Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

No description available.

Ornithine-$ delta$-biaminotransferase (OAT) is a mitochondrial matrix a enzyme which catalyzes the reversible conversion of ornithine to glutamic semialdehyde. In the retinoblastoma cell line Y79, the 5$ sp prime$ UTR of the OAT mRNA is alternatively-spliced. Previous work has shown that the two alternatively-spliced 5$ sp prime$ UTR are translated with different efficiencies. / The alternatively-spliced 5$ sp prime$ UTRs were isolated and subcloned. Expression vectors were constructed containing each of the OAT 5$ sp prime$ UTRs and the human growth hormone as a reporter gene. Transient transfection expression analysis in COS-1 cells confirmed that the 5$ sp prime$ UTRs conferred differential expression on the reporter gene. The shorter 5$ sp prime$ UTR was expressed at a level 3-fold higher than that of the longer 5$ sp prime$ UTR. / RNA mobility shift assays were performed to determine whether a transacting factor was interacting with one of the alternatively-spliced 5$ sp prime$ UTRs. A binding activity was detected that bound to the longer, exon 2-containing 5$ sp prime$ UTR with a significantly higher affinity than the shorter 5$ sp prime$ UTR. Competition studies demonstrated that this binding activity was specific for the longer 5$ sp prime$ UTR.

Optically pure aromatic D-amino acids, such as D-phenylalanine (D-Phe) and its derivatives, are high-value building blocks for the pharmaceutical industry. These compounds can be prepared using biocatalytic methods relying on various enzymes, including aminotransferases (ATs). ATs, also called transaminases (EC 2.6.1.X), are a subclass of pyridoxal 5′-phosphate-dependent enzymes that catalyze the transfer of the amino group from a donor substrate to a ketone acceptor. Synthesis of optically-pure amino acids using whole-cell biocatalytic cascades based on ATs possess several advantages compared to traditional chemical methods, including excellent enantioselectivity and increased process and step efficiency, which is achieved through the catalysis of multiple steps in one-pot reactions without requirement for intermediate work-ups, cofactor recycling, or toxic metals. However, enzyme biocatalysts typically need to be engineered to alter their substrate specificity or to increase their catalytic efficiency, which has limited their industrial application. Therefore, to facilitate the engineering process of ATs broadly and to produce aromatic D-amino acids, we developed a high-throughput assay for the testing of a broad range of ATs against libraries of potential substrates, and developed a biocatalytic cascade to produce optically pure aromatic D-amino acids.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Ornithine Aminotransferase (OAT) is expressed at high levels in liver, kidney, and retina. Tissue-specific regulation of OAT has been described for rat kidney and liver. To characterize OAT regulation in retinal lines, we studied OAT synthesis in retinoblastomas RB355 and Y79. / OAT transcription and mRNA levels in the two lines were similar, but 3-fold greater immunoreactive OAT protein and enzyme activity were observed for Y79. Characterization of polysome-associated OAT mRNAs indicated that they were translated less efficiently, due to decreased initiation, in RB355. Initiation factor eIF-4E mRNA and protein were reduced in RB355; eIF-4E overexpression in RB355 increased OAT translation and OAT protein to the level observed in Y79. / Estrogen and thyroid hormone increased OAT expression in both strains. Estrogen increased translational initiation, with no effect on transcription, whereas thyroid hormone was primarily a transcriptional activator. / An alternatively-spliced OAT mRNA was identified in these retinoblastomas; this mRNA was poorly translated and was not affected by eIF-4E overexpression, estrogen, or thyroid hormone. / This study has demonstrated several post-transcriptional regulatory mechanisms for OAT in retinal tissue.

Thesis (Ph. D.)--University of Hong Kong, 1993.

Made available in DSpace on 2014-06-11T19:28:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-02-22Bitstream added on 2014-06-13T18:34:45Z : No. of bitstreams: 1 silva_cj_me_jabo.pdf: 651711 bytes, checksum: 6f7b9a354cd661de53615cc81e866c62 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Embora esteja bem estabelecido pelos experimentos clássicos, qual são os fatores que limitam a produção, o funcionamento da planta na fase reprodutiva que envolve um complexo relacionamento tanto entre órgãos fonte e dreno de fotossintatos como do metabolismo do nitrogênio em ambos os tipos de órgãos, ainda permanece pouco esclarecido. Assim sendo, na fase de polinização foram impostas diferentes proporções de folhas (% de fonte) e de grãos (% de dreno) em plantas de milho para estudar o efeito destes tratamentos sobre o comportamento do metabolismo do nitrogênio em grãos, folhas e colmos, em diferentes etapas da fase reprodutiva da cultura e suas relações com a produção de massa seca, desenvolvimento de grãos, bem como desenvolvimento e senescência das folhas. Avaliou-se atividade de algumas enzimas, o teor dos principais metabólitos nitrogendados nas folhas, nos colmos e nos grãos em formação, bem como os reflexos destas variáveis sobre algumas características agronômicas aos 2, 10, 20 e 30 dias após a polinização (dap). Os resultados do presente trabalho permitiram esclarecer que a atividade da redutase do nitrato na folha não foi afetada pelas alterações nas proporções de fonte e dreno de fotossintatos. Os teores de N-total, N-nitrato e N-aminoácidos livres, nas folhas, colmos e endospermas foram mais intensamente afetados quanto mais drásticas foram as reduções de folhas ou grãos. As reduções da fonte e dreno promoveram aumentos significativos nos teores de N-total, N-nitrato e N-aminoácidos livres nas partes remanescentes analisadas. Os teores de proteína solúvel foram mais afetados nos grãos, onde os maiores valores foram encontrados aos 10 dap., nos tratamentos sem folhas e sem grãos... / Although it is well very established, for the classic experiments, which are the factors that limit the production, the operation of the plant in the reproductive phase that involves a compound so much relationship between organs source and fotossintatos drain as of the metabolism of the nitrogen in both types of organs, it remains unclear. Like this being, in the pollination phase different proportions of leaves were imposed (% of source) and of grains (% of drain) in corn plants to study the effect of these treatments on the behavior of nitrogen metabolism in grains, leaves and stems, in different stages during reproductive phase of the culture and your relationships with the production of dry mass, development of grains, as well as development and senescence of leaves. Enzymes activity were evaluated (NR, TGO and TGP), the level of main metabolites (N-total, N-nitrate, free amino acids and soluble protein) in the leaves, in the stems and in the grains in formation, as well as the reflexes of these varied on the agronomic characteristics (mass evaporates of leaves stems and grains), to the 2, 10, 20 and 30 days after the pollination (dap). The results of the present work allowed to clear that the activity of the nitrate reductase in the leaf was not affected by the alterations in the source proportions and photoassimilated drain. The levels of N-total, N-nitrate and free N-amino acids, in the leaves, stems and endosperms were more intensely affected the more drastic they were the reductions of leaves or grains. The reductions of the source and drain promoted significant increases in the levels of N-total, N-nitrate and free N-amino acids in the analyzed remaining parts. The soluble protein concentration was more affected in the grains, where the largest values were found to the 10 dap, in the treatments without leaves and grains... (Complete abstract, click eletronic address below).