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This study compared three indirect calorimetry determinations, as kcals/minute, over three consecutive days on 28 healthy, sedentary women of varying body composition. No significant within-individual variation for VO2, CO2, respiratory quotient (RQ), or Kcals/minute was found among the three days. A low coefficient of variation (3.4 +/- 3%) and a relatively small standard deviation in mean Kcals/day (1383 +/- 214) suggests possibly one or only a few measures are necessary for predicting resting metabolic rate (RMR) within a range applicable for clinical use. There were strong correlations of body weight and body composition variables (fat and LBM) with RMR. Knowing both LBM and fat mass increased the ability to predict RMR significantly over the prediction with either variable alone. The Harris Benedict equation over-predicted RMR by 11.1% compared to RMR measured by indirect calorimetry. When equations are based on body weight, rather than LBM, metabolic rate may be over-predicted in obese populations.

Phenylketonuria (PKU) is an inherited metabolic disease that affects about one in 10,000 of the population worldwide. In the classical form of the condition, the hepatic enzyme phenylalanine hydroxlase is absent or much reduced. If untreated, severe or profound mental handicap customarily results due to the accumulation of dietary phenylalanine (phe) which is neurotoxic. The mechanism by which phe impairs growth in the immature nervous system is little understood, but myelin metabolism appears to be disturbed. Treatment is by reduction of phe in daily food intake. Treatment should ideally begin in the neonatal period if intellectual loss is to be avoided. However, the safe range of phe concentrations during treatment and the age at which treatment can be discontinued without further damage being inflicted are uncertain. The studies reported in this volume investigated neuropsychological outcomes of treatment control and cessation factors. In addition, the question of whether executive functions are especially vulnerable to elevated phe concentrations during treatment was addressed. Patient samples conformed to the practice adopted in the West of Scotland regional centre for the management of PKU of maintaining dietary treatment until age 10 or beyond. Almost exclusively, negative findings emerged. These suggested that, if control of phe intake conforms to current UK recommendations for the preschool and primary years, neither global nor specific intellectual deficit result. Furthermore, the data supported the view that cessation of treatment at 10 years of age does not have harmful consequences. These findings have direct implications for the formulation of clinical policy on the treatment of PKU, but it must be recognized that the history of the successful treatment of PKU and mass screening for the disease spans a mere three decades. Thus, treatment outcome research to date is based only on children and young adults. In future investigations, a life-span approach will be required before the issues raised in this thesis can be finally settled.

published_or_final_version / Zoology / Master / Master of Science

Involvement of Cellular Retinoic Acid (CRABP) or Retinol (CRBP) Binding Proteins and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) receptors in the response of cultured cells to retinoic acid and 1,25(OH)₂D₃ was examined. A new method for saturation analysis of CRABP and CRBP was applied to human tumors, human neuroblastoma cells, which retinoic acid causes to differentiate, and a bioselected subline resistant to retinoic acid. These data suggest that CRABP may not mediate cell differentiation by retinoic acid. In other studies, 1,25(OH)₂D₃ receptors and bioresponses were characterized in cultured primate cells. Rhesus monkey kidney cells (LLC-MK₂) were resistant to 1,25(OH)₂D₃-dependent induction of 25(OH)D-24-hydroxylase enzyme. The ED₅₀ in LLC-MK₂ cells was 10-100 fold higher than in other cultured cells. This resistance resulted from a low affinity receptor. Since the LLC-MK₂ variant receptor did not differ in size from the wild type rhesus 1,25(OH)₂D₃ receptor, (Mᵣ = 52 kDa) a subtle alteration in the receptor likely caused the decreased ligand affinity. Also of interest was the possible cellular resistance to 1,25(OH)₂D₃, in the owl monkey (Aotus trivurgatus), which generally occurs in new world primates. Owl monkey kidney (OMK) cells had the same content of receptors for 1,25(OH)₂D₃ and sensitivity to this hormone as cells from the rhesus monkey (old world primate). The ED₅₀ for induction of 24hydroxylase was 2-3 nM in both the OMK cells and the rhesus monkey fibroblasts. Both cells contained 2300 high affinity receptor molecules per cell, which bound DNA and were characterized by immunoblot as 52 kDa proteins. 1,25(OH)₂D₃ treatment increased the content of 1,25(OH)₂D₃ receptors in OMK cells, by increasing the synthesis of receptor mRNA. These data indicate the owl monkey is not resistant to 1,25(OH)₂D₃, unlike other new world primates. This finding was confirmed independently by demonstration that the owl monkey maintained mean serum 1,25(OH)₂D₃ levels (29 pg/ml) in the range of old world primates (33 pg/ml) and humans, in contrast to the elevated 1,25(OH)₂D₃ in other new world primates (97-129 pg/ml). This result suggests the alteration of 1,25(OH)₂D₃-endocrine dynamics in new world primates occurred subsequent to the evolutionary divergence of the owl monkey.

Studies of aortic plaque reveal the presence of tissue macrophages filled with cholesteryl esters. To study lipoprotein metabolism of in vivo, maturated human macrophages, I isolated cells from human peritoneal effluent. Population analysis using cytochemistry showed substantial numbers of acid-esterase positive monocytic cells, lymphocytes, leukocytes and erythrocytes. Substantial variation in cell populations existed among patients. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (AcLDL) by high affinity, receptor-mediated processes. AcLDL degradation saturated at 15 ug protein/ml and LDL degradation saturated at 11 ug protein/ml. Positive correlation of the percentages of monocytic cells with the degradation values (LDL, r =.710; AcLDL, r =.725) and a degradation assay using cells isolated by Lymphoprep showed that the monocytic cells substantially contributed to the degradation of LDL. AcLDL degradation was calcium independent and inhibited by fucoidin. LDL degradation was calcium dependent and very low density lipoprotein and apoE-containing high density lipoprotein (HDL) competed with LDL for receptor uptake; apoE-free HDL, AcLDL and fucoidin did not reduce LDL degradation. Both receptors were pronase-sensitive and degradation was dependent upon lysosomal activity. ACAT activity analysis showed that pre-incubation of cells with LDL or AcLDL stimulated ACAT activity. ACAT activity was greatest for cells preincubated using AcLDL and fresh medium was necessary to maintain the ACAT activity values beyond 24 hrs. LDL-stimulated ACAT activity declined as time was increased above 24 hrs. Flow cytometry analysis of a total cell population and the Lymphoprep-isolated cells revealed a heterogenous T cell population, the presence of monocyte/macrophages, suggested that some of the cells present were activated and confirmed cytochemistry analysis demonstrating that Lymphoprep concentrated the mononuclear cells. Human peritoneal macrophages formed foam cells when incubated in the presence of AcLDL or LDL for 72 hrs. The formation of foam cells in the presence of LDL was dependent upon cell exposure time to the medium. Foam cell formation in the presence of LDL was accompanied by dense vacuolization and in the demonstrated absence of the oxidation of LDL the oil red O stainable material collected outside the vacuoles.

The principal substrate used by the normal adult human heart is free fatty acids, the remainder being, predominantly,carbohydrate. During failure, the heart becomes less reliant on fatty acid metabolism, possibly as a result of tissue hypoxia. Therefore, understanding hypoxic adaptation may explain the metabolic changes that occur during the development of heart failure.As peroxisome proliferator activated receptor alpha (PPARα) modulates cardiac fatty acid metabolism, the work in this thesis focused on the role of PPARα in cardiac metabolic adaptation to chronic hypoxia. It was found that isolated hearts from chronically hypoxic (11% O₂ for 3 weeks)mice were more glycolytic, had reduced PPARα expression and decreased fatty acid metabolism,but had normal function, determined using in vivocine-MRI. ³¹P MRS of isolated perfused mouse hearts showed a drop in PCr with hypoxia, but ΔG_ATP was not altered, indicating that metabolic reprogramming was sufficient to maintain ATP production and contractile function. Increased or decreasedPPARα expression, using a high fat diet or PPARα null mice, respectively, prevented metabolic adaptation to hypoxia and caused cardiac dysfunction. Hypoxia with high fat feeding was particularly deleterious, reducing ejection fraction by 9%,possibly due to increased mitochondrial uncoupling. PPARβ/δ and γ were not involved in the adaptation to hypoxia, and none were modified by PPARα stimulation or ablation. Cardiac VEGF and PDK1, prominent hypoxia-inducible factor (HIF) targets, were increased by hypoxia, indicating that HIF may have been involved in metabolic adaption. However, high fat feeding prevented VEGF accumulation during hypoxia, suggesting that impaired HIF signalling may have contributed to the maladaptive response to hypoxia. In order to determine the relationship between HIF and PPARα, HIFwas stabilised pharmacologically using FG2216/BIC in HL-1 cardiomyocytes, to show decreased PPARα expression and caused similar metabolic changes to those seen in the in vivo hypoxic heart. In conclusion, this study demonstrated that HIF downregulation of PPARα is crucial for metabolic adaptation and maintenance of cardiac function during chronic hypoxia. Similar metabolic changes that occur in end-stage heart failure may also be a response to increasing hypoxia.

The objective of this research was to study the pyrimidine salvage pathways of Rhizobium. Three approaches were used to define the pyrimidine salvage pathways operative in two species of Rhizobium, R. meliloti and R. leguminosarum . The first approach was to ascertain the pyrimidine bases and nucleosides that could satisfy the pyrimidine requirement of pyrimidine auxotrophs. Uracil, cytosine, uridine or cytidine all satisfied the absolute pyrimidine requirement. The second approach was to select for mutants resistant to 5-fluoropyrimidine analogues which block known steps in the interconversion of the pyrimidine bases and nucleosides. Mutants resistant to 5-fluorouracil lacked the enzyme uracil phosphoribosyltransferase (upp ) and could no longer use uracil to satisfy their pyrimidine requirement. Mutants resistant to 5-fluorocytosine, while remaining sensitive to 5- fluorouracil, lacked cytosine deaminase (cod) and thus could no longer use cytosine to satisfy their pyrimidine auxotrophy. The third approach used a reversed phase HPLC column to identify the products that accumulated when cytidine, uridine or cytosine was incubated with cell extracts of wild type and analogue resistant mutants of Rhizobium. When cytidine was incubated with cell extracts of Rhizobium wild type, uridine, uracil and cytosine were produced. This Indicated that Rhizobium had an active cytidine deaminase (cdd) and either uridine phosphorylase or uridine hydrolase. By dialyzing the extract and reincubating it with cytidine, uridine and uracil still appeared. This proved that it was a hydrolase ( nuh ) rather than a phosphorylase that degraded the nucleoside. Thus, Rhizobium was found to contain an active cytidine deaminase and cytosine deaminase with no uridine phosphorylase present. The nucleoside hydrolase was active with cytidine, uridine and to a far lesser extent with purines, adenosine and inosine. When high concentrations of cytidine were added to mutants devoid of hydrolase, cytosine was produced from cytidine - 5-monophosphate by the sequential action of uridine ( cytidine ) kinase and nucleoside monophosphate glycosylase. Both ft meliloti and ft leguminosarum had identical salvage pathways.

Wong Chi-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 119-132). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Figures --- p.vii / List of Tables --- p.ix / Chapter 1 --- Introduction / Chapter 1.1 --- Plant secondary metabolites --- p.1 / Chapter 1.2 --- Plant alkaloids --- p.6 / Chapter 1.3 --- Narciclasine --- p.12 / Chapter 1.3.1 --- Isoquinoline alkaloids --- p.12 / Chapter 1.3.2 --- Amaryllidaceae alkaloids --- p.14 / Chapter 1.3.3 --- Narcissus --- p.16 / Chapter 1.3.4 --- Narciclasine --- p.17 / Chapter 1.3.4.1 --- Isolation --- p.17 / Chapter 1.3.4.2 --- Biological and pharmaceutical functions --- p.20 / Chapter 1.3.5 --- High performance liquid chromatography (HPLC) --- p.23 / Chapter 1.3.6 --- In vitro protein synthesis --- p.24 / Chapter 1.3.6.1 --- Rabbit reticulocyte lysate --- p.25 / Chapter 1.3.6.2 --- Wheat germ extract --- p.25 / Chapter 1.3.6.3 --- Non-radioactive colorimetric detection system --- p.26 / Chapter 1.4 --- Objective --- p.28 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Plant materials --- p.29 / Chapter 2.2 --- Extraction of narcicalsine --- p.29 / Chapter 2.3 --- Distribution of NCS in Narcissus tazetta --- p.30 / Chapter 2.4 --- Stability test --- p.31 / Chapter 2.4.1 --- HPLC analysis --- p.31 / Chapter 2.4.1.1 --- HPLC system --- p.31 / Chapter 2.4.1.2 --- Analytical condition --- p.31 / Chapter 2.4.2 --- Seed germination assay --- p.32 / Chapter 2.5 --- Mode of action of NCS --- p.33 / Chapter 2.5.1 --- In vitro translation --- p.33 / Chapter 2.5.1.1 --- In vitro translation --- p.33 / Chapter 2.5.1.2 --- SDS-PAGE analysis --- p.33 / Chapter 2.5.1.3 --- Western blot analysis --- p.34 / Chapter 2.5.1.4 --- Colorimetric detection --- p.34 / Chapter 2.5.2 --- Assay of induction of a-amylase synthesis in aleurone cells of barley grains by GA3 --- p.36 / Chapter 2.5.2.1 --- Chemicals and reagents --- p.36 / Chapter 2.5.2.2 --- Reducing sugar assay --- p.37 / Chapter 2.5.3 --- Root tip smear --- p.43 / Chapter 2.5.3.1 --- Chemicals and reagents --- p.43 / Chapter 2.5.3.2 --- Assay --- p.43 / Chapter 2.6 --- Allelopathic test --- p.45 / Chapter 2.6.1 --- Soil planting --- p.45 / Chapter 2.6.1.1 --- Foliage spray --- p.45 / Chapter 2.6.1.2 --- Planting with Narcissus bulb --- p.45 / Chapter 2.6.2 --- Hydroponics --- p.46 / Chapter 2.7 --- Effect of NCS on plant cells via tissue culture --- p.48 / Chapter 2.7.1 --- Establishment of tissue culture system --- p.48 / Chapter 2.7.1.1 --- Initiation and maintenance of carrot callus --- p.48 / Chapter 2.7.1.2 --- Initiation and maintenance of tobacco callus --- p.49 / Chapter 2.7.1.3 --- Initiation and maintenance of Narcissus callus --- p.50 / Chapter 2.7.1.4 --- Optimisation of callus growth --- p.50 / Chapter 2.7.2 --- Effects of NCS --- p.51 / Chapter 2.7.3 --- Effect of tobacco extract on NCS --- p.51 / Chapter 2.7.3.1 --- Extraction of tobacco extract --- p.51 / Chapter 2.7.3.2 --- Bioassay --- p.52 / Chapter 2.8 --- Assay of effect of NCS on microorganisms --- p.53 / Chapter 2.8.1 --- Antibacterial activity --- p.53 / Chapter 2.8.1.1 --- Total bacterial count --- p.53 / Chapter A. --- Chemicals and reagents --- p.53 / Chapter B. --- Serial dilution --- p.54 / Chapter C. --- Assay --- p.54 / Chapter 2.8.1.2 --- Turbidity test --- p.55 / Chapter A. --- Bacteria --- p.55 / Chapter B. --- Chemicals and reagents --- p.55 / Chapter C. --- Assay --- p.55 / Chapter 2.8.2 --- Anti-fungal and anti-yeast activity --- p.56 / Chapter 2.8.2.1 --- Disc diffusion method --- p.56 / Chapter A. --- Fungi --- p.56 / Chapter B. --- Chemicals and reagents --- p.56 / Chapter C. --- Assay --- p.56 / Chapter 2.8.2.2 --- Tube dilution method --- p.57 / Chapter A. --- Yeast --- p.57 / Chapter B. --- Chemicals and reagents --- p.57 / Chapter C. --- Assay --- p.57 / Chapter 2.9 --- Statistical analysis / Chapter 3 --- Results / Chapter 3.1 --- Distribution of NCS in Narcissus tazetta --- p.59 / Chapter 3.2 --- Stability of NCS --- p.62 / Chapter 3.2.1 --- HPLC analysis --- p.62 / Chapter 3.2.2 --- Bioassay --- p.62 / Chapter 3.3 --- Mode of action of NCS --- p.66 / Chapter 3.3.1 --- In vitro translation --- p.66 / Chapter 3.3.2 --- Effect ofNCS on the induction of a-amylase synthesis in aleurone cells of barley grains by GA3 --- p.69 / Chapter 3.3.3 --- Root tip smear --- p.74 / Chapter 3.4 --- Allelopathic test --- p.77 / Chapter 3.4.1 --- Soil planting --- p.77 / Chapter 3.4.1.1 --- Foliage applications --- p.77 / Chapter 3.4.1.2 --- Planting with Narcissus bulb --- p.77 / Chapter 3.4.2 --- Hydroponics --- p.78 / Chapter 3.5 --- Effect of NCS on plant cells via tissue culture --- p.91 / Chapter 3.5.1 --- Optimisation of Narcissus callus growth --- p.91 / Chapter 3.5.2 --- "Effects of NCS on Narcissus, carrot and tobacco calli" --- p.91 / Chapter 3.5.3 --- Effect of tobacco extract on NCS --- p.91 / Chapter 3.6 --- Effect of NCS on microorganisms --- p.95 / Chapter 3.6.1 --- Antibacterial activity --- p.95 / Chapter 3.6.1.1 --- Total bacterial count --- p.95 / Chapter 3.6.1.2 --- Turbidity test --- p.95 / Chapter 3.6.2 --- Anti-fungal and anti-yeast activities --- p.95 / Chapter 3.6.2.1 --- Disc diffusion method --- p.95 / Chapter 3.6.2.2 --- Tube dilution method --- p.96 / Chapter 4 --- Discussion / Chapter 4.1 --- General properties --- p.103 / Chapter 4.2 --- Mode of action --- p.105 / Chapter 4.3 --- Other biological properties --- p.108 / Chapter 4.3.1 --- Allelopathic property --- p.108 / Chapter 4.3.2 --- Effect on other plants via tissue culture --- p.110 / Chapter 4.3.3 --- Effect on microoraganisms --- p.112 / Chapter 4.4 --- Further studies --- p.114 / Chapter 5 --- Conclusion --- p.115 / Appendix --- p.116 / References --- p.119