RESUMO
Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU-yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU-yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU-yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU-yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidroxibutirato Desidrogenase/metabolismo , Óperon , Salmonella typhi/genética , Salmonella typhi/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Repressão Catabólica , Humanos , Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/genética , Mutagênese Sítio-Dirigida , Salmonella typhi/crescimento & desenvolvimento , Febre Tifoide/microbiologiaRESUMO
An automatic flow procedure based on the multicommutation concept, comprising three-way solenoid valves, for the spectrophotometric determination of 3-hydroxybutyrate in animal serum and plasma is proposed. The 3-hydroxybutyrate was enzymatically converted to acetoacetate with the reduction of NAD+ to NADH monitored at 340 nm. It was possible to carry out up to 600 determinations without a significant decrease in the analytical signal, with 5 mg of 3-hydroxybutyrate dehydrogenase immobilized on porous silica beads and packed in a column. The system enabled 60 determinations/h of 3-hydroxybutyrate in the range of 10-150 mg L(-1), with a consumption of 0.9 mg of NAD+ and 200 microL of sample per determination. A detection limit of 2 mg L(-1) for both animal serum and plasma and coefficients of variation of 1.4% and 1.2% (n = 17), respectively, were determined. Animal serum and plasma samples were analyzed without previous treatment, the results of which agreed with those obtained using the conventional method (UV kit, Sigma).
Assuntos
Ácido 3-Hidroxibutírico/sangue , Bovinos/sangue , Ovinos/sangue , Espectrofotometria/métodos , Ácido 3-Hidroxibutírico/metabolismo , Animais , Concentração de Íons de Hidrogênio , Hidroxibutirato Desidrogenase/metabolismo , Masculino , Oxirredução , Sensibilidade e Especificidade , TemperaturaRESUMO
Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.
Assuntos
Abelhas/genética , DNA de Protozoário/análise , Técnica de Amplificação ao Acaso de DNA Polimórfico , Animais , Abelhas/enzimologia , Marcadores Genéticos , Genoma de Protozoário , Hidroxibutirato Desidrogenase/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
In the present work, the effect "in vivo' of increasing doses of RU 38486 upon the hepatic mitochondrial function of diabetic rats has been studied. At the same time, the action of adrenalectomy and corticosterone restitution on this function were comparatively demonstrated. The parameters measured were oxygen consumption with the substrates: 3-hydroxybutyrate (HB), succinate (Suc) and malate-glutamate (Mal-glut) in intact liver mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cyt.c oxid.) enzymes in broken liver mitochondria. The groups of animals studied were normal controls (N) and the following groups of diabetic rats: rats without any treatment (D), adrenalectomized rats (D+ADX), rats that were adrenalectomized and treated with corticosterone (D+ADX+C) and four groups treated with increasing oral doses of RU (in mg/kg body wt.), that is, 12.5 (D+RU1), 25.0 (D+RU2), 37.5 (D+RU3) and 50.0 (D+RU4). The results showed a tendency of increasing values of mitochondrial oxygen consumption in diabetic animals treated with RU. The favourable effect of increasing doses of RU on O2 consumption of diabetic rat liver mitochondria with each of the substrates showed a significant association as indicated by the values obtained for the correlation coefficients r (0.95, 0.97 and 0.99 according to the substrate HB, Succ or Mal-glut, respectively). Likewise, the correlation between the treatment with increasing doses of RU and the recovery of enzyme activities showed a significant dose-effect association with r 0.94 for HBD and r = 0.95 for Cyt.c oxid. Adrenalectomy showed a similar effect to treatment with the maximum dose of RU while corticosterone restitution gave measured values similar to those of the D group. In conclusion, the favourable, significant variation of the hepatic mitochondrial function of diabetic rats was demonstrated by the dose-dependent treatment with RU as seen by the correlation statistical study performed. At the same time, the pernicious effect that glucocorticoids exert upon such function in experimental diabetes was confirmed.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ácido 3-Hidroxibutírico , Adrenalectomia , Animais , Anti-Inflamatórios/farmacologia , Corticosterona/farmacologia , Diabetes Mellitus Experimental/enzimologia , Relação Dose-Resposta a Droga , Complexo IV da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Hidroxibutirato Desidrogenase/efeitos dos fármacos , Hidroxibutirato Desidrogenase/metabolismo , Hidroxibutiratos/metabolismo , Malatos/metabolismo , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Succinatos/metabolismo , Ácido SuccínicoRESUMO
In the present work we studied, in female chronic diabetic rats the effect of either the parenteral administration of tamoxifen (TAM) (500 micrograms.kg-1.day-1) for 15 days or the ovariectomy upon the respiration and oscillatory behaviour of intact mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cox) of disrupted liver mitochondria. The treatment with TAM as well as the ovariectomy of diabetic animals significantly increased the respiratory control (RC) and the state 3 (S3) of respiration of intact liver mitochondria with the three substrates assayed (3-hydroxybutyrate, malate-glutamate and succinate). Both treatments also lowered significantly the damped factors of the oscillatory variation of liver intact mitochondria of diabetic rats. Moreover, the two above-mentioned treatments restored the activities of HBD and Cox of liver disrupted mitochondria to normal values. The effect of estrogens at level of its receptors in the modulation of liver mitochondrial function and liver HBD and Cox activities in chronic diabetes is discussed.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/fisiologia , Ovariectomia , Tamoxifeno/farmacologia , Análise de Variância , Animais , Diabetes Mellitus Experimental/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Estradiol/fisiologia , Feminino , Hidroxibutirato Desidrogenase/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Consumo de Oxigênio , RatosRESUMO
In the present work the effects of corticosterone restitution were examined in female rats with chronic streptozotocin (SZ)-induced diabetes upon intact liver mitochondrial function and the activities of 3-hydroxybutyrate dehydrogenase (HBD), succinate dehydrogenase (SD) and cytochrome c oxidase (Cox) of the ruptured organelle. The liver mitochondrial function was analyzed by the respiration and the osmotic oscillatory behaviour. Respiration was measured by polarographic method and both the state 3 of active respiration (S3) and the respiratory control (RC) were determined using the following substrates: 3-hydroxybutyrate, succinate and malate-glutamate. The oscillatory behaviour was measured using as parameters the damping factors (DF) which are the ratios of amplitudes of two consecutive peaks or troughs of the spectrophotometrical tracings of this phenomenon. A group of control normal rats (N) and the following three groups of diabetic rats were studied: controls (D), adrenalectomized (D + ADX) and adrenalectomized with corticosterone restitution (D + ADX + C). The results of mitochondrial respiration showed that the mean values of S3 and RC decreased with the three substrates in the group D + ADX + C compared with D + ADX group (p < 0.001). This group demonstrated a significant increase of S3 and RC values of the respiration compared with the D group. The oscillatory behaviour of liver mitochondria of D + ADX + C group demonstrated a significant increase in the DF of peaks and troughs compared with D + ADX group. The values of DF of the latter group were not significantly different from the N group. The behaviour of the enzymes activities of ruptured liver mitochondria were different for each enzyme in the different groups of treated rats. Thus, in the D + ADX + C group the mean value of the activity of HBD significantly decreased, that of the Cox increased (p < 0.02) and that of SD did not show any variation compared with the corresponding values of the D + ADX group. Likewise, the mean value of HBD activity in this latter group was similar to that of the N group and that of Cox activity was lesser (p < 0.01) than that of the D group. The conclusion is drawn that corticosterone has significant additional diabetogenic effects upon biochemical functions of liver mitochondria in the SZ-induced diabetic state which could occur through the hormone cellular receptors.
Assuntos
Corticosterona/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroxibutirato Desidrogenase/metabolismo , Mitocôndrias Hepáticas/fisiologia , Succinato Desidrogenase/metabolismo , Adrenalectomia , Animais , Corticosterona/administração & dosagem , Diabetes Mellitus Experimental , Feminino , Mitocôndrias Hepáticas/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , RatosRESUMO
En el presente trabajo se describen los efectos de la restitución de corticosterona a ratas con diabetes inducida por estreptozotocina sobre a) la función de mitocondrias enteras de hígado y b) sobre las actividades enzimáticas de la 3- hidroxibutirato deshidrogenasa (HBD), citocromo c oxidasa (Cox) y succinato deshidrogenasa (SD) de mitocondrias que sufrieron ruptura por método físico. La función mitocondrial fue analizadas por la respiración y por el comportamiento oscilatorio osmótico de esas organelas. La respiración se midió por método polarográfico y se midieron el estado 3 de la respiración activa (S3) y el control respiratorio (CR) usando los siguientes sustratos: 3-hidroxibutirato, malato-glutamato y succinato. El comportamiento oscilatorio osmótico se midió usando como parámetro comparativo los coeficientes de amortiguación (CA), que son los cocientes de las amplitudes de dos picos o valles consecutivos obtenidos en el registro espectrofotométrico de dicho fenómeno. Se dispusieron un grupo de ratas normales no diabéticas (N) y los siguientes grupos de ratas diabéticas: controles (D), adrenalectomizadas (D + ADX) y adrenalectomizadas con restitución de corticosterona (D + ADX + C). Los resultados de la respiración mitocondrial mostraron que los valores medios de S3 y CR disminuyeron con los tres sustratos en el grupo D + ADX + C comparado con el grupo D + ADX (p < 0,001). Este grupo demostró, a su vez, un aumento significativo de los valores medios de S3 y CR de respiración con respecto al del grupo D. El comportamiento oscilatorio de mitocondrias enteras de hígado del grupo D + ADX + C demostró un significativo aumento de los CA de picos y valles comparado con los del grupo D + ADX. Los valores de CA del último grupo no fueron significativamente diferentes de los del grupo N. El comportamiento de las actividades enzimáticas de mitocondrias fraccionadas fueron diferentes para cada enzima según los diferentes tratamientos en los grupos de ratas diabéticas. En el grupo D + ADX + C el valor medio de la actividad HBD disminuyó significativamente, el de la Cox aumentó (p < 0,02) y el de la SD no mostró variación alguna con respecto a los correspondientes valores medidos de esas enzimas en el grupo D + ADX. Asimismo, el valor medio de la actividad HBD en este último grupo fue similar al del grupo N y el de Cox fue menor (p < 0,001) que el del grupo D. Se concluye que la corticosterona tiene un significativo efecto diabetogénico sobre la función bioquími
Assuntos
Animais , Feminino , Ratos , Corticosterona/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroxibutirato Desidrogenase/metabolismo , Mitocôndrias Hepáticas/fisiologia , Succinato Desidrogenase/metabolismo , Adrenalectomia , Consumo de Oxigênio , Corticosterona/administração & dosagem , Diabetes Mellitus Experimental , Mitocôndrias Hepáticas/enzimologiaRESUMO
En el presente trabajo se describen los efectos de la restitución de corticosterona a ratas con diabetes inducida por estreptozotocina sobre a) la función de mitocondrias enteras de hígado y b) sobre las actividades enzimáticas de la 3- hidroxibutirato deshidrogenasa (HBD), citocromo c oxidasa (Cox) y succinato deshidrogenasa (SD) de mitocondrias que sufrieron ruptura por método físico. La función mitocondrial fue analizadas por la respiración y por el comportamiento oscilatorio osmótico de esas organelas. La respiración se midió por método polarográfico y se midieron el estado 3 de la respiración activa (S3) y el control respiratorio (CR) usando los siguientes sustratos: 3-hidroxibutirato, malato-glutamato y succinato. El comportamiento oscilatorio osmótico se midió usando como parámetro comparativo los coeficientes de amortiguación (CA), que son los cocientes de las amplitudes de dos picos o valles consecutivos obtenidos en el registro espectrofotométrico de dicho fenómeno. Se dispusieron un grupo de ratas normales no diabéticas (N) y los siguientes grupos de ratas diabéticas: controles (D), adrenalectomizadas (D + ADX) y adrenalectomizadas con restitución de corticosterona (D + ADX + C). Los resultados de la respiración mitocondrial mostraron que los valores medios de S3 y CR disminuyeron con los tres sustratos en el grupo D + ADX + C comparado con el grupo D + ADX (p < 0,001). Este grupo demostró, a su vez, un aumento significativo de los valores medios de S3 y CR de respiración con respecto al del grupo D. El comportamiento oscilatorio de mitocondrias enteras de hígado del grupo D + ADX + C demostró un significativo aumento de los CA de picos y valles comparado con los del grupo D + ADX. Los valores de CA del último grupo no fueron significativamente diferentes de los del grupo N. El comportamiento de las actividades enzimáticas de mitocondrias fraccionadas fueron diferentes para cada enzima según los diferentes tratamientos en los grupos de ratas diabéticas. En el grupo D + ADX + C el valor medio de la actividad HBD disminuyó significativamente, el de la Cox aumentó (p < 0,02) y el de la SD no mostró variación alguna con respecto a los correspondientes valores medidos de esas enzimas en el grupo D + ADX. Asimismo, el valor medio de la actividad HBD en este último grupo fue similar al del grupo N y el de Cox fue menor (p < 0,001) que el del grupo D. Se concluye que la corticosterona tiene un significativo efecto diabetogénico sobre la función bioquími
Assuntos
Animais , Feminino , Ratos , Corticosterona/farmacologia , Mitocôndrias Hepáticas/fisiologia , Hidroxibutirato Desidrogenase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Succinato Desidrogenase/metabolismo , Corticosterona/administração & dosagem , Mitocôndrias Hepáticas/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Diabetes Mellitus Experimental , AdrenalectomiaRESUMO
Chronic diabetes induced by the injection of streptozotocin in male and female albino adult rats provoked significant alteration of liver mitochondrial function 30 or 35 days after administration of the drug. Thus, we obtained mean values of respiratory control (RC) and state 3 (S3) with 3-hydroxybutyrate as substrate 40 or 50% lower than those of non-diabetic animals. With other substrates (malate-glutamate, succinate) the decrease of RC and S3 in the diabetic animals was 20% or 30% of the normal mean values. The osmotic damped oscillations of mitochondria were measured as another parameter of the organella function. It was assayed with valinomycin as K+ ionophore and succinate as substrate. In diabetic rats of both sexes we found a significant increase of the mean damping factor of these oscillatory variations compared with normal values. The above-mentioned results indicate a lesser elasticity and an impaired K+ transport of mitochondria across the inner membrane in diabetic animals. Both reported parameters, respiration and oscillatory variations of liver mitochondria, were measured in normal non-diabetic rats and in the groups of diabetic rats referred to as follows: 1) intact (male and female), 2) gonadectomized (male and female), 3) oophorectomized with restitution of 17 beta-estradiol. Ovariectomized diabetic rats showed a significant increase in the values of the RC and S3 of liver mitochondria compared with intact female diabetic animals. The withdrawal of the ovarian hormone in female diabetic rats significantly decreased the values of the damping factors of the oscillatory mechanism and they were similar to the normal. The restitution of 17 beta-estradiol to oophorectomized diabetic rats resulted in a decrease of liver mitochondrial respiration. The damping factor of liver mitochondria of the oophorectomized diabetic rats treated with the estrogen showed values significantly higher than those of female diabetic animals without the hormone and similar to the values of the intact diabetic female rats. Castration of male rats did not produce any effect upon the liver mitochondrial RC and S3 or upon the mean damping factor of the oscillatory variation either. Then the castration of male diabetic rats did not modify the mitochondrial function. In contrast, the oophorectomy of diabetic animals produced amelioration of mitochondrial respiration and oscillatory behavior. The conclusion is drawn that in female rats the circulating 17 beta-estradiol produced a pernicious effect upon liver mitochondrial function in the experimental diabetic state.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Estradiol/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Orquiectomia , Ovariectomia , Consumo de Oxigênio/efeitos dos fármacos , Animais , Feminino , Hidroxibutirato Desidrogenase/metabolismo , Masculino , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Ratos , EstreptozocinaRESUMO
La diabetes crónica inducida por inyección de estreptozotocina (SZ) después de 30 a 35 días produjo significativas alteraciones de la función mitocondrial del hígado. Se comprobó: 1§) disminución significativa de los valores en el estado 3 (respiración activa con los sustratos 3-hidroxibutirato, malato-glutamato y succinato) y 2§) aumento significativo de los coeficientes de amortiguación de oscilaciones osmóticas. Los parámetros mencionados se midieron en ratas machos y hembras adultas, normales, y los siguientes grupos de ratas diabéticas:) a) machos y hembras enteras, b) machos y hembras gonadectomizados y c) hembras obariectomizadas com restitución de 17 ß-estradiol. Los resultados demonstraron que la ooforectomía de ratas diabéticas provocó aumento significativo de los valores del control respiratorio y del estado 3 de la respiración y disminución de los coeficientes de amortiguación hasta valores cercanos a los de ratas normales no diabéticas. La restitución de 17 ß-estradiol a ratas ooforectomizadas diabéticas volvió ambos parámetros de la función mitocondrial a valores semejantes a los de ratas enteras diabéticas. La orquidectomía de ratas diabéticas no demostró efecto alguno sobre los parámetros mitocondriales ensayados en comparación con machos enteros diabéticos. Se concluye que el endógeno de ratas hembras diabéticas actúa negativamente sobre las alteraciones de la función mitocondrial del hígado en la diabetes experimental crónica