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Methionine synthase as a potential therapeutic target
Kellovská, Kristýna ; Baszczyňski, Ondřej (advisor) ; Snášel, Jan (referee)
This thesis focuses on the enzyme methionine synthase (MS), which catalyzes methylation of homocysteine to produce methionine. Two main families of these enzymes are recognized in nature - cobalamin-dependent and cobalamin-independent. These two enzymes share no sequence homology, and they also use different catalytic mechanisms, substrates and cofactors. Cobalamin-dependent MS is found in humans, whereas cobalamin-independent MS is typical for plants and fungi. In humans, the enzyme provides a connection between folate and methionine cycle - two metabolic pathways which are crucial for example for DNA synthesis and S-adenosylmethionine-dependent biological methylations. Recently, the enzyme has been recognized as a potentially promising target for the development of chemotherapeutics and antifungal drugs, mainly based on its essentiality for the proliferation of cancer cells and both viability and virulence of pathogenic fungal species.
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Impact of the pre-analytical phase on enzymatic homocysteine determination
VOSÁTKOVÁ, Eva
This work deals with the stability of homocysteine in blood plasma, it emphasizes the importance of the preanalytical phase of laboratory examination, from the preparation of the patient for blood collection, to the insertion of samples into a fully automatic analyzer. The theoretical part describes homocysteine as such, the diagnostic significance of its examination and describes the laboratory environment. The experimental part monitors the concentration of homocysteine in samples that were processed after a different time interval from blood collection. The measured values of plasma homocysteine concentrations showed that with increasing time lag between sampling and processing itself, the stability of the analyte to be determined is impaired. The stability of plasma homocysteine was also verified in samples processed immediately after blood collection and left at room temperature.
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2-DE analysis of hepatocyte secretome under hyperhomocysteinemia
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Novák, Petr (referee)
Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these...
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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Folate and its role in early embryonic development of higher vertebrates.
Zahradníková, Hana ; Tlapáková, Tereza (advisor) ; Procházková, Michaela (referee)
Folate or vitamin B9 is an essential water-soluble nutrient that takes part in important cellular processes. These include amino acid metabolism, methylation, and nucleotide synthesis, the last two of which play a key role in early embryonic development. It was approximately 30 years ago when folic acid supplementation was confirmed to help prevent embryonic neural tube defects and since then an intake of 400 µg of folate a day has been recommended to all women of childbearing age. Despite this, folate deficiency is a relatively common phenomenon and during pregnancy can lead to congenital malformations and pregnancy complications. This bachelor thesis summarizes the latest findings on the association of neural tube defects and miscarriages with single-nucleotide polymorphisms in genes encoding folate metabolism enzymes in humans, focusing mainly on the gene encoding the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Furthermore, this thesis deals with potential mechanisms of the folate's protective effect which could participate in the prevention of neural tube defects and are studied mainly on model organisms. Key words: folate, embryo, neural tube defects, MTHFR, homocysteine, miscarriage, single-nucleotide polymorphisms
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef ; Jelínek, Ivan (advisor) ; Čabala, Radomír (referee) ; Cvačka, Josef (referee)
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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2-DE analysis of hepatocyte secretome under hyperhomocysteinemia
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Novák, Petr (referee)
Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these...
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Suplementace kreatinu u sportovce s hyperhomocysteinemií
Kohlíková, E. ; Petr, M. ; Navrátil, Tomáš ; Přistoupilová, K. ; Přistoupil, T. I. ; Pelclová, D. ; Žák, J. ; Heyrovský, Michael
One month's administration of creatine (CR) in the dose of 5 g/day to 11 young active sportsmen affected their daily amount of CR and creatinine excreted into urine. The probands were divided into 4 groups, according to the amount of CR found in urine, and of folates and of vitamin B12 determined in blood. The ganges of folates and vitamin B12 were mutually reciprocal. Each group utilized CR as donor of one- and two-carbon (1C and 2C) units by means of homocysteine (HoCySH), folates, and vitamin B12, in different metabolic pathways. In 10 men the CR administration was accompanied by an increase of HoCySH level in blood, while for the last man, with accidentally discovered hyperhomocysteinemia, after the month's CR administration the HoCySH level dropped by 50 %.
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Vliv suprafyziologických dávek kreatinu na homocysteinový metabolismus u sportovců
Kohlíková, E. ; Petr, M. ; Přistoupilová, K. ; Šenholdová, Z. ; Přistoupil, T. I. ; Pelclová, D. ; Žák, J. ; Heyrovský, Michael ; Navrátil, Tomáš
One month´s administration of creatine (CR) in the dose of 5 g/day to 11 young men affected their daily amount of CR, creatinine, and thiodiglycolic acid (TDGA) excreted into urine. The probands were divided into 4 groups, according to the amount of CR found in urine, and of folates and of vitamin B12 determined in blood. The changes of folates and vitamin B12 were mutually reciprocal, as well as muscle mass and body fat. Each group utilized CR as donor of one- and two-carbon (1C and 2C) units by means of homocysteine (HoCySH), folates, and vitamin B12, in different metabolic pathways.
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