|
| |
|
|
Cloning, expression and purification of human AKR1C2
Tobolová, Veronika ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Veronika Tobolová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C2 The coding sequence of AKR1C2 inserted in vector pOTB7 was multiplied by PCR and purified by alkali hydrolysis. Then it was ligated together with TOPO 2.1 vector. Prepared sequence inserted in vector TOPO 2.1 was transformed into the competent E. coli cells and multiplied. To verify these steps we did incubation of cell culture with ampicilin and incubation of coding sequence with restriction endonucleases.The samples inserted in vector TOPO 2.1 were sent to do sequencing. The next step was the digestion of the coding sequence inserted in vector TOPO 2.1 and the opening of purchased expressed vector pET-15b. Then a several attempts to ligation were made. The control of these steps was done by transformation of vector pET-15b with coding sequence into Hb101 cells, by incubation of transformed cells with antibiotics and by restriction sequence. Finally, the vector with the coding sequence was transformed into competent BL-21 cells and the expression was done. The result of expression we could observe after sonication and digestion of the cells by...
|
|
|
Versatile use of liquid chromatography and mass spectrometry in drug metabolism studies
Suchanová, Bohumila ; Wsól, Vladimír (advisor) ; Solich, Petr (referee) ; Lemr, Karel (referee)
Human organism has always been exposed to a vast array of chemicals encountered in the environment. Chemical revolution has significantly influenced biological evolution of humans leading to serious unpredictable toxicities. In response to continual chemical stress they have developed a variety of enzymes to transform these xenobiotics. Xenobiotics are mostly highly lipophilic and cannot readily be excreted from the body without metabolism to more hydrophilic, water-soluble metabolites. Not only environmental chemicals represent xenobiotics but also drugs, dietary components etc. Biotransformation studies play an important role in the drug discovery and development process. Usually data from drug metabolism is required before a new substance can advance towards the development stages of a new therapeutic agent. Data on metabolism is frequently used to optimize drug candidates, suggest more active compounds or support toxicology studies. The increased flux of new chemical entities into drug discovery has placed an increased need for fast and reliable information on the metabolism of these substances. Liquid chromatography coupled with mass spectrometry can meet demands for rapid drugs and metabolites analysis imposed by modern drug discovery strategies. This dissertation thesis presents an evidence...
|
|
|
Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines.
Kouklíková, Etela ; Novotná, Eva (advisor) ; Wsól, Vladimír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...
|
|
|
Purification and characterization of membrane-bounded reductases from human liver
Pelcová, Vendula ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Vendula Pelcová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of rigorous thesis: Purification and characterization of membrane-bound reductase from human liver. This study was focused on implementation of experimental techniques for purification of human liver microsomal fraction. There were chosen three basic steps of purification process. As a first step, ion-exchange chromatography was chosen followed by two purification steps on the base of gel filtration. Before purification the microsomal fraction had to be solubilized to release proteins from membrane bounds and conditions had to be optimized. Single fractions were subsequently incubated with specific substrate of reductases oracin and the quantity of metabolite 11-dihydroracin was assessed by achiral HPLC analysis. For the most active fractions the SDS-PAGE analysis was done to set size of unknown enzyme. By use of antibody against 11beta-HSD1 was axcleded the presence of this reductase in all analyses fractions. By use of described purification techniques we were successful to concentrate fraction with unknown human liver reductase.
|
|
|
Significance of genetic factors for prognosis and prediction of cancer therapy outcome.
Šůsová, Karolína ; Wsól, Vladimír (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Karolína Šůsová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Significance of genetic factors for prognosis and prediction of cancer therapy outcome Colorectal cancer (CRC) is one of the most common cancers in the world and also in the Czech Republic. There are many reasons for such a high prevalence and mortality. In general, we can say, that the level of awareness of risk factors and primary prevention is very low. For example, the inoperability of the tumor, caused by the diagnosis of CRC at a late stage, makes therapy of patients difficult and reduces survival. 5 - Fluorouracil and leucovorin are the gold standard in the treatment of CRC. The targeted therapy has a great success in the palliative therapy and prolongs survival. However, many patients do not respond to adjuvant and palliative therapy well. This failure is often caused by drug resistance. Efflux of cytotoxic drugs out of cells caused by transport proteins of cancer cells. ABC transporters remain among the many elements of drug resistance. Deregulation of gene expression of ABC transporters in tumor cells was discovered in connection with other types of cancer. The aim of this study is to find...
|
|
|
Cloning, expression and purification of human AKR1C4
Kosánová, Kateřina ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Kosánová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C4. AKR1C4 is one of four enzymes in men belonging to subfamily of aldo-keto reductases AKR1C. It is monomeric cytosolic protein with length of 323 amino acids expressed in liver. It plays an important role both in metabolism of endogenous and exogenous substances. It is involved in the metabolism of steroid hormones and bile acids and many drugs, for example tibolone and naltrexone. It also plays a role in activation of some cancerogenic substances, e.g. PAHs. cDNA of enzyme was delivered in cells of E. coli DH10B, in pDNR-LIB vector. After lysis of cells and isolation of plasmid, the coding sequence was amplified by PCR. Afterwards it was ligated into vector pET-28b, thanks to added restriction sites for Nde I and Xho I endonucleases in designed PCR primers. The recombinant plasmid prepared by this way was transformed by heat shock to cells E. coli HB101. After amplification of ligated plasmid it was transformed to E. coli BL21. Adjusted cells BL21 were used for expression of the protein. IPTG was used as induction reagent for overexpression. Pure...
|
|
|
Cloning, expression and purification of human AKR1C1
Vomočilová, Iva ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Iva Vomočilová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C1 This work is focused on synthesis of human AKR1C1. AKR1C1 coding sequence (cDNA), incorporated into plasmid pOTB7, was purchased and delivered in Escherichia coli cells. These cells with modified plasmid were multiplied in LB medium. After the multiplication plasmid was isolated and purified by the alkaline lysis process. Coding sequence for AKR1C1 was amplified by PCR method. The primers were designed in advance and contained restriction sites for XhoI and NdeI endonucleases. The results of PCR were validated by gel electrophoresis. Then the PCR product was purified on ultra-pure agarose gel. In the next step plasmid pET-28b(+) was used to insert prepared coding sequence. Plasmid was multiplied in competent cells E. coli HB101 and purified by the alkaline lysis process. Purified PCR fragment and plasmid were double digested by a pair of restriction endonucleases mentioned above. These digested fragments were purified and PCR fragment was put into the open vector pET-28b(+) by T4 DNA ligase enzyme. This modified plasmid was transferred into the...
|
|
|
Cloning and expression of human AKR1A1
Kořínková, Petra ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Kořínková Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning and expression of human AKR1A1 The aldo-keto reductase 1A1 (AKR1A1) is monomeric cytosolic enzyme from aldo- keto reductase superfamily (AKR) with carbonyl reducing activity. The members of AKR superfamily have an important role in reductive reactions in metabolism of some endogenous substrates and in the first phase of biotransformation of xenobiotics. Fifteen members of this large family have been found in the human body. These proteins are expressed in different tissues within all our organism. The highest concentrations of proteins have been detected in the hepatocytes and in the renal cells. The function of AKR1A1 is to catalyse reductive reactions of aromatic and aliphatic aldehydes to the respective alcohols. Among its important substrates belong mevalonate (endogenous substance) and some drugs like anthracykline antibiotics - doxorubicin and daunorubicin. AKR1A1 also participates in biotransformations of sorbitol and in the development of diabetes complications. Preparation of recombinant protein was performed in E. coli with using of pET28b(+) as an expression vector. Isolated cDNA was...
|
|
|
Characterization of human membrane-bound carbonyl reductase
Zvěřinová, Michaela ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Zvěřinová Supervisor: prof. Ing. Vladimír Wsól Ph.D. Title of diploma thesis: Characterization of a new human membrane-bound carbonyl reductase Reducing enzymes are involved in the metabolism of eobiotics and xenobiotics. Carbonyl reductases also belong to the group of reducting enzymes. These are grouped into three protein superfamilies, the SDR, AKR or MDR and they can be either soluble cytosolic or membrane-bound to endoplasmatic reticulum, mitochondria and peroxisomes. Recent research has focused mainly on characterization of microsomal carbonyl reducing enzymes. It has been decribed, only one microsomal carbonyl reductase in metabolism of xenobiotics, 11β-hydroxysteroid dehydrogenase 1. There is a discrepancy in metabolism of oracin between human liver microsomes and purified 11β-hydroxysteroid dehydrogenase 1 so an unknown microsomal carbonyl reductase participates in oracin biotransformation. This enzyme was purified. Carbonyl reductases have broad substrate specificity and also metabolize the strongest tobacco smoke carcinogen NNK. These enzymes reduce NNK to NNAL that can be excrected. The aim of this thesis is to optimize the method for determining metabolite NNAL...
|
guest ::
