RESUMEN
Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells.
Asunto(s)
Candida , Proteínas Fúngicas , Glucosafosfato Deshidrogenasa , Modelos Químicos , Oryza/química , Polisacáridos/química , Candida/enzimología , Candida/crecimiento & desarrollo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glucosafosfato Deshidrogenasa/biosíntesis , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/aislamiento & purificaciónRESUMEN
Cadmium chloride is an environmental toxicant implicated in human prostate carcinogenesis. The mechanism of its toxicity is far from fully understood. This study evaluates the effect of exposure to an oral non-carcinogenic dose of cadmium (15 ppm in drinking water for three months) on different parameters of the ventral prostatic lobe of normal and exposed rats. We analyzed the histology by optic light microscopy, activities of antioxidant enzymes (CAT, SOD, GPx and G-6-PDH), expression of iNOS and COX-2 by Western blot, expression of MT-I, MT-II, IGF-I, IGF-BP5 and rtert by RT-PCR. Histological changes were found: the height of the cells decreased, acinar lumen were enlarged and they lost the typical invaginations. Lipoperoxidation was increased in the Cd group and the antioxidant enzymes changed their activities: SOD increased, CAT and G-6-PDH decreased and GPx did not show variations. iNOS and COX-2 did not change their expressions. MT-I and IGF-BP5 mRNA increased while MT-II, IGF-I and rtert did not show variations. Cd exposure induces important morphological changes in the prostate, which could be a consequence of lipoperoxidation and oxidative stress, which are not related to iNOS and COX-2. The histology suggests an involution state of the gland, confirmed by the expression of IGF-I, IGF-BP5 and rtert.
Asunto(s)
Cadmio/toxicidad , Estrés Oxidativo/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Animales , Antioxidantes/metabolismo , Western Blotting , Catalasa/biosíntesis , Catalasa/genética , Ciclooxigenasa 2 , Glucosafosfato Deshidrogenasa/biosíntesis , Glucosafosfato Deshidrogenasa/genética , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Isoenzimas/biosíntesis , Masculino , Metalotioneína/biosíntesis , Metalotioneína/genética , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Próstata/efectos de los fármacos , ARN/biosíntesis , ARN/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
A strain of genetically modified Saccharomyces cerevisiae (S. cerevisiae) W303 181 was used to improve glucose-6-phosphate dehydrogenase (G6PDH) production in aerobic culture. Fed-batch cultures were carried out in a 5 L fermentor at variable values of the parameter K, namely, 0.2, 0.3, 0.5, 0.7, and 0.8 h(-)(1). The highest G6PDH production (1164 U/L) and specific activity (517 U/g(cell)) were obtained using the following conditions: glucose, 5.0 g/L; adenine, 8 microg/mL; histidine, 8 microg/mL; tryptophan, 8 microg/mL; temperature, 30 degrees C; inoculum, 1.28 g/L; pH, 5.7; agitation, 400 rpm; aeration, 2.2 vvm; and K, 0.2 h(-)(1). The exponential feeding pattern increased cell density (2.14 g/L), enzyme productivity (149.27), and biomass yield (0.18 g(glu)/g(cell)( )(mass)). The level of G6PDH in the genetically modified S. cerevisiae was approximately 4.1-fold higher than that found in a commercial strain.
Asunto(s)
Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/métodos , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/biosíntesis , Reología/métodos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , División Celular , Activación Enzimática , Glucosafosfato Deshidrogenasa/genética , Modelos Biológicos , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/citologíaRESUMEN
The elevated rate of oxygen consumption and high amount of polyunsaturated fatty acids make the central nervous system vulnerable to oxidative stress. The effect of Walker-256 tumor growth on oxi-reduction indexes in the hypothalamus (HT), cortex (CT), hippocampus (HC) and cerebellum (CB) of male Wistar rats was investigated. The presence of the tumor caused an increase in thiobarbituric acid reactant substances (TBARs) in the HT, CB and HC. Due to tumor growth, the activity of glucose-6-phosphate dehydrogenase increased in the HT and CB, whereas citrate synthase activity was reduced in the HT, CT and CB. Therefore, the potential for generation of reducing power is increased in the cytosol and decreased in the mitochondria of various brain regions of Walker-256 tumor-bearing rats. These changes occurred concomitantly with an unbalance in the brain enzymatic antioxidant system. The tumor decreased the activities of catalase in the HT and CB and of glutathione peroxidase in the HT, CB and HC, and raised the CuZn-superoxide dismutase activity in the HT, CB and HC. These combined findings indicate that Walker-256 tumor growth causes oxidative stress in the brain.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Carcinoma 256 de Walker/metabolismo , Animales , Anorexia/etiología , Neoplasias Encefálicas/complicaciones , Carcinoma 256 de Walker/complicaciones , Catalasa/biosíntesis , Catalasa/genética , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Citrato (si)-Sintasa/biosíntesis , Citrato (si)-Sintasa/genética , Citosol/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucosafosfato Deshidrogenasa/biosíntesis , Glucosafosfato Deshidrogenasa/genética , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Hipotálamo/metabolismo , Isoenzimas/biosíntesis , Isoenzimas/genética , Peroxidación de Lípido , Masculino , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Oxidación-Reducción , Estrés Oxidativo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abundant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase. As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up to 14-fold higher than the level of G6PD found in commercially available strains.
Asunto(s)
Glucosafosfato Deshidrogenasa/biosíntesis , Glucosafosfato Deshidrogenasa/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Aerobiosis , Reactores Biológicos , Biotecnología , Expresión Génica , Plásmidos/genética , ARN de Hongos/análisis , ARN de Hongos/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Recombinación GenéticaRESUMEN
1. The effect of administration of fish oil by gavage on key enzyme activities of glucose metabolism of the thymus, spleen, and mesenteric lymph nodes was investigated. 2. The activities of hexokinase, glucose-6-phosphate dehydrogenase, and citrate synthase in the lymphoid organs were markedly raised due to a daily administration of fish oil by gavage (0.4% of body weight). 3. These findings indicate that the therapeutic utilization of fish oil does affect the metabolism of the lymphoid organs, and possibly immune function; however, the mechanism involved remains to be investigated.
Asunto(s)
Citrato (si)-Sintasa/biosíntesis , Aceites de Pescado/farmacología , Glucosafosfato Deshidrogenasa/biosíntesis , Hexoquinasa/biosíntesis , Tejido Linfoide/enzimología , Animales , Aceites de Pescado/administración & dosificación , Masculino , RatasRESUMEN
Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzing sugar phosphorylation by ATP are reviewed. Hexokinases may exist, mainly in prokaryotes, as sugar-specific kinases (glucokinase, fructokinase, mannokinase) or as ubiquitous hexose-kinases which are relatively unspecific for the natural hexoses. Enzymes presenting intermediate specificity (e.g. mannofructokinases) have been also described. With a few exceptions, the molecular mass of a variety of hexokinases may be either 25 kDa, 50 kDa or 100 kDa. The smaller hexokinases have been found in some microorganisms whereas the 50 kDa enzymes are found (with only one exception) in most invertebrates and in a particular isozyme from vertebrates (hexokinase D). The 100 kDa enzymes are restricted to vertebrates (hexokinases A, B and C). These facts have led to the speculation that gene duplication events have played an important role in the evolutionary development of the hexokinases from present day organisms. The fact that the 100 kDa hexokinases are allosterically inhibited by the product, glucose 6-P, may indicate that a duplicated active site has evolved to a regulatory binding site. Comparisons of the amino acid sequence of a few peptides from hexokinase C are presented to support the gene duplication hypothesis. Also, partial sequence comparisons of vertebrate hexokinases with the sequences of two hexokinase isozymes from yeast show strong similarities suggesting a rather slow amino acid substitution rate of homologous genes.