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Cloning, expression and purification of human AKR1C3
Dudová, Radka ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Dudová Radka Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Cloning, expression and purification of human AKR1C3 The purification of the vector pOTB7 containing the coding sequence ( CDS ) of aldo-keto reductase 1C3 ( AKR1C3 ) in Escherichia coli ( E.coli ) cells was performed by an alkaline lysis method. The coding sequence was then amplified by polymerase chain reaction. Result accuracy of the demonstrated step was shown by verification of the size of the synthesized fragment and by a restrictive analysis with the restriction endonuclease PvuII. In the second step, the coding sequence was ligated into the Topo 2.1 vector which was transformed into competent E. coli cells. The cells were multiplied and Topo 2.1 vector with the inserted code sequence was obtained by the alkaline lysis method. The result was verified by comparing of the size of the Topo 2.1 vector without inserted CDS and the vector Topo 2.1 with the CDS on an agarose gel and the subsequent restrictive analysis with the restriction endonuclease HindIII. The coding sequence was verified by the sequenation. There was also carried out the subcloning of the CDS from the Topo 2.1 vector into the express...
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Cloning, expression and purification of human AKR1B1
Lundová, Tereza ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Lundová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1B1 Aldose reductase (EC 1.1.1.21) AKR1B1 is one of the 13 human enzymes of the AKR superfamily. All human AKRs are cytosolic and NADP(H) dependent. AKR1B1 plays an important role in metabolism of endogenous and exogenous substances. The main endogenous substrate is glucose. Its reduction to sorbitol is consistently linked to secondary diabetic complications. From xenobiotics metabolized by AKR1B1, daunorubicin, an anticancer drug from the group of anthracyclines, is reduced to daunorubicinol. This metabolite is less active than parent drug and is the cause of anthracycline related cardiotoxicity. At present, many projects are focused on AKR1B1 as a target enzyme and specific inhibitors of AKR1B1 are looking for. The recombinant protein of AKR1B1 was prepared in E. coli together with the pET expression system. First, cDNA for AKR1B1 in pOTB7 was isolated from E. coli. The coding sequence of AKR1B1 was amplified by a PCR. PCR was performed with Phusion Hot Start II polymerase and pair of forward and reverse primers, which contained NdeI and XhoI restriction...
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Inhibition Study of Human Membrane-bound Carbonyl Reductase
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Linda Laštovková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of thesis: Inhibition study of human membrane-bound carbonyl reductase. Carbonyl reductases are an important group of enzymes that participate in metabolism of both endogenous substances also xenobiotics. Potential anticancer drug oracin is one of such xenobiotics that is metabolised by various carbonyl reductases to two enantiomers of dihydrooracin. The new human microsomal carbonyl reductase also takes part in biotransformation of oracin. This enzyme was recently purified on Department of biochemical sciences (Faculty of Pharmacy in Hradec Králové). The aim of this study was to find some inhibitors of the new enzyme and distinguish it in terms of inhibitors from another microsomal carbonyl reductase 11β-hydroxysteroid dehydrogenase 1. Flavonoids are substances produced by plants, they have a different both positive and also negative effect on human organism. One of such effect is inhibition effect on diverse enzymes. Carbonyl reductases also fall in this group. It has been described inhibitory effect of different flavonoids on carbonyl reductases. Inhibition study of the new human micorosomal carbonylreductase was...
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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The impact of inflammation on muscarinic receptor subtypes expression - Immunohistochemical examination of lacrimal and salivary glands of rat and human
Székelyová, Anita ; Wsól, Vladimír (advisor) ; Soukup, Ondřej (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anita Székelyová Supervisor: Prof. Gunnar Tobin, O.D., Ph.D. Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The impact of inflammation on muscarinic receptor subtypes expression - Immunohistochemical examination of lacrimal and salivary glands of rat and human This project was focused on the characterization of muscarinic receptor subtype expression in lacrimal and salivary glands of the rat. Immunohistochemical methods were used to reveal the presence of the M1, M3 and M5 receptors and the expression of these subtypes was further studied under experimentally induced inflammatory conditions. The expression of the above mentioned muscarinic receptor subtypes was also studied in human labial glands, both healthy and with autoimmune sialadenitis compatible with Sjögren's syndrome. M1, M3 and M5 muscarinic receptors were found in the lacrimal and submandibular glands of the rat using immunoenzyme and immunofluorescent methods. The M5 receptor in the lacrimal gland was also detected on intracellular, possibly nuclear localization. The tissue with induced inflammation didn't show any significant differences in the muscarinic receptor expression. The immunofluorescent labeling of the...
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Optimization of purification of human membrane-bound carbonyl reductase.
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Bílková, Zuzana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Rudolf Andrýs Supervisor: prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Optimalization of purification of a human membrane-bound carbonyl reductase Carbonyl reductases are enzymes participating in metabolic pathways of various eobiotics and xenobiotics. Of all known enzymes metabolizing xenobiotics only 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is found in the microsomal membrane. It also contributes to metabolism of prochiral anticancer drug oracin, which main metabolic pathway is a carbonyl reduction on the position 11 leading to two enantiomers of (+) an (-) 11-dihydrooracin (DHO). Based on the discrepancy between microsomes and 11β-HSD1 stereospecifity of oracin reduction exist a hypothesis of participation an unknown microsomal enzyme in oracin metabolism. The aim of this study is to purify a new microsomal carbonyl reducing enzyme contributing in the biotransformation of oracin. Human liver microsomes were solubilised and desalted. The prepared sample was used for the first purification step on Q-Sepharose. Captured flow through fraction Q2 was loaded on Phenyl-sepharose. Captured suitable fraction P11 was used for third purification step by gel filtration. All...
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Human microsomal glutathione s-transferase
Hrnčiarik, Karel ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
The microsomal glutathione S-transferases (GST, EC 2.5.1.18) constitute a group of phase II detoxification enzymes present in eukaryotic cells and prokaryotic organisms. They play significant roles in defense against carcinogenic, toxic, and pharmacologically active electrophilic compounds. The microsomal glutathione S-transferase is a homotrimeric, membrane-bound enzyme, that catalyzes conjugation of electrophilic compounds with glutathione and reduction of lipid hydroperoxides. Peroxidase activity may be of importance for protection against lipid peroxidation under conditions of oxidative stress. A number of biochemical approaches have been used to study the properties of MGST, including enzymatic activity, sub-cellular distribution, substrate specificity, and proteins structure. MGST is considered a member of the MAPEG (membrane- associated proteins in eicosanoid and glutathione metabolism) superfamily of structurally and phylogeneticaily related enzymes, including MGST 1, MGST 2, MGST3, microsomal Ya-GST, MGST 1 Like 1, 5-lipoxygenase activating protein, and leukotriene C4 synthase. Enzymes in this superfamily are involved in detoxification, protection from oxidative stress, and synthesis of prostaglandin E and cysteinyl leukotrienes. On the basis of understanding funcitons MGST is possible...
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The Role of Reductases in Cancer.
Škarydová, Lucie ; Wsól, Vladimír (advisor) ; Hodek, Petr (referee) ; Bílková, Zuzana (referee)
Only a small attention was paid for long time to reducing enzymes, but today it is clear that these are an important part of the endogenous metabolism and also the phase I biotransformation of xenobiotics. The significant group of reducing enzymes are carbonyl reductases that belong to two superfamilies - short chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Their role in cancer is now intensively studied and their functions in cancer it is possible to divide into two main sections. It is known that carbonyl reductases play a substantial role in hormone-dependent cancers as prostate, breast or endometrial cancer. Active estrogens or androgens are important growth factors for these cancers because they evoke increasing of cell proliferation so that elevated possibility of mutations of important genes and development of cancer. Carbonyl reductases along with other enzymes (e.g. aromatase) participate in formation of these active sex hormones in extragonadal tissues, so an inhibition of such enzymes may be a target of anticancer therapy of hormone-dependent cancers. It is necessary to determine which enzymes are essential for the formation of active sex hormones in particular types of cancers. Besides hormone-dependent cancers, carbonyl reductases play also role in cancers that...
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Enzyme activity of recombinant enzymes. Influence of histidine tag.
Navrátil, Václav ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Václav Navrátil Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Enzymatic activity of recombinant enzymes. Evaluation of His- tag influence. AKR1B1, AKR1C1 and AKR1C3 are three representatives from 15 human enzymes of AKR superfamily, so far described. In the body they take part in a number of biochemical pathways and their substrates include both xenobiotics and endogenous compounds. Reduction of glucose to sorbitol catalyzed by AKR1B1 is associated with the development of secondary diabetic complications. AKR1B1 is also involved in cardiotoxic effect of the cytostatic drug daunorubicine. AKR1C1 is physiologically responsible for the conversion of progesterone to less efficient 20α- hydroxyprogesterone, thereby regulates progestational activity. Overexpression of AKR1C1 may be connected with resistance to certain cytostatic agents. AKR1C3 in human catalyzes a reaction in which testosterone and 5α-DHT is formed and it is also associated with development of several cancer types. Significant involvement of these enzymes in a number of pathological processes led to their closer study. The aim of this work was to evaluate influence of His-tag and chosen buffer on the...
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Purification and characterization of selected human microsomal reductase
Skarka, Adam ; Wsól, Vladimír (advisor) ; Živná, Lucie (referee)
Area of human liver microsomal reductases hasn't been fully explored yet. Purification and characterization of still undescribed enzymes is an important step in finishing of this task. There is separation and purification of this reductases described in my work, with usage of hydrophobic interaction chromatography on low pressure chromatography instrument ÄKTA purifier. There were two columns with different substrates used, Phenyl sepharose (low sub) and Octyl sepharose. Separation conditions were the same in both cases. Results obtained by incubation of fractions and measurement on HPLC showed significant differences between both columns and their substrates. But it isn't possible to determine, which column is better for purification, because big area still remain for adjustment and optimization of purification conditions.
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