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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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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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The effect of Hsp70 protein on the infection caused by Potato virus Y
Doričová, Vlasta ; Hýsková, Veronika (advisor) ; Hoffmeisterová, Hana (referee)
Whithin their natural environment, plants are subjected to a combination of stress conditions. Since potential interactions between signal pathways, plants respond to multiple stresses differently from how they do to individual stresses, activating a specific programme. Heat shock proteins (HSP70) overexpressed after heat shock influence the viral infection. On one side HSP70 can participate on refolding of aggregated or partially denaturated proteins, on the other side HSP70 can interact with viral proteins and facilitate propagation of viral replication complexes. In this work the effect of heat shock (42řC, 2. hours) applied before or after the inoculation of plants Nicotiana tabacum L. cv. Petit Havana SR1 with Potato virus Y on viral infection was detected. This effect was studied in two biological experiments. The amount of coat protein of PVYNTN and protein HSP70 were detected simultaneously with the activity assays of Hatch-Slack cycle enzymes, glycosidases and peroxidase. Both experimental approaches (heat shock applied before or after the inoculation by PVYNTN ) enhanced amount of the virus and in the 2nd experiment it accelerated infection development. Immediately after application of heat shock the amount of HSP70 was increased. The enhancement of HSP70 by viral infection occurred...
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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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Antioxidant properties of medicinal plants
Tupec, Michal ; Hýsková, Veronika (advisor) ; Poljaková, Jitka (referee)
During the evolution plants have developed rather efficient mechanisms for the re- moval of harmful compounds, which include e. g. the reactive oxygen species (ROS). These mechanisms are mainly based on the antioxidant effects of ascorbic acid, gluta- thione in intimate cooperation with superoxide dismutase, catalase and various perox- idases - the reduced coenzymes, NADH and NADPH, play a major role in the ROS elimination, too. However, in recent times, the attention has turned from these key compounds of primary metabolism to secondary metabolites. Interestingly, the phenolic com- pounds together with some terpenes and alkaloids exhibit antioxidant effects, too, and apparently they play an irreplaceable role in the life of a plant - besides their antiox- idant action they are also important in the fight against pathogens and herbivores, in attracting the pollinators or in the moderation of UV light effects. Natural products are also studied in their relation to man - a broad variety of effects is known including the antioxidant, anti-inflammatory and anticancer effects for the most part. In this work, extracts comprising mainly of low-molecular-weight compounds were obtained from 32 plants with either hot water, 80 %w aqueous methanol or just by plain juicing. The extracts were then examined by three...
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Laboratory tests for thyroid function assessment from the economic point of view
Stehlíková, Petra ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
In 2011 Recommendation for laboratory diagnostics of functional and autoimmune thyroid diseases was published. Its target is to recommend correct method when choosing thyroid examination parameters to general practitians and thus find a way how to save money on useless examinations which then can be used by hospital in other area. To verify physicians adhere to these recommendations file of VFN patients in Prague for year 2011 was used and statistically evaluated. Watched parameters included thyrotropin (TSH), free triiodothyronine (FT3), total triiodothyronine (T3), free thyroxine (FT4), total thyroxine (T4), thyroid peroxidase antibodies (anti - TPO), thyroglobulin antibodies (anti - Tg), thyroglobulin (Tg) and thyroxine - binding globulin (TBG). Basic test according to published recommendation should include only TSH and if out of reference limits then it should be followed by FT4 test or antidote assessment. Other parameters are unnecessary for initial examination, some of them are not used anymore or should be examined only by clinics specialized in endocrinology. Based on the file records many physicians are still ordering unnecessary examination parameters. In one year 13,8 % of unnecessary parameters out of 44 415 thyroid function examinations were determined. This is 1 095 013 points that could by...
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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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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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