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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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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.

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by Priscilla Miu-kuen Poon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / ACKNOWLEDGEMENT --- p.1 / ABSTRACT --- p.2 / CONTENTS / Chapter 1. --- INTRODUCTION --- p.4 / Chapter 1.1 --- Discovery of vitamin D / Chapter 1.2 --- Bioavailability of vitamin D and its metabolites / Chapter 1.3 --- Metabolism of vitamin D and its metabolites / Chapter 1.4 --- Mode of action of vitamin D / Chapter 1.5 --- Vitamin D-related diseases / Chapter 2. --- METHODS OF MEASURING VITAMIN D AND ITS METABOLITES --- p.32 / Chapter 2.1 --- Deproteinization / Chapter 2.2 --- Extraction / Chapter 2.3 --- Separation / Chapter 2.4 --- Quantitation / Chapter 3. --- OBJECTIVES --- p.51 / Chapter 4. --- MATERIALS & METHODS --- p.52 / Chapter 4.1 --- Materials / Chapter 4.2 --- General methods / Chapter 4.3 --- Blood collection / Chapter 4.4 --- Radioreceptor assay / Chapter 4.5 --- Serum treatment / Chapter 4.6 --- High Performance Liquid Chromatography (HPLC) / Chapter 4.7 --- Gas Chromatography-Mass Spectrometry (GC-MS) / Chapter 4.8 --- "Serum 1α,25-dihydroxyvitamin D3 analysis" / Chapter 4.9 --- Application of the established GC-MS method / Chapter 4.10 --- Study on hypercalcaemia of tuberculosis / Chapter 5. --- RESULTS --- p.66 / Chapter 5.1 --- Analysis of vitamin D3 standard / Chapter 5.2 --- "Analysis of 1α,25-dihydroxyvitamin D3 standard" / Chapter 5.3 --- Separation of vitamin D3 metabolites / Chapter 5.4 --- "Analysis of lα,25-dihydroxyvitamin D3 in serum samples" / Chapter 5.5 --- Study on hypercalcaemia of tuberculosis / Chapter 6. --- DISCUSSIONS --- p.118 / Chapter 6.1 --- Derivatization / Chapter 6.2 --- Optimization of GC-MS parameters / Chapter 6.3 --- Sample pre-treatment / Chapter 6.4 --- "GC-MS analysis of serum lα,25-dihydroxyvitamin D3" / Chapter 6.5 --- Study on hypercalcaemia of tuberculosis / Chapter 7. --- CONCLUSION --- p.129 / LIST OF ABBREVIATIONS --- p.131 / LIST OF FIGURES --- p.134 / LIST OF TABLES --- p.137 / REFERENCES --- p.139

The first three studies in this thesis address the mechanism for the aberrant fall in serum 1,25-dihydroxyvitamin D$ sb3$ (1,25-(OH)$ sb2$D$ sb3 rbrack$ and increase in renal 1,25-(OH)$ sb2$D$ sb3$-24-hydroxylase(24-hydroxylase) activity in X-linked hypophosphatemic mice (Hyp). The 24-hydroxylase is the first enzyme in the C-24 oxidation pathway that degrades the vitamin D hormone to its final inactivation product, calcitroic acid. We demonstrated that: (i) the aberrant increase in 24-hydroxylase activity in Pi-deprived Hyp mice is specific to the kidney and is the result of an increase in enzyme Vmax, immunoreactive protein and mRNA abundance; (ii) the increase in 24-hydroxylase mRNA in both Pi-deprived Hyp mice and 1,25-(0H)$ sb2$D$ sb3$-treated normal littermates can be ascribed to an increase in the transcriptional activity of the 24-hydroxylase gene; (iii) 24-hydroxylase transcripts in normal mice, Pi-deprived Hyp and normal mice and 1,25-(OH)$ sb2$D$ sb3$-treated normal mice are localized to the proximal tubule by in situ hybridization; and (iv) recombinant human growth hormone administration normalizes the aberrant increase in 24-hydroxylase but that this response is not sufficient to correct serum 1,25-(OH)$ sb2$D$ sb3$ levels in Pi-deprived Hyp mice. / The fourth study addresses the mechanism whereby EB 1089, an analogue of 1,25-(OH)$ sb2$D$ sb3,$ is less calcemic than the vitamin D hormone, while being more potent in its antiproliferative action. We demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25-(OH)$ sb2$D$ sb3$ for the vitamin D catabolic enzyme, 24-hydroxylase; and (ii) EB 1089 and 1,25-(OH)$ sb2$D$ sb3$ exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished