Clostridium perfringens was found to produce marked changes in its environment during growth. Decrease in electropotential, as well as decrease in pH, correlated with resumption of cellular activity on transfer into a defined medium. Eight amino acids were found to be essential for growth, while the remaining ten amino acids fell into four categories according to the degree of independence the organism had with respect to them. The form of the B-6 vitamin present in the medium had an important influence on the synthesis of several amino acids. The synthesis and degradation of amino acids was investigated further. The enzyme systems involved in these functions could be described as three major groups: those enzymes involved in synthesis of amino acids and amino compounds from glucose, the enzymes responsible for the interconversion of threonine, glycine and serine, and those involved with glutamic acid, proline and arginine metabolism. Uptake of amino acids by both resting and growing cell suspensions was found to be by an efficient, non-energy dependent diffusion mechanism. During growth approximately 5% of the exogenously supplied glucose was assimilated into cell material, primarily as cell wall or nucleic acid components. Molar growth yields from glucose were found to be significantly greater than the theoretical value. The calculated ratio of reduced to oxidized coenzymes indicated a characteristic trend over the logarithmic and stationary growth phases. The relationship between amino acid metabolism, glucose dissimilation and assimilation, and overall metabolite and energy balances has been discussed with a view to understanding the extent of control and influence C. perfringens has on its own metabolic functions and on its environment. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The effect of pyridoxine (PN) supplementation on the activities of erythrocyte alanine aminotransferase (EAlaAT) and aspartate aminotransferase (EAspAT) was observed in five men, aged 22 to 25 years. The subjects received a constant diet containing 1.34 mg of vitamin B-6 Monday through Friday of each week during this five-week study. Starting on day 6 of week 1, the subjects were given orally 5 mg PN daily, except on Tuesday and Thursday of each week when they were given either no PN or 2 mg of vitamin B-6 in the form of crystalline PN or as food. Basal and pyridoxal phosphate (PLP)-stimulated EAlaAT and EAspAT activities were determined weekly. Both basal and PLPstimulated activities of the two enzymes increased after only three days of PN supplementation and continued to increase throughout the four weeks of PN supplementation; percent stimulation by PLP added in vitro decreased concomitantly. It is suggested that the binding of PLP to erythrocyte apoaminotransferases may be another reservoir for vitamin B-6. / Graduation date: 1982

Ma Junhao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 111-123). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xi / Table of Contents --- p.xii / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter 1. --- Literature review --- p.1 / Chapter 1.1 --- Importance of amino acid metabolism --- p.1 / Chapter 1.1.1 --- Rice as the important source of essential amino acids --- p.1 / Chapter 1.1.2 --- Rice seeds are nutritionally incomplete --- p.2 / Chapter 1.1.3 --- The nitrogen source for aspartate family amino acid synthesis --- p.3 / Chapter 1.1.4 --- Synthesis of aspartate family amino acids in plants --- p.4 / Chapter 1.1.5 --- Regulation of the aspartate pathway at free amino acid level --- p.12 / Chapter 1.2 --- Regulation of amino acid metabolism during seed development --- p.14 / Chapter 1.3 --- Signalling system for nitrogen metabolism --- p.17 / Chapter 1.3.1 --- Nitrogen signalling in plants --- p.18 / Chapter 1.3.2 --- GCN signalling: another nitrogen signalling pathway --- p.21 / Chapter 1.3.2.1 --- Mechanism of GCN signalling pathway in yeast --- p.21 / Chapter 1.3.2.2 --- GCN in mammalian --- p.24 / Chapter 1.3.2.3 --- GCN in higher plant --- p.24 / Chapter 1.3.3 --- Relationship between carbon and nitrogen metabolic signaling in plants --- p.26 / Chapter 1.3.4 --- Paradigm for elucidating new signal transduction pathways --- p.29 / Chapter 1.4 --- Hypothesis of this thesis work --- p.30 / Chapter 2. --- Materials and Methods --- p.32 / Chapter 2.1. --- Materials --- p.32 / Chapter 2.1.1 --- Plants --- p.32 / Chapter 2.1.2 --- Bacterial strains and vectors --- p.32 / Chapter 2.1.3 --- Chemicals and reagents --- p.33 / Chapter 2.1.4 --- Buffer，solution and gel --- p.33 / Chapter 2.1.5 --- Commercial kits --- p.33 / Chapter 2.1.6 --- Equipments and facilities used --- p.33 / Chapter 2.1.6 --- Growth medium --- p.34 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Profiling genes expression pattern in developing rice seeds --- p.34 / Chapter 2.2.1.1 --- Growth conditions of rice --- p.34 / Chapter 2.2.1.2 --- Collection of developing rice seeds --- p.35 / Chapter 2.2.1.3 --- Total RNA extraction from rice seeds --- p.37 / Chapter 2.2.1.4 --- Total RNA extraction from plant leaf --- p.37 / Chapter 2.2.1.5 --- Gel electrophoresis --- p.38 / Chapter 2.2.1.6 --- First strand cDNA synthesis from rice total RNA --- p.39 / Chapter 2.2.1.7 --- Search for the coding sequence of rice genes related to amino acid metabolism --- p.39 / Chapter 2.2.1.8 --- Alignment of homologous coding sequence between family member genes --- p.42 / Chapter 2.2.1.9 --- Primer design --- p.42 / Chapter 2.2.1.10 --- Quantitation of total RNA and determination of internal control --- p.45 / Chapter 2.2.1.11 --- PCR to amplify the DNA fragments --- p.45 / Chapter 2.2.1.12 --- DNA Sequencing --- p.46 / Chapter 2.2.1.13 --- Generation and testing of single-stranded DIG-labelled DNA probes --- p.46 / Chapter 2.2.1.14 --- Northern blot --- p.47 / Chapter 2.2.1.15 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.48 / Chapter 2.2.2 --- Expression assay of selected genes in herbicide treated plants --- p.49 / Chapter 2.2.2.1 --- Growing conditions and herbicide treatments --- p.49 / Chapter 2.2.2.2 --- GCN2 homologue in Arabidopsis and rice --- p.51 / Chapter 2.2.2.3 --- RT-PCR to analyze the change in expression level of selected genes in herbicide treated plants --- p.53 / Chapter 2.2.3 --- Generation of transgenic Arabidopsis --- p.56 / Chapter 2.2.3.1 --- Preparation of T-vector for T-ligation --- p.56 / Chapter 2.2.3.2 --- Cloning of Arabidopsis GCN2 gene --- p.56 / Chapter 2.2.3.3 --- Transformation of the plasmid into DH5a competent cell --- p.57 / Chapter 2.2.3.4 --- Screening of right recombinants --- p.58 / Chapter 2.2.3.5 --- Construction of chimeric AtGCN2 genes --- p.59 / Chapter 2.2.3.6 --- Transformation of electro-competent Agrobacterium cell --- p.60 / Chapter 2.2.3.7 --- Transformation of Arabidopsis by vacuum infiltration --- p.61 / Chapter 2.2.3.8 --- Selection of hemizygous and homozygous transgenic plants --- p.61 / Chapter 2.2.3.9 --- Screening of the T3 transformants --- p.63 / Chapter 2.2.3.10 --- Expression analysis of homozygous AtGCN2 transgenic Arabidopsis --- p.63 / Chapter 3. --- Results --- p.63 / Chapter 3.1 --- Profiling genes expression pattern in developing rice seeds --- p.63 / Chapter 3.1.1 --- Quantification of total RNA from seeds at different developing stages --- p.63 / Chapter 3.1.2 --- DNA sequence analysis --- p.66 / Chapter 3.1.3 --- Profiling the gene expression in developing rice seeds --- p.67 / Chapter 3.1.3.1 --- Expression profiles of nitrogen assimilation related genes --- p.67 / Chapter 3.1.3.2 --- Expression profiles of aspartate pathway genes --- p.72 / Chapter 3.1.3.3 --- Expression profiles of branched-chain amino acid synthesis pathway genes --- p.78 / Chapter 3.2 --- Relationship between GCN2 and amino acid metabolism in plants --- p.82 / Chapter 3.2.1 --- GCN2 homologue in A. thaliana and rice --- p.82 / Chapter 3.2.2 --- GCN2 and amino acid starvation --- p.85 / Chapter 3.2.3 --- Effects of amino acid starvation on GCN2 expression --- p.90 / Chapter 3.2.3 --- Changes in the expression level AK and BCAT genes in herbicide treated rice and A. thaliana --- p.93 / Chapter 3.3 --- Characterization of GCN2 transgenic A. thaliana --- p.96 / Chapter 3.3.1 --- Construct of pBI121-AtGCN2 --- p.96 / Chapter 3.3.2 --- Construction of GCN2 transgenic A. thaliana --- p.96 / Chapter 3.3.3 --- Expression of GCN2 in transgenic A. thaliana --- p.97 / Chapter 3.3.4 --- Expression level changes of AK and BCAT in transgenic A. thaliana --- p.99 / Chapter 4. --- Discussions --- p.101 / Chapter 4.1 --- Expression pattern of selected metabolic genes in developing plant seeds --- p.101 / Chapter 4.1.1 --- Most genes studied displayed a similar pattern --- p.101 / Chapter 4.1.2 --- Regulation of gene expression in developing rice seeds --- p.105 / Chapter 4.2 --- GCN2 and its role in higher plants --- p.106 / Chapter 4.2.1 --- The existence of the GCN2 gene in rice --- p.106 / Chapter 4.2.2 --- GCN2 responses solely to amino acid starvation --- p.106 / Chapter 5. --- Conclusion and Prospective --- p.109 / Reference --- p.111 / Appendix I: Chemicals and reagents --- p.124 / "Appendix II: Buffer, solution and gel" --- p.126 / Appendix III: Commercial kits --- p.128 / Appendix IV: Equipments and facilities used --- p.128

A regulação dependente de Ca2+ da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnTl-157), 1-76 (TnTl-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191). Estudos das interações desses fragmentos com actina e tropomiosina demonstraram que: i) o fragmento TnTl-76 não se liga à tropomiosina ou a actina; ii) a região da TnT correspondente aos resíduos 158-191 liga-se à actina cooperativamente, mas não se liga à tropomiosina; iii) a região correspondente seqüência de aminoácidos 77-157 é necessária para a interação da TnT com o resíduo de aminoácido 263 da tropomiosina; iv) TnT77-191 ativa a atividade ATPásica da acto-miosina com a mesma intensidade que TnTl-191. Também observamos que TnTl-157, TnTl-76, TnT77-157, TnT158-91 e combinações de TnT158-191 com TnTl-157 e TnT77-157 não afetam a atividade ATPásica da acto-miosina. Concluímos que a região da TnT delimitada pelos aminoácidos 77 e 191 é essencial para a ativação da atividade ATPásica da actomiosina e que essa ativação é mediada pelas interações dessa região da TnT com a tropomiosina e a actina. / The Ca2+-regulation of the actomyosin ATPase activity at physiological ratios of actin, tropomyosin and troponin occurs only in the presence of troponin T. Our group has previously demonstrated that a recombinant polypeptide corresponding to the first 191 amino acids of TnT (TnTl-191) activates the aetomyosin Mg2+-ATPase activity in the presence of tropomyosin and in the absence of TnI/TnC. In order to further map and characterize this activation domain, we constructed a set of recombinant or synthetic TnT fragments, corresponding to amino acids 1-157 (TnTl-157), 1-76 (TnTl-76), 77-57 (TnT77-157), 77-191 (TnT77-191) and 158-191 (TnT158-191). Binding assays using these fragments demonstrated that: i) amino acids 1-76 of TnT do not bind to tropomyosin or actin; ii) amino acids 158-191 bind to actin cooperatively, but not to tropomyosin; iii) the sequence 77-157 is necessary for TnT\'s interaction with residue 263 of tropomyosin; iv) TnT77-191 on its own activates de actomyosin ATPase activity to the same extent as previously described for TnTl-191. TnT1-157; TnTl-76; TnT77-157; TnT158-191 and combinations of TnT158-191 with TnTl-157 or TnT77-157 showed no effect on the ATPase activity. We conclude that interactions of amino acids 77-191 of TnT with tropomyosin and actin are essential for the activation of actomyosin ATPase activity, and that this activation may be mediated in part by a direct interaction between TnT residues 158-191 and actin.

Typewritten copy Includes bibliographical references.

Graduation date: 1969

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