Graduation date: 1995

The nucleic acids digested by ruminants are essentially of microbial origin. Absorbed purines are extensively degraded and excreted in urine as allantoin, uric acid, xanthine and hypoxanthine. The measurement of the urinary purine derivatives could be used to estimate the uptake of purines and hence that of microbial protein. 1) Automated methods for measurements of purine derivatives were improved. A HPLC method for determining total adenine and guanine was used to measure the purine contents of mixed rumen bacteria and the digestibility of microbial purines in sheep. 2) Endogenous excretions of purine derivatives by sheep and cattle of different physiological states were measured using animals nourished by intragastric infusions of VFA and casein. The species difference in and the effects of changes in protein supply on the endogenous excretion were studied. 3) A microbial nucleic acid extract was infused into the abomasum of lambs at 6 levels. The subsequent urinary excretion of purine derivatives was examined. The results suggested that exogenous purines were utilised by the sheep. A model is proposed to describe the relationship between purine derivative excretion and purine uptake. 4) Allantoin was infused intravenously to sheep and the changes in plasma concentration, nephric tubular re-absorption and urinary excretion of allantoin were studied. Results showed that plasma allantoin was rapidly removed and a constant proportion of the allantoin entering the blood was excreted in urine of sheep. 5) Secretion of allantoin and uric acid into the gut via saliva was quantified in sheep. The possible decomposition of the allantoin in the rumen by microbes in the rumen fluid and in the rumen wall of sheep was examined <i>in vitro</i> and <i>in vivo</i>. Allantoin infused into the rumen or abomasum of sheep and cattle was not recovered in urine. 6) A model for calculation of microbial protein available to sheep is proposed. It is suggested that a different model should be used for cattle. These models were applied in two feeding experiments with sheep and steers.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2004. / Includes bibliographical references.

No description available.

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2006. / Includes bibliographical references.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2007. / Includes bibliographical references.

The presence of the purine nucleotide inosine monophosphate (IMP) was studied in direct relationship to the development and expression of cell mediated immunity in guinea pigs using DNCB or Histoplasma capsulatum as sensitizing antigens. The IMP content of T-cell enriched lymphocytic lysates was measured by isocratic high pressure liquid chromatography (HPLC). Intracellular IMP levels of cells from homologously skin tested sensitized animals were significantly increased one day after skin testing when compared to the concentrations found in these cells during the period following sensitization. Concurrent with these observations were the findings that the absolute lymphocyte counts and histoplasmin stimulated in vitro blastogenic responses increased following sensitization while the PHA-induced proliferative response decreased slightly. One day after skin testing, when IMP levels had increased, there was a slight decrease in lymphocyte numbers and a marked decrease in the PHA response. Cells collected at this time and cultured in vitro with histoplasmin responded with increased levels of protein production and increased IMP levels. These data suggest (1) the proliferative response of cells from sensitized animals appears to be associated with lower levels of intracellular IMP, and (2) sensitized cells stimulated in vivo with antigen appear to have characteristically higher IMP concentrations.

The discovery of cisplatin, cis-[pt(NH3)2Cl2], as an anticancer agent in 1969 by Rosenbert and his colleagues sparked interest in the area of metal complexes as chemotherapeutic agents. Anticancer dimetal complexes such as Re2(O2CCH2CH3)2Br4·2H2O are proposed to prevent replication of cancel cells by coordinating to the purine nucleobases in DNA. To investigate the interaction between dimetal compounds and DNA, dirhenium complexes coordinated to purine dinucleotides were isolated and analyzed. LC/MS, HPLC, 1H NMR, and UV-Visible spectroscopy were used to characterized complexes of Re2(O2CR)2X4·2H2O (R = CH3, CH2CH3; X= Cl, Br) with the purine dinucleotides dApG and dGpG. HPLC, UV-Vis, and 1H NMR are used to investigate the aquation of Re2(O2C2H3)2Cl4μ2H2O which may contribute to its biological activity. Upon reaction of Re22C2H3)2Cl4μ2H2O with dApG or dGpG, the intact dirhenium:dinucleotide complex is observed by LC/MS after two days. In both of these reactions, dirhenium:GMP complexes are also observes. 1H NMR studies show the appearance of new resonances in the aromatic region that cannot be attributed to starting material or hydrolyzed DNA fragments. These resonances are proposed to result from the formation of dirhenium:dinucleotide complexes. Additionally, MS spectra support the conclusion that a complex between the dinuclear rhenium complex and the purine dinucleotides of dApG and dGpG is formed after two days. A dirhenium:nucleotide product is also formed as a result of the dinucleotide hydrolysis.

Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.

Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas. / Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.