Intracellular dNTP pool sizes are highly asymmetric, with dGTP usually comprising 5 to 10% of the sum of the dNTP pools. The work presented in this dissertation addresses the question of whether the underrepresentation of dGTP is related to its potential to be oxidized by reactive oxygen species. 8-oxo-guanine is important in oxidative mutagenesis, and current evidence indicates that this lesion arises in DNA partly through oxidation of dGTP, followed by incorporation of 8-oxo-dGTP into DNA. The bacterial MutT protein and its mammalian homolog catalyze the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in vitro. It is a widely accepted premise that the primary function of these enzymes is to remove 8-oxo-dGTP from the nucleotide pool of cells so that it cannot be used as a substrate for DNA synthesis. However, this model has been called into question by observations that some mutT strains of E. coli display a mutator phenotype when grown anaerobically, and by kinetic studies that showed 8-oxo-dGTP to be a poor DNA polymerase substrate. In this study, the dNTP pools of mammalian cells cultured in varying oxygen conditions were measured, with the expectation that the dGTP pool would expand under low oxygen conditions if it were a target for damage by reactive oxygen species. HeLa cells cultured in 2% 0��� showed no change in the dGTP pool when compared to cells cultured in 20% 0���; however, in V79 cells, the dGTP pool did expand in 2% 0���. This result was not specific to the dGTP pool, as pools of dATP and dTTP also increased when V79 cells were cultured at 2% 0���. These results suggest that there may be increased turnover of the dGTP pool when cells are cultured in high oxygen, but these experiments did not address the reason for this oxygen-dependent change. In order to determine whether 8-oxo-dGTP accumulates to levels that are sufficient to cause mutagenesis in cells, an analytical method for the measurement of 8-oxo-dGTP from cell extracts was developed. By use of this method, which involves reversed-phase high performance liquid chromatography coupled with electrochemical detection, no 8-oxo-dGTP was detected in mutT E. coli cells, even when they were cultured in the presence of H���0���. The estimated upper limit of 8-oxo-dGTP in these cells is about 240 molecules per cell, which corresponds to an intracellular concentration of approximately 0.34 ��M. When 8-oxo-dGTP was added at this concentration to an in vitro DNA replication system in which replication errors could be scored as mutations, along with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect on the frequency of mutation. Therefore, the presence of 8-oxo-dGTP at physiologically relevant concentrations does not appear to be significantly mutagenic. The results presented in this dissertation suggest that the mechanism by which the MutT enzyme counteracts mutagenesis should be reevaluated. / Graduation date: 2003

Thesis (M.S.)--University of Delaware, 2009. / Principal faculty advisor: Kenneth L. Van Golen, Dept. of Biological Sciences. Includes bibliographical references.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-98). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Montana, 2008. / Title from title screen. Description based on contents viewed Aug. 19, 2008. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 140-161).

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 94-103). Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.)--Indiana University, 2006. / Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 181-239)

Abstract Ornithine decarboxylase (EC 4.1.1. 17) is the first and the rate-controlling enzyme in polyamine biosynthesis. It decarboxylates L-ornithine to form diamine putrescine. ODC activity in cells is strictly regulated and one of the central elements of ODC regulation is an inhibitory protein called antizyme. Antizyme binds to ODC, inhibits its activity and targets ODC for the proteasomal degradation. Essentiality of polyamines for the normal cell growth and proliferation is well known. Recently their roles in the regulation of several classes of cation channels have been discovered. Some of these channels are expressed abundantly in the brain, which has increased interest in the polyamine metabolism in the central nervous system. In this study guanosine 5'-triphosphate activatable ODC was detected in the rat brain lysates. This activation was more significant after antizyme was separated from ODC. GTP-activatable ODC was more resistant to heat and displayed higher Vmax than kidney ODC. Previously GTP-activatable ODC had been found in mammalian tissues only in some tumors. ODC and antizyme expression in brain was localized by in situ hybridization and immunocytochemistry. Both proteins displayed wide and largely overlapping expression patterns restricted to neurons. The proteins were localized predominantly to cytoplasm at the most brain regions, but antizyme had a main localization in nuclei in some regions of the brain. In addition, the role of one of the most highly conserved regions in eukaryotic ODCs was studied using site-directed mutagenesis. The aspartate-233 to valine mutation was made and detected to increase Km values for the cofactor PLP and the substrate L-ornithine as well as Ki value for the inhibitor DFMO. In another part of this study a transgenic mouse line expressing ODC under the control of viral promotor was generated. The most significant changes in ODC activity were detected in reproductive organs of male mice. The high number of infertile transgenic males supported earlier reports about the importance of balanced polyamine metabolism for spermatogenesis. Infertility of female mice was increased as well, but the involvement of polyamines remained unproven. Transgenic mice were prone to various pathological conditions such as inflammations and tumour formation, which may be due to deregulated polyamine metabolism.

When released extracellularly, the purine nucleoside guanosine (Guo) can exert a wide range of physiological effects in vitro and in vivo. Guo can induce the release of neurotrophic factors such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and can initiate the differentiation, growth and proliferation of neurons and glia. While structural and pharmacological evidence support the existence of a putative Guo binding site in the rat brain, there is a paucity of information on the mechanism through which Guo exerts these effects. Through bioinformatic research, our lab has identified an orphan G-protein coupled receptor as the first Guo receptor (termed G1R). The aim of this dissertation is to determine the mechanism of action of Guo using radioligand binding assays. It is hypothesized here that G1R is a distinct purinergic receptor for Guo. Using the calcium phosphate (CaP) co-precipitation (co-i.p.) method, Drosophila Schneider 2 (S2) cells were stably and transiently transfected with G1R recombinant cDNA. A series of binding assays using tritiated Guo ([3H]-Guo) showed no difference in binding between CaP transfection groups and wild S2 controls that do not endogenously express G1R, suggesting that the [3H]-Guo may not have a high binding affinity for the G1R binding site. Preliminary experiments using the Lipofectamine® 3000 to transfect S2 cells showed higher G1R mRNA expression as well as increased binding affinity to Guo when compared to the CaP transfected groups. This suggests that the results in the CaP mediated groups may be due to low transfection efficiency. In conclusion, transfections using the CaP method resulted in too low of a transfection efficiency to see a difference in binding affinity between wild S2 and transfected S2 cells. Findings from this work can be used to further examine the binding relationship of Guo to the G1R and optimize transfections using S2 cells and radioligand binding assays using purine based compounds. / Thesis / Master of Science (MSc)