RESUMEN
Increasing attention has been paid in the past decade to assessing the toxicological effects of nanoparticles and finding a protectant; thus, the current study aimed to investigate the protective effect of the mitochondria-targeting drug methylene blue (MB) against copper oxide nanoparticle (CuO-NP)-induced neurobehavioral toxicity in rats. For this purpose, twenty rats were allocated to four equal groups (n = 5). The negative control group received distilled water intraperitoneally (IP) and Tween 80 (10 %) orally. The CuO-NP group was given a dose of 100 mg/kg of CuO-NPs, administered orally, and the positive control group was treated with 1 mg/kg MB intraperitoneally (IP). The final group was concurrently exposed to CuO-NPs and MB for 14 consecutive days. At the end of the study, each group was neurobehaviorally blind tested relative to other experimental animals, then brain tissue markers were determined and a histopathological examination was conducted. The results showed that supplementation with CuO-NPs induced neurobehavioral alterations; increased Cu content in the brain; and enhanced lipid peroxidation (malondialdehyde [MDA]), protein peroxidation (protein carbonyl [PC]), and DNA oxidative damage (8-hydroxy-2-deoxyguanosine [8-OH-dG]) compared to other treatments. In addition, a decrease was noted in the mitochondrial dehydrogenases' (aldehyde dehydrogenase 2 [ALDH2], and glutamate dehydrogenase [GDH]) activity in Cu-exposed rats. The histopathological findings revealed shrunken, pyknotic, and hypereosinophic cortical neurons and increased immune positive brown staining of caspase-3 protein, indicating apoptosis. Co-treatment with methylene blue ameliorated the neurotoxic effects of CuO-NPs; therefore, MB evidently had a powerful modulatory effect against the neurotoxicity of nano-Cu oxide via its antioxidant and mitochondrial protection properties.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/efectos de los fármacos , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cobre/toxicidad , Inhibidores Enzimáticos/farmacología , Glutamato Deshidrogenasa/efectos de los fármacos , Azul de Metileno/farmacología , Nanopartículas/toxicidad , Síndromes de Neurotoxicidad/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Cobre/administración & dosificación , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Masculino , Azul de Metileno/administración & dosificación , Nanopartículas/administración & dosificación , RatasRESUMEN
Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.
Asunto(s)
Ciclo del Ácido Cítrico/efectos de los fármacos , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Compuestos Onio/farmacología , Compuestos de Tritilo/farmacología , Animales , Línea Celular , Citrato (si)-Sintasa/efectos de los fármacos , Citrato (si)-Sintasa/metabolismo , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/efectos de los fármacos , Isocitrato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/efectos de los fármacos , Malato Deshidrogenasa/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/enzimología , Músculo Esquelético/enzimología , Complejo Piruvato Deshidrogenasa/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Ratas WistarRESUMEN
Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the cassette mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl- CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA.
Asunto(s)
Cisteína/química , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Palmitoil Coenzima A/farmacología , Regulación Alostérica/efectos de los fármacos , Sustitución de Aminoácidos , Sitios de Unión , Glutamato Deshidrogenasa/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Isoenzimas/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismoRESUMEN
Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.
Asunto(s)
Acetatos/farmacología , Antraquinonas/metabolismo , Ciclopentanos/farmacología , Oxilipinas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rubia/metabolismo , Antraquinonas/análisis , Compuestos de Bifenilo/farmacología , Ciclo del Carbono , Supervivencia Celular , Células Cultivadas , Eritritol/análogos & derivados , Eritritol/metabolismo , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Isocitrato Deshidrogenasa/efectos de los fármacos , Isocitrato Deshidrogenasa/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Compuestos Onio/farmacología , Vía de Pentosa Fosfato/efectos de los fármacos , Inmunidad de la Planta , Prolina/análisis , Prolina/efectos de los fármacos , Rubia/citología , Rubia/enzimología , Rubia/crecimiento & desarrollo , Transducción de Señal , Fosfatos de Azúcar/metabolismo , Factores de TiempoRESUMEN
Magnesium is one of the essential elements for plant growth and cerium is a beneficial element for plant growth. However, the effects of the fact that cerium improves the nitrogen metabolism of plants under magnesium deficiency is poorly understood. The main aim of the study was to determine the role of cerium in the amelioration of magnesium-deficiency effects in spinach plants. Spinach plants were cultivated in Hoagland's solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present media and magnesium-deficient media. Spinach plants grown in the magnesium-present media and magnesium-deficient media were measured for key enzyme activities involved in nitrogen metabolism such as nitrate reductase, nitrite reductase, glutamate dehydrogenase, glutamate synthase, urease, glutamicpyruvic transaminase, and glutamicoxaloace protease transaminase. As the nitrogen metabolism in spinach was significantly inhibited by magnesium deficiency, it caused a significant reduction of spinach plant weight, leaf turning chlorosis. However, cerium treatment grown in magnesium-deficiency media significantly promoted the activities of the key enzymes as well as the contents of the free amino acids, chlorophyll, soluble protein, and spinach growth. It implied that Ce3+ could partly substitute for magnesium to facilitate the transformation from inorganic nitrogen to organic nitrogen, leading to the improvement of spinach growth, although the metabolism needs to be investigated further.
Asunto(s)
Cerio/farmacología , Nitrógeno/metabolismo , Spinacia oleracea/efectos de los fármacos , Spinacia oleracea/metabolismo , Alanina Transaminasa/metabolismo , Aspartato Aminotransferasas/metabolismo , Activación Enzimática/efectos de los fármacos , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Sintasa/metabolismo , Deficiencia de Magnesio , Nitrato-Reductasa/metabolismo , Nitratos/metabolismo , Nitrito Reductasas/metabolismo , Ureasa/metabolismoRESUMEN
The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of L-glutamine and L-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since L-glutamine and L-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.
Asunto(s)
Amoníaco/metabolismo , Arginina/metabolismo , Glutamina/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Vena Porta/metabolismo , Urea/metabolismo , Amoníaco/sangre , Cloruro de Amonio/farmacología , Animales , Arginina/análisis , Arginina/sangre , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/análisis , Glutamina/sangre , Mucosa Intestinal/efectos de los fármacos , Hígado/efectos de los fármacos , Sus scrofa , Urea/agonistas , Urea/sangreRESUMEN
AIM OF THE STUDY: The effects of administration of aqueous extract of Fadogia agrestis (Schweinf. Ex Hiern) stem on some testicular function indices of male rats (Rattus norvegicus) and their recovery potentials for 10 days were investigated. MATERIALS AND METHODS: Rats were grouped into four: A, B, C and D where A (the control) received orally 1 ml of distilled water (the vehicle), B, C and D (the test groups) received orally on daily basis graded doses of 18, 50 and 100mg/kg body weight of the plant extract, respectively, for 28 days. RESULTS: Compared with the control, extract administration for 28 days at all the doses resulted in significant increase (P<0.05) in percentage testes-body weight ratio, testicular cholesterol, sialic acid, glycogen, acid phosphatase and gamma-glutamyl transferase activities while there was significant decrease (P<0.05) in the activities of testicular alkaline phosphatase, acid phosphatase, glutamate dehydrogenase and concentrations of protein. Recoveries were made by the animals on some of the testicular function indices mainly at 18 mg/kg body weight. CONCLUSIONS: The alterations brought about by the aqueous extract of Fadogia agrestis stem are indications of adverse effects on the male rat testicular function and this may adversely affect the functional capacities of the testes. The recovery made at the dose of 18 mg/kg body weight as used in folklore medicine suggests that it does not exhibit permanent toxicity at this dose.
Asunto(s)
Extractos Vegetales/toxicidad , Rubiaceae/química , Testículo/efectos de los fármacos , Fosfatasa Ácida/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Administración Oral , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Glucógeno/metabolismo , Masculino , Ácido N-Acetilneuramínico/metabolismo , Nigeria , Extractos Vegetales/administración & dosificación , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Ratas , Testículo/metabolismo , gamma-Glutamiltransferasa/efectos de los fármacos , gamma-Glutamiltransferasa/metabolismoRESUMEN
According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate-metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as > or = 20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients.
Asunto(s)
Antipsicóticos/uso terapéutico , Plaquetas/enzimología , Glutamato Deshidrogenasa/sangre , Glutamato-Amoníaco Ligasa/sangre , Esquizofrenia/sangre , Esquizofrenia/tratamiento farmacológico , Adulto , Benzodiazepinas/uso terapéutico , Femenino , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Humanos , Cinética , Masculino , Persona de Mediana Edad , Olanzapina , Valores de Referencia , Esquizofrenia/enzimologíaRESUMEN
The specific activity of recombinant Pyrobaculum islandicum glutamate dehydrogenase (pis-GDH) expressed in Escherichia coli is much lower than that of the native enzyme. However, when the recombinant enzyme is heated at 90 degrees C or exposed to 5 M urea, the activity increases to a level comparable to that of the native enzyme. Small-angle X-ray scattering measurements revealed that the radius of gyration (R(g,z)) of the hexameric recombinant enzyme was reduced to 47 A from 55 A by either heat or urea, and that the final structure of the active enzyme is the same irrespective of the mechanism of activation. Activation was accompanied by a shift in the peaks of the Kratky plot, though the molecular mass of the enzyme was unchanged. The activation-induced decline in R(g,z) followed first-order kinetics, indicating that activation of the enzyme involved a transition between two states, which was confirmed by singular-value decomposition analysis. When the low-resolution structure of the recombinant enzyme was restored using ab initio modeling, we found it to possess no point symmetry, whereas the heat-activated enzyme possessed 32-point symmetry. In addition, a marked increase in the fluorescence emission was observed with addition of ANS to the inactive recombinant enzyme but not the active forms, indicating that upon activation hydrophobic residues on the surface of the recombinant protein moved to the interior. Taken together, these data strongly suggest that subunit rearrangement, i.e., a change in the quaternary structure of the hexameric recombinant pis-GDH, is essential for activation of the enzyme.
Asunto(s)
Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/metabolismo , Estructura Cuaternaria de Proteína , Pyrobaculum/enzimología , Rastreo Diferencial de Calorimetría , Clonación Molecular , Activación Enzimática , Glutamato Deshidrogenasa/efectos de los fármacos , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/metabolismo , Urea/farmacología , Difracción de Rayos XRESUMEN
Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.
Asunto(s)
Depuradores de Radicales Libres/farmacología , Glucosafosfato Deshidrogenasa/efectos de la radiación , Glutamato Deshidrogenasa/efectos de la radiación , Animales , Ácido Benzoico/farmacología , Partículas beta , Bovinos , Radicales Libres/química , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/efectos de los fármacos , Manitol/farmacología , Protectores contra Radiación/farmacología , Agua/químicaRESUMEN
Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k(inact) of 2.7 min(-1) and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP+, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 microM. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD+-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in alpha helices and beta sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.
Asunto(s)
Aluminio/toxicidad , Glutamato Deshidrogenasa/efectos de los fármacos , Aluminio/metabolismo , Quelantes/farmacología , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/toxicidad , Humanos , Concentración de Iones de Hidrógeno , Ácidos Nucleicos/metabolismoRESUMEN
Data on liver enzyme elevations were collected in a retrospective study of 7,263 treatment courses with haloperidol, clozapine, perphenazine, and perazine. Charts of 233 patients hospitalized between 1980 and 1992 at Tübingen University Psychiatric Clinic were selected because clinically relevant increases of liver enzymes had been detected during monotherapy with one of the four examined neuroleptics. At least one hepatic enzyme (mostly alanine aminotransferase [ALAT]) exceeded the established reference range of 3-fold elevations of ALAT, aspartate aminotransferase, gamma-glutamyl transpeptidase, and glutamate dehydrogenase and 2-fold elevations of alkaline phosphatase (AP) during monotherapy with clozapine in 15%, perazine in 7.6%, perphenazine in 4%, and haloperidol in 2.4% of the cases. If all liver enzyme abnormalities with any elevation greater than the conventional upper limits are considered, incidences were as follows: clozapine, 78%; perphenazine, 62%; perazine, 59%; and haloperidol, 50%. Testing for overall differences within the four neuroleptics resulted in significantly different incidences of liver enzyme elevations (chi2 test,p < 0.0001). Threefold increases of AP (>540 U/L) were seen in three patients receiving haloperidol (0.3%) only. Twofold increases of AP (>360 U/L) were distributed as follows: clozapine, 1%; haloperidol, 0.8%; perazine, 0.3%; and perphenazine, 0.1%. Only in the group with 1-fold elevations of AP (>180 U/L) were the differences within the drug regimens significant (clozapine, 40.3%; haloperidol, 33.2%; perphenazine, 23.4%; and perazine, 23.1%; chi2 test, p < 0.0001). In the period under study, no instance of icterus occurred.
Asunto(s)
Antipsicóticos/farmacología , Hígado/efectos de los fármacos , Esquizofrenia/enzimología , Alanina Transaminasa/efectos de los fármacos , Alanina Transaminasa/metabolismo , Antipsicóticos/uso terapéutico , Aspartato Aminotransferasas/efectos de los fármacos , Aspartato Aminotransferasas/metabolismo , Distribución de Chi-Cuadrado , Clozapina/farmacología , Clozapina/uso terapéutico , Femenino , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Haloperidol/farmacología , Haloperidol/uso terapéutico , Humanos , Hígado/enzimología , Masculino , Perazina/farmacología , Perazina/uso terapéutico , Perfenazina/farmacología , Perfenazina/uso terapéutico , Estudios Retrospectivos , Esquizofrenia/tratamiento farmacológico , Estadísticas no Paramétricas , gamma-Glutamiltransferasa/efectos de los fármacos , gamma-Glutamiltransferasa/metabolismoRESUMEN
Reactive oxygen species generated by xanthine oxidase during reperfusion of ischemic liver might in part be responsible for ischemic organ injury. In normothermic ischemia/reperfusion rat model, we investigated whether allopurinol pretreatment improved ischemia-induced mitochondrial dysfunction. Rats were subjected to 60 min of hepatic ischemia and to 1 h and 5 h of reperfusion thereafter. At 18 h and 1 h before ischemia, the animals received 0.25 mL of either saline or allopurinol (50 mg/kg) i.p. In saline-treated ischemic rats, serum aspartate aminotransferase levels increased significantly at 5 h (4685 +/- 310 IU/L) and were significantly reduced with allopurinol pretreatment. Similarly, mitochondrial lipid peroxidation was elevated in the saline-treated ischemic group, but this elevation was prevented by allopurinol. In contrast, mitochondrial glutamate dehydrogenase activity and ketone body ratio decreased in the saline-treated group, but this decrease was also inhibited by allopurinol. Hepatic ATP levels in the saline-treated rats were 42% lower 5 h after reperfusion. However, treatment with allopurinol resulted in significantly higher ATP levels. Allopurinol treatment preserved the concentration of AMP in ischemic liver but inhibited the accumulation of xanthine in reperfused liver. Our findings suggest allopurinol protects against mitochondrial injury, which prevents a mitochondrial oxidant stress and lipid peroxidation and preserves the hepatic energy metabolism.
Asunto(s)
Alopurinol/farmacología , Inhibidores Enzimáticos/farmacología , Isquemia/metabolismo , Hígado/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/efectos de los fármacos , Metabolismo Energético , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Cetonas/sangre , Peroxidación de Lípido , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Xantina Oxidasa/antagonistas & inhibidoresRESUMEN
The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
Asunto(s)
Arginina/farmacología , Glutamato Deshidrogenasa/genética , Pseudomonas aeruginosa/genética , Regulación Alostérica , Secuencia de Aminoácidos , Secuencia de Bases , Ácido Cítrico/farmacología , Clonación Molecular , Inducción Enzimática , Regulación Bacteriana de la Expresión Génica , Glutamato Deshidrogenasa/efectos de los fármacos , Ácido Glutámico/farmacología , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutación , NAD , Regiones Promotoras Genéticas , Conformación Proteica , Pseudomonas aeruginosa/enzimología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Male Sprague-Dawley rats (8 per group) were administered a single oral dose of cyclosporine A (10, 30 and 50 mg/day) for 5 days or vehicle (corn oil, 1.5 mL/kg) and urinary enzymes excretion was monitored. Only minor changes in enzymuria were observed in the 10 and 30 mg/kg group. However, in the 50 mg/kg group, nephrotoxicity was evident by significant increase in the excretion of N-acetyl-beta-D-glucosaminidase (NAG), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH on day 2 of treatment. As chemotherapeutic drug interaction with cyclosporine A (CyA) is thought to aggravate its nephrotoxicity, the effect of combined CyA (30 mg/kg) and the antibiotic gentamicin (50 mg/kg) for 5 days was investigated. Gentamicin alone caused a significant enzymuria, whilst co-treatment of rats with CyA gave rise to increased changes in enzymuria on days 1 and 2, between the groups receiving gentamicin+vehicle and those receiving CyA+gentamicin. This was particularly marked by significant changes in LDH excretion. In contrast these observed differences were not paralleled by changes in serum creatinine and other functional parameters. Treatment with gentamicin, appears to enhance CyA nephrotoxicity, but only in the first 2 days, after this there was no significant differences between the two groups. Our data suggest that urinary enzyme measurements could serve as a valuable non-invasive means of monitoring renal performance in animals or humans who may be exposed to combination of drugs. CyA is found not to potentiate the nephrotoxic effect of gentamicin in the animal model used in this study. It therefore appears safe to use the combined therapy particularly in the treatment of transplant patients.
Asunto(s)
Ciclosporina/toxicidad , Enzimas/orina , Gentamicinas/toxicidad , Inmunosupresores/toxicidad , Riñón/efectos de los fármacos , Riñón/enzimología , Acetilglucosaminidasa/efectos de los fármacos , Acetilglucosaminidasa/orina , Administración Oral , Análisis de Varianza , Animales , Ciclosporina/administración & dosificación , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Enzimas/efectos de los fármacos , Gentamicinas/administración & dosificación , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/orina , Inmunosupresores/administración & dosificación , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/orina , Masculino , Probabilidad , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.
Asunto(s)
Archaeoglobus fulgidus/química , Enzimas/química , Glicerofosfatos/farmacología , Rubredoxinas/química , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/efectos de los fármacos , Animales , Archaeoglobus fulgidus/fisiología , Biomasa , Clonación Molecular , Clostridium/metabolismo , Desulfovibrio/metabolismo , Estabilidad de Medicamentos , Estabilidad de Enzimas , Enzimas/efectos de los fármacos , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/efectos de los fármacos , Glicerol/farmacología , Glicerofosfatos/síntesis química , Glicerofosfatos/aislamiento & purificación , Calor , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/efectos de los fármacos , Músculo Esquelético/enzimología , Fosfatos/farmacología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Rubredoxinas/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Thermococcus/enzimologíaRESUMEN
Glutamate dehydrogenase (GDH) isomerizes in response to pesticides and environmental chemicals, but the biochemical basis of the isomerization is not known. Clearer understanding of the isomerization would permit expansion of its utility in the diagnosis of the responses of plant tissues to challenged environments. Peanut plants were treated with different rates of Basagran (3-(1-methylethyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide), Bravo 720 (tetrachloroiso-phthalonitrile), and Sevin XLR Plus (1-naphthyl N-methylcarbamate). Free solution isoelectric focusing, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fractionated the peanut seed GDH to its constituent subunits and degradation polypeptides. After western transfer to nitrocellulose membrane, the GDH subunits and degradation polypeptides were immunodetected with anti-GDH. The pesticide treatments did not induce increased proteolytic activity, but induced about 50% degradation of the GDH, whereas the GDH of the control peanut suffered only about 25% degradation, thus showing that the degradation rate was about double the rate of de novo synthesis in the pesticide treatments. The heavy displacement of the GDH subunit equilibrium toward degradation explains the biochemical basis of the isomerization reaction.
Asunto(s)
Arachis/enzimología , Glutamato Deshidrogenasa/química , Plaguicidas/farmacología , Endopeptidasas/metabolismo , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , IsomerismoRESUMEN
The activity of aspartate aminotransferase, glutamate dehydrogenase in the liver of rats in 1, 7 and 15 days after gamma irradiation effect of the dose of 0.5 Gy on the background of consumption by animals of sodium nitrate, sodium nitrite and nitrosodiethylamine was studied. The combined influence of chemical agents and gamma irradiation modified the effects of nitro compounds-xenobiotics on processes of the synthesis and dissociation of the glutamic acid as well as the intensity of transamination of the reamination by aspartate aminotransferase.
Asunto(s)
Aspartato Aminotransferasas/efectos de los fármacos , Aspartato Aminotransferasas/efectos de la radiación , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/efectos de la radiación , Ácido Glutámico/metabolismo , Ácido Glutámico/efectos de la radiación , Nitrocompuestos/farmacología , Aminación/efectos de los fármacos , Aminación/efectos de la radiación , Análisis de Varianza , Animales , Aspartato Aminotransferasas/metabolismo , Desaminación/efectos de los fármacos , Desaminación/efectos de la radiación , Rayos gamma , Glutamato Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/efectos de la radiación , Masculino , Ratas , Factores de TiempoRESUMEN
The effect of orthovanadate, vanadyl sulphate and vanadyl acetylacetonate on glutamate dehydrogenase activity was studied in liver mitochondria and isolated hepatocytes of rabbit. In permeabilized mitochondria with free access of substrates and drugs to glutamate dehydrogenase, orthovanadate and vanadyl sulphate at 200 microM concentrations decreased both glutamate synthesis and glutamate deamination by 80 and 50%, respectively, while vanadyl acetylacetonate was less potent. In view of kinetic data obtained at various ammonium concentrations, orthovanadate appeared to be a competitive inhibitor (Ki = 40 +/- 3 microM), while vanadyl sulphate was a non-competitive one (Ki = 147 +/- 10 microM). In contrast to orthovanadate, vanadyl sulphate augmented the inhibitory action of increased above 0.5 mM 2-oxoglutarate concentrations. All these effects on the enzyme activity were partially reversed in the presence of L-leucine and ADP, which are allosteric activators of glutamate dehydrogenase. Moreover, all compounds studied suppressed both glutamate formation and glutamate deamination in isolated hepatocytes incubated under various metabolic conditions, as concluded from decreased rates of glutamate and urea synthesis, respectively. In view of these observations it seems likely that vanadium-containing compounds may be potent inhibitors of glutamate metabolism in liver.
Asunto(s)
Glutamato Deshidrogenasa/efectos de los fármacos , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Compuestos de Vanadio/farmacología , Animales , Desinfectantes/farmacología , Glutamato Deshidrogenasa/metabolismo , Hidroxibutiratos/farmacología , Hipoglucemiantes/farmacología , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/enzimología , Pentanonas/farmacología , Conejos , Vanadatos/farmacologíaRESUMEN
During the progression of certain degenerative conditions, including myocardial ischemia-reperfusion injury, mitochondria are a source of increased free-radical generation and exhibit declines in respiratory function(s). It has therefore been suggested that oxidative damage to mitochondrial components plays a critical role in the pathology of these processes. Polyunsaturated fatty acids of membrane lipids are prime molecular targets of free-radical damage. A major product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is highly cytotoxic and can readily react with and damage protein. In this study, the effects of HNE on intact cardiac mitochondria were investigated to gain insight into potential mechanisms by which free radicals mediate mitochondrial dysfunction. Exposure of mitochondria to micromolar concentrations of HNE caused rapid declines in NADH-linked but not succinate-linked state 3 and uncoupled respiration. The activity of complex I was unaffected by HNE under the conditions of our experiments. Loss of respiratory activity reflected the inability of HNE-treated mitochondria to meet NADH demand during maximum rates of O2 consumption. HNE exerted its effects on intact mitochondria by inactivating alpha-ketoglutarate dehydrogenase. These results therefore identify a potentially important mechanism by which free radicals bring about declines in mitochondrial respiration.