The effect of temperature on the oxidative-decarboxylation of isocitrate by the soluble NADP+-linked isocitrate dehydrogenase (NADP-IDH, EC 1.1.1.42) from rainbow trout (Salmo gairdnerii) liver has been investigated. A particular interest was given those properties of the enzyme which might help to explain the temperature-independent function of the Krebs cycle and the large increase in fatty acid synthesis known to occur during low temperature acclimation. Within the thermal range experienced by rainbow trout, control of catalysis by this enzyme is temperature-independent. Acclimation to an altered thermal regime is accompanied by an increase in the relative proportion of the slowest migrating isozyme of liver NADP-IDH on starch-gel electrophoresis. These cold- and warm-isozyme variants display different and adaptive Km-temperature relationships, and allow for temperature-independent modulation of enzyme activity through the entire thermal range this species is likely to encounter in nature. Other trout species, including the brook (Salvelinus frontinalis), lake (Salmo namaycush) and their hybrid, the splake trout, were investigated for similar responses. The elaboration of enzyme variants in brook and splake trout are complexly regulated by temperature changes, but the lake trout genome contains a single gene coding for liver NADP-IDH which is not affected by temperature. Catalysis by the trout liver enzyme is modulated not only by temperature, but also ADP and ɣ-KGA. Both of these metabolites alter the Km of DL-isocitrate; at physiological ADP concentrations, the Km is reduced as it is with ɣ-KGA below 0.05 mM, but at higher ɣ-KGA concentrations it is markedly increased. These two controls suggest this enzyme may be important for the Krebs cycle oxidation of isocitrate. The availability of a purified NADP-IDH from pig heart allowed a study of the kinetic properties of homologous enzymes from both a poikilotherm and a homeotherm. Even though the molecular weights, Ea values, substrate, cofactor and inhibitor specificities are similar, subtle changes in enzyme structure and/or conformation as identified by electrophoresis, may result in the observed differences in temperature characteristics. These apparent adaptive enzyme responses are of importance to the rainbow trout which lives in a fluctuating thermal regime, but not the pig which does not experience these changes. In vivo, the response of enzymes to temperature fluctuations may be quite different to those seen in vitro. The locus(i) coding for rainbow trout liver NADP-IDH was found to contain a large amount of heterogeneity; in fact, seven distinct phenotypes were found to coexist in one hatchery population. The kinetics of three of these phenotypes were investigated and it was found that by increasing the number of slow moving isozymes, an increase in Km(DL-isocit) at high assay temperatures occurs. This suggests that irrespective of changes in the cellular milieu, isozymal content can determine the Km-temperature response. The data from this study suggest that changes in enzyme-substrate affinity with temperature as a result of either the temperature directed production of enzyme variants and/or their genetic expression, are important in controlling the catalytic activity of NADP-IDH from the eurythermal rainbow trout. Also, unlike the mammalian enzyme, the trout liver enzyme may be important in both fatty acid synthesis and the Krebs cycle oxidation of isocitrate. / Science, Faculty of / Zoology, Department of / Graduate

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Chan, Chi Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 103-127). / Abstracts in English and Chinese. / Thesis Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / 摘要 --- p.iv / Abstract --- p.vi / List of Abbreviations --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Table of Content --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Classification of the aldehyde dehydrogenase superfamily --- p.1 / Chapter 1.2 --- Structures and catalytic mechanism of ALDH --- p.4 / Chapter 1.3 --- Multiple functions of ALDH --- p.8 / Chapter 1.4 --- Antiquitin - background and recent discoveries --- p.12 / Chapter 1.5 --- Aim of study --- p.19 / Chapter Chapter 2 --- Mitochondrial and Cytosolic Localizations of ALDH7A1 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Cell culture --- p.26 / Chapter 2.2.2 --- Subcellular fractionation --- p.26 / Chapter 2.2.3 --- Western blot analysis --- p.27 / Chapter 2.2.4 --- Flow cytometric analysis of mitochondria in WRL68 cells --- p.28 / Chapter 2.2.5 --- Transient transfection of various EGFP constructs --- p.29 / Chapter 2.2.6 --- Immunofluorescence staining --- p.31 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Presence of ALDH7A1 in cytosol and mitochondria in WRL68 cells --- p.33 / Chapter 2.3.2 --- Mitochondrial-targeting N-terminal sequence in ALDH7A1 --- p.34 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In silico and in vitro subcellular localization studies on ALDH7A1 --- p.40 / Chapter 2.4.2 --- Significance of mitochondrial and cytosolic localizations of ALDH7A1 --- p.45 / Chapter 2.4.3 --- Comparison of animal ALDH7A and plant ALDH7B enzymes --- p.48 / Chapter Chapter 3 --- "ALDH7A1: A Potential Regulator for Cell Growth, Cell Cycle and a Potential Biomarker for Cancer (Stem) Cells" / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Cell synchronization --- p.55 / Chapter 3.2.2 --- Semi-quantitative determination of DNA amount in synchronized cells --- p.55 / Chapter 3.2.3 --- Total protein extraction --- p.55 / Chapter 3.2.4 --- Western blot analysis --- p.57 / Chapter 3.2.5 --- Immunofluorescence staining --- p.57 / Chapter 3.2.6 --- Expression and purification of ALDH7A1 and its mutant --- p.57 / Chapter 3.2.7 --- Kinetic analysis of ALDH7A1 and its mutant --- p.58 / Chapter 3.2.8 --- Generation of native ALDH7 A1 and mutant for transfection --- p.58 / Chapter 3.2.9 --- Generation of stable cell line transfectants --- p.59 / Chapter 3.2.10 --- 2D cell culture and ultra-low attachment cell culture --- p.59 / Chapter 3.2.11 --- Collection of total cell lysates --- p.60 / Chapter 3.2.12 --- Western blot analysis --- p.60 / Chapter 3.2.13 --- Growth analysis --- p.61 / Chapter 3.2.14 --- Aldefluor assay --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Expression level of ALDH7A1 at different phases of the cell cycle --- p.62 / Chapter 3.3.2 --- Subcellular distribution of ALDH7A1 in synchronized cells --- p.64 / Chapter 3.3.3 --- Changes in the expression level of key cell cycle regulators and the growth rate after ALDH7A1 knockdown --- p.68 / Chapter 3.3.4 --- Absence of catalytic activity in the purified ALDH7A1 mutant C302S --- p.68 / Chapter 3.3.5 --- Over-expression of ALDH7A1 variants in HEK293 cells --- p.73 / Chapter 3.3.6 --- Growth rates of cells overexpressing different ALDH7A1 variants --- p.73 / Chapter 3.3.7 --- Expression level of ALDH7A1 in various 2D cell types and stem-like cells --- p.76 / Chapter 3.3.8 --- Aldefluor assay on cells over-expressing different ALDH7A1 variants --- p.79 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Nuclear localization of ALDH7A1 --- p.82 / Chapter 3.4.2 --- Potential role of ALDH7A1 in cell cycle --- p.86 / Chapter 3.4.3 --- Non-catalytic role of ALDH in cell growth and development --- p.86 / Chapter 3.4.4 --- Relationship between ultra-low attachment culture and stem-like cells --- p.89 / Chapter 3.4.5 --- Up-regulation of ALDHs in cancer and CSCs and the evaluation of applicability of Aldefluor assay in CSC isolation --- p.93 / Chapter 3.4.6 --- Comparison on ALDH7A1 expression level in primary and stem-like cells --- p.98 / Chapter Chapter 4 --- Future Prospects / References --- p.103

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Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 101-112).

Recent epidemiological data have implicated prenatal events in the development of cardiovascular disorders. Thus low birth weight strongly predicts the later occurrence of hypertension, type II diabetes mellitus, syndrome X and deaths from ischaemic heart disease. The mechanism linking prenatal events and later disease is not clear, although maternal malnutrition has been advocated. We have advanced the hypothesis that glucocorticoids might be important as they retard foetal growth and programme offspring hypertension in rats. The foetus has been thought to be protected from the 2-10 times higher maternal glucocorticoid levels by the placental enzyme 11B-hydroxysteroid dehydrogenase (11B-HSD), which is present in many tissues and in humans catalyses the conversion of the active glucocorticoid cortisol to inert cortisone (corticosterone to 11-dehydrocorticosterone in rats). The precise role of 11B-HSD as a barrier to maternal glucocorticoids during prenatal life has not been fully characterised. The role of 11B-HSD in controlling prenatal glucocorticoid exposure in humans and animals has thus been examined. Two isoforms of 11B-HSD exist, type 1, a widespread NADP dependent reversible enzyme and type 2, a high affinity NAD dependent dehydrogenase found mainly in placenta and kidney. 11B-HSD was found in abundance in the ovary and placenta. The main site of immunohistochemical staining and expression of mRNA (11B-HSD-1) in the rat ovary was in the oocyte. 11B-HSD was oxidative, inactivating corticosterone. In both rat placenta in-vitro (11B-HSD-2), and human placenta in-vitro and ex-vivo (11B-HSD-2) the bioactivity was also predominantly oxidative. The lowest placental enzyme activity at term (and hence the greatest foetal glucocorticoid exposure) was found in the smallest rats with the largest placentas, i.e. those in human studies who would be predicted to develop the highest adult blood pressures (birth weight vs. placental 11B-HSD activity: n = 56; r = 0.46; p < 0.0005). A method to examine 11B-HSD function in fresh intact human placentas was developed (ex-vivo dual circuit cotyledon perfusion) which allows close approximation to the in-vivo situation. The majority of cortisol, from low to high nanomolar concentrations, infused through the maternal circulation was metabolised to inert cortisone by the time it reached the foetal circulation, although considerable individual variation was observed. 118-HSD was the only significant contributor to placental cortisol metabolism at physiological maternal concentrations and inhibition of 118-HSD with either the liquorice constituent glycyrrhetinic acid or its hemi-succinate, carbenoxolone, resulted in abolition of the glucocorticoid barrier, allowing maternally administered cortisol to pass unmetabolised through the placenta. In a prospective study, on 16 normal primiparous women whose placentas were studied with this technique, a positive and significant correlation was found between the effectiveness of 118-HSD and offspring birth weight (r = 0. 67; p < 0. 005). The relationship between placental 118-HSD effectiveness in-vivo and term cord blood osteocalcin (a sensitive marker of glucocorticoid exposure) was prospectively examined in 19 women. Cord blood levels of the bone specific protein osteocalcin were determined with radioimmunoassay. The lowest cord blood osteocalcin levels were found in the foetuses whose placental 118-HSD barrier function was poorest (r = 0.58; p < 0.02), (and had presumably had the greatest glucocorticoid exposure), suggesting that term cord blood osteocalcin levels might be a useful predictor of hypertension, ischaemic heart disease and possibly metabolic bone disease. The findings presented in this thesis represent direct evidence that 118-HSD is the barrier to maternal glucocorticoids, its effectiveness correlating with foetal growth in rats (in-vitro), in humans (ex-vivo), and in-vivo with human cord blood osteocalcin levels (osteocalcin may be a marker of glucocorticoid exposure). In the light of studies on pregnant rats in which administration of exogenous glucocorticoids or 118-HSD inhibitors reduces birth weight and programmes hypertension in the offspring, it is reasonable to propose that increased foetal glucocorticoid exposure consequent upon attenuated placental 118-HSD function may play a role in intrauterine programming of later hypertension.

published_or_final_version / Chemistry / Master / Master of Philosophy

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