RESUMEN
BACKGROUND: The molecular mechanisms of cellular changes responsible for diabetic gastroparesis, primarily seen in middle-aged women, still remain incompletely defined. We hypothesized that a decrease in the expression, dimerization, and post-translational modification of neuronal nitric oxide synthase alpha (nNOSα) is estrogen mediated and responsible for the gender-specific prevalence of this malady. METHODS: We induced diabetes by injecting male and female rats with streptozotocin. Male diabetic rats without gastroparesis were then injected with estrogen for 3 weeks and evaluated for gastroparesis development. Gastric tissues were analyzed for the elucidation of biochemical events associated with diabetes and gastroparetic dysfunction. RESULTS: Although male diabetic, gastroparetic (either streptozotocin- or estrogen-induced) rats exhibited similarity in disease pathology to that of females, the molecular mechanisms of development were different. Our results indicate that slow gastric emptying in both male diabetic, gastroparetic rat groups was not associated with the level of expression of nNOSα in gastric tissues. However, nNOSα dimerization, which reflects nNOSα activation, did decline slightly in the antrum of diabetic males with estrogen-induced gastroparesis, suggesting a possible estrogen role. Females with diabetic gastroparesis, in contrast, demonstrated significantly impaired levels and dimerization of nNOSα in the antrum and pylorus. Although the precise mechanism remains unknown, nNOSα dimerization impairment in female antrum is apparently associated with reduced phosphorylation of Ser(1416) in the activation loop of nNOSα. CONCLUSION: Taken together, these results demonstrate that gender and estrogens may be leading factors, through molecular changes involved in nitric oxide synthesis down-regulation, within the antrum and pylorus of female diabetic, gastroparetic rats.
Asunto(s)
Diabetes Mellitus Experimental , Estradiol/metabolismo , Gastroparesia/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Western Blotting , Dimerización , Femenino , Vaciamiento Gástrico , Gastroparesia/fisiopatología , Masculino , Fosforilación , Ratas , Factores SexualesRESUMEN
BACKGROUND: Diabetic gastroparesis in human and animal models suggest different developmental causes in females vs males. Previously, we demonstrated that although male and female diabetic gastroparetic rats exhibited similarity in disease pathology, molecular mechanisms were different: slow gastric emptying in male diabetic gastroparetic rats was not associated with the level of expression and dimerization of neuronal nitric oxide synthase α in gastric tissues, as was demonstrated in females. Male gastroparesis may involve other mechanisms, such as oxidative stress. We hypothesize that sustained increased reactive oxygen species (ROS) and degradation of MAP kinase phosphatase-1 with subsequent unregulated activation of c-Jun N-terminal kinase and p38MAP kinase pathways are associated with gastroparesis in a male diabetic rat model. METHODS: Using a male rat model of diabetic gastroparesis, we analyzed serum and pyloric tissue for ROS and antioxidant enzyme levels using ELISA; MAP kinase phosphatase-1, c-Jun N-terminal kinases, and p38MAP kinase levels utilized western blotting techniques and phospho-specific antibodies. KEY RESULTS: Both diabetic and diabetic gastroparetic rats demonstrated overproduction of ROS. However, loss of MAP kinase phosphatase-1, a MAP kinase pathway negative regulator, with subsequent activation of c-Jun N-terminal kinase 2 and p38MAP kinase pathways, were observed only in diabetic gastroparetic rats. Diabetic rats without gastroparesis had no significant pathway activation. CONCLUSIONS & INFERENCES: These results suggest that sustained, increased ROS and degradation of MAP kinase phosphatase-1, with subsequent unregulated activation of c-Jun N-terminal kinase and p38MAP kinase pathways, are likely to be factors in diabetic gastroparesis phenotype in a male diabetic rat model.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Gastroparesia/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Diabetes Mellitus Experimental/complicaciones , Modelos Animales de Enfermedad , Gastroparesia/complicaciones , Masculino , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Helicobacter pylori causes disease manifestations in humans including chronic gastric and peptic ulcers, gastric cancer, and lymphoid tissue lymphoma. Increasing rates of H. pylori clarithromycin resistance has led to higher rates of disease development. Because antibiotic resistance involves modifications of outer membrane proteins (OMP) in other Gram-negative bacteria, this study focuses on identification of H. pylori OMP's using comparative proteomic analyses of clarithromycin-susceptible and -resistant H. pylori strains. Comparative proteomics analyses of isolated sarcosine-insoluble OMP fractions from clarithromycin-susceptible and -resistant H. pylori strains were performed by 1) one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein separation and 2) in-gel digestion of the isolated proteins and mass spectrometry analysis by Matrix Assisted Laser Desorption Ionization-tandem mass spectrometry. Iron-regulated membrane protein, UreaseB, EF-Tu, and putative OMP were down-regulated; HopT (BabB) transmembrane protein, HofC, and OMP31 were up-regulated in clarithromycin-resistant H. pylori. Western blotting and real time PCR, respectively, validated UreaseB subunit and EF-Tu changes at the protein level, and mRNA expression of HofC and HopT. This limited proteomic study provides evidence that alteration of the outer membrane proteins' profile may be a novel mechanism involved in clarithromycin resistance in H. pylori.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/análisis , Claritromicina/farmacología , Farmacorresistencia Bacteriana , Helicobacter pylori/química , Helicobacter pylori/efectos de los fármacos , Proteoma/análisis , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Western Blotting , Electroforesis en Gel de Poliacrilamida , Humanos , Proteoma/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.