In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 95-99). Description: xii, 99 leaves : ill. ; 29 cm. / Blood oxygen carrying capacity, myoglobin levels and LDH isozyme compositions in the heart, gastrocnemius and pectoralis muscles were determined in Common Murre adults and during maturation of the chick at sea. Oxygen stores in the chick (hemoglobin, hematocrit, muscle myoglobin) increased significantly with growth. High levels of the aerobic isozyme, LDH 1, were maintained throughout maturation in the heart. All five LDH isozymes were maintained at similar levels in the gastrocnemius muscle. The pectoralis showed an increase in LDH 1, 2, 3, and 4, yet retained relatively high levels of LDH 5 throughout maturation. Upon leaving the nesting colony, metabolic capacities in the heart and gastrocnemius of the chick are similar to those of adults. The chick pectoralis tissue, however, gains aerobic capacities with maturation which is concomitant with the needed capacity for aerial and aquatic flight upon fledging.

Made available in DSpace on 2014-06-11T19:33:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-01-29Bitstream added on 2014-06-13T21:06:49Z : No. of bitstreams: 1 childs_gmf_dr_jabo.pdf: 764394 bytes, checksum: e911809aa7ff8c7e97896f6faa8e8727 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Funep / Monsanto / Avaliou-se o efeito de oito herbicidas em duas concentrações (2 e 10 vezes a dose media recomendada por hectare) sobre a microbiota de solo. Os herbicidas (bentazon, metolachlor, trifluralin, imazethapyr, imazethapyr+lactofen, haloxyfop-methyl, glyphosate e chlorimuron-ethyl) foram selecionados em função dos resultados de um estudo prévio. Como bioindicadores de atividade se utilizou: respiração microbiana, quantificando-se a emissão de CO2 aos 2, 4, 8, 12, 16, 20, e 24 dias de incubação, mineralização de nitrogênio, atividade da enzima desidrogenase e a hidrólise do diacetato de fluoresceína (FDA), aos 8 e 28 dias. Também se estimou diversidade microbiana. Bentazon e a mistura de imazethapyr+lactofen, na maior concentração, e o haloxyfop-methyl nas duas concentrações, apresentaram efeitos inibitórios na respiração microbiana, embora diferentes em época e duração do efeito. Na mineralização de nitrogênio não foi possível detectar efeitos dos tratamentos. Nenhum dos tratamentos herbicidas afetou a hidrólise do FDA. A atividade da desidrogenase mostrou comportamento variável aos 8 e 28 dias, com resultados de inibição e de estimulo. Somente o herbicida metolachlor 10x causou inibição na quantidade de fungos, os restantes efeitos detectados resultaram em incrementos dos microrganismos. A única correlação significativa encontrada foi entre a atividade da desidrogenase e a respiração basal aos oito dias de incubação. / The effects of eight herbicides on soil microorganism activity were evaluated at two concentrations: 2x and 10x the recommended doses for each product in soybean. These herbicides were selected through the results of a prior experiment were 17 herbicides were tested. The selected herbicides were: bentazon, metolachlor, trifluralin, imazethapyr, imazethapyr+lactofen, haloxyfop-methyl, glyphosate e chlorimuron-ethyl. To study the microorganism activity the following parameters were measured: CO2 soil production until 28 days of incubation, nitrogen mineralization rate, dehydrogenase and FDA activities, at 8 and 28 days of incubation. The functional microbe diversity also was investigated. Bentazon (10x) and the mix imazethapyr+lactofen (10x) and haloxyfopmethyl at both concentrations, reduced the CO2 production, although with differences in timing and duration. No effects of the herbicides could be detected on nitrogen mineralization or FDA hydrolyses. Variable effects involving inhibition or stimulation were detected in the dehydrogenase activity according on herbicide, concentration and period of incubation. Only metolachlor (10x) had inhibition effects on the soil fungi population; the other herbicides promoted increases on microorganism populations. Only dehydrogenase activity and soil respiration at 8 days correlated significatively.

Inorganic sulfur-oxidising Bacteria are present throughout the Proteobacteria and inhabit all environments of Earth. Despite these facts they are still poorly understood in terms of taxonomy, physiology, biochemistry and genetics. Using phylogenetic and chemotaxonomic analysis two species that were erroneously classified as Thiobacillus trautweinii spp. in 1921 and 1934 are in fact novel chemolithoheterotrophic species for which the names Pseudomonas trautweiniana sp. nov. and Achromobacter starkeyanus sp. nov. are proposed, respectively. These species were found to oxidise thiosulfate in a “fortuitous” manor when grown in continuous culture and increases in maximum theoretical growth yield (YMAX) and maximum specific growth rate (μMAX) were observed. Cytochrome c linked thiosulfate-dependent ATP production was confirmed in both species, confirming “true” chemolithoheterotrophy. Evidence is presented that the ATP concentration governs the benefits of chemolithoheterotrophy. There were significant changes in enzyme activities, including enzymes of the TCA cycle that might be affecting amino acid synthesis. This is strong evidence that chemolithoheterotrophy gives a strong physiological boost and evolutionary advantage over strictly heterotrophic species. An autotrophic species that was historically placed in Thiobacillus was also shown to be a novel species for which the name Thermithiobacillus parkerianus sp. nov. is proposed. The enzyme profiles of Thermithiobacillus parkerianus differed significantly between different inorganic sulfur growth substrates and was the first time all TCA cycle enzymes were assayed in a member of the Acidithiobacillia. The properties of thiosulfate dehydrogenase varied significantly between Pseudomonas sp. Strain T, Achromobacter sp. Strain B and Thermithiobacillus sp. ParkerM both in terms of optimal parameters and the effect of inhibitors. This evidence adds to the increasing body of work indicating there to be at least two thiosulfate dehydrogenases present in the Bacteria.

Resumo: O presente trabalho descreve e compara duas metodologias enzimáticas de dosagem de etanol (métodos UV e colorimétrico), que utilizam desidrogenase alcoólica (álcool: NAD+: oxidoredutase EC 1.1.1.1) de fermento de panificação (Mauri Brasil Ind. Comp. e Imp. Ltda), adquirida no comércio na forma desidratada. A atividade da álcool desidrogenase (ADH) presente no extrato bruto de levedura de panificação, da ordem de 5,66 U/mL, foi utilizada nos ensaios colorimétricos, enquanto que nos ensaios no ultravioleta (UV), atividades ao redor de 30 U/mL foram obtidas através da otimização das condições de extração e purificação parcial da ADH. A estabilidade da ADH foi mantida durante 2 meses, na forma liofilizada a 4oC (retenção de 96,2% de sua atividade), na presença de 1 mM de azida de sódio. A mesma preparação enzimática, reconstituída em PEG 15% e armazenada durante 12 meses em freezer (-18oC), apresentou retenção de 50% de sua atividade até 2 meses. O método ultravioleta de dosagem de etanol (detecção na faixa de 2,3 x 10-4 g/L a 6,91 x 10-3 g/L ou 5 æM a 150 æM) baseia-se na conversão enzimática do etanol a acetaldeído, através de reação de óxido-redução, tendo o NAD+ como aceptor de elétrons. O NADH formado pela reação é quantificado com leituras espectrofométricas a 340 nm, conforme descrito por Gattás (2002). Uma preparação enzimática parcialmente purificada e diluída foi utilizada na quantificação de etanol em diferentes bebidas, mostrando desvios dos teores alcoólicos de no máximo 2,1% quando comparados às especificações do produtor. O ensaio de etanol em vinho foi realizado com recuperação da ordem de 99,25% em amostra contendo, originalmente, 249,65 g/L de etanol. O método colorimétrico de dosagem de etanol (detecção na faixa de 4,6 x 10-2 g/L a 23,0 x 10-2 g/L ou 1000 æM a 5000 æM)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work describes and compares two enzymatic methodology of ethanol dosage (UV and colorimetric methods), that use alcohol dehydrogenase (alcohol: NAD+: oxidoredutase EC 1.1.1.1) of baker’s yeast (Mauri Brasil Ind. Comp. e Imp. Ltda) acquired in the commerce in the dry form. The activity of the alcohol dehydrogenase (ADH) at around 5.66 U/mL present in the crude extract of baker’s yeast was used in the colorimetric assay, whereas activities at around of 30 U/mL were obtained through the optimization of the extraction conditions and partial purification of ADH in the ultraviolet assay (UV). The stability of liophilized ADH at 4ºC was maintained for 2 months (retention of 96.2% of activity) in the presence of 1 mM sodium azide. The same enzymatic preparation reconstituted in PEG 15% and stored for 12 months in freezer (-18ºC) presented retention of 50% of your activity up to 2 months. The ultraviolet method of ethanol dosage (detection range of 2.3 x10-4 g/L to 6.91 x 10-3 g/L or 5 æM to 150 æM) is based on the enzymatic conversion of ethanol into acetaldehyde, through oxido-reduction reaction with NAD+ as the aceptor of electrons. The NADH formed by the reaction was quantified spectrophotometrically at 340 nm, as described by Gattás (2002). A partially purified and diluted enzymatic preparation was used for ethanol quantification in different beverages, showing alcoholic contents deviations up to 2.1% when compared to the specifications of the manufacturer. The ethanol assay in wine was accomplished with a recovery at around 99.25% in sample originally containing 249.65 g/L of ethanol. The colorimetric method of ethanol dosage (detection range of 4.6 x 10-2 g/L to 23.0 x 10-2 g/L or 1000 æM to 5000 æM) uses the color reagents system MTT/PMS dissolved in saline phosphate buffer (PBS), determined spectrophotometrically at 570-655 nm... (Complete abstract, click electronic address below) / Orientador: Edwil Aparecida de Lucca Gattás / Coorientador: Maristela de Freitas Sanches Peres / Banca: Rubens Monti / Banca: Luis Henrique Souza Guimarães / Mestre

Gracias a la resurrección de varias enzimas de nuestros antepasados primates se han identificado varias mutaciones que ocurrieron hace, aproximadamente, 10 millones de años, las cuales le confirieron a nuestros antepasados una capacidad mucho mayor para metabolizar etanol. Este episodio de evolución enzimática coincidió con un cambio climático global asociado con la reducción de los bosques africanos y la transición de nuestros antepasados de un estilo de vida arbórea a un estilo de vida terrestre en el cual la fruta altamente fermentada era más común. Estos estudios sugieren que la evolución de las enzimas de nuestros antepasados pueden haberles permitido explotar una fuente alternativa de alimento cuando la comida era escasa. / We have resurrected ancient enzymes from our primate ancestors and identified several mutations occurring approximately 10 million years ago that endowed our ancestors with an enhanced capacity to metabolize ethanol.  This episode of enzyme evolution coincided with a global climate change associated with shrinking African forests and our ancestor’s transition from an arboreal lifestyle to a terrestrial lifestyle where highly fermented fruit is more common. These studies suggest that the evolution of our ancestor’s enzymes may have enabled our ancestors to exploit an alternative source of nourishment during a time when food was scarce.

Orientadores: Fumio Yokoya, Wilma Aparecida Spinosa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T23:38:20Z (GMT). No. of bitstreams: 1 SantosJunior_Vitoriodos_D.pdf: 1117180 bytes, checksum: 0701207136023e01100a6834c8566c3b (MD5) Previous issue date: 2009 / Resumo: As bacterias aceticas sao utilizadas na producao de vinagre. O requerimento nutricional das bacterias aceticas e pouco conhecido e a reproducao desses microrganismos e muito dificil. Os reatores de fermentacao acetica submersa promovem condicoes de estresse fisiologico: acidez (acima de 10% p/v), teor alcoolico (2% p/v) e oxigenacao (0,4 vvm). Esses parametros sao considerados extremos e indicam a necessidade de ativacao de rotas metabolicas especificas, para a manutencao da capacidade de conversao alcool-acido acetico e divisao celular. Este estudo tem como objetivo contribuir na avaliacao qualitativa e quantitativa do efeito de minerais sobre a produtividade de acido acetico, que e definida como a porcentagem de acido acetico produzido em 24 horas. Os experimentos foram conduzidos com linhagem de Acetobacter sp., proveniente de industria produtora de vinagre. As etapas do trabalho foram: ativacao do reator com nutriente padrao; teste de efeitos dos minerais, empregando como ferramenta estatistica um delineamento fatorial incompleto (2(5-1)), isolamento e identificação dos microrganismos, utilizando provas bioquimicas classicas e analise molecular da expressao de RNA mensageiro, por meio da tecnica do DDRT/PCR, com utilizacao de primers da enzima alcool desidrogenase (ADH). Com os resultados obtidos a partir de um ensaio fatorial incompleto para a linhagem estudada, foram selecionados como minerais que aumentam a produtividade, sao eles: o ferro, o molibdenio e o manganes e, ainda, como minerais que diminuem a produtividade, o zinco e o boro. A linhagem isolada do reator apresentou todas as características bioquimicas do genero Acetobacter e a analise molecular evidenciou a expressão do gene para a enzima alcool desidrogenase em todos os experimentos / Abstract: The acetical bacteria are used in vinegar production. The nutritional requirement of acetical bacteria is poorly known, and the maintenance of these microorganisms is too difficult. The reactors of submerged acetical fermentation promote conditions of physiologic stress: acidity (up to 10% p/v), alcohol (2%p/v) and oxygenation (0,4 vvm).These parameters are considerate extremes and indicate the necessity of the activation of specifics metabolic to the maintenance of alcohol-acetic acid transformation capacity and cell division. This research has in view to contribute to the qualitatively and quantitatively evaluation of minerals effects on the productivity of acetic acid, which is defined as the acetic acid percentage produced until 24 hours. These experiments were conducted with of Acetobacter sp. strain, proceeding from vinegar industries. The work steps were: activation of the reactor with standard nutrient; minerals effects tests using the fractional factorial design (25-1) as statistic tool; isolation and identification of the microorganisms, employing classical biochemistry proofs and molecular analysis of the RNA messenger expression, through DDRT/PCR techniques, applying alcohol dehydrogenase enzyme primers. With the results obtained from the fractional factorial design for the strains studied, were selected minerals as the iron, the molybdenum and the manganese that increase productivity and, yet, minerals as zinc and boron that decrease it. The reactor isolated strain presented all biochemical characteristics of Acetobacter genus and the molecular analysis evidenced the expression of the gene for the enzyme alcohol dehidrogenase in all experiments / Doutorado / Doutor em Ciência de Alimentos

Abstract Multifunctional enzyme type 2 (MFE-2) catalyses the second and the third reactions in the eukaryotic peroxisomal β-oxidation cycle, which degrades fatty acids by removing a two-carbon unit per each cycle. In addition to the 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase activities, mammalian MFE-2 has also a sterol carrier protein type 2-like (SCP-2L) domain. In contrast, yeast MFE-2 has two (3R)-hydroxyacyl-CoA dehydrogenases, one 2-enoyl-CoA hydratase 2 and no SCP-2L domain. The physiological roles of yeast (3R)-hydroxyacyl-CoA dehydrogenases (A and B) were tested by inactivating them in turn by site-directed mutagenesis and testing the complementation of Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2) with mutated variants of Sc MFE-2. Growth rates were lower for fox-2 cells expressing only a single functional domain than for those expressing the Sc MFE-2. Kinetic studies with purified Candida tropicalis MFE-2 and its mutated variants show that dehydrogenase A catalyzes the reaction more efficiently with the medium- and long-chain substrates than dehydrogenase B, which in turn is the only one active with the short chain fatty acids. The structural basis of the substrate specificity difference of these two dehydrogenases was solved by X-ray crystallography together with docking studies. Protein engineering was used to produce a stabile, homogenous recombinant protein of C. tropicalis dehydrogenases in one polypeptide. The heterodimeric structure contains the typical fold of the short-chain alcohol dehydrogenase/reductase (SDR) family. Docking studies suggest that dehydrogenase A binds medium chain-length substrates as bended, whereas short chain substrates are dislocated, because they do not reach the hydrophobic contacts needed for anchoring the substrate to the active site, but are instead attracted by L44. Dehydrogenase B has a more shallow binding pocket and thus locates the short chain-length substrates correctly for catalysis. Thus the data provide clues for structural basis of the different substrate specificities. The molecular basis of the patient mutations of MFE-2 (DBP deficiency) was studied using the recently solved crystal structures of rat (3R)-hydroxyacyl-CoA dehydrogenase, human 2-enoyl-CoA hydratase and SCP-2L. The predicted effect of the mutations on protein structure could in several cases be explained, and these data supported the conclusion that a genotype-phenotype correlation exists for DBP deficiency.

Abstract The biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens. In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence. In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2. The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer.

Corticosterone in foetal tissues after injection of the mother with ¹⁴C-corticosterone was determined by acetylation. with ³H-acetic anhydride and crystallization to constant specific activity. The corticosterone content of whole foetal tissue varied between gestational days 13 and 17 from 641 to 300 ng/g respectively. The specific activity of foetal hormone recovered remained essentially constant; after a 15-minute pulse this was as much as one-fourth that of maternal hormone. However, placenta, head and liver showed distinctly different patterns of metabolism, which changed greatly during this time in head and liver, with a decrease in the conversion of corticosterone to 11-dehydrocorticosterone and a rise in foetal liver 113-hydroxysteroid:NADP oxidoreductase activity. This mitochondrial enzyme, Km=33yM, pH optimum 6, which reduces the 11-dehydro metabolite to the biologically active 116-OH compound, increased sharply, raising the relative amount of the latter in foetal tissues from 15 to 91% during this period. One day after removal of maternal adrenals, foetal corticosterone was normal and maternal levels close to normal, indicating ability of foetal adrenals to function. Maternal hormone, however, crossed to the foetus readily and it was considered most likely that, normally, the maternal source predominates. Regardless of origin, foetal or maternal, however, the hormone is maintained in different foetal tissues in a distinct and different manner. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate