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Metabolism of carcinogenic o-nitroanisol and its metabolite o-nitrophenol and two environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone
Svobodová, Martina ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Souček, Pavel (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Metabolism of carcinogenic o-nitroanisole, its metabolite o-nitrophenol and environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone Summary of PhD Thesis RNDr. Martina Svobodová Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Martina Svobodová Introduction -1- INTRODUCTION 2-Nitroanisole 2-Nitroanisole (2-methoxynitrobenzene, 2-NA, figure 1) is an important industrial pollutant and a strong carcinogen for rodents causing neoplastic transformation in the urinary bladder and, to a lesser extent, in the spleen, liver and kidney [19, 30, 31] . 2-NA is also a toxic compound, causing anemia. 2-NA is used primarily as a precursor in the synthesis of o-anisidine (2-methoxyaniline), which is an intermediate in the production of many azo dyes. This compound is used in pharmaceutical industry as an intermediate in the synthesis of some medicaments [30, 31] . In spite of potent rodent carcinogenicity of 2-NA, this chemical is weakly mutagenic in the Ames test with the Salmonella typhimurium. This carcinogen also exhibits a low activity in cytogenetic tests. It induces a slight increase in chromosomal aberration and in sister chromatid exchanges, but only at high concentrations [31] . 2-nitroanisole may be...
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Eukaryotic and prokaryotic nitric oxide synthase - structure-function studies
Mikula, Ivan ; Martásek, Pavel (advisor) ; Entlicher, Gustav (referee) ; Král, Vladimír (referee)
Nitric oxide (NO) is an important signaling molecule in organisms. It plays a role in wide spectrum of physiological and pathophysiological processes, including vasodilatation, neurotransmission and host defense. The gaseous molecule of NO is produced by oxidative reaction catalyzed by proteins from the family of nitric oxide synthases (NOSs). Three NOS isoforms were identified in mammals, endothelial (eNOS), neuronal (nNOS) and inducible or immunologic (iNOS). Some bacteria harbor genes coding for proteins homologous to the mammalian NOS oxygenase domain and showing NO-producing activity in vitro. NO generated by pathologic organisms such as B. anthracis and S. aureus is supposed to play a critical role in the pathophysiological processes during the infection. Comparative study of bacterial NOS-like proteins and mammalian NOSs confirmed their principal similarity, but also revealed differences in the interactions of distinct bacterial proteins and mammalian NOS isoforms with different analogs of substrate L-arginine and various ligands. On the basis of the kinetics measurement of NO-rebinding a second NO-binding site in the active center of NOS was predicted. Further, the regulation of NO dynamic and release from the protein by the active site Hbonding network connecting the heme, the substrate and BH4...
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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Entlicher, Gustav (referee)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Recombinant aspartic proteases of blood-feeding parasites
Váchová, Jana ; Konvalinka, Jan (advisor) ; Entlicher, Gustav (referee)
The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.
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Liver gangliosides in cholestasis induced by bile duct ligation.
Hynková, Barbora ; Entlicher, Gustav (advisor) ; Ledvinová, Jana (referee)
Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17α− ethinylestradiol induced cholestasis, but the increase of b-series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatis induced bile duct ligation (HO-1 activator- hemine, HO-1 inhibitor- Sn-mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals, but a decrease of some a- and b- series gangliosides was observed. In group with activated HO-1 total sialic acid increased, but the composition of gangliosides...
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alpha5beta1 integrin as a therapeutical target for cerebral tumors
Bartík, Petr ; Entlicher, Gustav (advisor) ; Stiborová, Marie (referee) ; Rauch, Pavel (referee)
INTRODUCTION Cancer is a leading cause of death in Europe taking yearly about 1.7 million of lives. Modern medicine hav! made considerable progress in treatment of some type of cancer such as cancers of prostate, breast and skin with the 5-years survival rate óver 90%o. However. current treatment options (surgery, radiotherapy, chemotherapy) are apparently less effective against other kinds of"uncli with bad prognosis such as cancer of lung and brain. Some of the most lethal cancers are high gňa" gliomas, which account for the most of cerebral tuňo,.. Ťn.y u.. djft.."' rapidly growing' highly inťrltrative and abundantly vascularized. These óháracteristics together with the delicate place of their incidence result in bad prognosis oť patients diagnosed with these cancers (Lefranc et al., 2006). Despite some recent advances the current non-targeted therapies only slightly improve patients post diagnosis survival rates ňd -aggróssive gliomas remain incurable disease. That is wtry tňJneuro- oncologists call for novel and more efficient treatment modalities. The hallmarks of tumor progression are the capacities of tumor cells to multiply rapidly, invade 'urrounáing tissue and stimulate endothelial cells to form new vasculature to nourish them. Therefore the aims of novel therapies should be to block the...
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Effects of valproic acid and its combinations with cytostatic agents on tumor cells in vitro
Hinďoš Hřebačková, Jana ; Dyr, Jan (advisor) ; Vávrová, Jiřina (referee) ; Entlicher, Gustav (referee)
Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...
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