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Hemoprotein Nitric Oxide Synthase in Aplysia Californica
Buganová, Michaela ; Martásek, Pavel (advisor) ; Pelouch, Václav (referee) ; Druga, Rastislav (referee)
Nitric oxide (NO) plays a crucial role in neuronal signaling in a variety of eukaryotic and prokaryotic organisms. Nitric oxide synthases (NOS) are heme-containing monooxygenases that catalyze the oxygen dependent oxidation of L-arginine to NO and L-citrulline. The NO produced by NOS activity is a gaseous molecule that diffuses easily through membranes and acts inter or intracellularly. NO activates metal-containing enzymes, including soluble guanylate-cyclase (sGC) that increase levels of the messenger molecule cyclic 3,5-guanosine monophosphate (cGMP) (1, 2) which in turn mediate various pathophysiological or physiological functions in neurons. Nevertheless, many aspects of nitrergic neurons and NO function in the central nervous system (CNS) are unclear. The aim of research described in this thesis was to characterize neuronal NOS, proteins metabolically linked to NOS and NO signaling pathways in the CNS of Aplysia cali/ornica (Aplysia), a popular experimental model in cellular and system neuroscience. The biochemical characteristics of Aplysia NOS (AcNOSj described here revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence. A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia Polyclonal anti-rat nNOS antibodies hybridized with a putative purified...
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The role of NSD(P)H oxidase in signal transduction
Vejražka, Martin ; Štípek, Stanislav (advisor) ; Pelouch, Václav (referee) ; Zima, Tomáš (referee)
Reactive oxygen species are usually assumed as dangerous, cytotoxic substances included in pathogenesis of variety of diseases due to their ability to damage biomolecules. However, ROS also play an indispensable role in many physiological processes. Low concentrations of ROS are involved in signal transduction as a part of pathways regulating protein phosphorylation, gene expression, NO availability or intracellular Ca2+. They are important for cell cycle control and apoptose. In the last years, the role of ROS in regulation of vascular tonus is intensively studied. NAD(P)H oxidases are supposed the major source of ROS. These are enzymes similar to phagocyte NADPH oxidase, a key enzyme of phagocyte respiratory burst in immune response. We have studied NAD(P)H oxidase of vascular wall in normotensive and hypertensive rat. Activity of superoxide production by NAD(P)H oxidase differ between cells from normotensive and hypertensive rat. Control of NAD(P)H oxidase activity is also changed in hypertensive animals, at least with respect to the effect of angiotensin II and uric acid. In our experimental work, we encountered unexpected properties of apocynin, a known inhibitor of phagocyte NADPH oxidase. We proved that apocynin can increase ROS production. The reason is that NAD(P)H oxidase is not inhibited directly...
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The Role of Human Sco1, Sco2, Surf1 and Oxa1l in the Biogenesis of the Mitochondrial Oxidative Phosphorylation System
Stibůrek, Lukáš ; Zeman, Jiří (advisor) ; Entlicher, Gustav (referee) ; Pelouch, Václav (referee)
Disertační práce byla vypracována v Laboratoři pro studium mitochondriálních poruch, Kliniky dětského a dorostového lékařství a 1.LF UK v Praze. Studium mitochondriální biogeneze a jejích poruch, a to především s ohledem na biogenezi systému oxidativní fosforylace (OXPHOS), zaznamenalo v uplynulém desetiletí mimořádný pokrok. Znalost kompletní sekvence lidského genomu, spolu s výsledky elegantních kvasinkových studií zaměřených na identifikaci respiračně významných genových produktů, dovolila molekulární identifikaci a následné studium biochemické podstaty celé řady lidských mitochondriálních patologií. Tyto studie odhalují molekulární a biochemické mechanismy etiopatogeneze těchto chorob a současně přinášejí nové unikátní informace o lidské mitochondriální biogenezi jako takové. Výsledky a původní publikace prezentované v této disertační práci se týkají snahy o pochopení molekulárních rolí lidských asemblačních faktorů Sco1, Sco2, Surf1 a Oxa1l v biogenezi multimerních membránových komplexů lidského systému OXPHOS. Prezentovaná data ukazují, že lidské asemblační faktory Sco1 a Sco2 fungují vysoce tkáňově specifickým způsobem na úrovni odlišných posttranslačních kroků maturace podjednotky Cox2 cytochrom c oxidázy (CcO). Prezentovaná data dále naznačují, že oba výše zmíněné lidské SCO proteiny, a asemblační...
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Examining the significance of peptides regulating the intake of food and the nutritional state of children and adolescents
Bronský, Jiří ; Průša, Richard (advisor) ; Pelouch, Václav (referee) ; Hyánek, Josef (referee)
Introduction: Regulation of food intake and nutritional status is mediated by complex interactions of regulatory peptides of the central nervous system, gastrointestinal tract and adipose tissue. These systems are connected by feedback loops which inform the centre about amount of ingested food and energy reserves in the organism. Dysfunction of any of these regulatory areas may lead to changes in nutritional status of the organism. Methods: We used radioimmunoassay to measure plasma levels of orexin A, total ghrelin and serum levels of leptin and enzyme immunoassay to measure serum levels of adiponectin in healthy subjects and in children with obesity, anorexia nervosa, Crohn's disease and celiac disease and we evaluated the influence of nutritional therapy on these levels. Moreover, we evaluated relationship of these regulatory peptides to other biochemical and anthropometrical factors of nutritional status. We also measured plasma levels of total and unreduced amylin by enzyme immunoassay with immunofluorescence detection in adult patients with osteoporosis, type II diabetes mellitus and in the control group. Results: During reduction of body weight in obese children and adolescents, there were statistically significant changes of plasma orexin A levels and total ghrelin levels, but we haven't seen any...
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Sites and Consequences of Mitochondreal ROS Production
Mráček, Tomáš ; Houštěk, Josef (advisor) ; Pelouch, Václav (referee) ; Kolarov, Jordan (referee)
The thesis consists of 8 articles (6 published and 2 submitted), which may be divided into two major lines. The first (articles 1-4) deals with mitochondrial glycerophosphate dehydrogenase. We have shown that this enzyme is capable of massive ROS production and in the presented works we studied in more detail this ROS production as well as some aspects of enzyme biogenesis, so as to better understand its physiological significance. The second line (articles 5-8) represents our studies on the potential involvement of ROS in pathogenesis of mitochondrial diseases. 1. Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide; Drahota Z., Chowdhury SKR., Floryk D., Mráček T., Wilhelm J., Rauchová H., Lenaz G., Houštěk J.; J Bioenerg Biomembr. 2002; 34(2):105-13. Indeed, this was first article to demonstrate ROS production by mammalian mGPDH. We studied oxidation of glycerophosphate by brown adipose tissue mitochondria, which are known to contain high amounts of mGPDH. We found significant GP-dependent AA- stimulated production of H2O2. To further verify our findings, we used three independent methods for H2O2 measurement - fluorescence detection with p-hydroxyphenylacetic acid, antimycine A - insensitive oxygen consumption, and luminometric...
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