Overzicht externe adviesorganen van de centrale overheid

Political Scienc

Regulation of Hepatic Steroid Metabolism by Protein Kinase C

The oxidative metabolism of both drugs and steroids occurs mainly as a consequence of utilising the hepatic monooxygenase system (Kuntzman et al, 1964). It has been shown that the activities of various hepatic enzymes are not constant, but are subject to regulation by the endocrine system, and also by hormones. The main classes of hormones which have been implicated in the regulation of hepatic metabolism are; (a) androgens, as extensively reviewed by Skett & Gustafsson (1979) and Gustafsson and coworkers (1980); (b) the pituitary hormones, including prolactin (Colby et al, 1974) and growth hormone (Colby et al, 1974; Mode et al, 1981); and (c) the pancreatic hormones, particularly insulin (Kato & Gillette, 1965; Kato et al, 1970). As the different hormonal pathways within the body are subject to complex interactions, and since some hormones have different secretion profiles in male and female animals, the precise mechanism by which hormones act to regulate the activity of hepatic enzymes has not yet been fully elucidated. One mechanism by which hormones can produce their intracellular effects is by stimulating the hydrolysis of membrane-bound phospholipids. The consequence of this hydrolysis is that two intracellular second messengers are produced, inositol-1,4,5-trisphosphate, which causes an elevation in intracellular free calcium levels, and 1,2-diacylglycerol, which interacts with phosphatidylserine and calcium to activate the phosphorylating enzyme, protein kinase C. Activation of protein kinase C by receptor-mediated inositol phospholipid hydrolysis, relays information across the cell membrane to regulate a variety of intracellular processes, and hormonal activation of protein kinase C in the liver has been reported. Vasopressin is known to affect hepatic enzymes, such as glycogen synthetase (Blackmore et al, 1986a; Ahmad et al, 1984) and glycogen phosphorylase (Barritt et al, 1988), and vasopressin also has general effects upon hepatic events, especially a1-mediated adrenergic effects (Garcia-Sainz et al, 1986). Angiotensin II has also been reported to have hepatic effects mediated through activation of protein kinase C (Garcia-Sainz et al, 1986; Garcia-Sainz & Hernandez-Sotomayor, 1987). Recently, some actions of insulin have also been attributed to protein kinase C activation (Acevedo-Duncan et al, 1989; Cooper et al, 1987; Gomez et al, 1988), although evidence implicating hepatic effects of insulin through protein kinsae C are still contradictory. Recently, it has been demonstrated that, in a reconstituted microsomal membrane system, protein kinase C, and protein kinase A, are capable of phosphorylating cytochrome P450, the terminal oxidase in hepatic monooxygenases (Pyerin et al, 1983; Pyerin et al, 1987). It seemed possible, therefore, that stimulation of inositol phospholipid hydrolysis, and therefore of protein kinase C and subsequent phosphorylation of cytochrome P450, or other proteinaceous components, may be one mechanism by which hepatic monooxygenase activity could be regulated by hormones. The proposed aim of the present study was to try to determine whether or not activation of protein kinase C in the rat liver could influence the activity of hepatic monooxygenases, and to try to elucidate the underlying mechanism of any effect which was observed. This study was conducted using isolated rat hepatocytes in an attempt to avoid complex hormonal interactions, and to try to represent a more physiological situation than is often encountered during in vitro studies. To activate protein kinase C directly, tumor-promoting phorbol esters and synthethic diacylglycerols were used. Phorbol esters are reported to act solely through protein kinase C (Niedel et al, 1983; Castagna et al, 1982) and they are convenient probes of protein kinase C activity in isolated cell systems. Hormonal activation of protein kinase C, via stimulation of inositol phospholipid hydrolysis, was investigated with vasopressin and angiotensin II, and the role of elevating intracellular calcium was investigated by the use of the ionophore A23187. The effect of activating protein kinase C upon the metabolism of the endogenous steroid 4-androstene-3,17-dione was ascertained after various times of incubation with the different compounds. It was found that activation of protein kinase C with all of the compounds, except angiotensin II, produced a time-dependent inhibition in the activity of all of the enzymes metabolising 4-androstene-3,17-dione. This inhibition of enzymes activity was not sex-dependent, males and female were both affected to the same extent. With vasopressin, a biphasic pattern of enzymes inhibition was observed, an effect which implies a dependency on diacylglycerol initially, with the later effect being more dependent upon calcium. (Abstract shortened by ProQuest.)

ME/CFS

“ME/CFS: Causes, Clinical Features and Diagnosis” addresses the early stages of ME/CFS and underlying predisposing factors. It considers the plight of the individual patient, and also the impact of the illness on society as a whole, which is considerable, in terms of both costs and social disruption. Patients and their families and carers frequently experience discrimination and difficulties accessing care. This volume will be of particular interest to those undertaking scientific research and those providing clinical care for ME/CFS patients, as well as to social policy analysts, policy makers and governments, and specialists in social research and medical education. There is a major focus on shortcomings in terms of medical education, resulting in considerable gaps in knowledge and understanding of the condition among many doctors. International comparisons indicate that these problems are encountered in many countries. This is particularly topical at a time when Long Covid-19 has moved post-viral syndromes to the forefront of the political agenda, and confronted society with new challenges in this area on a hitherto unprecedented scale. The volume addresses the many points of similarity between Long Covid-19 and ME/CFS. Mitigation of the illness is also addressed, through espousal of a more patient-centred approach to care, and through consideration of the scope for prevention. Sixty-nine authors from seventeen European countries, and from Canada and the USA have contributed to this volume, which is a truly international collaboration

Mitochondriale Zellatmung ist essenziell für die frühe B Zell Entwicklung und die humorale Immunantwort

The prevention and treatment of cancer and autoimmune diseases but also the development and improvement of vaccines require a deep understanding of the underlying molecular immunological processes. The past few years have shown that metabolism plays a crucial and decisive role for several immune cell types. Within the B cell lineage, the pro B to pre B cell transition is accompanied by a metabolic downregulation and a switch towards oxidative metabolism. Conversely, activated B cells upregulate oxidative and glycolytic metabolism, while anergy is induced via metabolic suppression. Yet, a detailed analysis of mitochondrial metabolism at central and peripheral B cell differentiation stages are missing. Therefore, in this thesis I tested the hypothesis that mitochondrial respiration is essential for early B cell development and peripheral activation, and that consequently mitochondrial function is ensured and selected for at certain checkpoints. The establishment of a mouse line that expresses a dominant-negative mutant of the mitochondrial helicase Twinkle (DNT) in conjunction with IRES-GFP specifically in B cells using the Cre-loxP system, resulted in decreased mitochondrial (mt) DNA abundance in B cells. Since essential respiratory chain subunits are encoded in mtDNA, these cells consequently have impaired mitochondrial respiration. This enabled the analysis of mitochondrial respiration specifically in early B cell development and during B cell activation by using mb1Cre and CD23Cre mice, respectively. Expression of DNT in B cell progenitors via mb1Cre revealed an essential requirement of mtDNA replication and mitochondrial respiration at the pro to pre B cell transition, as these mice exhibited a developmental block at the pre B cell stage. Furthermore, transitional B cells with higher mtDNA content are selected into the pool of marginal zone B cells. This is in accordance with other results obtained in this thesis that showed the highest mtDNA content in marginal zone B cells and plasma cells, especially in the bone marrow. In accordance, the generation of plasma cells and basal as well as induced humoral immune responses to T cell- dependent and -independent antigens are highly dependent on functional mtDNA replication and mitochondrial respiration in B cells, as shown by the analysis of DNTxCD23Cre mice. The decreased oxidative phosphorylation in lipopolysaccharide activated B cells led to an aberrant flux of the TCA cycle and to decreased abundance of saturated phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 expression were reduced, whereas pAMPK, HIF1α, and glycolysis were increased. In summary, mitochondrial respiration in B cells is essential for early B cell development in the bone marrow at the pro to pre B cell transition. Furthermore, marginal zone B cells and BM plasma cells have the highest mtDNA content in the B cell lineage. Lastly, replication of mtDNA, which provides essential subunits of the respiratory chain, in mature B cells is crucial for B cell activation, subsequent plasma cell differentiation, and consequently the humoral immune response.Sowohl die Vorbeugung und Behandlung von Krebs und Autoimmunerkrankungen als auch die Entwicklung und Verbesserung von Impfstoffen setzen ein tiefgreifendes Verständnis von zugrunde liegenden molekularen, immunologischen Prozessen voraus. Studien aus jüngerer Vergangenheit konnten zeigen, dass der Stoffwechsel eine essenzielle und entscheidende Rolle in diversen Immunzelltypen spielt. Innerhalb der B Zell Linie wird der Stoffwechsel während der pro zu prä B Zell Transition runter reguliert und wechselt zu überwiegend oxidativen Stoffwechselwegen. Aktivierte B Zellen erhöhen sowohl oxidative als auch glykolytische Stoffwechselwege, wohingegen Anergie in B Zellen durch Suppression des Stoffwechsels erreicht wird. Jedoch fehlen bisher detaillierte Analysen der Rolle des mitochondrialen Stoffwechsels an zentralen und peripheren Kontrollpunkten während der B Zell Differenzierung. Daher teste ich in dieser Arbeit die Hypothese, dass die mitochondriale Zellatmung eine essenzielle Rolle während der frühen B Zell Entwicklung und während der peripheren Aktivierung spielt, und dass folglich B Zellen mit funktionellen Mitochondrien an bestimmten Kontrollpunkten selektiert werden. Die Cre-loxP vermittelte Generierung einer Mauslinie, die eine dominant-negative Mutante der mitochondrialen Helikase Twinkle (DNT), gekoppelt an IRES-GFP, spezifisch in B Zellen exprimiert, führt zu reduzierter mitochondrialer DNA in B Zellen. Da essenzielle Untereinheiten der Atmungskette im mitochondrialen Genom kodiert sind, ist die mitochondriale Zellatmung folglich gestört in diesen Zellen. Das ermöglicht die Untersuchung der mitochondrialen Zellatmung während der frühen B Zell Entwicklung durch Kreuzung mit mb1Cre Mäusen und während der B Zell Aktivierung durch Kreuzung mit CD23Cre Mäusen. Die mb1Cre vermittelte DNT Expression in pro B Zellen zeigte, dass mitochondriale Zellatmung eine essenzielle Rolle während der pro zu prä B Zell Transition spielt, da diese Mäuse in diesem Stadium einen Entwicklungsblock aufwiesen. Außerdem werden transitionale B Zellen mit funktioneller Replikation der mitochondrialen DNA in das Kompartment von Marginalzonen B Zellen selektiert. Damit übereinstimmend konnte in dieser Dissertation gezeigt werden, dass Marginalzonen B Zellen und Plasma Zellen den höchsten Gehalt an mitochondrialer DNA innerhalb der B Zell Linie aufweisen. Die Analyse von DNT x CD23Cre Mäusen zeigte, dass die Generierung von Plasma Zellen und humorale Immunantworten, sowohl basal als auch T Zell-abhängig oder -unabhängig induziert, stark von funktioneller Replikation der mitochondrialen DNA und mitochondrialer Zellatmung abhängig sind. Reduzierte oxidative Phosphorylierung in Lipopolysaccharid-aktivierten B Zellen führt zu aberrantem Fluss des Citratzyklus und verringerten Vorkommen an Phosphatidat. Folglich waren mTORC1 Aktivität und BLIMP1 Expression verringert, wohingegen pAMPK, HIF1α, und Glykolyse erhöht waren. Zusammenfassend konnte im Rahmen dieser Arbeit gezeigt werden, dass mitochondriale Zellatmung in B Zellen eine essenzielle Rolle während der frühen B Zell Entwicklung im Knochenmark spielt. Außerdem zeigen Marginalzonen B Zellen und Plasma Zellen den höchsten Gehalt an mitochondrialer DNA innerhalb der B Zell Linie auf. Und schließlich ist die Replikation mitochondrialer DNA in reifen B Zellen essenziell für die B Zell Aktivierung und darauffolgende Plasma Zell Differenzierung, und somit folglich für die humorale Immunantwort

Accumulation of pyruvate by isolated rat liver mitochondria

1. 1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane

Accumulation of pyruvate by isolated rat liver mitochondria

1. 1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane