1 Session 7: Molecular Basis of Diseases Lectures L7.1 Molecular investigations in medical practice Anna Tylki-Szymańska The Children s Memorial Heath Institute, Warsaw, Poland Anna Tylki Szymanska The ability to perform molecular tests for diagnostic and prognostic purposes in medical practice dates back to the eighties of the last century. Molecular tests have proved crucial in the diagnosis of certain diseases and particularly important in identification of the carrier state in pre-symptomatic patients, in the family and even population wide screening tests. Initially, the molecular research was associated with hope of finding a simple relationship between genotype and phenotype. But along with the data gathering and experience gaining it was proved that the molecular tests only bring some information about the potential of the disease phenotype. They cannot form the basis for accurate prediction of the clinical course of illness. Association of the defect type in the gene with the dysfunction of its product, e.g. enzyme protein, allowed for a better understanding of biochemical processes in many metabolic disorders. Surprisingly, the molecular studies have brought a lot of interesting and surprising information about the founder effects and migration in the area of Europe. L7.2 Molecular basis of diabetes mellitus Krzysztof Zabłocki, Dorota Dymkowska, Katarzyna Wierzbicka, Beata Drabarek, Sylwia Wojciechowska Nencki Institute of Experimental Biology, Department of Biochemistry, Warsaw, Poland Krzysztof Zablocki Diabetes mellitus (DM) is a serious incurable metabolic disease which affects approximately 1% o the global human population and the increase in incidence of diabetes is expected to double in a generation. DM is subdivided into two major classes: type 1 diabetes (T1D) accounts for 10% of all diabetes and type 2 (T2D) accounts for 80-90% of all diabetic patients. While an etiology of T1D is relatively clear and attributed to the autoimmune damage to the beta cells, pathophysiology of T2D is poorly understood. It is thought that changes of life style, particularly western-type diet, leading to obesity and hypertension are an important cause of rapid increase of diabetes prevalence. Together with environmental and behavioral factors a genetic background seems to have an important but not well understood impact on susceptibility of individuals to insulin resistance and diabetes. Although the pathophysiology of T2D is very complex and heterogeneous, in some cases diabetes may result from well defined mutations or be secondary to specific genetic disorders. Among a number of concepts concerning mechanism of insulin resistance and T2D, the mitochondrial hypothesis is broadly discussed and accepted. There is a growing body of evidence indicating that an apparently slight mitochondrial dysfunction might increase (probably together with other factors) the probability of insulin resistance and T2D development. The focus of this lecture is to present and discuss modern opinions on diabetes mellitus with special emphasis on T2D.
2 45 th Annual Meeting of the Polish Biochemical Society 113 L7.3 Hepatitis C virus: epidemiology, molecular biology and antiviral therapy Anna Boguszewska-Chachulska 1 Genomed, Ponczowa 12, Warsaw, Poland 2 Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, Warsaw, Poland Anna Boguszewska Chachulska About 180 million people worldwide are chronically infected with Hepatitis C virus (HCV), an agent responsible for 50 76% of all cases of liver cancer, and for two thirds of all liver transplants in the developed world (WHO, 2007). HCV remains one of the most important viral threats to human health in spite of advanced studies on its life cycle and encouraging results of studies on the development of new drugs. Mechanisms of viral entry, replication and virion formation are dissected in view of obtaining new anti-hcv drugs. The present treatment for HCV infection, however, is still limited to the pegylated interferon-α (IFN-α) in combination with ribavirin, with response rates between 40 and 50% for genotype 1, the most prevalent genotype in Europe and the United States and up to 80% for genotypes 2 and 3. The development of antiviral agents directly targeting the viral life cycle seems to be the most promising therapeutic strategy, as it should block HCV replication and thus spread of infection. This goal could be achieved by direct inhibition of viral enzymes involved in the replication process, such as the NS3/4A protease protein, exerting both serine protease and RNA helicase/ NTPase activities, or the NS5B protein - the RNA-dependent RNA polymerase. A few drugs targeting the NS3/4A protease, or the NS5B polymerase, are already in advanced clinical trials and are expected to be approved for the HCV treatment in a few years time. However, even the introduction of new directly acting antivirals targeted against the viral protease and polymerase will not solve one of the major problems encountered when treating HCV infections - that of viral breakthroughs due to the emergence of HCV mutants resistant to the treatment applied, observed also during the clinical trials with a protease inhibitor, telaprevir, a prospective new drug. Still, as an enzyme indispensable for HCV replication, that unwinds double-stranded forms of RNA and allows viral replication and translation to occur, the NS3 helicase/ntpase represents a tempting target for specific anti-hcv drug design. Another advantage is that NS3 helicase does not possess close homologs among human cellular enzymes. The NS3 helicase inhibitors will be discussed as potential components of a multidrug therapy against HCV. L7.4 Dysregulation of cellular calcium homeostasis a cause or a result of neurotoxicity Ludmila Zylinska Department of Molecular Neurochemistry, Medical University, Lodz, Poland Ludmila Zylinska Calcium ions belong to a group of the most effective signaling molecules that regulate physiological events in every type of the cells. To keep the intracellular free Ca 2+ concentration at safe, nanomolar resting level, the cells developed a dynamic homeostatic system that equilibrates a release from intracellular calcium stores, controls fluxes across plasma membranes and buffering capacity of cellular proteins. Dysregulation of calcium homeostasis and concomitant cellular derangement are the important mediators of neurotoxicity. Altered membrane excitability, overload or deficiency of neurotransmitters may result in substantial changes in expression of calcium-regulated genes, thereby either trigger some adaptive mechanisms or lead to cell death. Among transporters engaged in calcium flux, the key role is played by channels, exchangers and pumps; however only the last group is energy-consuming and use one molecule of ATP for transport of one or two calcium ions. Disturbances in cytosolic calcium levels by deregulation of different homeostatic control mechanisms have been observed in a number of neuropathological conditions including brain ischemia, asphyxia, and several neurodegenerative disorders. Triggering of the specific pathway activated by Ca 2+ depends on spatio-temporal cooperation of a variety of components in the plasma membrane, endoplasmic reticulum and mitochondria, which serve to amplify the Ca 2+ signals. Calcium overload disrupts the mitochondrial function by decreasing ATP synthesis, increasing the production of reactive oxygen/nitrogen species and finally, leads to enhancement of apoptosis or necrosis. Acknowledgements: Supported by the grants , and from the Medical University of Lodz.
3 114 Abstracts L7.5 Cell cycle dysregulation in Alzheimer s disease Urszula Wojda Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland Urszula Wojda Alzheimer s disease (AD) is the most common dementia in the elderly characterized by progressive neuronal loss. Majority of AD cases occur sporadically and have unknown etiology (SAD). The Abeta peptide deposition in the neuritic plaques is one of the major AD hallmarks. However, the role of Abeta as a primary driver in AD progression arouses controversy. Mounting evidence indicates that AD pathogenesis is triggered by genomic instability and reentry into the cell cycle of differentiated, postmitotic neurons. In SAD neurons, activation of cell cycle proteins accompanied by partial or complete DNA replication without signs of mitosis was demonstrated. It is supposed that at the early stages of SAD, activation of cell cycle proteins may represent a protective response to stress factors, allowing neurons survive due to additional alleles of genes produced by DNA replication. The price for survival, however, is the appearance of pathological changes characteristic of neurodegeneration and higher susceptibility of these neurons to death triggered by stress factors later in the SAD process. Our recent data suggest that some of the cell cycle changes can be also observed in human peripheral cells such as lymphocytes. Our results from PCR arrays involving 90 cell cycle genes and from immunobloting experiments in the EBV-immortalized lymphocytes from SAD patients showed most significant changes in the expression of genes and proteins engaged in the control of G1/S cell cycle phases. The most striking difference occurred in p21 protein level, which was significantly elevated in SAD comparing to control lymphocytes. Alterations in the expression of cell cycle genes and p21 increase observed for SAD lymphocytes were accompanied by prolongation of G1 phase with simultaneous shortening of S phase, as demonstrated by flow cytometry and BrdU-labeling. These results emphasize that cell cycle dysregulation is found not only in AD neurons, but also in peripheral lymphocytes. Thus, easily accessible human lymphocytes have a potential diagnostic value in AD. L7.6 Mechanisms of delayed postischaemic neuronal death and regeneration Barbara Zabłocka, Małgorzata Beręsewicz, Joanna E. Kowalczyk Molecular Biology Unit, Mossakowski Medical Research Centre, Pawińskiego 5, Warsaw, Poland Barbara Zablocka Ischaemic stroke is a common and devastating disease and its frequency is on the increase in aging populations. It occurs when the blood supply to the brain is disturbed (due to cerebral thrombosis, embolism or cardiac arrest) starving neurones of oxygen and rapidly causing some cells to die and leaving others damaged. Unfortunately, no treatment is available while the human and the economic costs of this disease are enormous. The major problem in adult ischaemic stroke is that the pathological events quickly become self-sustaining and, within hours, any treatment becomes ineffective. Neuronal injury after transient ischaemia is induced by a combination of hypoxia, hypoglycemia, glutamate-dependent excitotoxicity as well as free radical damage. An extracellular glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca 2+ overload, reorganization of protein complexes at excitatory synapses forming postsynaptic density (PSD), changes in the activity of protein kinases and cytochrome c release from mitochondria. A biphasic increase in cytosolic cytochrome c in the hippocampi following transient global ischaemia in gerbils was demonstrated. It takes part in ischemic signal propagation via endoplasmic reticulum and amplification of apoptotic cascade. But, transient ischaemic insult stimulate complex changes which play a role in neurodegeneration as well as in tissue remodeling and repair. It was shown that brain ischaemia induces changes in endogenous expression of the specific alternatively spliced IGF-1 variant (called MGF or IGF-1Ec). In the post-ichaemic hippocampus the levels of both MGF mrna and the peptide were increased selectively in neurones resistant to the ischaemic insult. It suggests that MGF may form a part of a novel neuroprotective mechanism in the brain. In addition, the endogenous production of MGF by stressed neurones suggests that strategies to specifically up-regulate expression of this isoform or gene therapy targeting MGF to neurones could be viable approaches.
4 45 th Annual Meeting of the Polish Biochemical Society 115 Oral Presentations O7.1 Laminopathies and molecular background of disease phenotypes are we ready for a development of universal stem cell and gene therapy? Magdalena Zaremba-Czogalla, Katarzyna Kozioł, Magda Dubińska-Magiera, Maria Żeleźnik, Janusz Wiśniewski, Ryszard Rzepecki Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland Magdalena Zaremba CZogalla The term laminopathies defines a group of human degenerative disorders which are associated with mutations in genes coding for nuclear lamina (LMNA, LMNB1, LMNB2) and nuclear envelope proteins (EMD, LAP2, LBR) and which include systemic disorders and tissue restricted diseases. To date, more than 340 unique mutations have been reported from more than 1000 patients. Laminopathies arise mainly from missense and frameshift mutations of lamin genes or defective posttranslational processing of pre-lamin A. It is still unclear yet how specific mutations result in a particular tissue specific laminopathy phenotype considering the fact that A and B type lamins are expressed in nearly all differentiated cell types. Many different mutations can give rise to the same clinical conditions. On the other hand the same single mutation can result in different phenotypes probably depending on genetic background of particular patient. It might be explained by the two hypothesis: structural and gene expression hypothesis. There is also possibility that in some cases, effects of the mutated LMNA gene are altered by mutations in one or more other genes. Considering the gene therapy approach for the laminopathies we face several problems. They can be roughly divided into two groups. The first set of problems and difficulties is associated with not fully known molecular mechanisms of diseases and interactions between proteins of interest and other proteins. The second set of problems concerns the enormous difficulties associated with the design of the more or less universal and efficient gene therapy protocol for possible treatment. We consider two main strategies for a development of therapy of laminopathies. One may involve the ex vivo gene therapy of hematopoietic stem cells or mesoangioblasts of the patient using replication deficient retrovirus vector and transdifferentiation into muscle stem cells followed by systemic delivery. The second may be the in vivo gene therapy directed specifically to muscle stem cells or myotubes using fully gutted, specifically pseudotyped retrowirus vectors. Acknowledgements: The research was supported by Wroclaw Research Centre EIT+ under the project Biotechnologies and advanced medical technologies - BioMed (POIG /08) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). O7.2 Plasma proteome changes in chronic kidney disease-related arteriosclerosis. The analysis of low-abundant proteins Magdalena Łuczak 1, Dorota Formanowicz 2, Marek Figlerowicz 1 1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, Poznań, Poland; 2 Department of Clinical Biochemistry, Poznań University of Medical Sciences, Grunwaldzka 6, Poznań, Poland Magdalena Łuczak Chronic kidney disease (CKD) is a progressive loss of renal function. CKD can be divided into five stages (CKD1-5) based on glomerular filtration rate (GFR). The latter describes the flow rate of filtered fluid through the kidney. Arteriosclerosis is one of the most serious and frequent complications occurring in patients suffering from CKD. It was also found to be the major cause of mortality in patients with mild to moderate and end-stage renal disease. Moreover, the risk of cardiovascular events increases with the reduction of renal function. On the other hand, renal dysfunction is one of the most serious complications of cardiac surgery, and often cardiovascular disease (CVD) patients have a kidney function decline. Thus, the close connection between kidney dysfunction and arteriosclerosis is undeniable. However, some of the symptoms are different or even opposite for CKD-related arteriosclerosis (CKDA) and for classical CVD. To increase our knowledge on the mechanisms underlying CKDA development we attempted to identify the proteins specifically accumulating in plasma of patients in different stages of the disease. In addition, the results of our analysis were confronted with analogous data obtained for patients with CVD and normal renal function. The studies involved 60 patients: 15 patients in the initial stage of CKD with the first CKDA symptoms (CKD 1-2), 15 pre-dialyzed patients (CKD 3-4), 15 patients with end-stage renal disease and full CKDA symptoms (CKD 5) and 15 patients with CVD and normal renal function and 20 healthy volunteers as controls. Earlier, it was suggested that in case of human blood plasma the disease-specific biomarkers usually occur among proteins of low abundance. Accordingly, our studies were focused on this fraction of molecules. After the depletion of high-abundant proteins with commercially available MARS-Hu7 columns plasma samples were subjected to a standard proteomic analysis (2DE and MALDI-ToF-MS). The undertaken experiments revealed quantitative differences in the accumulation of several proteins. As a result, biomarkers characteristic for the particular stages of CKD and differentiating between CKDA and CVD were identified.
5 116 Abstracts O7.3 The -675_-674insG polymorphism of the PAI- I gene in determining the predisposition to ischaemic stroke in children I. Żak 1, T. Iwanicki 1, A. Balcerzyk 1, E. Emich-Widera 2, I. Kopyta 2, E. Pilarska 3, K. Pienczk-Ręcławowicz 3, M. Nowak 1 1 Department of Biochemistry and Medical Genetics, Medyków 18, , Katowice, Poland; 2 Department of Neuropediatrics Medical University of Silesia, Katowice, Poland; 3 Department of Neurology, Developmental Neurology Clinic, Medical University of Gdańsk, Gdańsk, Poland Tomasz Iwanicki Background: Stroke in children is relatively rare in comparison to adults. Incidence of this disease entity are approximately six cases in every children per year. The most often stroke impairs are neurological functions and children`s social abilities. The cause of the one- third of cases remains unknown but as a multifactorial disease stroke has a number of possible risk factor. Hypercholesterolemia, oxidative stress or disorders in coagulation system can increase a risk of ischaemic stroke presence. In recent years, epidemiological studies have shown that abnormalities in some hemostatic parameters may help to predict the risk for ischemic events. Plasminogen Activator Inhibitor I (PAI-I) is a glycoprotein attending an important role in fibrinolysis. Its main function is to inhibit a synthesis of plasmin- an enzyme dissolving fibrine. High levels of PAI-I seems to influence processes of smooth muscle cell proliferation, plaque, and matrix remodeling in the direction of promoting atherothrombosis. The -675_-674insG is a insertion/ deletion polymorphism located in the promoter of PAI- I gene. 4G allele produces up to 6 times mrna in vitro and is associated with high plasma PAI-I activity in vivo. The aim of this study was to evaluate possible association between PAI-I gene polymorphism and the childhood stroke. Material and methods: The study population included 70 affected children and 133 healthy children as a control group. Stroke children were also analyzed with their biological patients (n=140). The age of patients in acute phase of ischaemic stroke was 8.65± The -675_-674insG polymorphism was genotyped with PCR- RFLP method. The Transmission/ Disequilibrium Test (TDT) was used for the analysis of specific allele transmission from heterozygous patients to their affected children. The statistical analysis was performed with Statistica 7.1 and EpiInfo software. Results: There were 57 informative trios for the -675_- 674insG polymorphism of PAI- I gene. Test TDT did not show a preferential transmission of any alleles from heterozygous parents to their affected children (χ 2 = 0.00, p= 1.00). There was no statistical significance in the genotypes and alleles frequencies between stroke children and control group (4G/ 5G: χ 2 = 0.00, p= 0.99). Conclusion: The obtained results did not reveal any association between -675_-674insG polymorphism of PAI-I gene and ischaemic stroke in children. O7.4 Expression of vascular endothelial growth factor and its receptors in human neointima Andrzej Małkowski 1, Radosław Kowalewski 2, Marek Gacko 2, Krzysztof Sobolewski 1 Medical University of Białystok, 1 Department of Medical Biochemistry, Poland; 2 Department of Vascular Surgery and Transplantology, Białystok, Poland Andrzej Małkowski Endothelial dysfunction is a key factor underlying many vascular pathologies. Invasive surgical procedures, such as angioplasty or peripheral vascular by-pass grafting, result in mechanical damage of the arterial wall, which initiates numerous cellular and molecular mechanisms. Disturbed vessel wall homeostasis leads to proliferation and migration of smooth muscle cells, which often exceeds the repair process and results in extensive neointima formation and arterial stenosis. Vascular endothelial growth factor (VEGF) is one of the main factors that stimulate angio- and vasculogenesis. It stimulates cell division, their migration, and increases the permeability of capillaries. Furthermore VEGF rouses cells to increased extracellular matrix metabolism. In experimental models it was observed that exogenous VEGF, intensifying the proliferation of endothelial cells, reduces neointima formation. However increased VEGF expression in the neointima was found in various animal models. The aim of the study was to evaluate VEGF-A and its two receptors (VEGFR1 and VEGFR2) in the neointima, when compared to normal aortas. The studied material consisted of 12 neointima samples, that were obtained from patients with chronic limb ischaemia in the age range years, after aorto-femoral by-pass reconstructions. The tissue samples were collected from distal anastomoses during secondary operations performed due to graft occlusion 6 to 24 months after primary procedure. Normal aortas without macroscopically visible lesions, that were harvested from age-matched organ donors were the control material. Expression and content of VEGF-A, VEG- FR1 and VEGFR2 were evaluated with RT-PCR, Western blot and ELISA methods respectively. We have observed in human model, that artery injury followed by neointima formation shows increase in expression of VEGF-A and its receptors type 1 and 2. It has confirmed data obtained from in vitro and animal experiments that VEGF is one of the key agents involved in neointima formation. The overexpressed VEGF-A in the neointima influences many gene expression via VEGFRs, including these involved in cell proliferation, extracellular matrix metabolism and artery wall remodeling.
6 45 th Annual Meeting of the Polish Biochemical Society 117 Posters P7.1 Multi-sumoylation of Usp protein - heterodimerization partner of EcR from Drosophila melanogaster Katarzyna Bielska 1, Justyna Seliga 1, Elżbieta Wieczorek 1, Rainer Niedenthal 2, Andrzej Ożyhar 1 1 Department of Biochemistry, Wroclaw University of Technology, Wroclaw, Poland; 2 Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Hannover, Germany Katarzyna Bielska Reversible posttranslational modification is a rapid and efficient system to control the activity of proteins. Modifiers range from small chemical moieties to proteins. Small ubiquitin-like modifier (SUMO) belongs to a family of a proteins that have been found to be covalently attached to lysine residues of specific target proteins. Sumoylation modulates important functional properties, depending on the protein substrate. The ultraspiracle protein (Usp), which together with the ecdysone receptor (EcR) forms a functional EcR/Usp complex in Drosophila melanogaster, provides an ideal model system for construction of gene switches in humans. However, there is no information about sumoylation of Usp. To investigate this modification of Usp we used Ubc9 fusion-directed sumoylation (UFDS) system (Jakobs et al., 2007) which is based on fusion of conjugating enzyme Ubc9 with substrate protein. We prepared constructs coding fusion proteins where Ubc9 was attached to the N- or C-terminus of Usp (Ubc9-Usp, Usp- Ubc9), expressed them in human embryonic kidney cells (HEK293) and analyzed the protein extracts by western blotting with an Ubc9 antibody. The results revealed that both Ubc9-Usp and Usp-Ubc9 are sumoylated by SUMO1 and SUMO3, however Usp-Ubc9 less efficiently. We used bioinformatic tools (SUMOplot, SUMOsp and PCI- SUMO) to predict and score potential sumoylation sites in Usp. Using this data, we prepared some point mutants. To determine region of the modification, we also prepared fragments of Usp including A/B region, DNA binding domain (DBD) and ligand binding domain (LBD) fused to Ubc9. For A/B region and LBD we showed that there are potential residues that can be sumoylated. Identification of Usp sumoylation sites is currently underway. References: Jakobs A, Himstedt F, Funk M, Korn B, Gaestel M, Niedenthal R (2007) Nucleic Acids Res 35: e109. Acknowledgements: This work was supported by the Wroclaw University of Technology. P7.2 Effect of high-carbohydrates diet on the level of mrna expression for selected elongases and desaturases in liver tissue of rats Jagoda Drąg, Anna Goździalska, Anna Gawędzka, Jerzy Jaśkiewicz Department of Analytical Biochemistry, Faculty of Pharmacy, Collegium Medicum, Jagiellonian University, Kraków, Poland Jagoda Drąg Hepatic lipids contain fatty acid derived from diet or from endogenous synthesis. Both sources provide saturated and n-9 unsaturated fatty acid. In addition, diet is the only source of essential polyunsaturated n-3 and n-6 fatty acids. In human body the structure of fatty acids is modified by fatty acid elongases and desaturases. The activity of these enzymes is regulated by different factors including diet, hormones and transcription factors. Fatty acid profile of the body is determined by the activity of elongases and desaturases. Qualitative and quantitative changes of fatty acids affect structure and function of cell s membrane. Development of knowledge on enzymes modifying the composition of fatty acid molecules may be helpful in elucidating the pathomechanism of different diseases. The aim of our study was determine the influence of the high-carbohydrate diet on hepatic gene expression for Elovl 2, Elovl 5, Δ5 desaturase, Δ6 desaturase, Scd1 and Scd2. We demonstrated that mrna expression level of Elovl 5, Δ5 desaturase, Δ6 desaturase and Scd2 in liver of rats fed high-carbohydrate diet was similar to the levels of mrna for the same enzymes in liver of animals fed standard diet. Statistically significant difference was shown between mrna expression of Elovl2 and Scd1. High carbohydrate diet increases the level of glucose and triggers insulin synthesis and release which in turn cause the activation of transcription factor called SREBP. It is also known that ChREBP is required for the carbohydrateinduced transcriptional activation of several additional enzymes involved in fatty acid synthesis, most notably SCD-1. High carbohydrate diet augments the synthesis of monounsaturated fatty acids which are major components of triglycerides that are transported by blood and stored in adipose tissue. Obtained results suggest that the analysis of SCD1 mrna expression together with other biochemical markers can be added to diagnostic panel of diabetes and obesity. References: 1. Wang Y et al. (2005) J Lipid Res 46: Popeijus et al. (2008) Int J Obes 32: Shanmugam M et al. (2009) Nutr Metab 6: Iizuka K et al. (2008) Endocr J 55: Moon YA et al. (2009) J Lipid Res 50: Weickert MO et al. (2006) Diabetologia 49: Dentin R et al. (2006) J Nutr 136: Dentin R et al. (2005) Biochimie 87:
7 118 Abstracts P7.3 The effect of physical effort with incremental load on the lipid profile in the blood serum of non-athletes Wioleta Dudzinska 1, Anna Lubkowska 1,2 1 Faculty of Natural Sciences of Szczecin University, Department of Physiology, Szczecin, Poland; 2 Pomeranian Medical University, Department of Biochemistry and Medical Chemistry, Szczecin, Poland Wioleta Dudzi?ska The aim of this study was to assess the influence of physical activity with increasing load on the serum lipid profile in non-athletes. The study was carried out on 22 healthy men, aged 21.9±2.33 years. The subjects underwent a single test on an ergometer (Kettler X-7, Germany), with progressively increasing load. The test began with resistance of 70W, and the participants were asked to maintain 70 revolutions per minute. The effort was continued with increasing load (by 20W every 3 minutes) until refusal, that is until the participant was not possible to continue at the required rate of revolutions. A Polar S610 heart rate monitor (Polar, Finland) was used to record a resting heart rate and its changes during exercise. In order to assess the uptake of oxygen during exercise, an Oxycon gas analyzer was used (Jaeger, Germany). Blood samples were taken from the cubital vein three times: before the test, during 3 minutes after the completion of the exercise, and in the 30th minute after the completion of the exercise. The determinations of the studied lipid parameters (triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol) were performed in blood serum using enzymatic methods. In the studied group, an exercise with incremental load, continued until refusal, lead to a significant increase in triglycerides in the 30th minute of rest, a significant increase in lipoprotein HDL and HDL/LDL ratio, and a decrease in cholesterol/hdl ratio. A single physical exercise favorably modified the concentration of anti-atheroscerotic lipoproteins. P7.4 The Membrane-Type Matrix Metalloproteinases (MT1-MMP and MT2- MMP) of the umbilical cord in preeclampsia Zofia Galewska 1, Lech Romanowicz 1, Stefan Jaworski 2, Edward Bańkowski 1 1 Departments of Medical Biochemistry and 2 Gynecology, Medical University of Białystok, Białystok, Poland Zofia Galewska The vascular system of mother and placenta plays an important role in the intrauterine development of the fetus. Preeclampsia is a conditions characterized by systemic vascular endothelial dysfunction. Our previous research demonstrated that preeclampsia - associated accumulation of collagen and proteoglycans in the umbilical cord tissues is a result of increased biosynthesis and decreased degradation of these components. Metalloproteinases are enzymes engaged in degradation of collagen and protein cores of proteoglycans, including those which bind peptide growth factors. The Membrane-Type Matrix Metalloproteinases (MT-MMPs), with the exception of MT4-MMP, are classical extracellular matrix (ECM)-degrading endopeptidases, and such they exhibit a broad spectrum of substrate specificity degrading one or more ECM components. We used Western Immunoblot method and immunoenzymatic assay (ELISA) for detection of metalloproteinases. We found that the human umbilical cord arteries, vein and Wharton s jelly of control and preeclamptic newborns contained MT1-MMP (MMP-14) and MT2-MMP (MMP-15). Free latent form of both investigated MT-MMPs was not detected in all investigated tissue extracts. The indicated molecular weight of them (about 54 kda) corresponds to free active form of respective MMP. The umbilical cord tissues of control and preeclamptic subjects contain both enzymes in a form of complexes with other extracellular matrix components. Furthermore, we observed a distinct increase in the amount of MT1-MMP in preeclamptic umbilical cord vessel walls and significant decrease in preeclamptic Wharton s jelly in comparison to control materials. It may be one of the mechanism of extracellular matrix remodelling in the umbilical cord of preeclamptic newborns.
8 45 th Annual Meeting of the Polish Biochemical Society 119 P7.5 Characterization of the activation mechanism of human HtrA2 protease Dorota Żurawa-Janicka, Mirosław Jarząb, Agnieszka Lubomska, Przemysław Glaza, Barbara Lipińska Department of Biochemistry, University of Gdansk, Gdansk, Poland Mirosław Jarząb Human HtrA2 protein belongs to the HtrA family of stress-induced serine proteases, characterised by the presence of a conserved trypsin-like protease domain and at least one PDZ domain at the C-terminal end. Under physiological conditions HtrA2 promotes cell survival by the maintenance of mitochondrial homeostasis. In stressful conditions HtrA2 functions as a proapoptotic factor, triggering cell death. Disturbancesin its function contribute to the development of Parkinson s disorder and cancer. Analysis of crystal structure of HtrA2 trimers was a basis of a model of the HtrA2 regulation wherein C-terminal fragments of PDZ domains interact with protease domains through van der Waals contacts and block the access to the protease active sites. The aim of the study was to characterize the mechanism of thermal activation of HtrA2 and find out which residues are involved in stabilization of the active site architecture. We generated several point mutation-containing HtrA2 mutants using site-directed mutagenesis. Hydrophobic amino acid residues theoretically involved in the stabilization of active site architecture have been substituted by hydrophilic amino acids:v226k, F331Y, and I329N within the protease domain, and V325D in the PDZ domain. The wthtra2 and mutated variants of HtrA2 were overproduced in bacterial cells and purified. Their proteolytic activity was characterized in the range from 20 to 55 o C. While proteolytic activity of the wthtra2 increased gradually with temperature, mutated HtrA2s: F331Y, I329N, V325D were unable to degrade the substrate. The proteolytic activity of HtrA2V226K was higher than the activity of wthtra at all temperatures tested. It suggests that these residues play an important role in regulation of the HtrA2 proteolytic activity. Their hydrophobic features are probably indispensable for proper architecture of the HtrA2 active site. Understanding of the molecular basis of HtrA2 regulation will be helpful for pathogenesis studies and generation of new therapeutic strategies for diseases connected to HtrA2 dysfunction. P7.6 Lipid droplet-like structure formation in stressed endothelial and adipose stromal vascular fraction (SVF) cells in presence of TNFα and fatty acids Beata Kiec-Wilk 1, Anna Knapp 1, Magdalena Korczynska 1, Urszula Czech 1, Joanna Goralska 1, Agnieszka Sliwa 1, Anna Gruca 1, Horst Robenek 2, Gerd Schmitz 3, Aldona Dembinska-Kiec 1 1 Departament of Clinical Biochemistry Collegium Medicum, Jagiellonian University; Cracow, Poland; 2 Leibniz Institute for Arteriosclerosis Research, University of Muenster; Muenster, Germany; 3 Department of Clinical Biochemistry, University Hospital Regensburg, Germany Beata Kiec In 20 40% adipose tissue consists of the mesenchymal stromal vascular fraction (SVF) containing preadipocytes, and in 10 20% of preendothelial cells. The di-/dedifferentiation of SVF is poorly understood. Free fatty acids(ffa) and insulin resistance are responsible for lipotoxicity and lipid droplets (LD) formation. Nutritional overload induces endoplasmic reticulum (ER)-stress, changes in mitochondrial membrane potential, LD formation accompanied by autophagy important for survival and protection from apoptosis. Study is aimed to find the sequence of the gene expression/protein biosynthesis and the lipid content changes characteristic for the LD formation and disappearance in preadipocytes and HUVEC does not accumulating lipids. Methods: HUVEC and SVF were cultured in EBM with PA, AA, EPA (30 μm), VEGF, or L-Arginine (SIGMA) for 24 hours (SVF), when with PA or TNFα (5 ng/ml) for 1, 4 and 24 h (HUVEC). Changes in gene expression was analyzed by microarray. Metabolic activity of mitochondria were analyzed by ATP production and oxygen requirement (Oxygraf 2-K Oroboros). The changes DY, PAT protein and selected ER chaperones were followed by the fluorescence microscopy imaging (Bioimager BD) and by freezefracture technique combined with replica immunolabeling for high-resolution imaging. Results: VEGF, as well EPA and AA inhibited, when PA promoted differentiation SVF to adipocytes. SVF metabolism (oxygen consumption and ATP) was higher than HUVEC. FFA did not significantly change oxygen consumption, but ATP generation was decreased by PA and OA. TNF induced expression of adipophyllin HUVEC. Microarray analysis revealed an induction of intracellular substrate transporters, metabolism and angiogenesis genes, as well as induction of ER-shock chaperones. The electron-microscopic technique of freeze-fracture replica immunolabeling illustrates the localization of the proteins p61er, GRP78, and GRP94 on the E-face of the ER membrane, the E-face of the inner nuclear membrane and in the lumen of the ER. Conclussion. The ER shock associates formation of the LD-like structures in lipid not accumulating HUVEC. Acknowledgements: Supported by EU F7 HEALTH :LIPIDOMICNET.
9 120 Abstracts P7.7 Modulation of PPAR-gamma in mice influences EPC functions. Jerzy Kotlinowski 1, Anna Grochot-Przęczek 1, Magdalena Kozakowska 1, Ewa Zuba-Surma 1, Jakub Zimoch 1, Rafał Derlacz 2,3, Laszlo Nagy 4, Józef Dulak 1, Alicja Józkowicz 1 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Cracow, Poland; 2 Adamed Ltd, R&D Department, Pieńków, Poland; 3 University of Warsaw, Faculty of Biology, Department of Metabolic Regulation, Warsaw, Poland; 4 University of Debrecen, Medical and Health Science Centre, Department of Biochemistry and Molecular Biology, Debrecen, Hungary Jerzy Kotlinowski PPARγ nuclear receptor is a target for thiazolidinediones (TZD), the commonly used insulin sensitizers. TZD can also improve some functions of endothelial progenitor cells (EPC) in diabetic patients, but the role of PPARγ in this pathway has not been proven. Our aim was to check how modulation of PPARγ activity affects EPC functions in healthy and diabetic mice. Experiments were performed on the 12-week old mice with different expression of PPARγ (wild type PPARγ WT, heterozygous PPARγ HT, and heterozygous with knock-out in monocytes PPARγ HT/Mo- ) or on 10-week old wild type and diabetic db/db mice. EPC were characterized as the KDR, Sca-1 and lectin positive cells. We found that reduced expression of PPARγ had no effect on percentage of EPC in the bone marrow: in all animals tested the KDR, Sca-1 and lectin positive progenitors constituted approximately 0.014% of cells. Furthermore, PPARγ expression did not affect the EPC fraction in the blood of healthy mice. However, we observed significantly reduced mobilization of EPC 24 h after hind limb ischemia in PPARγ HT and PPARγ HT/Mo- animals: the number of EPC in the blood of wild type individuals raised by 6.8-fold, while in mice with reduced PPARγ levels this increase ranged from 2.2 to 2.8-fold. Analysis of the gene expression pattern in EPC suggested that this effect may be associated with down-regulation of receptors for VEGF, the major factor responsible for EPC mobilization. What is more, EPC from PPARγ HT/Mo- mice produced in vitro significantly less SDF-1a, whereas expression of VEGF did not depended on PPARγ level. Importantly, percentage of EPC was significantly reduced in the bone marrow of diabetic mice (to 0.009%). Reduction related to blood glucose levels suggested that diabetes influences the number of EPC according to its severity. These defect was fully reversed by an oral application of TZD (rosiglitazone or pioglitazone, 10 mg/kg, 28 days), however loss of the correlation between blood glucose and EPC at the end of the study indicated that TZD did not act directly by lowering glucose levels. In in vitro assays, EPC isolated from db/db mice displayed impaired migratory and angiogenic potential, also restored by rosiglitazone (10 μmol/l, 24 h) in a PPARγ-dependent manner. Thus, PPARγ is an important regulator of EPC mobilization and its activation may be used to improve the EPC functions. P7.8 Nuclear lamina and nuclear envelope proteins multiple roles in mitotic entry and exit, new implications for a development of laminopathy Katarzyna Kozioł, Magdalena Zaremba-Czogalla, Magda Dubińska-Magiera, Ryszard Rzepecki Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Katarzyna Kozioł The cell nucleus is separated from the rest of the cell by nuclear envelope (NE). It includes two lipids bilayers (outer and inner nuclear membrane ONM, INM respectively), nuclear pores and the nuclear lamina (NL). ONM is functionally similar to ER membranes, INM has specific composition and functions. NL is composed of lamins and is located between INM and the peripheral chromatin. Lamins together with nuclear envelope proteins plays important role in maintenance of the nuclear shape, spacing of nuclear pore complexes, organization of chromatin, DNA replication, regulation of transcription factors, epigenetics, DNA repair, transcription, cell cycle regulation, cell development and differentiation, nuclear migration and apoptosis. Recent studies have provided evidences in support of nuclear proteins function in virus infection, tumorogenesis, mitosis and for linking the nucleoplasm to all major cytoskeletal networks. Mutations in nuclear proteins genes may cause a wide range of heritable human diseases collectively called laminopathies/envelopathies. During mitosis entire structure of cell nucleus is remodeled, NE and NL are depolymerized and mitotic spindle forms allowing separation of chromosomes into daughter cells. Then the NE and NL reassembles around separated chromatids. We analyzed the localization, distribution and dynamics of selected NE and NL proteins. In Xenopus we analyzed XLAP2 proteins, lamins, BAF, membranes and tubulin. In HeLa cells we analyzed distribution of emerin, LAP2, lamins, dynein, TPX2, pericentrin, tubulin and membrane fractions. We find that emerin, LAP2, XLAP2 and lamins together with membranes of NE transiently associate with mitotic spindle microtubules and mitotic spindle matrix both in Xenopus and mammalian cells. Knockdown of such proteins is either lethal, as in the case of XLAP protein or affects mitosis resulting in abnormal phenotypes (emerin, LAP2). Similarly overexpression of protein domains necessary for interaction with mitotic apparatus results in abnormal mitosis. We suggest that abnormal mitosis may be the additional feature of cells with laminopathy and may contribute to manifestation of disease phenotype in patients.
10 45 th Annual Meeting of the Polish Biochemical Society 121 P7.9 Lysenin, a toxin which specifically recognizes sphingomyelin in rafts and affects signaling of immunoreceptor FcγIIA Magdalena Kulma, Katarzyna Kwiatkowska, Andrzej Sobota Nencki Institute of Experimental Biology, Laboratory of Plasma Membrane Receptors, Warsaw, Poland Magdalena Kulma Sphingolipids and cholesterol of the plasma membrane are organized into microdomains, named rafts. Within rafts distinct tyrosine kinases and scaffolding proteins are anchored allowing formation of signaling platforms of immunoreceptors, including FcγRIIA. To analyze the dynamics and functions of rafts in FcγRIIA signaling we developed a probe, GST-lysenin, a toxin which selectively recognized membrane sphingomyelin, one of the major constituent of lipid rafts. Interaction of GST-lysenin with membranes of various composition was examined by measurements of surface plasmon resonance. This analysis revealed that GST-lysenin bound 2-fold stronger to SM/DOPC than to SM/DPPC liposomes. Incorporation of cholesterol to the liposomes facilitated formation of sphingomyelin-rich domains and improved the binding of GST-lysenin. Binding of GST-lysenin to sphingomyelin membranes was correlated with protein oligomerization and pore assembly. To analyze the effect of lipid membrane composition on the pore formation by lysenin, we measured efflux of carboxyfluorescein from liposomes of various compositions. We found that GST-lysenin released carboxyfluorescein 2.5- fold more effectively from SM/DOPC/cholesterol liposomes than from SM/DPPC. Liposomes without sphingomyelin were insensitive to lysenin. The data suggest that GST-lysenin binds preferentially to membranes of lipid composition corresponding to that of rafts. To analyze interaction of lysenin with the plasma membrane, U937 cells pretreated with lysenin were solubilized in Triton X-100 and fractionated by gradient centrifugation. GST-lysenin formed oligomers (hexamers) upon binding to cell which were detected exclusively in isolated raft fraction. The size exclusion chromatography of lysates of cells pretreated with GST-lysenin followed by FcγRIIA activation showed that the toxin induced formation of large complexes of activated FcγIIA. Binding of the toxin to intact U937 cells affected FcγRIIA signaling as indicated by enhancement of phosphorylation of FcγRIIA and proteins of signaling cascade. The data indicate that incorporation of GSTlysenin to the sphingomyelin-rich microdomains induces the formation of large signaling platforms in the plane of the plasma membrane and facilitates signaling of FcγRIIA receptor. The data indicate that GST-lysenin can be an useful tool to study organization of sphingomyelin in the plasma membrane and the role of the lipid in the receptor signaling. P7.10 Expression of gene encoding TGF-β and genes encoding its receptors in amelanotic melanoma cell cultures C-32 exposed to Photodynamic Therapy (PDT) with Photolon Dariusz Kuśmierz, Małgorzata Latocha, Aleksandra Zielińska, Monika Rybarz, Elektra Sliupkas-Dyrda Department of Cell Biology, Medical University of Silesia, Jedności 8, Sosnowiec, Poland Dariusz Kuśmierz The transforming growth factor beta (TGF-β) family of proteins are a signaling molecules with unique and potent immunoregulatory properties. TGF-β is produced mainly by macrophages, neutrophiles, thrombocytes and activated T and B lymphocytes. Its immunosupressive properties are i.a consequence of its ability to inhibit: T and B lymphocytes and NK cells proliferation, MHC class II expression, Tc lymphocytes development and IgM and IgG antybodies production. Frequently, effect of TGF-β on immune response is more efficient than in case of immunosupressive drugs. TGF-β may exert tumor promoter activities at later stages of carcinogenesis. Most human tumors secrete large amounts of TGF-β, which directly influences the microenvironment and promotes tumor growth, invasiveness and capacity to form metastases. Elevated levels of the TGF-β in plasma of cancer patients contribute to systemic immunosupression promoting tumorgenesis. Increased expression and secretion of different TGF-β isoforms in melanoma cell lines was observed in several studies. TGF-β1 is secreted by normal melanocytes and melanomas at various stage, while TGF-β2 and TGF-β3 are secreted only heterogenously in nevi and melanoma. The aim of the study was to investigate the expression of gene encoding transforming growth factor β (TGF-β) and genes encoding its receptors in amelanotic melanoma cell cultures C-32 exposed to PDT with a Photolon as a photosensitiser. The photosensitizer was applied to the melanoma cell cultures at a concentration of 0.01 mg/ml. After 1h of incubation, cell cultures were irradiated with laser light (l=662nm) at three doses: 5, 10, 20 J/cm 2. Next, after incubation time of 3h, total RNA was extracted with TRIzol reagent (Invitrogen). Spectrophotometry was performed to analyse the extracted total RNA for concentration and purity. To analyze the relative changes in examined genes expression the real-time PCR was performed. PCR products were detected in 8% polyacrylamide gel electrophoresis. The results obtained in our study show a very low levels of expression of TGF-β and its receptors TGF-β RI, TGF-β RII i TGF-β RIII in amelanotic melanoma cells C-32. In melanoma cells previously subjected to PDT-Photolon a statistically significant decrease of mrna copy number of TGF-β and all its receptors were observed.
11 122 P7.11 Abstracts P7.12 Endocytosis as a component of LPS signaling in macrophages Maciej Czerkies 1, Galyna Kleveta 1,2, Andrzej Sobota 1, Katarzyna Kwiatkowska 1 1 Nencki Institute of Experimental Biology, Lab. Plasma Membrane Receptors, Warsaw, Poland, 2 Ivan Franco Lviv National University, Department of Biochemistry, Ukraine Katarzyna Kwiatkowska Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. Upon bacterial infection LPS activates macrophages triggering production of cytokines and other mediators of inflammatory responses. If excessive host reactions towards LPS take place, deadly systemic inflammation named sepsis develops. Stimulation of macrophages with LPS engages CD14 and TLR4 receptors and triggers two signaling pathways with one originating from the plasma membrane and the second one linked presumably to endocytosis. In order to reveal whether endocytosis is a component of LPS signaling, CD14 and TLR4 distribution in LPS-stimulated J774 cells was examined. At ng/ml LPS did not induce changes of the CD14 cell surface level nor affected the total pool of CD14, as indicated by flow and laser scan cytometry. On the contrary, the amount of the exofacial radio-labeled TLR4 was transiently diminishing by about 50% in LPS-treated cells. Under the influence of dynasore, an inhibitor of GTPase dynamine and hence clathrindependent endocytosis, the reduction of the cell-surface TLR4 level was augmented and degradation of TLR4 took place. Simultaneously, LPS-induced signaling exemplified by NFκB and Erk1/2 phosphorylation and cytokine production was inhibited. Confocal microscopy revealed that during LPS stimulation macrophages polarized. Co-localization of CD14 and TLR4 was limited to the leading edge of stimulated cells and the Pearson s correlation of CD14 and TLR4 co-labeling reached only 0.41 in relation to 1 as the maximal value. The data indicate that in LPS induces transient co-localization of CD14 and TLR4 followed by TLR4 internalization. However, TLR4 was dispensable for an uptake of 1 μg/ml LPS-Alexa Fluor 488 while function blocking anti-cd14 antibody abolished LPS internalization. Similarly, dextran sulfate, a ligand of scavenger receptors, inhibited internalization of 1 μg/ml LPS. Contrary to TLR4 endocytosis, the uptake of LPS was up-regulated by dynasore but required participation of the actin cytoskeleton and was negatively regulated by ceramide. We hypothesize that TLR4 undergoes clathrin-dependent endocytosis together with a small signaling pool of LPS while CD14 remains on the cell surface and can transfer higher amounts LPS to scavenger receptors which mediate actin-dependent uptake of LPS leading to its detoxification. Role of heme oxygenase-1 in ochratoxin A-induced kidney damage Anna Stachurska 1, Aleksandra Sierpniowska 1, Christine Boesch-Saadatmandi 2, Gerald Rimbach 2, Alicja Jozkowicz 1, Jozef Dulak 1, Agnieszka Loboda 1 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Kraków, Poland; 2 Christian Albrechts University Kiel, Institute of Human Nutrition and Food Science, Kiel, Germany Agnieszka Loboda The main target organ of ochratoxin A (OTA) is kidney. It is also hepatotoxic in rats and teratogenic in a number of species including mice and potentially human. Heme oxygenase-1 (HO-1), an enzyme involved in heme degradation, possessing anti-apoptotic, immunomodulatory and anti-oxidant properties, has been recently suggested to play an important role in kidney pathology. To assess the role of HO-1 in OTA-induced toxicity, HO-1 +/+, HO-1 +/ and HO-1 / mice were injected with 2.5 mg/kg/bw OTA i.p., every other day for 20 days. 38% of mortality was observed only in OTA-injected HO-1 / animals, however after OTA delivery all mice lost their weights. Moreover, HO1 / mice had larger kidneys compared to other mice and OTA potently reduced the size of kidneys in HO1 / mice. The expression of several genes important for kidney functioning was changed after OTA treatment. Vascular endothelial growth factor (VEGF) level was downregulated in OTA-injected animals. Expression of p21, a protein which mediates cell cycle G1 phase arrest in response to stress, as well as genes encoding proteins involved in apoptosis (e.g. Bax, PUMA) was potently elevated by OTA. Level of mrna for MDM2, negative regulator of p53 suppressor protein, was increased in HO-1 +/+ and HO-1 +/ but not in HO-1 / animals. Interestingly, OTA induced expression of NF-E2-related factor-2 (Nrf2), the major factor regulating the antioxidant response, and its target gene HO-1. However, expression of catalase and superoxide dismutases (SOD-1, 2) was diminished by OTA delivery. Additionally, expression of genes encoding enzymes involved in glutathione metabolism, like glutathione reductase (GSR) and glutathione S-transferase (GSTA) was attenuated and elevated after OTA, respectively. OTA may also affect the expression of profibrotic genes. We observed induction of expression of tissue inhibitor of metalloproteinases-1, 2 (TIMP-1, 2) as well as transforming growth factors β (TGFβ1 and TGFβ2), with the fold of induction the highest in HO-1 / mice. OTA also diminishes expression of markers of epithelial-mesenchymal transition (EMT) cadherin 1 (CDH1) in HO-1 +/+ and CDH2 in HO-1 +/ animals. Taken together, toxic effect evoked by OTA in kidneys is reflected in alterations in various pathways. Lack of HO-1 enhances susceptibility to OTA but the mechanism needs further investigation. Acknewledgements: Supported by the grant No N N from the Polish Ministry of Science and Higher Education.
12 45 th Annual Meeting of the Polish Biochemical Society 123 P7.13 Disruption of sphingomyelin metabolism in plasma membrane inhibits receptor FcγIIA signaling Anna Łukasik, Katarzyna Kwiatkowska, Andrzej Sobota Nencki Institute of Experimental Biology, Departament of Cell Biology, Warsaw, Poland Anna Łukasik Sphingomyelin (SM) is a structural component of the plasma membrane, asymmetrically distributed in the bilayer but being concentrated in the extracellular leaflet of the membrane. There, together with cholesterol forms tightly packed microdomains termed lipid rafts. The rafts are considered as a signaling platforms as long as they are collecting elements necessary for signal transduction, like activated receptors and protein tyrosine kinases. Previous studies in our laboratory show that stimulated receptor FcγIIA associates with sphingolipid/cholesterol domains. At the onset of the receptor activation, SM hydrolysis in the extracellular part of the plasma membrane takes place leading to formation of ceramide. The hydrolysis of SM is mediated by acid sphingomyelinase (ASMase), enzyme being extruded onto the cell surface during FcγRIIA stimulation and increasing ceramide level. On the other hand, in the plasma membrane sphingomyelin synthase 2 (SMSase 2) is also located which use ceramide as a substrate to rebuild SM pool. In this study we modulated activities of these two enzymes in U937 cells diminishing their gene expression by RNA interference technique. Down-regulation of ASMase caused significant decrease in level of protein phosphorylation triggered by FcγRIIA activation. Similar disturbances in phosphorylation cascade appeared in cells deficient in SMSase 2 activity. We measured the quantity of SM in the plasma membrane to examine changes ensuing from perturbationes at the metabolism of the lipid. Surprisingly, silencing of SMSase 2 as well as ASMase gene expression correlated with decrement in SM level. At this conditions, the receptor association with rafts was examined. We assessed accumulation of the activated receptor in detergent resistant membrane fractions containing raft-derived membranes. We found that down-regulation of ASMase and SMSase 2 facilitated association of activated FcγRIIA with rafts. Furthermore, both ASMase and SMSase 2 depletion favored formation of bigger complexes containing stimulated receptor, as shown by size exclusion chromatography of detergent cells lysates. All together, the results indicate that disturbances in sphingomyelin metabolism influence negatively phosphorylation cascade triggered by FcγRIIA activation, but do not perturb association of the receptor with rafts. P7.14 Role of G/2G MMP-1 and 372 C/T TIMP-1 gene polymorphisms in pathogenesis of open angle glaucoma Łukasz Markiewicz 1, Karolina Przybyłowska 1, Anna K. Kurowska 2, Anna Kamińska 2, Jerzy Szaflik 2, Ireneusz Majsterek 1 1 Department of Clinical Chemistry and Biochemistry, Medical University in Lodz, Lodz, Poland; 2 Department of Ophthalmology and Eye Clinic Of 2nd Medical Faculty in Warsaw, Medical University of Warsaw, Warsaw, Poland Łukasz Markiewicz Extracellular matrix remodeling process mediated by matrix metalloproteinases (MMPs) is an important determinant factor for the development of glaucoma. Well-balanced activity of MMPs and tissue inhibitors of matrix metaloproteinases (TIMP s) seems to be crucial for maintaining proper outflow of humor aquosus from eye anterior chamber. The main aim of this study was an evaluation of the G/2G MMP-1 and 372 C/T TIMP-1 gene polymorphisms association with the development of primary open angle glaucoma (POAG) in Polish patients. DNA obtained from blood samples of 99 adult patients with POAG and 107 control subjects were enrolled in our study. The polymorphism analyses were performed using the polymerase chain reaction, restriction fragment length polymorphism (RFLP-PCR). An association between genetic polymorphisms and the risk of POAG were estimated by χ 2 test and logistic regression. Performed research of the G/2G MMP-1 gene polymorphism showed increased chance of POAG occurrence in case of 2G2G genotype (OR 2.81, 95% CI , p<0.01) as well as 2G allele (OR 2.31, 95% CI , p<0.01). Analysis of 372 C/T TIMP-1 showed increased chance of POAG occurrence in case of C allele (OR 1.70, 95% CI , p<0.01). In conclusion we suggested that G/2G MMP-1 and 372 C/T TIMP-1 gene polymorphisms might be associated with the risk of primary open angle glaucoma in Polish population. Acknowledgements: This work was supported by grant N N from Polish Ministry of Science and Higher Education.
13 124 Abstracts P7.15 Analysis of consequently changed signal a novel method for analyzing microarray data Jakub Mieczkowski, Karolina Swiatek- Machado, Bozena Kaminska The Nencki Institute of Experimental Biology, Laboratory of Transcription Regulation, Department of Cell Biology, Warsaw, Poland Jakub Mieczkowski A common microarray analysis focuses on a long list of differentially expressed genes. Extracting biological information from such list is a major challenge. Moreover widely used single gene-based statistics has some limitations, e.g it neglects interactions between genes or cannot detect multivariate changes. Additionally, frequently two lists of differentially expressed genes obtained by two different groups studying the same biological condition may contain a small number of overlapping genes. On the other hand, it is known that deregulation in a group of functionally related genes, such as signaling pathway, plays an essential role in many diseases. A microarray technology allows to shift attention from gene-based analysis to analysis of gene categories, in particular signaling pathways. Since most of the existing methods of pathway analysis treat pathways as an ordinary set of genes, neglecting genes interactions, novel pathway-specific approaches are desired. We propose a novel analysis of consequently changed signal (ACCS) for investigation of signaling pathways. Our method combines microarray gene-specific data with established knowledge of gene interactions. The ACCS allows to pinpoint pathways within which signal is importantly changed under specific conditions. We illustrate our method using expression datastes previously published in biologically oriented papers. This guarantees an unbiased control for paths pointed by our method. In our work we used signaling pathways from KEGG (Kanehisa et al., 2010, Nucleic Acids Res 238: D355 D360 ). By combining expression data and pathway information, ACCS may facilitate analysis of microarray data. P7.16 Amino trasferase activity in the cerebral cortex of rats with intermittent alcohol intoxication M. N. Kurbat, V. V. Lelevich Grodno State Medical University, Department of Biochemistry, Grodno, Belarus Kurbat Mikhal Not the last role in the mechanisms of ethanol dependence syndrome is a violation of amino acid metabolism in the CNS, therefore, would seem appropriate fully integrated study of the level of metabolism in different models of experimental alcoholism. The purpose of this study to determine the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cerebral cortex of rat brain in a state of intermittent alcohol intoxication. Experiments were carried out on white male rats, with a mass at the beginning of the experiment g, contained on a standard diet vivarium with free access to water. Ethanol (25% solution) was administered intragastrically twice a day at a dose of 3.5 g/kg b.w., period of alcohol addiction was 4 days, and cancel 3 days. Cycles of alcoholism/cancellation repeated 2 and 4 times. Decapitation of animals was carried out on 14 and 28 days of the experiment, which allowed the dynamics to study the development of this form of alcohol intoxication. Determination of transaminase activity in brain cortex tissue was performed by kinetic method. The experimental results showed that in all experimental groups the activity of key enzymes of transamination of amino acids (ALT and AST) differ from those in intact animals. The degree of change depends on the duration of receipt of alcohol and number of cycles of alcohol addiction and correlated with published data on enhancing the activity of cytoplasmic fractions of ALT and AST and mitochondrial AST during prolonged alcohol abuse among animals. Change of studied enzymes may also be related to the disruption of metabolic adaptation, modification of enzyme kinetics and related disorders with vitamins (especially B6) under the influence of chronic ethanol proceeds into the organism. Undoubtedly, the destabilization of aminotransferase activity in the CNS may be one reason of metabolism disturbances in the brain in case of intermittent alcohol consumption, because studied enzymes are the link between carbohydrate, protein, amino acid and energy metabolism.
14 45 th Annual Meeting of the Polish Biochemical Society 125 P7.17 Synergistic effect of CYBA gene A640G polymorphism and hypercholesterolemia increases the risk of coronary artery disease Paweł Niemiec 1, Iwona Żak 1, Tomasz Nowak 1, Jolanta Krauze 2 1 The Medical University of Silesia, School of Health Care, Department of Biochemistry and Medical Genetics, Medyków 18, Katowice, Poland; 2 The First Department and Clinic of Cardiology, Ziołowa 45/47, Katowice, Poland Tomasz Nowak Background: Oxidative stress is implicated in the pathogenesis of coronary artery disease (CAD). NAD(P)H oxidases are the main source of superoxide in the vasculature. The p22phox peptide encoded by the CYBA gene is a critical component of NAD(P)H oxidases and a functionally relevant A640G and C242T polymorphisms of the CYBA gene modulate generation of reactive oxygen species. This report focuses on the interactions between A640G CYBA polymorphism and traditional risk factors of CAD like hypercholesterolemia and cigarette smoking. Aims: Aim of the study was to analyze whether A640G CYBA and C242T polymorphisms and the CYBA haplotypes are associated with CAD and its traditional risk factors. Methods: The study population of white Polish Caucasians included: 93 patients and 78 controls. The CYBA gene polymorphisms were genotyped using PCR-RFLP method. Statistical analyses were performed using Statistica 6.0 software. Gene-traditional risk factors interactions were analyzed using 4x2 tables, with the Rothman s synergy index (SI) as a determinant of the power of interaction. Results: Case-control study did not reveal any statistically significant differences in genotypes and alleles frequencies of A640G polymorphism between analyzed groups. However, there is a tendency to the prevalence of 242 T allele and TT 242 genotype among patients with CAD. The differences of haplotypes frequencies of both CYBA gene polymorphisms did not show statistical significance, although there is a tendency to the prevalence of G 640 +T 242 haplotype in the patients group (39.1 vs 31.6). The hypercholesterolemic carriers of 640 G allele were more frequent in the patients group than in the controls (43% vs 15.4%, p<0.0001). The risk of CAD associated with the presence of hypercholesterolemia was about eight-fold higher in 640 G allele carriers than in AA 640 homozygous subjects (SI=7.71). Conclusion: The A640G polymorphism can not be considered as an independent risk factor of CAD, but it modulates the risk of CAD associated with hypercholesterolemia. P7.18 Effect of tau and MAP2 on PrP-induced oligomerization of tubulin Katarzyna M. Osiecka, Hanna Nieznanska, Krzysztof Nieznanski Nencki Institute of Experimental Biology, Department of Biochemistry, Pasteur 3, Warsaw, Poland Katarzyna Osiecka Microtubules (MT) are cytoskeletal structures composed of tubulin which are involved in many cellular processes such as chromosome segregation, cell division, intracellular transport, secretion, cell motility and determination of cell morphology. Dynamic instability of MT that is cycling between assembly and disassembly is crucial to fulfill their function in the cell, hence it is precisely regulated by numerous MT-associated proteins (MAPs). Dysregulation of the balance between assembly and disassembly may result in cell death as it takes place in Alzheimer disease in which hyperphosphorylation of tau abolishes its ability to stabilize MT. In previous studies we have demonstrated that prion protein (PrP) - a key molecule engaged in fatal neurological disorders called prion diseases interacts with MT/ tubulin which leads to oligomerization of tubulin and consequent inhibition of MT assembly. Molecular mechanism of PrP neurotoxicity may include this phenomenon. In this report we present an effect of MAPs on PrP-induced oligomerization of tubulin. Taking advantage of turbidimetry we have shown that MAPs fraction isolated from pig brain inhibits PrP-induced oligomerization of tubulin. Further studies with purified tau and MAP2 - major constituents of MAPs fraction have demonstrated that both proteins affect formation of the oligomers. Moreover, we have found that tau phosphorylation modulates the effect of tau on PrPinduced oligomerization of tubulin. Based on the above results we propose that hyperphosphorylation of tau - a key event observed in Alzheimer disease promotes PrPinduced oligomerization of tubulin which exacerbates disruption of microtubular cytoskeleton.
15 126 P7.19 Abstracts P7.20 Enolase epitopes as plasmonogen-binding receptors in parasites in silico analysis Michał Piast University of Arts and Sciences, Department of Public Health and Environmental Sciences, Kielce, Poland Michał Piast Enolase [EC ] is a glycolytic pathway enzyme, catalyzing 2-phospho-glycerate dehydratation. Enolase performs several functions, ie. an eye tau-crystallin protein, surface receptor for the binding of plasminogen, Myc-binding protein, etc. Enolase ability to act as a plasminogen-binding protein in bacteria and parasites has been recently studied. In this research, selected enolase amino acid sequences from bacterial and parasites organisms were chosen for analysis together with human alpha enolase to check for possible antibody binding sites. Data analysis involved ClustalX sequence alignment, MEGA4 evolutionary analysis and SIFT analysis to find the most conserved parts of molecules. According to previous work on alpha-enolase-like proteins, they share common sequence DLDFKSPDDPSRYISP, located on the external part of molecules, binding to monoclonal antibody. Our research indicate strong sequence similarity between human alpha enolase and four analysed parasites F. hepatica, S. japonicum, P. falciparum and T. gondii (average of 79.75% similarity). These four molecules share identical string 257-YDLDFK-262 with human alpha enolase. Furthermore, latter part of string, after position 262, consists of amino acids predicted as tolerated substitutions. Sequences from E. histolytica, M. balamuthi and analysed bacteria show strong difference in indicated region. Theoretical model of F. hepatica enolase was conducted in SWISS-MODEL using existing human alpha enolase as template. Model quality was checked with anolea, verify3d and Ramachandran plot. Previously indicated string, possibly involved in plasminogen binding is located on external part of molecule. Results shown might have implication in understanding process of enolase-plasminogen interaction and might prove useful in treating parasite induced diseases. References: 1. Arza et al. (1997) Thromb Haemost 78: Chumchua et al. (2008) Bioinformation 3: Short-term pre- or post-treatments with TNF-α, IFNα and IFNγ do not affect insulin-dependent expression of MRF and MyHC in C2C12 myotubes Barbara Pijet 1, Maja Pijet 1, Anna Litwiniuk 1, Arkadiusz Orzechowski 1,2 1 Warsaw University of Life Sciences (SGGW), Faculty of Veterinary Medicine, Department of Physiological Sciences, Nowoursynowska 159, Warsaw, Poland; 2 Polish Academy of Sciences, Mossakowski Medical Research Center, Department of Cell Ultrastructure, Pawinskiego 5, Warsaw, Poland Barbara Natalia Pijet Skeletal muscle cachexia is associated with muscle loss. NF-κB activated by TNF-α may lead to proteolysis of muscle proteins. Furthermore, STAT kinases could play a significant role in alteration of TNF-α action by IFNs. The main symptom of accelerated protein catabolism in muscle seems to be the reduced expression and/or degradation of skeletal, fast myosin heavy chain (MyHC). The aim of the present study was to examine the effect of insulin and/or IFNs on the TNF-α-dependent activity of NF-κB, STAT kinases, and protein levels of MyHC protein in C2C12 myotubes analyzed by immunoblotting. TransAm multiplate test was used to examine transcriptional activity. IFNs increased TNF-α-stimulated degradation of IκB protein, the NF-κB translocation to nuclei, and it s DNAbinding capacity. Surprisingly, long-term (48 h) exposure of 6-days C2C12 myotubes to TNF-α and IFNγ alone, or in combination did not alter expression of MyHC. IFNα alone and together with TNF-α elevated expression of MyHC in 8-days myotubes. The protein level of myogenin in 8-days old myotubes was decreased merely in the longterm treatment with TNF-α. MyoD expression level remained unchanged. 24-hours pretreatment of 5-days old C2C12 myotubes with insulin strongly enhanced expression of MyHC in the 8th day of differentiation. This effect was not altered by 48-hours exposure to cytokines (TNF-α, IFNγ alone or with TNF-α). Myogenin and MyoD expression did not change, either. Cytokines in 24-hours pretreatment prior insulin administration also had no impact on MyHC and selected MRF expression levels in 8-days myotubes. Cytokines used in short-term application after longterm exposure to insulin did not affect insulin-dependent expression of MyHC. Insulin action was apparently dominant. These results indicate that insulin participates in myogenesis from C2C12 cells by contributing to the elevated expression of MyHC and modulation of NF-κB activity. Acknowledgements: This work was supported by the grants No N /0759 and No N from the Ministry of Science and Higher Education, Poland.
16 45 th Annual Meeting of the Polish Biochemical Society 127 P7.21 Down-regulation of CDK4 and CDK6 genes by RNA interference as therapeutic approach for Alzheimer s disease Danuta Piotrzkowska, Małgorzata Sierant, Milena Sobczak, Barbara Nawrot Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Sienkiewicza 112, Lodz, Poland Danuta Piotrzkowska Alzheimer s disease (AD) is an incurable, neurodegenerative brain disease that destroys memory and thinking skills. Several concepts were proposed to explain pathogenesis of AD, among them the most widespread the β-amyloid cascade hypothesis (reviewed in ). Some investigators link the neuronal cells death in AD and reactivation of the cell cycle. They related abnormal activation of the cell-cycle components such as the cyclin-dependent kinases (CDK4, CDK6, CDC2), cyclins (B1, D) and cdk inhibitors of the INK4 family in pathologically affected neurons in the AD brain . Arendt et al. suggested that aberrant activation of the Ras-MAPK signaling pathway triggers reactivation of the cell cycle. Usually, in postmitotic and terminally differentiated neurons, the cell-cycle activity is arrested by the enrichment of cdk inhibitors, and neurons are locked in the G0 phase. At the beginning of neuropathogenesis, neurons leave the G0 phase and progress until the S phase. Very likely that neurons die at the G1-M transition. The aim of our studies was to down-regulate the expression of two kinases CDK4 and CDK6 in neural cells through RNA interference-based gene silencing. Thus, sixteen sirnas of sequences complementary to human, mouse and rat transcripts of CDK4 and CDK6 were designed and synthesized in house. Their silencing activity was screened in human (HeLa, SH-SY5Y), mouse (Neuro2a, RAW 264.7) and rat (PC12) cell lines. The mrna and target protein levels in cells transfected with screened sirnas were determined by quantitative RT-PCR (Roche) and by Western blot analyses, respectively. The cells transfected with non-silencing sirna (100% expression of target gene) were used as a control. For CDK4 protein an average 60 90% lowering of mrna level and 60-80% lowering of protein were observed, while for CDK6 corresponding values of 50 80% and 60 85% were found. The sequences of the most powerful sirna silencers were used to design shrna and micro RNA inserts, which were subsequently cloned into psilencer 2.0-U6 (Ambion) plasmid downstream of the U6 promoter and into pegfp-c1 (BD Biosciences) plasmid downstream of the CMV promoter. These constructs down-regulated the CDK4 and CDK6 genes to 30 70% and 30 60% (mrna) and 21 95% and 20 65% (protein), respectively. References: 1. Nawrot B, Sierant M, Paduszynska A (2009) RSC Drug Discovery 3: Yang Y, Mufson EJ, Herrup K (2003) J Neurosci 23: P7.22 The effect of different nitric oxide availability on high fat diet induced metabolic changes in mice Urszula Razny 1, Beata Kiec-Wilk 1,3, Anna Polus 1, Grzegorz Dyduch 2, Bogdan Solnica 1, Lukasz Wator 1, Maciej Malecki 3, Romana Tomaszewska 2, John P. Cooke 4, Aldona Dembinska-Kiec 1 Jagiellonian University Medical College, 1 Department of Clinical Biochemistry, Kopernika 15a, Cracow, Poland; 2 Department of Pathomorphology, Grzegorzecka 16, Cracow, Poland; 3 Department of Metabolic Diseases, Kopernika 15, Cracow, Poland; 4 Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA , USA Urszula Razny Background: High fat diet impairs NO bioavability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. Accordingly, the purpose of this study was to investigate the effect of high fat diet on metabolism in transgenic mice with decreased (enos / mice ) and increased (DDAH overexpressing mice) NO bioavailability. Materials and Methods: Transgene enos /, DDAH, and WT mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and insulin were monitored. The 3D matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis. Gene expression in adipose tissues was analyzed by microarray and Real Time PCR. Results: enos / mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose level was observed. Fasting insulin and cholesterol levels remains unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-regulated, whereas in enos / mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were upregulated. Conclusions: Our results indicate that response to a high fat diet is modulated by NO availability. Increased NO availability attenuates some HFD-induced alterations in metabolism and gene expression associated with insulin resistance. Acknowledgements: This study was supported by: K/PBW/000048, K/ZDS/000623, NuGO FP
17 128 Abstracts P7.23 Altered sphingolipid composition in human Wharton s jelly of preeclamptic newborns Lech Romanowicz, Edward Bańkowski Department of Medical Biochemistry, Medical University of Białystok, Białystok, Poland Lech Romanowicz Wharton s jelly is a myxomatous substance surrounding the umbilical cord vessels to protect them against extension, bending, twisting and compression. Preeclampsia (hypertension, edema, proteinuria) is the most common pregnancy-associated pathological syndrome. It is accompanied by significant alterations in the umbilical cord composition. Here we describe the sphingolipids of Wharton s jelly from 10 newborns delivered by healthy mothers and from 10 babies of mothers with preeclampsia. Thin layer chromatography, solid phase extraction and high-performance liquid chromatography were employed. Control tissues were abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases were distinctly lower. Preeclampsia is associated with a significant increase in sphingomyelins, ceramides, sphingosine, sphinganine, 4-OH-sphinganine, sphingosine 1-phosphate and glycosylated sphingosine. Furthermore, a decrease in sphinganine 1-phosphate was found. Stearate (C18:0) is the dominating fatty acid in sphingomyelins and ceramides of control tissue. In contrast to that preeclamptic material contained the highest amount of laurate (C12:0) in sphingomyelins and myristoleate (C14:1) in ceramides. Sphingolipids and some sphingoid bases are bioactive molecules which contribute to regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of Wharton s jelly resulting in remodeling of its composition. P7.24 The cytotoxic and mutagenic effect of methylmethane sulfonate in M13 phage replicated in Escherichia coli wild and alkb strains Karol P. Ruszel 1, Paweł Kowalczyk 2, Beata Sokołowska 1, Agnieszka M. Maciejewska 1, Jarosław T. Kuśmierek 1 1 Department of Molecular Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland, 2 Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Warsaw, Poland Karol Ruszel We have evaluated the role of AlkB dioxygenase in repair of DNA alkylation damage in E. coli cells. Using M13mp18 phage lacz system, we compared the survival and mutagenesis of MMS treated phage DNA in E. coli wild-type or alkb mutant. Single stranded DNA of M13mp18 was modified in vitro by MMS and subsequently DNA samples were transfected into E. coli strains JM105 wild type (as a control) and JM105 alkb (adapted or not by MMS). The obtained DNA of mutants was isolated and sequenced using the Sanger method with primer BT3 for lacz gene. We have found that MMS treated M13mp18 DNA had lower survival and higher mutation frequency in alkb cells than in the wild type. Preliminary results of sequencing of mutants showed that the most frequent mutations are simultaneous big deletions of 93 (DM15 deletion of F lacz) and 54 (M13 polylinker) nucleotide fragments of phage DNA. These deletions are more frequent when phage is replicated in non adapted alkb or in adapted alkb than in adapted wt strain. The most frequent base substitution in non adapted wild type were the G=>A transitions followed by C=>A and T=>A transversions. Among the frameshifts, insertions of a single C or single A and deletions of a single A or G in runs of GA, were detected. In non adapted alkb strain, among the frameshifts, additions of a single A, single C, single T were observed. The detected base substitutions were frequent G=>A transitions in runs of GA (as in wild type), C=>A, A=>C, G=>C transversions and A=>G transition. In adapted alkb we observed mainly frameshifts such as: additions of single A and single C, deletion of single G. Among the base substitutions: G=>A transitions and G=>C transversions were also observed. The effect was more prominent than in the adapted wild type.
18 45 th Annual Meeting of the Polish Biochemical Society 129 P7.25 Recombinant form of EBA-140 a novel ligand of Plasmodium falciparum expression and purification Joanna Rydzak, Ewa Jaskiewicz Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Immunochemistry, Wrocław, Poland Joanna Rydzak Malaria causes considerable morbidity in some tropical areas what results in upwards of half a billion episodes of clinical malaria and the deaths of more than a million children each year in sub-saharan Africa alone . Plasmodium falciparum, malaria parasite, causes the most severe form of malaria in humans. That parasite can use multiple host receptors to invade human red blood cells (rbc) . Two main invasion pathways has been identified: the sialic acid dependent and the sialic acid independent. Most studies indicate that rbc glycophorins serve as host receptors for invasion via the sialic acid-dependent pathway . The EBA- 140 is a member of DBL protein family of P. falciparum that has been shown to be a novel ligand for human erythrocyte glycophorin C. It shares structural homology with EBA-175, another P. falciparum DBL ligand. The conserved binding domain of each member of DBL protein family, called Region II, consists of two subdomains F1 and F2. The F2 domain seems to be more important for receptor binding . We have cloned Region II of EBA-140 ligand from genomic DNA of P. falciparum clone Dd2. Expression of the recombinant form of EBA-140 RII was carried out in two bacterial expression systems using the following vectors: pet28a + (Novagen), pmalp2x and pmalc2x ( New England BioLabs). The recombinant form of EBA- 140 RII was purified from cytoplasm, periplasm and inclusion bodies on Nti-Agarose for the pet28a + system or by affinity chromathography on amylose for pmal system, respectively. The purified recombinant form of EBA-140 RII will be used in successive studies on parasite ligand and erythrocyte receptor interaction. References: 1. Cowman AF, Crabb BS (2006) Cell 124: Li X (2008) Biochem Biophys Res Commun 376: Thompson JK et al. (2001) Mol Biol 41: Pandey KC et al. (2002) Mol Biochem Parasitol 123: P7.26 Acid sphingomyelinase is translocated onto the cell surface upon activation of immunoreceptor FcγRIIA Agata Samonek, Andrzej Sobota Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland Agata Samonek Fcγ receptor IIA (FcγRIIA) is expressed exclusively in human cells of the immunodefense system and mediates phagocytosis of pathogens coated with IgG. Upon activation the receptor is phosphorylated which triggers downstream signaling pathways. Our previous results showed that activated immunoreceptor FcγRII in U937 cells undergoes clustering and is recruited from glycerophospholipid-rich environment of the plasma membrane to sphingomyelin/cholesterol enriched microdomains, called lipid rafts. The interaction between activated receptors and rafts seems to be facilitated by ceramide, a product of sphingomyelinase activity. In this study we demonstrated that during FcγRII activation acid sphingomyelinase (ASMase) is translocated onto the cell surface. This was concluded on the basis of measurement of activity of ASMase detected at the cell extracellular leaflet of the plasma membrane. We observed elevation of ASMase activity (~1.5-fold) during FcγRII activation. The profile of ASMase activity in the course of FcγRII activation was correlated with generation of the cell surface ceramide and decreasing level of sphingomyelin in crude plasma membrane fraction. To examine if translocation of ASMase is a specific response to FcγRII activation, the activity of this enzyme was estimated in U937 cells after cross-linking of transferrin receptor, CD55 and glycoproteins and glycolipids by concanavalin A. These types of cell stimulation did not induce translocation of ASMase onto the cell surface. Electron microscopy performed on isolated membrane sheets showed translocation of ASMase onto the cell surface during FcγRII cross-linking. This enzyme was accumulated in electron-dense structures in the plane of the plasma membrane in which FcγRII and NTAL (lipid raft marker) were also concentrated. These structures seem to be assembled by coalescence of lipid rafts. Taken together, the data indicate that activation of immunoreceptor FcγRII induces translocation of acid sphingomyelinase onto the cell surface where ceramide is generated and this process seems to be specific for activation of immunoreceptor FcγRII in U937 cells.
19 130 P7.27 Abstracts P7.28 Effect of sodium azide and sodium nitrite on rat liver aldehyde dehydrogenases Valentina I. Satanovskaya Institute of Pharmacology and Biochemistry of the NAS of Belarus, Grodno, Belarus Valentina Satanovskaya Purpose: Hypoxia induced by various factors is accompanied by appearance of reactive oxygen species, free radicals and activation of lipid peroxidation (LPO). The toxic aldehydes produced (malondialdehyde, 4-oxynonenal, etc.) are metabolized by the system of aldehyde dehydrogenases (ALDH) occurring as a variety of isoforms. Methods: Male Wistar rats weighing g were used in the experiment. The hemic hypoxia was induced by administration of sodium nitrite (50 mg/kg, i.p., 4 days), whereas tissue hypoxia by injections of the cytochrome oxidase inhibitor, sodium azide (10 mg/kg, i.p., 4 days). Liver tissue was assayed for the activities of NAD-and NADP-dependent ALDH with various Km for aldehydes. Results: Both the substances did not affect markedly the NAD-dependent with high and low Km ALDH forms but activated NADP-dependent ALDH with high Km for acetaldehyde: control mu, sodium azide , p<0.02; sodium nitrite , p<0.01. In using aromatic aldehyde (benzaldehyde) as substrate, activation by sodium nitrite was found: 2.8±0.05 mu control; 2.8±0.1 sodium azide; 3.4±0.2, p<0.01, sodium nitrite. This enzyme form (NADP-dependent with high and low K m to aliphatic and aromatic aldehydes) belongs to the Class ALDH-3 which is expressed during tumor growth. Some authors (Reisdorph R et al., 1998) show that expression of ALDH-3 genes is regulated by a receptor of polycyclic hydrocarbons (cancerogenes), and by dimer of this receptor with a hypoxia-inducible factor 1 alpha (HIF-1- alpha), or by LPO products. Conclusions: Our data suggest that systemic intoxication by nitrates and azide can induce neoplastic changes in the liver. References: Reisdorph R, Lindhal R et al. (1998) Biochem Biophys Res Commun 249: Sumoylation of the Drosophila 20-hydroxyecdysone receptor Justyna Seliga 1, Katarzyna Bielska 1, Elzbieta Wieczorek 1, Rainer Niedenthal 2, Andrzej Ozyhar 1 1 Department of Biochemistry, Wroclaw University of Technology, Wroclaw, Poland; 2 Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Germany Justyna Seliga The 20-hydroxyecdysone (20E) coordinates crucial biological processes in insects such as development and reproduction. It acts through a specific nuclear receptor complex, 20-hydroxyecdysone receptor (EcR) and ultraspiracle (USP). EcR and its ligands are absent in vertebrates and thereby used in commercial ecdysone-inducible expression systems. They are also regarded as building elements of potential gene switches with future therapeutic usage. But EcR action is still poorly understood. Now its activity regulation sets the stage for a new research field, especially as far as posttranslational modifications are concerned. Till now there has been no study about EcR sumoylation - attachment of the small ubiquitin-like modifier protein (SUMO) that plays a vital role in regulating functions of wide range of cellular proteins. First we performed bioinformatic analysis of sumoylation sites of Drosophila melanogaster EcR with four predictors: PCI-SUMO, SUMOplot TM, SUMOpre, SUMOsp 2.0. Then to confirm SUMO conjugation to EcR we used the Ubc9 fusion-directed SUMOylation (UFDS) system. We prepared a set of constructs coding all three isoforms of EcR from Drosophila melanogaster EcR-A, EcR-B1 and EcR-B2 fused with sumoylation E2 enzyme Ubc9 on N- or C-termini. After expression in HEK293 line, cell extracts were fractionated by SDS-PAGE, followed by immunobloting with anti-ubc9 antibodies. Electrophoretic mobility changes for all proteins were observed suggesting modification by SUMO, both endogenous and coexpressed. Sumoylaion pattern is different depending on location of Ubc9 on N- or C-termini and specific for each isoform but common for SUMO1 and SUMO3. It is also preserved while EcR is coexpressed with its dimerization partner Usp. In order to identify sumoylation sites we prepared another series of constructs with cdna of domains and fragments of Drosophila melanogaster EcR which revealed regions associated with modification by SUMO protein. Acknowledgements: This work has been supported by the Polish Ministry of Science and Higher Education Grant N /2827 and The Wroclaw University of Technology.
20 45 th Annual Meeting of the Polish Biochemical Society 131 P7.29 Oxidative RNA damage and its potential role in cellular responses to radiation M. Skonieczna 1, S. Student 1, A. Cieślar-Pobuda 1, R. Herok 2, R. Jaksik 1, J. Rzeszowska-Wolny 1,2 1 Institute of Automatic Control, Silesian Uiversity of Technology, Gliwice, Poland; 2 Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Branch Gliwice, Poland Magdalena Skonieczna mrna stability is an element of the regulation of gene expression, and its modulation is important for proper responses to inter- and intra-cellular signaling and cellular reactions to environmental changes. The structure of transcripts and the presence of nucleotide sequence motifs that are specifically targeted by proteins or mirnas are crucial for this modulation. Damage introduced in RNA by different factors can influence the specific interactions between RNA and modulators of its stability. One such factor could be oxidative damage to cellular macromolecules by exposure to ionizing radiation. Accumulation of 8-oxo- 7,8-dihydroguanosine (8-oxoG) in RNA can be measured experimentally by high pressure liquid chromatography with electrochemical detection (HPLC-EC). In the present work we followed the changes in 8-oxoG levels in RNA of colon cancer HCT 116 p53 +/+ and p53 / cells exposed to ionizing radiation (IR). Cells grown in DMEM-F12 medium supplemented with 10% FBS, under standard CO 2, humidity and temperature were irradiated by 4 Gy of X-rays from a Clinac GMV system, and at different times after irradiation RNA was isolated using a Total RNA Isolation Kit (A&A Biotechnology). The level of 8-oxoG in RNA from irradiated cells changed with time, reaching the highest value a short time (1 h) after irradiation. Transcript profiles measured in parallel by the microarray method showed high numbers of up-regulated mrnas that were enriched in mirna-targeted sites and ARE sequence motifs. Our results are consistent with the hypothesis that after irradiation, this changed abundance of many transcripts enriched in mirna-targeted and protein binding AU-rich motifs reflects modulation of mrna-mirna and mrna-protein interactions due to radiation-induced damage to RNA by reactive oxygen species. This mechanism could be implicated more generally in cellular responses to stresses where ROS levels increase. Acknowledgements: This work was supported by grant No. N N from the Polish Ministry of Education and Science and No. PBU-48/Rau1/2009 from the Silesian University of Technology. P7.30 Tumor necrosis factor in varicose veins and varicose veins complicated by thrombophlebitis Radosław Kowalewski 1, Andrzej Małkowski 2, Krzysztof Sobolewski 2, Marek Gacko 1 1 Medical University of Białystok, Department of Vascular Surgery and Transplantology, Białystok, Poland; 2 Medical University of Białystok, Department of Medical Biochemistry, Białystok, Poland Krzysztof Sobolewski Varicose veins are among the most common features of chronic venous insufficiency (CVI), which exerts a high impact on patient quality of life. The disease pathogenesis is still not clearly understood. Although valvular incompetence, alterations in smooth muscle cells (SMCs) arrangement, and changes in extracellular matrix (ECM) composition are the most commonly noted factors affecting the development of varicose veins, the exact mechanisms through which they interact in disease pathogenesis remain unknown. Extensive extracellular matrix remodeling of the vein wall is involved in the pathogenesis of varicose veins. The process is controlled by numerous factors. Among them tumor necrosis factor α (TNF-α) is one of the most important cytokines involved in proliferation and growth of cells responsible for inflammatory and immunological response. Furthermore TNF-α influences synthesis many other factors, which can modulate the ECM metabolism. The aim of the study was to evaluate TNF-α and its two receptors (TNF-R1 and TNF-R2) in the wall of varicose and varicose veins complicated by thrombophlebitis, when compared to normal ones. Segments of normal, varicose and varicose veins complicated by thrombophlebitis were collected during varicose veins surgery in 17 patients (9 women and 8 men, years age range). Expression and content of TNF-α, TNF-R1 and TNF-R2 were evaluated with RT-PCR, Western blot and ELISA methods respectively. Decreased TNF-α expression were observed in the wall of varicose veins. However in the varicose veins complicated by thrombophlebitis its expression was significantly higher in comparison with the wall of normal veins. The evaluation of TNF receptors type 1 and 2 showed slight differences in their expression, both in varicose and varicose veins complicated by thrombophlebitis walls. The overexpressed TNF-α in the wall of thrombophlebitis varicose veins via TNF receptors may influence gene expression of enzymes involved in extracellular matrix metabolism and play a role in vein wall remodeling, as well as in the disease pathogenesis.
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