that sugars repress degradation of storage carbohydrates, proteins and lipids by catabolic repression. Regulatory role of sugars is studied almost only in plants which seeds are rich in starch or lipids. Protein seeds are not investigated in those experiments. Activity of acyl-CoA oxidase (β-oxidation) and PEP carboxykinase (gluconeogenesis) was assayed in isolated embryo axes, excised cotyledons as well as in seedlings axes and cotyledons growing in vitro for 96 hours in darkness on Heller’s medium with 60 mM sucrose or without the sugar. We used seeds of three lupine species which differ significantly in storage lipid content. Yellow lupine contains ca. 6% of lipid in dry mass, white lupine 7–14% and Andean lupine up to 20%. Both enzymes were more active (calculated per gram fresh weight) in excised and seedling cotyledons than in embryo axes. Acyl-CoA oxidase in organs of yellow lupine was more active in sugar deficiency, whereas in white and Andean lupine the enzyme was more active in organs growing on medium supplemented with sucrose. PEP carboxykinase was more active in organs fed with sucrose in each of the three lupine species. The results are very difficult to interpret. The increase in acyl-CoA oxidase activity in sugar-deficient conditions can be explained upon catabolic repression, but only in yellow lupine. It is striking that the activity of acyl-CoA oxidase in white and Andean lupine as well as activity of PEP carboxykinase in each of the three lupine species is higher in organs growing on medium with sucrose. Probably this caused by specific features of protein seeds, in which nitrogen metabolism is complex and there is intensive degradation of storage lipids in conditions of appropriate supply of tissues in basic respiratory substrate. This issue, however, requires further investigations with the use of molecular biology techniques. Acknowledgements: This work was supported by grant no. 2 P06A 004 29 from science fundings in years 2005-2008. Iwona Radziejewska1, Małgorzata Borzym-Kluczyk2, Joanna Wosek1 1Department of Medical Chemistry, 2Department of Pharmaceutical Biochemistry, Medical University of Białystok, Białystok, Poland e-mail: Iwona Radziejewska <> Ascitic fluid is a pathologic fluid which is accumulated within the abdominal cavity. It is observed especially in patients with cirrhosis, other severe liver diseases and many cancers. Malignant ascites represent about 10% of all cases. They are a manifestation of advanced malignant disease that is associated with a poor diagnosis and significant morbidity. The exact mechanisms of ascites formation are poorly understood. It is suggested that lymphatic obstruction, immune modulators, vascular permeability factors and metalloproteinases are involved in these mechanisms. Because of a big variability of the ascitic fluids, they have not been well characterized so far. One of their components is a soluble form of MUC 1 mucin, highly O-glycosylated glycoprotein, carrier of many various glycoform structures. It is known that in cancers glycosylation of MUC 1 is altered, oligosaccharides are reduced in chain lengths, the protein core is more densely glycosylated, very often a relatively higher amount of sialic acid can be observed. The main goal of our study was characterization of glycoform structures of ascitic fluids taken from patients with different clinical events. To perform this ELISA tests with biotinylated lectins where used. Results could be considered as useful in discrimination between benign and malignant diseases.
  17. 17. The Congress of Biochemistry and Cell Biology — Abstracts 220 2008 P9.23 P9.24 Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVECs) The conversion of cholesterol into derivatives by the enzyme(s) located in the lysosomal membranes of human placenta Joanna Wosek1, Halina Porowska1, Krzysztof Wnuczko2, Marek Szczepański2 1Department of Medical Chemistry, 2Department of Neonatology, Medical University of Białystok, Białystok, Poland e-mail: Joanna Wosek <> Mucins are high-molecular-weight O-glycosylated proteins (50–80% of their mass is due to O-linked carbohydrate chains) that participate in the protection, lubrication, and acid resistance of the epithelial surface. Each organ or tissue exhibits specific pattern of MUC gene expression. MUC1 mucin is a type I transmembrane glycoprotein, which level increases with malignant transformation. MUC1 is normally expressed at the apical borders of glandular epithelial cells, by contrast, the polarization of MUC1 expression is lost in carcinoma cells. Proposed functions for MUC1 include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and protection of epithelial surfaces against infection. Although expression of transmembrane mucins was originally thought to be restricted to epithelial tissues, in some recent studies MUC1 and MUC4 was detected in several types of endothelial cells. This fact directed us to search of MUC1 mucin on the surface of human umbilical vein endothelial cells (HUVECs). The expression of MUC1 mucin in HUVE cells was examined by the method of Western blotting, ELISA and flow cytometry. The influence of TNF-α and interferon-gamma on the expression and shedding of MUC1 as well as on cell adhesion to ECM proteins was also tested. Our experiments confirmed the expression of MUC1 on the surface of HUVECs, which was increased after treatment with cytokines, like as α2β1 and α5β1 integrins expression in the cells treated with TNF-α. Culture media contain MUC1 glycoprotein in greater quantity than lysates, and molecular weight of this protein was higher in medium than in lysate. Shedding of MUC1 to medium was increased after incubation with cytokines. TNF-α treatment caused a decrease in sialic acid level and T antigen level was not changed. The adhesion to fibronectin was slightly increased in cells treated with TNF-α, but adhesion to collagen type I was not changed. Vascular endothelial cells may play an important role in angiogenesis, a critical process in wound-healing, inflammation, embriogenesis cancer and development. Expression of adhesion and anti-adhesion molecules plays an important role in the interaction of tumor cells with vascular endothelial cells during tumor invasion and metastasis. The presence of transmembrane mucin MUC1 in HUVECs may have implication in interactions with different type of cells in physiological and pathological processes. Katarzyna Roszek, Wioletta Werner, Michał Komoszyński Biochemistry Department, Institute of General and Molecular Biology, Nicolaus Copernicus University, Toruń, Poland e-mail: Katarzyna Roszek <> It is well known that cholesterol plays a crucial role in maintaining the proper fluidity of the membranes. We adapted the enzymatic CHOD/PAP method of cholesterol quantification [1] to the examination of cholesterol metabolism in the lysosomal membranes. Our data indicate that in the lysosomal membranes of human placenta there is an enzyme(s) converting cholesterol into its derivatives. Addition of 5 mM cholesterol to the lysosomal membranes and incubation for 10 to 120 minutes effected in the linear decrease in cholesterol concentration. It depended on the time of incubation and the amount of membranes used. These data suggest the enzymatic conversion of cholesterol into its derivatives. Optimum pH for the examined reaction was 5.0. The decrease in cholesterol concentration was also catalyzed by the partially purified preparation of cholesterol sulphate sulphohydrolase [2], what excludes the non-enzymatic interactions between lysosomal membranes and cholesterol. Besides, cholesterol sulphate sulphohydrolase may be a part of the multienzymatic complex involved in regulation of cholesterol content in the lysosomal membrane.Our studies with HPLC method were focused on the qualitative analyses of emerging cholesterol derivatives. The results revealed that a steroid derivative with more hydrophilic properties appeared simultaneously with the decrease in cholesterol concentration. Currently, our research aims at the identification of this compound. Concluding remarks: 1/ The concentration of membranous cholesterol is precisely controlled since it influences the stability of the membranes. 2/ Enzymatic conversion of cholesterol may be the simple way for the regulation of cholesterol content and lysosomal membranes fluidity. References: 1. Richmond W (1973) Clin Chem 19: 1350–1356. 2. Roszek K, Gniot-Szulżycka J (2008) J Steroid Biochem Mol Biol (in press).
  18. 18. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 221 P9.25 P9.26 Dose-dependent effect of catechin on Pyruvate Dehydrogenase Kinase (PDK) activity in rats Does obestatin play a functional role in metabolism of white adipocytes? Małgorzata Tyszka-Czochara1, Joanna Gdula-Argasińska1, Sylwia BobisWozowicz2, Ewa Leśko2, Beata Bystrowska3, Marcin Majka2, Jerzy Jaskiewicz1 Dawid Szczepankiewicz, Ewa PruszyńskaOszmałek, Maciej Sassek, Marek Skrzypski, Iwona Hertig, Agnieszka Wojtkowiak, Wojciech Szlachcic, Paweł Maćkowiak, Krzysztof W. Nowak 1Department Department of Animal Physiology and Biochemistry, University of Life Sciences, Poznań, Poland e-mail: Dawid Szczepankiewicz <> of Analitycal Biochemistry, 2Department of Transplantology, 3Department of Toxicology, Faculty of Pharmacy, Collegium Medicum, Jagiellonian University, Kraków, Poland e-mail: Malgorzata Tyszka-Czochara <mtyszka@poczta. fm> Catechin is a polyphenolic compound found in a human diet, especially in beverages such as tea. Current interest of biological activities of polyphenolic compounds is due to their antioxidant properties. Anti-hepatotoxic action of Catechin has been reported. Additionally, it was suggested that Catechin may influence carbohydrate and lipid metabolism but the molecular mechanism of influence on metabolic pathways is still unclear. It was demonstrated in previous work that Catechin affects both Pyruvate Dehydrogenase Kinase (PDK) activity and PDK isoenzymes protein amount in rats hepatocyte culture but the range and features of the influence in vivo has not been studied yet. PDK phosphorylates and causes inactivation of Pyruvate Dehydrogenase Complex (PDH). The irreversible reaction of decarboxylation of Pyruvate to AcetylCoA catalysed by PDH links glycolysis with Citric Acid Cycle and, indirectly, with Fatty Acids synthesis in a liver. Precisely balanced carbohydrate and lipid metabolism is critical to the whole body homeostasis. Therefore PDH Complex is one of the key enzymes which influences glucose as well as lipid metabolism. On the other hand PDK is a subject to regulation by dietary factors and xenobiotics. Therefore it is of interest to find out if Catechin influences PDK activity in vivo. Aim of the study: This study investigated the dose-dependent effect of Catechin on PDK in rats in order to find out how a diet enriched with established doses of polyphenolic compound influences PDK activity in a rat liver. Diet and animals: Wistar male rats were fed ad libidum for 14 days with a chow diet and each day were administred Catechin at several doses ranging from 0.5 mg/kg body weight to 200 mg/kg body weight. Control rats were administred dissolvent only. After two weeks of experiment rats were sacrificed and livers were collected. PDK activity assay: PDK activity was measured by Arylamine Acetyltransferase coupled assay. The activity of PDK was expressed as first order rate constant of inactivaction of the PDH complex by the PDK over period of incubation of probe with ATP. Results: It was established that Catechin caused a significant decrease in PDK activity after administration of 2.5 mg of phenolic acid/kg body weight as well as 1 mg/kg body weight when compared to the control group. The dose of polyphenol 2.5 mg/kg body weight was the most effective in the reduction of PDK activity. In contrast, a higher dose of Catechin, 200 mg/kg body weight/day, increased PDK activity. Ghrelin and obestatin are encoded by the preproghrelin gene and originate from posttranslational processing of the preproghrelin peptide. Obestatin like a ghrelin is present in the stomach, but in contrast to ghrelin, obestatin inhibits appetite. Preliminary, it was found that obestatin acts by GPR39 receptor. However, it was presented that obestatin may acts by other type of receptor like glukagon-like peptide 1 receptor (GLP-1R). In our study, we analyzed obestatin action on lipolysis and lipogenesis in isolated rat adipocytes. We also studied changes of expression of putative obestatin receptors: GPR39 receptor and GLP-1 receptor in adipocytes. Moreover, we investigated expression of ghrelin receptor (GHSR-1a). Adipocytes were isolated from epididymal fat pad of Wistar rats according to the method of Rodbell (1964). Isolated adipocytes (106 cells/ml) were incubated with KBRHEPES buffer supplemented with obestatin at concentration 1, 10 and 100 nM, in the absence (basal) or presence of adrenalin. Incubations were carried out at 37oC for 120 min. The lipolysis was determined by measuring the level of released glycerol in incubation medium. Lipogenensis was studied as the incorporation of [U-14C] glucose into lipids. Adipocytes (106 cells/ml) in the same medium as described above without (basal) or with insulin, in the additional presence of [U-14C] glucose. Fraction of lipids was added to scintillation liquid for β-counting. Total RNA was isolated from adipocytes using TriPure reagent (Roche). Reverse transcription was undertaken using ImProm-II Reverse Transcription System (Promega). The cDNA was amplified by multiplex PCR using primers and universal probes (Roche) for GHSR-1a, GPR39 and GLP-1R and internal standard GAPDH. Analysis was made using LightCycler Software Version 4.5 and was based on the relative quantification method with efficiency correction. Our results for the first time present that obestatin influences on lipolysis and lipogenesis. In our study, we found that glycerol concentration was significantly higher in comparison to the control with higher concentration of obestatin in adrenalin-stimulated adipocytes. We also observed that obestatin significantly inhibited lipogenesis in both basal and insulin-stimulated adipocytes. In addition, we presented expression of GHSR-1a. We also found, that GPR39 and GLP-1R expression was present in adipocytes. Moreover, we observed that GPR39 had significantly higher expression after obestatin stimulation in comparison to the control.
  19. 19. The Congress of Biochemistry and Cell Biology — Abstracts 222 2008 P9.27 P9.28 Nuclear hormone receptors LXR and PPAR in the regulation of glucose and lipid homeostasis The role of peroxisome proliferator-activated receptors (PPARs) in the regulation of adipokine gene expression in isolated mature adipocytes Zdzislaw Kochan Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Zdzislaw Kochan <> The liver X receptors (LXRα and β) and peroxisome proliferator-activated receptors (PPARα, β/δ, and γ) are members of the nuclear hormone receptor family, a large family of ligand-dependent transcription factors that coordinate gene expression in response to hormonal and metabolic signals. The binding of ligand to these receptors usually induces the release of corepressors and the recruitment of coactivators, thus triggering the transcription of target genes. Downstream targets of LXR include genes involved in the regulation of cholesterol homeostasis: 7α-hydroxylase (CYP7A1), the lipid scavenger receptors (SR-B1 and FAT/CD36), and members of the ABC family of membrane transporters (ABCA1, ABCG1, ABCG5 and ABCG8). Moreover, LXRs and PPARs activate the transcription of genes that affect uptake of free fatty acids–lipoprotein lipase (LPL) and fatty acid transport protein (FATP); stimulate de novo lipogenesis and promote lipid storage in the form of triacylglycerols fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase (SCD1). Among direct targets of these nuclear receptors is also a major regulator of lipogenesis–sterol regulatory element binding protein 1c (SREBP-1c). In adipose tissue, PPARγ induces the expression of genes which promote recycling of intracellular fatty acids and glycerol–glycerol kinase (GyK) and phosphoenolpyruvate carboxykinase (PEPCK). Both LXRs and PPARs appear to serve as lipid sensors, as they are activated by oxidized cholesterol and naturally occurring fatty acids or fatty acid derivatives, respectively. Moreover, it has recently been demonstrated that LXR may bind glucose, thus integrating glucose sensing and regulation of lipid metabolism. Considering their role in the regulation of glucose and lipid homeostasis, LXRs and PPARs may represent promising therapeutic targets for many common disorders, including obesity, type 2 diabetes, hyperlipidaemia, and atherosclerosis. Joanna Karbowska Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Joanna Karbowska <> Nuclear receptors from the PPAR family are ligand-activated transcription factors involved in the regulation of gene expression. So far, three isoforms of PPARs have been described: PPARα, which is most abundant in the liver, kidney and heart; the ubiquitously expressed PPARβ/δ; and PPARγ, which is highly expressed in fat cells. PPARγ is adipocyte predominant transcription factor and ultimate effector of adipogenesis. During adipocyte differentiation, the genes encoding adiponectin, resistin and leptin are induced; thus to examine the sole effect of PPAR activation on these adipokines we measured gene expression and protein release of adiponectin, resistin and leptin in isolated mature fat cells incubated with agonists for PPARα, PPARβ/δ and PPARγ. Mature adipocytes were prepared from epididymal fat pads of male Wistar rats by collagenase digestion. Freshly isolated cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 25 mM glucose and 10% fetal bovine serum and treated with PPAR agonists for 24 h at 37°C, 5% CO2. Total RNA was then extracted from adipocytes and the mRNA expression of adiponectin, resistin and leptin was quantified by realtime RT-PCR. Leptin gene expression in mature adipocytes was downregulated by PPARγ ligands. In contrast, PPARγ ligands induced gene expression of resistin in these cells. Moreover, this effect was more pronounced when dual agonists of PPARα and PPARγ were used, confirming the role of PPARα in the regulation of resistin transcription. Unexpectedly, agonists of PPARγ had no effect on adiponectin gene expression in isolated rat fat cells. These results indicate that ligand-dependent activation of PPARs modulates the endocrine functions of isolated mature adipocytes in a different manner than in adipocyte cell lines.
  20. 20. 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology Vol. 55 223 P9.29 P9.30 Effect of food restriction on glycogen synthase activity in rat heart Effect of orexin A on lipid metabolism in isolated rat’s white adipocytes Ewa Kossowska, Dorota Kazimierska, Paulina Pawłowska Tatiana Wojciechowicz, Marek Skrzypski, Ewa Pruszyńska-Oszmałek, Dawid Szczepankiewicz, Przemysław Kaczmarek, Maciej Sassek, Emilia Kotowska, Krzysztof W. Nowak Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland e-mail: Ewa Kossowska <> Myocardial glycogen may be considered as a depot form of glucose. It is generally belived to be synthesized by myocardiocytes under conditions of rich glucose supply. Cardioprotective effect of glycogen have been proposed as a protective mechanisms against ischemic heart stress. Glycogen is directly synthesized from UDPG by glycogen synthase-enzyme existing in two forms: “I”(active), and “D”(less active, requiring G-6-P for its further activation). During fasting the amount of myocardial glycogen increas. Our previous studies demonstrated an increase in myocardial glycogen content in rats kept 30 days at the restricted diet (50% of “ad libitum” diet) in comparison with rats at the unrestricted diet (control group). After subsequent two days of refeeding the content of the myocardial glycogen in the investigated group of rats was increased. The aim of the present study was to investigate how the restriction diet alone as well as in combination with the subsequent refeeding can modify glycogen synthase activity in the hearts of rats. The experimental results showed stimulation of both forms of glycogen synthase by the restricted diet. In cardiomyocytes of the rats refeeded after the previous limited fasting, the activity of the enzyme was higher than in not refeeded rats. The observed increase of glycogen amount in cardiomyocytes of rats kept at described experimental conditions seems probably be a result of glycogen synthase induction. Department of Animal Physiology and Biochemistry, University of Life Sciences, Poznań, Poland e-mail: Tatiana Wojciechowicz <tatianawojciechowicz@> Orexin-A and orexin-B (hypocretins), hypothalmic neuropeptides, were identified in neural cells of the lateral hypothalmic area (LHA). Both peptides are coded by the same gene (HCRT). They are widely distributed throughout the body, interacting with two G protein-coupled receptors (GPCR), orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Orexins play a predominant role in feeding and sleep behavior. Orexins and their receptors were also found in the peripheral tissues including white adipose tissue. In our study we focused on effects of orexin A on lipid metabolism (lipolysis and lipogenesis) in isolated rat’s adipocytes. Adipocytes were isolated from epididymal fat pad of Wistar rats according to the method of Rodbell (1964). Isolated adipocytes (106 cells/ml) were incubated with KBR-HEPES buffer supplemented with orexin A at concentration 1, 10 and 100 nM, in the absence (basal) or presence of epinephrine. Incubations were carried out at 37oC for 120 min. After incubation media were explanted and stored at –80oC. The lipolysis was determined by measuring the level of released glycerol in incubation medium using a commercially available colorimetric kit. Lipogenensis was studied as the incorporation of [U-14C] glucose into lipids. Adipocytes (106cells/ml) were placed in the medium supplemented with orexin A at concentration 1, 10 and 100 nM and without (basal) or with insulin, in the additional presence of [U-14C] glucose. Fractions of lipids were extracted using Dole’s solution and added to scintillation liquid for β-counting. Our results indicate that orexin A influences on lipolysis and lipogenesis. In our study, we found that glycerol concentration was significantly lower in group treated with orexin A when compared to the control. This effect was observed both, in basal and epinephrine-stimulated lipolysis. We also observed that orexin A stimulated lipogenesis in both basal and insulin-stimulated adipocytes.
  21. 21. The Congress of Biochemistry and Cell Biology — Abstracts 224 P9.31 Lipids and insoluble carbohydrates in thallus cells of the Antarctic lichens Irena Giełwanowska1,2, Ewa Gojło1, Ryszard Górecki1, Maria Olech2,3 1Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland; 2Department of Antarctic Biology, PAS, Warszawa, Poland; 3Zdzisław Czeppe Department of Polar Research and Documentation, Institute of Botany, Jagiellonian University, Kraków, Poland e-mail: Irena Giełwanowska <i.gielwanowska@uwm.> Antarctic lichens are organisms adapted to life under the conditions of extremely low temperatures, dehydration and solar radiation. They carry out the process of photosynthesis with the minimum of light and at a temperature below 0oC, or even below the temperature of ice nucleation in the cellular fluids of the thallus. Lichens stimulate ice deposition in intercellular spaces, just like vascular plants. They transform freezing loosely bound water into tightly bound water, which does not freeze, in order to avoid freezing of the intracellular water. We studied the ultrastructure of cell walls and protoplasts of cells in lichen thalli and the location of insoluble polysaccharides and other metabolites in six species of Antarctic lichens. These are Bryoria forsteri, Caloplaca regalis, Cetraria aculeata, Ramalina terebrata, Sphaerophorus globosus and Usnea antarctica. The lichens were collected in the vicinity of the Polish H. Arctowski Antarctic Station on King George Island (South Shetland Islands) during Arctic summer (2006/2007) at two times: during a sunny warm (about 9–11oC) and cloudy cool (–1–0oC) day. Several-millimetre thallus fragments were fixed in Carnoy’s fixer (for the PAS reaction to insoluble polysaccharides) and in 3.5% glutaraldehyde in 0.5M phosphate buffer at pH 7.0–7.2 for the anatomical (in light) and ultrastructural (in transmission electron microscope) observations. In all the studied lichen species, both in the thalli collected on a sunny and cloudy day, algal cells had a similar protoplast structure. The occurrence of a wide space between the cell wall and the protoplast, as well as a very tight arrangement of all cell organelles, was their most characteristic feature. PAS positive starch grains, areas of cytoplasm with lipid grains and huge drops of dense osmophilic material were visible in the central part of the photobionts. This material periodically accumulated on the protoplast periphery, in the area close to the wall. The same material filled intercellular spaces in thalli and occurred inside the mycobionts. 2008