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The influence of stress on NADP-dependent enzymes in higher plants.
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Dračínská, Helena (referee)
Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...
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Phospholipase D Beta in hormonal signaling of Arabidopsis Thaliana
Krutinová, Hana ; Ryšlavá, Helena (advisor) ; Hýsková, Veronika (referee)
Plants, as well as all other living organisms, have to react to environmental changes and are forced to put up with the danger that comes from their environment. As a response to this danger, a sophisticated defense system, which moderates responses to stress cues, comes from the outside as well as created inside the plant itself, has evolved in plants. The signaling in this system is mediated by a number of phytohormones, which include salicylic acid, jasmonic acid, abscisic acid, ethylen and auxins. The phytohormone signaling results in a transcription of specific genes. One of the essential components of this signaling network is the phospholipid signaling system, where enzymes called phospholipases play a major role. These enzymes catalyze the hydrolysis of phospholipids, which are part of cellular membranes and products of the hydrolysis can act as signal transferring molecules. In this thesis we studied the potential engagement of phospholipase Dβ isoform into signaling pathways, using an Arabidopsis thaliana mutant plant with mutations in both genes coding phospholipase Dβ. Firstly, we verified that wild type phospholipase Dβ mRNA is not being transcribed in the mutant plants. Subsequently, we compared mutant and wild type plants' reactions to mechanical wounding, pathogen infection and...
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Integration of pharmaceuticals with higher plants as a model of phytoremediation
Chupíková, Alice ; Smrček, Stanislav (advisor) ; Hýsková, Veronika (referee)
The presence of pharmaceuticals in wastewater, which are not eliminated in sewage treatment plant process and thus get easily into rivers and aquatic environment in general, constitutes a severe problem to the whole society. The research into the removal of pharmaceuticals from the environment began about twenty years ago. Phytoremediation represents one of the most promising wastewater treatment methods. It is based on the ability of plants to remove xenobiotics from their environment and sequester them, build them into their tissues or degrade them. Many plant species have been tested as potential phytoremediation agents including maize (Zea mays L.) and sunflower (Helianthus annuus L.). These two plant species were also used for phytoremediation experiments described in this project. The sunflower BELEM variety and the maize G1 variety were cultivated in sterile media containing carbamazepine or its metabolite 10,11-epoxycarbamazepine. The same experiments were carried out with ibuprofen and acetaminophen for comparison. The antiepileptic drug carbamazepine belongs to the group of compounds, which are, due to their high stability, poorly degraded in the environment. Its metabolite 10,11-epoxycarbamazepine was synthesized by epoxidation of the double bond of carbamazepine using m-chlorperoxybenzoic acid...
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Isolation of proteins from bovine oviductal fluid by affinity chromatography on DNA cellulose
Litváková, Rozálie ; Liberda, Jiří (advisor) ; Hýsková, Veronika (referee)
Oviductal fluid was collected from cow oviducts. Obtained material was lyophilized and separated by affinity chromatography on DNA-cellulose. Proteins in non-binding and eluted fractions were identified electrophoretically, while those in the eluted fraction were further determined through application of the MALDI-TOF MS method. The complete oviductal fluid demonstrated antimicrobial activity. Furthermore, glycosidase activity of proteins isolated from oviductal fluid was also tested. A column has been designed and prepared in order to avoid the possibility of glycosidase activity leading to degradation. In czech.
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NADP-dehydrogenases of medicinal plants.
Bartošová, Martina ; Hýsková, Veronika (advisor) ; Kubíčková, Božena (referee)
Medicinal plants usually contain metabolites formed by secondary biosynthetic pathways for example phenylpropanoid and shikimate pathway, which depend on NAPDH availability. In this work total content of phenolic compounds and flavonoids was determined in 10 medicinal plants common for Czech Republic. Activity of enzymes providing NADPH was determined as well. These enzymes were glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (oxalacetate decarboxylating) (NADP-ME, EC 1.1.1.40). The activity of shikimate dehydrogenase (SDH, EC 1.1.1.25), the main enzyme of shikimate pathway, was also determined. Extraction methods were optimized for each assessment. Applicable source for NADP-dependent enzymes are black radish (Raphanus sativus L. var. niger), garlic (Allium sativum L.) and mint (Mentha piperita L). The highest content of phenolic compounds and flavonoids was determined in sage (Salvia officinalis), lemon balm (Melissa officinalis L.) and Mentha citrata. Extraction with methanol was more effective than extraction with hot water. More extraction methods and studies are necessary for characterization of individual compounds and principals on which they work. Key words: medicinal plants, NADP-dependent enzymes, phenolic...
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Abundance of NADP-dependent enzymes in plants
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Ryšlavá, Helena (referee)
NADPH is an important molecule in plant metabolism. It is an indispensable substrate for many processes; participates in redox balance of the cell and it is a part of antioxidant defense system in plants. In this work, activities of selected NADP-dependent enzymes producing NADPH, NADP-malic enzyme (oxalacetate decarboxylating) (NADP-ME, EC 1.1.1.40), NADP-isocitrate dehydrogenase (decarboxylating) (NADP-ICDH, EC 1.1.1.42), glukosa-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and shikimate dehydrogenase (SDH, EC 1.1.1.25) in various plants and their parts (leaves, fruits, seeds) were established and compared. The aim of this work was to select suitable model plants for testing the effect of abiotic stress on NADP-dependent enzymes and also to select the best source for purification of these enzymes. Cucumber (Cucumis sativa L.), pea (Pisum sativum L.), or water-lily (Nymphaea) were chosen as suitable water-receptive model plants and Capsella bursa-pastoris as xerophilic plant from 19 analyzed plants. From group of 39 plants the most suitable for purification of NADP-ME were roots of Chinese radish (Raphanus sativus var. Major), which were simultaneously a rich source of other analyzed NADP-enzymes. The best source of NADP-ICDH was poppy (Papaver somniferum), or quinoa Chenopodium quinoa. For...
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Connection between abiotic stress and NADP-dependent enzymes in plants
Plisková, Veronika ; Hýsková, Veronika (advisor) ; Kubíčková, Božena (referee)
Salt stress in plants is accompanied with decreased growth caused by lowered leaf water potential, closed stomata and thus reduced photosynthesis producing NADPH. However, NADPH is indispensable coenzyme for biosynthetic processes (e.g. biosynthesis of osmotically active compounds decreasing influence of stress) and for antioxidant system. In this study it was examined, whether NADP-dependent enzymes: glucose-6- phosphate dehydrogenase (G6PDH), NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME) and NADP-shikimate dehydrogenase (SDH), producing NADPH are involved in plant defence response against 100 mM NaCl. Three different model plants were used: Pisum sativa L., Phaseolus vulgaris L. and Cucumis sativa L. While in leaves of pea and bean plants salt stress caused rather decreased activities of particular NADP-dependent enzymes (especially of G6PDH and NADP-ICDH), in leaves of cucumber 9th and 11th day of stress condition were found increased activities of all studied enzymes. Simultaneously the Na+ content in salt-stressed plants was analysed by atomic emission spectrometry.
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Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes
Mizerovská, Jana ; Stiborová, Marie (advisor) ; Mareš, Jaroslav (referee) ; Hýsková, Veronika (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes Summary of PhD Thesis RNDr. Jana Mizerovská Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Jana Mizerovská Introduction 1 INTRODUCTION 3-Nitrobenzanthron, precursor of 3-ABA The nitroaromatic 3-nitrobenzanthrone (3-nitro-7H-benz de anthracen-7-one, 3-NBA) occurs in diesel exhaust and in airborne particulate matter(8, 9, 14) . 3-NBA is most likely formed during the atmospheric reaction of benzanthrone with nitrogen oxides, especially in the presence of ozone, or during imperfect burning of diesel. 3-NBA exhibits extremely high mutagenic activity(9, 14) and is also a genotoxic carcinogen causing lung tumors in rats(14) . 3-NBA is also evaluated to be a potential carcinogen for humans(14, 1, 9, 19) . The genotoxicity of 3-NBA was documented by the detection of specific 3-NBA-derived DNA adducts in vitro, in human cell lines and also in vivo in rats and mice(12, 13, 15, 2) . The predominant DNA adducts formed by 3-NBA after its metabolic activation by reduction of the nitro group are 2-(2'-deoxyguanosin-N2 -yl)-3- aminobenzanthrone and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone(9, 14) and these are most probably responsible for the...
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Plant alkaloid sanguinarine and its derivatives
Tůmová, Lucie ; Hýsková, Veronika (referee) ; Stiborová, Marie (advisor)
This work is summarizing actual knowledge about sanguinarine and quaternary benzo[c]fenanthridine alkaloids. The quaternary benzo[c]fenanthridine alkaloids were found in roots plants Sanguinaria canadensis and Macleaya cordata. This plants are used in tradicional Chinese medicine for its antimycotic, antibacterial and anti-inflammatory activities since antiquity. Regarding to possibility quaternary benzo[c]fenanthridine alkaloids to induce apoptosis these investigated such as possible agents for cancer treatment. The quaternary benzo[c]fenanthridine alkaloids interact with DNA and proteins. They are able to intercalate to the DNA. The alkaloids can be used like fluorescence DNA probe. Metabolism by sanguinarine and next quaternary benzo[c]fenanthridine alkaloids has not yet completely determinated. The first step sanguinarine detoxication is its conversion to less toxic dihydrosanguinarine. Sanguinarine oxidation is mediated by cytochrome P450 1A1. Key words: Quaternary benzo[c]fenanthridine alkaloids, sanguinarine, apoptosis, intercalate, heterogenous substances, enzymes, cytochrom P450
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