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Protease of bovine follicular fluid
Drahokoupil, Marek ; Liberda, Jiří (advisor) ; Hýsková, Veronika (referee)
This bachelor thesis deals with isolation and partial characterisation of cow protease follicular fluids. At first, the follicular fluid was isolated according to molecular weights by means of gel chromatography on Sephadex G-100. As another method of separation there was selected the ion exchange chromatography on Sephadex DEAE. For the characterization of resulting fractions SDS electrophoresis, assessment of proteolytic activity of the proteolytic substrate azakasein, zymography and differential SDS electrophoresis were used. The collected fractions of the follicular fluid defined by the gel chromatography method had the relative molecular weights raging 114 000 - 131 000 in fraction pattern I, 44 000 - 51 000 in fraction pattern II, 151 000 - 204 000 in fraction pattern III, 57 000 - 99 in fraction pattern IV, 14 000 - 38 000 in fraction pattern V and 180 - 500 in fraction pattern VI. From the results of the proteolytic activity I decided to work further only with fraction patterns III, IV and V collected from the gel chromatography and with the pattern of the delayed fraction PBS II collected from the ion exchange chromatography. These patterns reported the highest specific activity. Inhibitors were used to determine which type of protease occurs in thee chosen fractions. In the fraction...
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Identification of antimicrobila peptides in spider venom
Benýšek, Jakub ; Tichá, Marie (advisor) ; Liberda, Jiří (referee)
Still increasing resistance to antibiotics leads to the need to find new active compounds with antimicrobial properties. This work is focused on the occurrence, chemical and physical description, mechanism of action and biological activity of such substances, found in spider venom. The second part is focused on isolation and identification of compounds with these properties from the venom of wild bees and a one spider. A novel peptide was isolated and identified from venom of bee Trachusa byssina. This novel peptide possess antimicrobial properties and low hemolytic activity. Molecular weight was estimated to 1749,9 ? 0,1contains 16 amino acids and is amidated on its C-terminus. Its primary structure GILSVLKNLLKKHMAS-NH2 was determined by using Edman degradation and ESI-QTOF mass spektrometry.
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The study of fluids and secretions from reproductive tracts of pig (Sus scrofa f. domestica) and cattle (Bos primigenius f. taurus)
Dráb, Tomáš ; Liberda, Jiří (advisor) ; Jonáková, Věra (referee) ; Ulčová-Gallová, Zdeňka (referee)
Interactions between proteins and saccharide moieties play an indispensable role in mammalian reproduction as they stand behind of such processes as maturation and mutual recognition of gametes and sperm oviductal reservoir formation. In my dissertation thesis I focused on activities of glycosidases from bovine and porcine follicular fluids and their changes connected with follicle development. Activities of five glycosidases were detected in tertiary and preovulatory follicles in both species. The most active enzymes were α-L-fucosidase in cow and α-D-mannosidase in sow and both enzymes also demonstrated the most pronounced increase in their activities during follicle maturation. Interestingly, both α-L-fucose in cow and α-D-mannose in sow were described as saccharides responsible for the formation of the sperm oviductal reservoir and we offered a hypothetical mechanism of synchronisation between sperm release from their reservoir with the time of ovulation based on a surge of activities of corresponding follicular glycosidases through the oviduct. Subsequently, it was demonstrated that β-D-galactosidase and α-D-mannosidase affect sperm-zona pellucida binding in pig, as they both decrease interaction between sperm receptors for zona pellucida and zona pellucida. This may explain the observation...
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Properties of plant extracts
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Plants synthesize a wide variety of compounds called secondary metabolites. These compounds comprise products of the phenylpropanoid pathway (flavonoids, stilbens, anthocyanines and tannins), isoprenoids, and alkaloids. Since plant secondary metabolites seem to affect human health in a positive way, decrease the risk of some diseases such as cancer, diabetes type 2 and disorders of the cardiovascular system, their studies attract a lot of attention. The mechanism of the effect of individual compounds on human health has not been clarified yet, the positive effect is probably caused by their antioxidant capacity. In this work 22 extracts from different kinds of fruit and vegetables were prepared. The total content of phenolic compounds and flavonoids, the antioxidant capacity by methods FRAP (ferric ion reducing antioxidant power) and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6- sulphonic acid)] were determined. High content of phenolic compounds was established in the extract from strawberry, cherry and kiwi, the highest antioxidant capacity was found in the extract from strawberry. Another studied attribute of extracts from fruit and vegetables was their influence on the activity of digestive enzymes pancreatic lipase and α-amylase. The extracts from lemon, red currant, jostaberry, lime and...
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Influence of cytokines on peripheral metabolism of glucocorticoids
Vitáčková, Kateřina ; Pácha, Jiří (advisor) ; Liberda, Jiří (referee)
11β-hydroxysteroid dehydrogenase (11βHSD) participates in prereceptor metabolism of glucocorticoids by conversion of their active forms (cortisol or corticosterone) to inactive forms (cortisone or 11-dehydrocorticosterone), thereby regulating concentration and activity of glucocorticoids in organism. 11βHSD type 1 (11βHSD1) activates glucocorticoids from their biologically inactive keto-forms, whereas the role of 11βHSD type 2 (11βHSD2) is their biological reinactivation. Previous in vivo studies showed that mRNA expression and enzyme activity of 11βHSD1 or 2 can be modified by influence of these cytokines, for example IL-1β and TNF-α. The aim of this study was to determine whether these cytokines play a role in regulation of 11βHSD expression in distal colon organ cultures and whether the changes of 11βHSD2 expression are induced by a direct effect of cytokines on epithelial cells. Total RNA was isolated from all samples and RNA was transformed to cDNA by reverse transcription. Subsequently mRNA of 11βHSD1 or 2 and the given housekeeping gene was measured by Q-PCR. Incubation of distal colon organ cultures with TNF-α (10 ng/ml; 48 hours) caused significant increase of 11βHSD1 expression, but no influence on 11βHSD2 expression was observed. In contrast, the expression of 11βHSD2 but not 11βHSD1 was...
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