No description available.

Rhizobium aspartate transcarbamoylase (ATCase; EC 2.1.3.2) was previously believed to be similar to the Pseudomonas ATCase which has been studied extensively. To facilitate the study of the Rhizobium ATCase a pyrimidine-requiring mutant of R. meliloti was isolated and used in the purification of the enzyme.

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.

Tsoi Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 82-104). / Text in English; abstracts in English and Chinese. / Tsoi Sze Man. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of study --- p.1 / Chapter 1.2 --- Cerebellum --- p.2 / Chapter 1.2.1 --- General Structure of cerebellum --- p.3 / Chapter 1.2.2 --- Cell types of cerebellar cortex --- p.4 / Chapter 1.2.2.1 --- Basket cells --- p.5 / Chapter 1.2.2.2 --- Stellate cells --- p.6 / Chapter 1.2.2.3 --- Purkinje cells --- p.6 / Chapter 1.2.2.4 --- Granule cells --- p.7 / Chapter 1.2.2.5 --- Golgi cells --- p.8 / Chapter 1.2.2.6 --- Unipolar brush cells --- p.9 / Chapter 1.2.2.7 --- Deep cerebellar nuclear neurons --- p.11 / Chapter 1.2.3 --- The neuronal circuitry of the cerebellum --- p.12 / Chapter 1.2.4 --- Cerebellar function --- p.14 / Chapter 1.3 --- Cyclic nucleotide-gated (CNG) channels --- p.16 / Chapter 1.3.1 --- Molecular characterization of CNG channels --- p.16 / Chapter 1.3.2 --- Functional properties of CNG channels --- p.19 / Chapter 1.3.3 --- Expression of CNG channels in brain --- p.21 / Chapter 1.3.4 --- CNG channel and neuronal plasticity --- p.23 / Chapter 1.4 --- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels --- p.26 / Chapter 1.4.1 --- Molecular characterization of HCN channels --- p.27 / Chapter 1.4.2 --- Functional properties of HCN channels and Ih current --- p.29 / Chapter 1.4.3 --- Modulation by cyclic nucleotides --- p.31 / Chapter 1.4.4 --- Expression of HCN channels in brain --- p.33 / Chapter 1.4.5 --- Physiological roles of Ih current in central nervous system --- p.35 / Chapter 1.5 --- Aims of study --- p.38 / Chapter Chapter 2 --- Material and Methods --- p.39 / Chapter 2.1 --- Immunohistochemistry Experiments --- p.39 / Chapter 2.1.1 --- Animal preparation --- p.39 / Chapter 2.1.2 --- Tissue preparation --- p.39 / Chapter 2.1.3 --- Primary and secondary antibodies --- p.40 / Chapter 2.1.4 --- Immunofluroescence staining --- p.41 / Chapter 2.1.5 --- Confocal laser scanning microscopy and data processing --- p.41 / Chapter 2.2 --- Whole cell patch clamp recordings --- p.42 / Chapter 2.2.1 --- Brain slice preparation and identification of the cerebellar Purkinje neurons --- p.42 / Chapter 2.2.2 --- Whole cell voltage- and current-clamp recordings --- p.43 / Chapter 2.2.3 --- Drug solutions and delivery --- p.44 / Chapter 2.2.4 --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Expression of Various Cyclic Nucleotide-Gated (CNG) Channel Subunits in Rat Cerebellum --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Results --- p.46 / Chapter 3.2.1 --- Immunoreactivity of CNGA1 in cerebellum --- p.46 / Chapter 3.2.2 --- Immunoreactivity of CNGA2 in cerebellum --- p.47 / Chapter 3.2.3 --- Immunoreactivity of CNGA3 in cerebellum --- p.47 / Chapter 3.3 --- Discussion --- p.48 / Chapter Chapter 4 --- Expression of Various Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Subunits in Rat Cerebellum --- p.53 / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Results --- p.53 / Chapter 4.2.1 --- Immunoreactivity of HCN 1 in cerebellum --- p.53 / Chapter 4.2.2 --- Immunoreactivity of HCN2 in cerebellum --- p.55 / Chapter 4.2.3 --- Immunoreactivity of HCN3 in cerebellum --- p.55 / Chapter 4.2.4 --- Immunoreactivity of HCN4 in cerebellum --- p.55 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Electrophysiological Recordings of Cyclic Nucleotide-Gated Ionic Conductance in Rat Cerebellar Purkinje Neurons --- p.59 / Chapter 5.1 --- Introduction --- p.59 / Chapter 5.2 --- Results --- p.59 / Chapter 5.2.1 --- Effect of cyclic nucleotides on the membrane potential of cerebellar Purkinje neurons --- p.59 / Chapter 5.2.2 --- Ionic conductance of the cyclic nucleotide-induced inward current --- p.61 / Chapter 5.2.3 --- The mechanism of the cyclic nucleotide-induced inward current --- p.61 / Chapter 5.2.3.1 --- Site of action --- p.62 / Chapter 5.2.3.2 --- Involvement of CNG channels and HCN channels --- p.63 / Chapter 5.2.3.3 --- Involvement of protein kinase A (PKA) and protein kinase G (PKG) --- p.65 / Chapter 5.2.3.4 --- Involvement of inwardly rectifying potassium (Kir) channels and transient receptor potential (TRP) channels --- p.65 / Chapter 5.2.4 --- Effect of cyclic nucleotides on Ih current in Purkinje neurons --- p.67 / Chapter 5.3 --- Discussion --- p.68 / Chapter Chapter 6 --- Concluding remarks References --- p.78 / References --- p.82

Wu, Feng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 38-44). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Acknowledgment --- p.iv / Table of Contents --- p.v / List of Figures --- p.vii / List of Abbreviations and Symbols --- p.xi / Abstract (English version) --- p.xii / Abstract (Chinese version) --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Significance of DNA CCTG repeats --- p.1 / Chapter 1.2 --- Objectives of this work --- p.2 / Chapter 1.3 --- DNA structure --- p.3 / Chapter 2 --- Materials and Methods --- p.5 / Chapter 2.1 --- Sample design --- p.5 / Chapter 2.2 --- Sample preparation --- p.5 / Chapter 2.3 --- NMR spectroscopy --- p.6 / Chapter 2.4 --- Resonance assignment --- p.7 / Chapter 3 --- NMR Structural Studies of (CCTG)3 --- p.9 / Chapter 3.1 --- Overview --- p.9 / Chapter 3.2 --- NMR resonance assignments --- p.9 / Chapter 3.3 --- Formation of two-residue CT-loop in the middle repeat of (CCTG)3 --- p.12 / Chapter 3.4 --- C-bulge and T.T mispair in (CCTG)3 hairpin stem region --- p.13 / Chapter 3.5 --- Summary --- p.15 / Chapter 4 --- NMR Structural Studies of (CCTG)4 --- p.16 / Chapter 4.1 --- Overview --- p.16 / Chapter 4.2 --- Conformational exchange in (CCTG)4 --- p.16 / Chapter 4.3 --- Formation of two-residue CT-loops in different repeats of (CCTG)4 --- p.17 / Chapter 4.4 --- Mutational studies of (CCTG)4 --- p.19 / Chapter 4.4.1 --- Mutational studies on the 1st repeat of (CCTG)4: (CCTG)4-C2T --- p.19 / Chapter 4.4.2 --- Mutational studies on the 2nd repeat of (CCTG)4：(CCTG)4-C6T --- p.21 / Chapter 4.4.3 --- Mutational studies on the 3rd repeat of (CCTG)4：(CCTG)4-C 10T --- p.26 / Chapter 4.4.4 --- Mutational studies on the 4th repeat of (CCTG)4: (CCTG)4-C14T --- p.28 / Chapter 4.5 --- Summary --- p.33 / Chapter 5 --- Conclusions and Future Works --- p.35 / References --- p.38

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

No description available.

Serine transhydroxymethylase (STHM), a pyridoxal 5'- phosphate requiring enzyme is indirectly involved in pyrimidine deoxynucleotide metabolism. A decrease in the activity of this enzyme could lead to altered deoxycytidine (dC) metabolism. This study was undertaken to determine if a vitamin B-6 deficiency affects dC metabolism. The effect of a vitamin B-6 deficiency on the activity of STHM in liver, thymus, spleen and bone marrow was examined. In addition, the effect of a vitamin B-6 deficiency on urinary excretion of dC was examined. The effect of a vitamin B-6 deficiency on the urinary excretion and tissue retention of ³H label from ip injected ³H-dC was monitored. Rats were assigned in groups of six to one of four treatment groups: ad libitum control (ALC), pair fed control (PFC), ad libitum deficient (ALD) or meal fed deficient (MFD). At the end of weeks 2 and 6, rats from each treatment group received an ip injection of ³H-dC. Urines were collected for 24 hours following the ip inhibited due to lack of cofactor, then dTMP levels would fall. In an attempt to increase the concentration of dTMP, enzymes active in the conversion of dC and dCMP to dUMP would be expected to increase. Thus, dC salvage pathways would increase and dC synthesis would decrease as metabolism shifts toward production of deoxythymidine triphosphate (dTTP). The result would be lower urinary dC excretion. The present study was undertaken to explore the relationship between vitamin B-6 and pyrimidine deoxynucleotide metabolism. There were four hypothesis tested: Vitamin B-6 deficient rats will excrete less urinary dC than either ad libitum or pair fed controls; vitamin B-6 deficient rats will excrete a lower percentage of labeled dC in urine than control rats; vitamin B-6 deficient rats will incorporate less labeled dC into DNA than control rats but may retain more label in tissues as dC metabolites; activity of STHM from tissues of vitamin B-6 deficient rats will be lower than that from the control rats. / Graduation date: 1990

Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have been able to independently manipulate the nucleotide pools and study its effects on the enzymes in the biosynthetic pathway. The enzyme asapartate transcarbamoylase was repressed 5-fold when exogenous uridine was high and cytidine was low. The enzyme dihydroorotate was repressed 9-fold when uridine was high. These results suggest that a uridine compound may be the primary repressing metabolite for the enzymes encoded by pyrB and pyrC. This is the first study to be done with the proper necessary mutants in the biosynthetic pathway of P aeruginosa. In the past it has been impossible to vary the internal UTP and CTP pools in this organism.

An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.