Wong, Kin Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 86-98). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ABSTRACT (ENGLISH) --- p.ii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vi / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.x / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Antibiotics market and β-lactam antibiotics --- p.1 / Chapter 1.2. --- Semi-synthetic cephems --- p.1 / Chapter 1.3. --- Conversion of CPC to 7-ACA --- p.3 / Chapter 1.4. --- Chemical production versus enzymatic bioconversion --- p.5 / Chapter 1.5. --- Industrial two-step bioconversion of CPC --- p.11 / Chapter 1.6. --- Phylogenetics and physiological roles of DAAO --- p.15 / Chapter 1.7. --- Yeast DAAOs are suitable candidates for enzymatic bioconversion --- p.17 / Chapter 1.8. --- Structural and mechanistic studies of DAAOs --- p.18 / Chapter 1.9. --- "Modifications of pkDAAO, RgDAAO and TvDAAO" --- p.25 / Chapter 1.10. --- Objectives of the study --- p.26 / Chapter CHAPTER 2 --- HOMOLOGY MODELLING / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Sequence alignment and selection of homologs --- p.28 / Chapter 2.2.2. --- Generation of three-dimensional TvDAAO model --- p.28 / Chapter 2.3. --- Results --- p.29 / Chapter 2.4. --- Discussion --- p.33 / Chapter CHAPTER 3 --- "MUTAGENESIS, EXPRESSION, PURIFICATION AND SCREENING OF MUTANTS" / Chapter 3.1. --- Introduction --- p.38 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Cloning of TvDAAO mutants / Chapter 3.2.1.1. --- Preparation of competent E. coli --- p.39 / Chapter 3.2.1.2. --- Transformation of E. coli --- p.40 / Chapter 3.2.1.3. --- Agarose gel electrophoresis and gel-purification --- p.41 / Chapter 3.2.1.4. --- Plasmid extraction --- p.42 / Chapter 3.2.1.5. --- Site-directed mutagenesis of TvDAAO --- p.42 / Chapter 3.2.2. --- Heterologous expression and purification of mutants / Chapter 3.2.2.1 --- Shake flask fermentation --- p.45 / Chapter 3.2.2.2. --- Cell harvest and disruption --- p.45 / Chapter 3.2.2.3. --- Purification of WT and mutants --- p.47 / Chapter 3.2.2.4. --- Determination of protein concentration --- p.47 / Chapter 3.2.2.5. --- SDS-PAGE --- p.48 / Chapter 3.2.3. --- Screening of mutants --- p.48 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Preparation of purified TvDAAO mutants --- p.50 / Chapter 3.3.2. --- Evaluation of activity and thermostability --- p.50 / Chapter 3.4. --- Discussion --- p.53 / Chapter CHAPTER 4 --- ENZYME KINETICS / Chapter 4.1. --- Introduction --- p.57 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Standard assay --- p.58 / Chapter 4.2.2. --- Determination of kinetic parameters --- p.59 / Chapter 4.2.3. --- Inhibitory studies --- p.59 / Chapter 4.2.4. --- Effects of pH --- p.60 / Chapter 4.2.5. --- Heat treatments --- p.60 / Chapter 4.2.6. --- CD measurements --- p.61 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Time progress curve analysis --- p.61 / Chapter 4.3.2. --- Kinetics of WT and mutants --- p.62 / Chapter 4.3.3. --- Temperature-dependent and time-dependent thermostability --- p.67 / Chapter 4.3.4. --- Secondary structure measurements --- p.71 / Chapter 4.4. --- Discussion --- p.71 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.83 / BIBLIOGRAPHY --- p.86

No description available.

The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

Photocopy of typescript. / Digitized by Kansas Correctional Industries

No description available.

No description available.

No description available.

Since glucose is an essential nutrient for normal fetal growth and development, the impact of reduced maternal dietary glucose supply, on amniotic fluid (amf) amino acid composition was investigated. Furthermore, this study investigated whether any resulting changes in the concentrations of amf amino acids could be predictive of fetal growth and metabolic status. Pregnant rat dams were fed isocaloric diets containing graded levels of dietary glucose (0, 12, 24 and 60%) and the amf amino acid content was analysed on gestational days (gd) 18.5 to 21.5. Carbohydrate restriction produced significant increases in the concentrations of amf isoleucine (on gd 21.5), tryptophan (on gd 18.5 and 21.5) and 3-methylhistidine (on gd 20.5 and 21.5). An interaction between diet and day of gestation modified amf taurine levels such that dams fed low carbohydrate diets showed significant increases in amf taurine as pregnancy progressed. Specific amf amino acids correlated with fetal growth parameters and fetal tissue glycogen reserves indicating the ability of amf composition to reflect fetal distress under conditions of compromised maternal nutritional status. A greater statistical predictability of amf constituents was obtained with fetal growth parameters than with fetal tissue glycogen reserves. These results suggest that amf amino acids are better predictors of fetal growth status than of fetal metabolic status.

Biotin is a water soluble vitamin that is mainly used as a cofactor in carboxylation reactions by a class of enzyme known as biotin-dependent carboxylases. In order to act as a cofactor, the biotin molecule has to be covalently attached to a lysine residue by an enzyme called holocarboxylase synthetase (HCS). Inherited deficiency of the biotin-dependent propionyl-CoA carboxylase (PCC) results in the inborn error of metabolism propionic acidemia. Mutations in either the alpha (PCCA gene) or beta (PCCB gene) subunit of the enzyme have been shown to cause propionic acidemia. Mutation analysis of the PCCB gene have revealed several mutations. However, few PCCalpha mutations have been described. The first goal of this thesis was to determine the molecular etiology of alpha subunit deficiency at the mRNA as well as at the protein level. I found that most mutations destabilized either the mRNA or the protein. Two other mutations were found to affect the biotinylation of PCCalpha, defining residues important for the folding of the domain or for interaction with HCS. The second part of my thesis was to study in more details the interactions between HCS and the biotinylation domain of PCCalpha, represented by the last 67 amino acids of the subunit (p-67). I expressed and purified p-67 from Pichia pastoris. I compared p-67 with the E. coli biotinylation domain (BCCP87) as substrates for the E. coli orthologous enzyme BirA, using steady-state as well as stopped-flow kinetics. I noticed some differences between these two substrates and how it might relate to the biotinylation reaction. I generated N-terminal and C-terminal deletions of HCS and I tested their activity in vivo and in vitro using purified susbtrates. I was able to map the minimal sequence requirement for HCS activity to the last 348 amino acids of the enzyme. I also found that some longer HCS were either almost or totally inactive or some that were active showed a differential activity towards the different susb

Short/branched chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase (ACD) family of enzymes, catalyzes the oxidation of branched chain fatty acids and the branched chain amino acids isoleucine and valine. This research project focuses on expression studies of the SBCAD gene. Northern blot analysis detected two SBCAD mRNA species of 2.7 and 6.5 kb in various human tissues and cell types. A single 4.1 and 2.0 kb SBCAD message was detected in rat and pig tissues, respectively, revealing a species difference in SBCAD mRNA size. Studies of human and rat SBCAD tissue-specificity and relative abundance, at both the RNA and protein levels, identified liver and kidney as the tissues with the highest levels of SBCAD expression, establishing a unique tissue-specific expression pattern that is not seen among the other members of the ACD family. Furthermore, a fetal and adult difference in SBCAD expression was observed in human kidney, suggesting that the SBCAD gene may be developmentally regulated in some tissues. Finally, an attempt was made to isolate and characterize the SBCAD promoter region in order to provide valuable data for future SBCAD promoter studies.