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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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