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Enzyme kinetics and regulation of shikimate dehydrogenase from parsley roots.
Šmeringaiová, Ingrida ; Hýsková, Veronika (advisor) ; Kalendová, Alžběta (referee)
In plants the shikimate pathway followed by the phenylpropanoid pathway leads to the formation of not only aromatic amino acids but also a plenty of secondary metabolites. The crucial enzyme shikimate dehydrogenase is there a part of bi-functional protein 3-dehydroquinate dehy- dratase/shikimate dehydrogenase (DHD/SDH; EC 4.2.1.10 and EC 1.1.1.25). Whereas the regulation of the shikimic pathway is complex, little is known about the feedback regulation of plant SDH. The aim of this project was to find plant source with high SDH activity and to find out, if and in which way this SDH is regulated by compounds of phenylpropanoid metabolism, specifically by simple polyphenols. SDH from parsley root (Petroselinum crispum) has been prepared by 3-step purification to final specific activity 470 mol.min-1 .mg-1 . The enzyme exhibited one band after both isoelectric focusing and red native electrophoresis using detection of the activity. Relative molecular mass of native enzyme was determined by gel chromatography (Mr 60 000) and red native electrophoresis (Mr 63 000). Isoelectric point pI 4.5 was established by isoelectric focusing. Optimal pH for the reaction catalyzed by SDH was determined in rage pH 9.5 - 10.0. The kinetic parameters (Km, Vmax) of SDH for substrate shikimate, and coenzyme NADP+ were...
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Substituated Poly(ethylene Glycols) as Drugs Carriers
Pravda, Martin ; Hrabálek, Alexandr (advisor) ; Dittrich, Milan (referee) ; Kalendová, Andrea (referee)
The aim of this dissertation thesis was preparation of drug delivery systems for polyene antimycotics, especially amphtoricin B(AmB). Series of pH-sensitive conjugates of AmB with substituated poly(ethylene glycols) (PEG) have been synthesized and characterized for this purpose. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AmB gave conjugates containinga pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. The stability of prepared conjugates under the different physiological conditions was studied. Phosphate buffers (pH = 7,4 or 5,5) were used as model media. Stability of conjugates in human blood plasma and human blood serum was examinated. The imine linkage is split to give free AmB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights ofthe PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4...
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Anticorrosive properties of silicotungstic acid and phosphotungstic heteropolyacid in the paint films
Nechvílová, K. ; Kalendová, A. ; Stejskal, Jaroslav
The aim of this work was to provide an initial characterisation of heteropolyacids and examine their behaviour and properties as pigments in paints using different concentrations of the pigment and filler. Solvent-based epoxy-ester resin served as the binder. The paints were applied to steel and glass panels for mechanical and corrosion resistance testing. The organic coatings were exposed to a corrosive atmosphere with NaCl. This test was completed with electrochemical measurement. The parameters examined included the paint film's mechanical and anticorrosion efficiency. The environments in which the new coatings are most efficient were also sought.
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Enzyme kinetics and regulation of shikimate dehydrogenase from parsley roots.
Šmeringaiová, Ingrida ; Hýsková, Veronika (advisor) ; Kalendová, Alžběta (referee)
In plants the shikimate pathway followed by the phenylpropanoid pathway leads to the formation of not only aromatic amino acids but also a plenty of secondary metabolites. The crucial enzyme shikimate dehydrogenase is there a part of bi-functional protein 3-dehydroquinate dehy- dratase/shikimate dehydrogenase (DHD/SDH; EC 4.2.1.10 and EC 1.1.1.25). Whereas the regulation of the shikimic pathway is complex, little is known about the feedback regulation of plant SDH. The aim of this project was to find plant source with high SDH activity and to find out, if and in which way this SDH is regulated by compounds of phenylpropanoid metabolism, specifically by simple polyphenols. SDH from parsley root (Petroselinum crispum) has been prepared by 3-step purification to final specific activity 470 mol.min-1 .mg-1 . The enzyme exhibited one band after both isoelectric focusing and red native electrophoresis using detection of the activity. Relative molecular mass of native enzyme was determined by gel chromatography (Mr 60 000) and red native electrophoresis (Mr 63 000). Isoelectric point pI 4.5 was established by isoelectric focusing. Optimal pH for the reaction catalyzed by SDH was determined in rage pH 9.5 - 10.0. The kinetic parameters (Km, Vmax) of SDH for substrate shikimate, and coenzyme NADP+ were...
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Functions of actin and myosin 1c in the cell nucleus and in the cytoplasm
Kalendová, Alžběta ; Hozák, Pavel (advisor) ; Binarová, Pavla (referee) ; Forstová, Jitka (referee)
Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...
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Substituated Poly(ethylene Glycols) as Drugs Carriers
Pravda, Martin ; Hrabálek, Alexandr (advisor) ; Dittrich, Milan (referee) ; Kalendová, Andrea (referee)
The aim of this dissertation thesis was preparation of drug delivery systems for polyene antimycotics, especially amphtoricin B(AmB). Series of pH-sensitive conjugates of AmB with substituated poly(ethylene glycols) (PEG) have been synthesized and characterized for this purpose. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AmB gave conjugates containinga pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. The stability of prepared conjugates under the different physiological conditions was studied. Phosphate buffers (pH = 7,4 or 5,5) were used as model media. Stability of conjugates in human blood plasma and human blood serum was examinated. The imine linkage is split to give free AmB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights ofthe PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4...
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