No description available.

by Yu-Wai Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 122-129.

Abstract Fetal alcohol syndrome (FAS) is a common cause of mental retardation and is attributable to the teratogenic effects of alcohol exposure in utero in individuals with genetic susceptibility. The Coloured communities from the Western and Northern Cape regions have some of the highest recorded incidence rates (~70 affected children per 1000 live births) in the world. The candidate genes selected for this study belong to the family of alcohol dehydrogenase genes that code for enzymes which metabolise alcohol. The ADH1B and ADH1C genes have previously been examined in the Western Cape Coloured community and the enzyme encoded by the allele ADH1B*2 was significantly associated with protection against the development of FAS. ADH4, a new candidate gene, was selected due to its role in both the alcohol and retinol metabolic pathways. A case-control genetic association study was performed to examine the potential roles of the ADH1B, ADH1C and ADH4 genes in the etiology of FAS in two Coloured populations from the Northern and Western Cape. Single nucleotide polymorphisms found within the candidate genes were typed by PCR-based methods in samples from the FAS children, their mothers and controls. Significant associations were observed in the Western Cape cohort but were not replicated in the Northern Cape. Allelic association tests revealed that ADH1B*2 may be a protective marker as it occurred more commonly in the controls than the mothers (p= 0.038). The alleles of the polymorphic variant, ADH4.8, have been shown to influence the promoter activity of ADH4 (the ‘A’ allele has been shown to increase the activity of the promoter when compared to the ‘C’ allele as the same position). The alleles of this polymorphic marker were significantly associated with the risk for FAS. The ‘A’ allele was shown to occur more commonly in the mothers and FAS-affected children (p= 0.002 and 0.035 respectively) when compared to the controls, suggesting a role in disease susceptibility while the ‘C’ allele was shown to occur more commonly in the controls. Itwas also observed that ADH1B and ADH4.8 when examined together in a haplotype demonstrated an association with susceptibility to the disease. While the 2-C haplotype (ADH1B-ADH4.8) was shown to be associated with protection against the development of FAS, the 1-A haplotype was associated with increased susceptibility. The results suggest that mothers with the common ADH1B*1 allele and presumably a normal ADH1B function but an increased level of ADH4 (allele ‘A’) as a result of the promoter mutation, will, when the blood alcohol concentration is high, have an increased risk of having a child with FAS. Conversely when the mothers have a faster alcohol metabolising rate due to the allele ADH1B*2 and normal levels of ADH4 protein (allele ‘C’), the circulating alcohol in the blood is removed efficiently resulting in maternal protection against developing the disease. This study has also highlighted the genetic diversity within individuals of the South African Coloured population. Haplotype analysis and logistic regression revealed that the Western and Northern Cape Coloured communities are genetically different and as a result, the samples could not be pooled for analysis. Although the two groups of controls were genetically diverse, haplotype analysis revealed that the sample of mothers and FAS-affected children were not statistically different between the provinces thus possibly suggesting a similar genetic etiology for the disease. The results from this study suggest that the ADH genes do play a role in the pathogenesis of FAS.

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No description available.

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Geok-yen Yeo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 225-233). / Title page --- p.i / Members of Thesis Advisory Committee --- p.ii / Abstract --- p.iii -iv / Acknowledgments --- p.v / Dedication --- p.vi / Table of Contents --- p.vii -xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-31 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Fermentation --- p.1 / Chapter 1.3 --- Growth in Escherichia coli --- p.3 / Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3 / Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7 / Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8 / Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12 / Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13 / Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15 / Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17 / Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24 / Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25 / Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28 / Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28 / Chapter 1.6 --- Aims of this study --- p.30 / Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90 / Chapter 2.1 --- Bacterial strains --- p.32 / Chapter 2.2 --- Plasmids --- p.32 / Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32 / Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33 / Chapter 2.3 --- Bacterial culture media and solutions --- p.39 / Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39 / Chapter 2.3.2 --- L-Broth + MOPS --- p.39 / Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40 / Chapter 2.3.4 --- SOB and SOC media --- p.41 / Chapter 2.3.5 --- M9 Glucose medium --- p.42 / Chapter 2.3.6 --- Terrific Broth (TB) --- p.42 / Chapter 2.3.7 --- Rich Broth (RB) --- p.43 / Chapter 2.3.8 --- Antibiotic solutions --- p.43 / Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44 / Chapter 2.5 --- Isolation of chromosomal DNA --- p.45 / Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45 / Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48 / Chapter 2.6 --- Isolation of plasmid DNA --- p.50 / Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50 / Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54 / Chapter 2.6.2. --- A Boiling method --- p.54 / Chapter 2.6.2. --- B Alkaline Lysis method --- p.55 / Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56 / Chapter 2.7 --- Purification of DNA --- p.59 / Chapter 2.7.1 --- Ethanol precipitation --- p.59 / Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59 / Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61 / Chapter 2.8 --- DNA cloning techniques --- p.63 / Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63 / Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65 / Chapter 2.8.2. --- A Gel loading buffer --- p.66 / Chapter 2.8.2. --- B Electro-elution of DNA --- p.67 / Chapter 2.8.3 --- Size fractionation --- p.68 / Chapter 2.8.3. --- A Salt gradient fractionation --- p.68 / Chapter 2.8.3. --- B Sucrose gradient --- p.70 / Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71 / Chapter 2.8.5 --- Ligation of vector and insert --- p.72 / Chapter 2.8.6 --- Preparation of competent cells --- p.73 / Chapter 2.8.7 --- DNA transformation --- p.75 / Chapter 2.8.7.A --- By heat shock --- p.75 / Chapter 2.8.7.B --- By electroporation --- p.75 / Chapter 2.9 --- Screening for adhC transformants --- p.78 / Chapter 2.9.1 --- Screening for adhC clones --- p.78 / Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79 / Chapter 2.10 --- Confirmation of adhC clones --- p.80 / Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80 / Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80 / Chapter 2.10.3 --- Plasmid size determination --- p.82 / Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82 / Chapter 2.10.5 --- Physiological study of adhC clones --- p.83 / Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84 / Chapter 2.11 --- The dye-binding method of protein determination --- p.87 / Chapter 2.12 --- Special procedures --- p.88 / Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88 / Chapter 2.12.2 --- Sequencing reactions --- p.89 / Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122 / Chapter 3.1 --- Introduction: Cloning strategy --- p.91 / Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93 / Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93 / Chapter 3.3 --- Restriction mapping of the adh clones --- p.101 / Chapter 3.4 --- Subcloning the adhC insert --- p.110 / Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110 / Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115 / Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121 / Chapter 3.5 --- Remarks concerning the clones --- p.121 / Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.1.1 --- Choice of sequencing strategy --- p.123 / Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124 / Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125 / Chapter 4.2.1 --- Study of adh clone EPR --- p.125 / Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126 / Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127 / Chapter 4.2.4 --- Construction of a nested deletion library --- p.138 / Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177 / Chapter 5.1 --- Nucleotide sequencing --- p.149 / Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150 / Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151 / Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151 / Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152 / Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152 / Chapter 5.4.1 --- Sequence homology of the structural gene (nucleotide # 223- #28%) : Two nucleotide changes revealed in DNA sequence of the structural gene adhE of Escherichia coli --- p.153 / Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153 / Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155 / Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156 / Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156 / Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156 / Chapter 5.5.2 --- Isoelectric point prediction --- p.156 / Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203 / Chapter 6.1 --- Introduction --- p.178 / Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178 / Chapter 6.1.2 --- Choice of assay method --- p.179 / Chapter 6.1.3 --- Points to consider for ADH assay --- p.179 / Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181 / Chapter 6.2.1 --- Plate cultures --- p.181 / Chapter 6.2.2 --- Overnight liquid cultures --- p.183 / Chapter 6.2.3 --- Batch liquid cultures --- p.183 / Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190 / Chapter 6.2.5 --- Comparison of ADH activity --- p.192 / Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197 / Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197 / Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201 / Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220 / Chapter 7.1 --- Cloning of the adhC mutation --- p.204 / Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204 / Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207 / Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208 / Chapter 7.2 --- DNA sequence of the adhC clones --- p.211 / Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211 / Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213 / Chapter 7.3 --- Findings concerning the adhC mutation --- p.217 / Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217 / Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218 / Chapter 7.4 --- Conclusions --- p.220 / APPENDICES --- p.221 -224 / REFERENCES --- p.225 -233

by Ho Ka-Pong, Bosco. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 81-100). / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- PURIFICATION OF HUMAN CLASS I LIVER ADH --- p.19 / Chapter CHAPTER 3: --- DISSOCIATION AND REASSOCIATION OF HUMAN CLASS I ADH BY FREEZE/THAW TECHNIQUE --- p.36 / Chapter CHAPTER 4: --- "DISSOCIATION AND REASSOCIATION OF HUMAN CLASS I ADH BY USING UREA, GdmCl,HIGH SALT AND LOW pH" --- p.51 / Chapter CHAPTER 5: --- GENERAL DISCUSSION --- p.77 / REFERENCES --- p.81

Dihydromyricetin (DHM), also ampelopsin, is a flavonoid compound which exhibits a broad spectrum of positive effects on the human body. Herbal extracts containing this compound have been widely used in traditional Chinese medicine mainly for their hepatoprotective properties. DHM also helps with alcohol intoxication and reduces the signs of hangover or abstinence. Given the fact that the mechanism of DHM effects on the ethanol metabolism has not been clarified yet, the effect of dihydromyricetin on the expression and activity of alcohol dehydrogenase (ADH), one of the most important enzymes involved in ethanol metabolism, was therefore studied in this thesis. The cultivation conditions of primary hepatocytes which were isolated from unpretreated and ethanol-pretreated rats and subsequently exposed to EtOH and DHM were optimized. While determining the degree of cell damage caused by EtOH in the presence of DHM, no significant trend in the protective effect of DHM was found. On the other hand, the protective effect of ethanol in hepatocytes cultivated in EtOH and DHM was detected by technique of ELISA (the determination of alanine transaminase). The Western blot technique followed by immunodetection did not detect the induction of ADH expression in hepatocytes. Furthemore, the modulation effect of...

Butane-grown Pseudomonas butanovora oxidized butane by a soluble butane monooxygenase through the terminal pathway yielding 1 -butanol as the predominant product. Alcohol dehydrogenases (ADHs) involved in butane oxidation in P. butanovora were purified and characterized at the biochemical, genetic and physiological levels. Butane-grown P. butanovora expressed a type I soluble quinoprotein 1 -butanol dehydrogenase (BOH), a soluble type II quinohemoprotein 1 -butanol dehydrogenase (BDH) and an NAD���-dependent secondary ADH. Two additional NAD���-dependent secondary ADHs were also detected in cells grown on 2-butanol and lactate. BDH was purified to near homogeneity and characterized. BDH is a monomer of 66 kDa consisting of one mole of pyrroloquinoline quinone (PQQ) and 0.25 mole of heme c as the prosthetic groups. BOH was partially purified and its deduced amino acid sequence suggests a 67-kDa ADH containing a PQQ as a cofactor. BOH and BDH exhibited high activities and preference towards I -butanol and fair preference towards butyraldehyde. While BDH could not oxidize 2-butanol, BOH is capable of 2-butanol oxidation and has a broader substrate range than that of BDH. Genes encoding BOH and BDH and their deduced amino acid sequences were identified. BOH and BDH mRNAs and 1-butanol oxidation activity were induced when cells were exposed to butane. Primary C��� and C��� alcohols were the most effective inducers for boh and bdh. Some secondary alcohols, such as 2-butanol, were also inducers for BOH mRNA, but not for BDH mRNA. Insertional inactivation of boh or bdh affected unfavorably, but did not eliminate, butane utilization in P. butanovora. The P. butanovora mutant strain with both boh and bdh genes disrupted was unable to grow on butane and 1-butanol. This result confirmed the involvement of BOH and BDH in butane and 1-butanol metabolism in P. butanovora. Roles of B011 and BDH in butane and 1-butanol metabolism were further studied at the physiological level. There are no substantial differences between BOH and BDH in the mRNA expressions in response to three different 1- butanol levels tested and in their abilities to respond to 1-butanol toxicity. Different bioenergetic roles of BOH and BDH in butane and 1-butanol metabolism were suggested. A model of 1 -butanol- dependent respiratory systems was proposed where the electrons from 1 -butanol oxidation follow a branched electron transport chain. The role of BOH was suggested to function primarily in energy generation because B011 may couple to ubiquinone with the electrons being transported to a cyanide-sensitive terminal oxidase. BDH may be more important in the detoxification of 1 -butanol because the electrons from BDH may be transferred to a terminal oxidase system that is less sensitive to cyanide, which is not capable of energy generation. / Graduation date: 2003