Candida albicans is a dimorphic fungus exhibiting either a budding yeast or hyphal phase. A shift from the yeast phase to the hyphal phase can generally be induced by increasing the temperature of incubation from 25°C to 37°C. This shift occurs over a four hour period as approximately 90% of the yeast cells form germ tubes during this time.Interestingly, the specific activity of S-adenosylmethionine synthetase increases during the shift in vegetative cell types and begins to decrease after the 4 hour period. Utilizing the protein synthesis inhibitor tricodermin, we have demonstrated that the increase in specific activity requires de novo protein synthesis.SAM synthetase was characterized (in vitro) by kinetic analysis and response to putative inhibitors. The yeast phase enzyme had an apparent Km of 0.17 mM for methionine, 0.14 mM for ATP and is inhibited by dimethyl-sulfoxide (DMSO), methionine sulfone and methionine sulfoxide. The hyphal phase enzyme has an apparent Km of 0.06 mM for methionine, 0.02 mM for ATP and its activity is enhanced by the three inhibitors. This preliminary data suggests the presence of isozymes in Candida albicans and the possibility of morphology predominant form.The in vivo studies revealed that the addition of methionine inhibited enzyme activity. In addition, 1 mg/ml cycloleucine (in the presence of methionine) induced the activity of this enzyme, indicating that SAM (along with methionine) is a co-effector of enzyme activity and/or synthesis.Ball State UniversityMuncie, IN 47306

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on July 29, 2008). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xiii, 175 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 161-175).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 77-92).

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

A leishmaniose é uma doença parasitária de ampla distribuição e de difícil tratamento. Recentemente foi identificada a atividade leishmanicida de tamoxifeno, levando à proposta de sua utilização como alternativa para o tratamento de leishmaniose. Neste trabalho, propusemo-nos a caracterizar a atividade de ceramida sintase (CerS) em L. amazonensis e testar a interferência do tamoxifeno nesta enzima. Identificamos, no genoma de L. amazonensis, uma ORF que codifica uma proteína similar à CerS de Saccharomyces cerevisiae, com seis domínios transmembrana e um motivo Lag1 característico. A caracterização da atividade enzimática in vitro mostrou que a enzima reconhece esfingosina/esfinganina e palmitoil/miristoil CoA como precursores. A enzima não é sensível à fumonisina B2 ou a tamoxifeno. Isto indica que esta droga não atua através da inibição da CerS. A caracterização completa desta enzima fornecerá dados valiosos sobre o metabolismo de esfingolipídeos nestes protozoários. / Leishmaniasis is a widely distributed parasitic disease with difficult treatment. The leishmanicidal activity of tamoxifen was recently identified, leading to its proposal as an alternative treatment for leishmaniasis. In this work, we aimed at characterizing the activity of ceramide synthase (CerS) in L. amazonensis and testing whether this enzymatic activity in inhibited by tamoxifen. We have identified, in the L. amazonensis genome, an ORF which encodes a protein similar to the Saccharomyces cerevisiae CerS, with six transmembrane domains and a characteristic Lag1 motif. The characterization of the in vitro enzymatic activity showed that the enzyme recognizes sphingosine/sphinganine as well as palmitoyl/miristoyl CoA as precursors. This enzyme is not sensitive to fumonisin B2 or tamoxifen. This indicates that this drug does not act through the inibition of CerS. The complete characterization of this enzyme will provide valuable information about the sphingolipid methabolism of these protozoa.

Hartree-Fock, Moller-Plesset, and density functional theory calculations have been carried out using 6-31+G(d), 6-31+G(d,p) and 6-31++G(d,p) basis sets to study the properties of low-barrier or short-strong hydrogen bonds (SSHB) and their potential role in enzyme-catalyzed reactions that involve proton abstraction from a weak carbon-acid by a weak base. Formic acid/formate anion, enol/enolate and other complexes have been chosen to simulate a SSHB system. These complexes have been calculated to form very short, very short hydrogen bonds with a very low barrier for proton transfer from the donor to the acceptor. Two important environmental factors including small amount of solvent molecules that could possibly exist at the active site of an enzyme and the polarity around the active site were simulated to study their energetic and geometrical influences to a SSHB. It was found that microsolvation that improves the matching of pK as of the hydrogen bond donor and acceptor involved in the SSHB will always increase the interaction of the hydrogen bond; microsolvation that disrupts the matching of pKas, on the other hand, will lead to a weaker SSHB. Polarity surrounding the SSHB, simulated by SCRF-SCIPCM model, can significantly reduce the strength and stability of a SSHB. The residual strength of a SSHB is about 10--11 kcal/mol that is still significantly stable compared with a traditional weak hydrogen bond that is only about 3--5 kcal/mol in any cases. These results indicate that SSHB can exist under polar environment. Possible reaction intermediates and transition states for the reaction catalyzed by ketosteroid isomerase were simulated to study the stabilizing effect of a SSHB on intermediates and transition states. It was found that at least one SSHB is formed in each of the simulated intermediate-catalyst complexes, strongly supporting the LBHB mechanism proposed by Cleland and Kreevoy. Computational results on the activation energy for catalyzed and uncatalyzed model reactions shows that strong hydrogen bonding between catalyst and the substrate at the transition state can significantly reduce the activation energy. This implies that LBHBs are possibly playing a crucial role in enzyme catalysis by supplying significant stabilizing energy to the reaction transition state.

This work was done with the purpose of evaluating different forms of soybean milk processing, the product acceptance by the public, and to do a study on the feasibility for the production of milk at a small scale to be used as a nutritional supplement in school breakfasts. The soybean milk was prepared with 2 varieties "(Cristalina and Doko)" and two periods of enzymatic inactivation (Before and After) of the grain mush. The "organoleptic" quality was evaluated through surveys and its posterior statistical analysis. Parameter quality was also considered just as did the microbiologic analysis and the conservation tests. The surveys showed that the products obtained were of regular acceptance. The statistical results indicate that the best treatment was that of the variety "doko" with its enzymatic inactivation previous to the trituration. The degree of microbiologic contamination is moderate, it is within the ranges permitted by human consumption. The conservation tests showed that soybean milk without conservatives can have, if refrigerated, a duration similar to that of cow's milk. The financial economic analysis showed that it is possible for the installation of small rural soybean milk processing plants (VAN=2058.68, TIR=34.8). Finally, it is concluded that soybean milk can be constituted as part of a fundamental basic food to lighten the high malnutrition present in the rural and urban areas of our country.

O veneno de Bothops jararaca contém uma série de componentes, entre eles as metaloproteinases hemorrágicas jararagina e bothropasina. Os cDNAs dessas toxinas mostram 97% de identidade. As diferenças, distribuídas ao longo de seus cDNAs, sugerem que estes mRNas não resultam de splicing alternativo. O objetivo deste trabalho foi caracterizar os genes codificadores da jararagina e bothropasina pela identificação de exons e introns no DNA genômico. DNA foi extraído do sangue de um exemplar de B. jararaca; os primers para PCR foram baseados nos cDNAs publicados. Os produtos de amplificação foram clonados e seqüenciados revelando a sequência dos genes TOX1 com 12535 pb e TOX2 com 12268 pb. Quatorze exons e treze introns foram identificados em ambos os genes. Comparação entre as sequências mostrou pontos de mutação, inserções e deleções nos exons, e principalmente nos introns dos dois genes. Este constitui o primeiro relato na literatura sobre a identificação de exons e introns nos genes codificadores de jararagina e bothropasina. / The Bothops jararaca venom contains a number of components, including hemorrhagic metalloproteinases as jararhagin and bothropasin. The cDNA of these toxins show 97% identity. The differences distributed along the cDNAs length suggest that these mRNAs do not result from alternative splicing. This study aimed to characterize the genes that encode for jararhagin and bothropasin through the identification of exons and introns in genomic DNA. DNA was extracted from the blood of a B. jararaca specimen; PCR primers were based on published cDNA sequences. Amplification products were cloned and sequenced revealing the TOX1 gene is about 12,535 bp long, and TOX2 is 12,268 bp. Fourteen exons and thirteen introns were identified in both genes. Comparison of the sequences showed point mutations, insertions and deletions in exons, and particularly in introns. This is the first report in the literature on the identification of exons and introns in genes encoding for jararhagin and bothropasin.