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Development od methods for analysis of interactions of the antiviral drug ritonavir with lanosterol-14- α-demethylase of pathogenic yeasts
Burdová, Tereza ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Ritonavir is an HIV protease inhibitor, and has been used as a part of drug cocktails for highly active antiretroviral therapy (HAART). Oropharyngeal candidiasis is one of the most common opportunistic infections among AIDS patients. The research showed that those who were treated with cocktails containing HIV protease inhibitors including ritonavir suffered from oropharyngeal candidiasis to a much lesser extent than patients who received drugs without protease inhibitors. The reason may be the fact that HIV protease inhibitors also inhibit the proteases of pathogenic yeasts, despite high Ki. Another explanation may be that some of these inhibitors block lanosterol-14-α- demethylase, a key enzyme in the biosynthesis of ergosterol, which is an important component of yeast membranes. Aim of the thesis is the isolation of the microsomal fraction of the pathogenic yeasts Candida albicans and Candida parapsilosis and optimization of methods for analysis of the inhibition of yeast lanosterol-14-α- demethylase by ritonavir. Key words: ritonavir, Candida albicans, Candida parapsilosis, lanosterol-14-α- demethylase, microsomal fraction
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Isolation and characterization of microsomal fraction of fungus Pleurotus ostreatus and its role in the degradation of 17α-ethinylestradiol
Valášková, Petra ; Černá, Věra (advisor) ; Hodek, Petr (referee)
A synthetic hormone 17α-ethinylestradiol (EE2) which is a component of hormonal contraception pills has been identified as a main component of the endocrine-disrupting compounds (EDc). EDc are substances that mimic natural hormones in their action. Recently their amount especially in the groundwater and the surface water has been increased, which results in a negative impact on the hormonal system especially of aquatic organisms. Since it is not easy to replace these substances from the environment by conventional techniques other possibilities of their biodegradation are examined. White rot fungi, which are able to degrade lignin in nature, have promising biodegradation abilities towards many pollutants. These fungi contain a wide range of non-specific extracellular and intracellular enzymes that play an important role in the degradation. This bachelor thesis was targeted on the study of a white rot fungus, Pleurotus ostreatus, and especially on the degradation potential of its intracellular enzymes in the biodegradation of EE2. Initially, the ability of fungi Pleurotus ostreatus to degrade EE2 in vivo was tested. During the 48 hour incubation there was replaced 95,5 % of EE2. However, the role of cytochromes P450 (CYPs) in a metabolism of EE2 was not confirmed in this experiment by reason that an...
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
HAA are compounds which are showing numerous carcinogenic impacts on studied animals even human cells. These carcinogenes arise during the heat processing of meat or during (cigarette) smoking. Activation of these compounds is required to their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP) especially subfamily 1A1 and 1A2. As a consequence of activation with these enzymes are created N-hydroxylamines, which weakly reacting with DNA. For better formation of DNA aducts one more activation is essential. More reactive acetate and sulphate esters arise by second activation from N- hydroxylamines. The esters are produced by sulphotranspherase (SULT) even N- acetotranspherase (NAT). When we affect these enzymes we could positive control the formation of carcinoma. Caffeic acid is considered as a strong inhibitor of one SULT subfamily (phenolic sulfotranspherase P - PST). On the other side as a good inhibitor of NAT is considered (known) quercetin. (in czech) Key words: Heterocyclic amine, biotransformation, cytochrome P450, sulfotransferase, N-acetyltransferase
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The Bachelor Thesis deals with an activation of heterocyclic aromatic amines (HAA) which have numerous carcinogenic effects on studied animals and human cells. These carcinogens are formed during the heat processing of meat and during the smoking. However, further transformation of the compounds is required to gather their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP), especially its forms 1A1 and 1A2. The products of this activation - N-hydroxylamines - are further activated in conjugation reactions. In this work, we were focused on the transformation of N-hydroxylamines to more reactive acetate esters and sulphate esters, which is catalyzed by sulphotranspherase (SULT) and N-acetyltranspherase (NAT), respectively. The affection of these enzymes can control the formation of carcinoma. For example, some dietary compounds, such as caffeic acid and quercetin, are the most common inhibitors of these enzymes: caffeic acid is considered as a strong inhibitor of phenolic sulphotranspherase (P-PST), whereas quercetin is a good inhibitor of NAT. On the other hand, some dietary compounds can also induce an opposite effect: for instance, phenol acids induce the P-PST. (in Czech) Key words: Heterocyclic aromatic amines, biotransformation, cytochrome P450,...
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The effect of selected endocrine disruptors on the cytochromes P450 1A1 and 2C
Klusoňová, Zuzana ; Bořek Dohalská, Lucie (advisor) ; Linhartová, Lucie (referee)
Many currently produced chemicals reveal specific properties which allow them to be referred to as endocrine disruptors (ED). These substances exhibit an exogenic hormone activity and usually act as antagonists or agonists of endogenic hormones. The exogenic EDs studied in this work were 17α-ethinylestradiol (EE2) and benzoapyrene (BaP). 17β-estradiol (E2), a typical endogenic hormone, was also included to the study. In the presented work, the effect of these EDs and their combinations on the expression and specific activities of cytochromes P450 (CYP) 1A1 and 2C was determined. First, the microsomal fraction (MF) of liver, kidney and lung of rats premedicated with these compounds or without premedication was isolated. CYP expression was assessed by the Western blot analyses in these MF samples. Moreover, CYP1A1 and CYP2C specific activities were evaluated. It was found that premedication of rats with BaP increased CYP1A1 expression in all above mentioned organs. Whereas BaP strongly induced rat CYP1A1, EE2 and E2 were almost without this effect. But, when these disruptors were administered to rats with BaP, they supported its potency to induce CYP1A1. Further, CYP2C11 expression and its specific activity were gently increased by premedication of rat with EE2 and its combination with BaP....
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Study of the metabolism of 17α-ethinylestradiol by cytochromes P450
Valášková, Petra ; Černá, Věra (advisor) ; Levová, Kateřina (referee)
A synthetic estrogen 17α-ethinylestradiol (EE2) is the main active component of the hormonal contraceptive pills. The rise of consumption of hormonal contraceptives has increased the risk of the back negative effects of EE2 to aquatic organisms. EE2 belongs to the endocrine disruptive compounds known for mimicking natural hormones. A more detailed examination of the transformation of this compound in vivo and in vitro can contribute to a better understanding of its negative effects. This master thesis is therefore devoted to the study of the metabolism of EE2 in two selected model organisms. The ligninolytic fungus Pleurotus ostreatus is the type of fungi with promising biodegradation ability to a lot of pollutants. These properties have led to numerous studies of the degradation potential of P. ostreatus towards EE2, with the possibility of removing this compound from the environment. EE2 has been degraded by the fungus P. ostreatus in vivo resulting in one hydroxylated metabolite, which estrogenic activity is in need for further study. In vitro studies were carried out with a microsomal fraction isolated from the mycelium of this fungus. The conversion of EE2 in vitro via CYPs dependent on NADPH has not been demonstrated, however using KHP as a cofactor, there was one metabolite of EE2 found,...
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
HAA are compounds which are showing numerous carcinogenic impacts on studied animals even human cells. These carcinogenes arise during the heat processing of meat or during (cigarette) smoking. Activation of these compounds is required to their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP) especially subfamily 1A1 and 1A2. As a consequence of activation with these enzymes are created N-hydroxylamines, which weakly reacting with DNA. For better formation of DNA aducts one more activation is essential. More reactive acetate and sulphate esters arise by second activation from N- hydroxylamines. The esters are produced by sulphotranspherase (SULT) even N- acetotranspherase (NAT). When we affect these enzymes we could positive control the formation of carcinoma. Caffeic acid is considered as a strong inhibitor of one SULT subfamily (phenolic sulfotranspherase P - PST). On the other side as a good inhibitor of NAT is considered (known) quercetin. (in czech) Key words: Heterocyclic amine, biotransformation, cytochrome P450, sulfotransferase, N-acetyltransferase
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The Bachelor Thesis deals with an activation of heterocyclic aromatic amines (HAA) which have numerous carcinogenic effects on studied animals and human cells. These carcinogens are formed during the heat processing of meat and during the smoking. However, further transformation of the compounds is required to gather their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP), especially its forms 1A1 and 1A2. The products of this activation - N-hydroxylamines - are further activated in conjugation reactions. In this work, we were focused on the transformation of N-hydroxylamines to more reactive acetate esters and sulphate esters, which is catalyzed by sulphotranspherase (SULT) and N-acetyltranspherase (NAT), respectively. The affection of these enzymes can control the formation of carcinoma. For example, some dietary compounds, such as caffeic acid and quercetin, are the most common inhibitors of these enzymes: caffeic acid is considered as a strong inhibitor of phenolic sulphotranspherase (P-PST), whereas quercetin is a good inhibitor of NAT. On the other hand, some dietary compounds can also induce an opposite effect: for instance, phenol acids induce the P-PST. (in Czech) Key words: Heterocyclic aromatic amines, biotransformation, cytochrome P450,...
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Isolation and characterization of microsomal fraction of fungus Pleurotus ostreatus and its role in the degradation of 17α-ethinylestradiol
Valášková, Petra ; Černá, Věra (advisor) ; Hodek, Petr (referee)
A synthetic hormone 17α-ethinylestradiol (EE2) which is a component of hormonal contraception pills has been identified as a main component of the endocrine-disrupting compounds (EDc). EDc are substances that mimic natural hormones in their action. Recently their amount especially in the groundwater and the surface water has been increased, which results in a negative impact on the hormonal system especially of aquatic organisms. Since it is not easy to replace these substances from the environment by conventional techniques other possibilities of their biodegradation are examined. White rot fungi, which are able to degrade lignin in nature, have promising biodegradation abilities towards many pollutants. These fungi contain a wide range of non-specific extracellular and intracellular enzymes that play an important role in the degradation. This bachelor thesis was targeted on the study of a white rot fungus, Pleurotus ostreatus, and especially on the degradation potential of its intracellular enzymes in the biodegradation of EE2. Initially, the ability of fungi Pleurotus ostreatus to degrade EE2 in vivo was tested. During the 48 hour incubation there was replaced 95,5 % of EE2. However, the role of cytochromes P450 (CYPs) in a metabolism of EE2 was not confirmed in this experiment by reason that an...
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Biodegradation of polychlorinated biphenyls by white - rot fungi and their enzymes
Linhartová, Lucie ; Bořek Dohalská, Lucie (advisor) ; Černá, Věra (referee)
Polychlorinated biphenyls (PCB) represent relevant persistent organopollutants of the environment and the estimated amount of PCB released into the environment is 750000 metric tons. White-rot fungi have been studied for long time due to their degradative potential toward various aromatic pollutants and it is known that these fungi are able to decompose PCB in vivo. Biodegradation of PCB by the fungus Pleurotus ostreatus was studied in the frame of this work. A high degradative efficiency of P. ostreatus was observed in the first set of experiments, even in the presence of relative high amount of added PCB. Fungus was able to transform 780±50 µg out of the intial amount 1000 µg in 20 ml of a cultivation media within 42 days. A decrease in toxicity was recorded during the degradation that suggests the suitability of this organism for a practical use in decontamination. In vitro experiments with purified laccase induced with Cu2+ from this fungus did not prove any participation of the enzyme in the first step of PCB transformation. The enzyme did not show an ability to degrade PCB even after purification from cultivation media containing PCB. It was found that the first step of PCB transformation can be performed by an intracellular process with microsomal fraction. A degradation of 44-67% was observed for...
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