Guillermo Ceballos-Reyes

Glutamate (Glu) is a major excitatory neurotransmitter involved in epilepsy. Glu is synthesized by glutamate dehydrogenase (GDH, E.C. 1.4.1.3) and dysfunction of the enzymatic activity of GDH is associated with brain pathologies. The main goal of this work is to establish the role of GDH in the effects of antiepileptic drugs (AEDs) such as valproate (VALP), diazepam (DIAZ) and diphenylhydantoin (DPH) and its repercussions on oxygen consumption. Oxidative deamination of Glu and reductive amination of aketoglutarate (αK) in mice brain were investigated. Our results show that AEDs decrease GDH activity and oxygen consumption in vitro. In ex vivo experiments, AEDs increased GDH activity but decreased oxygen consumption during Glu oxidative deamination. VALP and DPH reversed the increase in reductive amination of αK caused by the chemoconvulsant pentylenetetrazol. These results suggest that AEDs act by modulating brain GDH activity, which in turn decreased oxygen consumption. GDH represents an important regulation point of neuronal excitability, and modulation of its activity represents a potential target for metabolic treatment of epilepsy and for the development of new AEDs.

... Jos?? Arnold Gonz??lez-Garrido1, Silvia Garrido-Llanos2, Guillermo Manuel Ceballos-Reyes3, Jos?? Rub??n Garc??a-S??nchez3 1 Centro de Investigaciones Biom??dicas-UV, Doctorado en Ciencias Biom??dicas (DCB-UV), Xalapa, Veracruz, M??xico. ...

ABSTRACT In this work we analized the effects of caveolin-1 scaffold peptide (82-101) on angiotensin II-induced intracellular calcium kinetics and the coexpression of caveolin-1, AT1-AT2 receptors and estrogen receptor ((). Angiotensin II through AT1 and AT2 receptors activation induce several effects in the vasculature, like vasoconstriction and synthesis of vasodilator factors. Caveolae, flask-shaped invagitations in the cells membrane have been related with several signalling molecules, i.e, G-proteins, regulation/transport of calcium, regulation/transport of cholesterol, PI3K-Akt. Caveolin-1 develop a scaffold zone in caveolae related to "preassembled signalling complexes". Using Human Vascular Smooth Muscle in culture we assay the effects of the scaffold peptide of caveolin-1 on intracellular calcium kinetics induced by angiotensin II and, the coexpression of AT1 receptor subtype and estrogenic receptor (() by immunofluorescence (confocal microscopy) and immunoblots. Our results showed that scaffold peptide of caveolin-1 inhibits angiotensin effects. AT1, caveolin-1 and estrogenic receptor (() colocalized. We also found AT2 receptor subtype immunoexpression in these cells and colocalization of AT2, caveolin-1 and estrogenic receptor (().

During Chronic Obstructive Pulmonary Disease (COPD) progression, the intracellular antioxidant defence in RBCs must preserve the integrity of the plasmalemma through NADPH+ generation to obtain a sufficient number of reduced non-protein SH-groups. Here, we studied the activities of enzymes in RBCs that are related to glutathione metabolism under conditions of increasing oxidative stress, which are associated with COPD progression, by increasing cellular damage in vitro with PM2.5, a ROS generator. The study included 43 patients, who were separated according to their GOLD classification into moderate and severe groups, along with 11 healthy volunteers (HV). Blood samples were analysed for G6PD, GAPDH, GPx, and GR. The results showed significant decreases in the oxidation of the G6PD, GR and GPx proteins, resulting in decreased enzymatic activity. By contrast, an increase (p<0.05) in GAPDH was observed, suggesting a pool of ATP on the membrane. However, it is evident that RBCs are ...

Hematological toxicity and oxidative stress are common in cancer patients. Antioxidant supplementation has been shown to decrease oxidative stress, but there is still controversy on this topic. The aim of this study was to determine the effect of antioxidant supplementation on oxidative stress, hematological toxicity, and quality of life (QoL) in cervical cancer patients. Randomized, single-blinded controlled trial in women with cervical cancer treated with radiotherapy and chemotherapy with cisplatin. Subjects were randomly assigned to receive antioxidant supplement or placebo supplement. Plasma concentrations of malondialdehyde, free carbonyls, and blood biometry were measured. EORTC quality of life questionnaire was applied before and after oncology treatment. Student's t test for independent samples and X (2) for categorical variables were performed. One hundred three patients were randomly assigned to receive treatment with antioxidants 49 (48 %) or placebo 54 (52.40 %). At the end of the oncology treatment, hemoglobin levels were maintained, and global QoL was better only in the supplemented group (p < 0.025). Antioxidant supplementation in patients treated with chemotherapy and radiotherapy apparently decreased oxidative stress, maintained hemoglobin levels, and improved QoL; however, more studies are needed to study the long-term effect of this intervention.

Oxidative stress damage to biomolecules has been implicated in several diseases including diabetes mellitus. In the present study, we investigated the effect of oxidative stress in whole blood (WB) from diabetic patients (n = 60) on recombinant human insulin. Insulin was incubated with WB obtained from diabetic patients (DP) who had hyperglycemia (>300 mg/dL) or from 41 healthy volunteers (HV). Whole blood of DP, unlike WB of HV, induced higher values of formazan (142%), dityrosines (279%), and carbonyls (58%) in the insulin residues. Interestingly, the insulin modified by WB of DP showed less hypoglycemic activity in rat (30%) in comparison with insulin incubated with WB of HV. The incubation of insulin in WB from DP induces chemical changes in insulin and a decrease in its biological activity, events that might be associated with the high levels of oxidative stress markers found in the plasma of these patients.

ABSTRACT 17Beta-estradiol (E2) plays an important role in Ca2+ fluxes in several cell types. It has been proposed that some of its effects are of nongenomic origin E2 at vascular smooth muscle level can block calcium entry through L-type calcium channels, this mechanism cannot include vascular endothelial cells (VECs), in which increases in the intracellular calcium concentration ([Ca2+]i) are necessary to NO synthesis. We used male rat aorta ECs in culture loaded with fura-2 and a fluorescence imaging system to evaluate the short-term effects of E2 on [Ca2+]i kinetics. We explored the participation of the intracellular steroid receptor on the effects induced by E2, using tamoxifen (1 microM) and ICI 182,780 (10 microM). Our results showed that E2 (like bradykinin) induced an increase in [Ca2+]i. Such agonist-like effects showed a biphasic curve behavior. The 17beta-estradiol effects were not modified by the presence of the intracellular estradiol-receptor antagonist tamoxifen, but it is blocked in the presence of the ICI 182,780. The 17beta-estradiol effects were obtained even with restriction of steroid-free diffusion into cells (17beta-estradiol-bovine serum albumin). Phospholipase Cbeta activity is involved in these effects, because U-73122, a PLCbeta inhibitor, blocked E2 effects. All E2 effects were of rapid onset (milliseconds), exerted at the membrane level, and of rapid offset. We conclude that estradiol can influence the endothelium physiologic responses through effects of nongenomic origin.

Aromatase CYP19 catalyzes the synthesis of estrogen from androgens in a tissue-specific manner. This enzyme is present in several tissues, including gonads, brain and fatty tissue. More recently, its presence has been described in vessels. Here, we describe the expression of aromatase in human uterine artery and compare its expression with that found in arteries of estrogen-dependent uterine leiomyomata from women. To do this, we employed immunohystochemical and in situ hybridization techniques. We used, a polyclonal antibody raised against the carboxyl terminus of aromatase (ARO) and RNAm probes, of the exon 1 of ARO. We found an increased immunoreactivity of ARO in uterine arteries of patients with leiomyoma as compared with control group. Probe showing positive signal in skin fibroblasts (1b), showed positive hybridization signal in normal artery, while probes with positive signal in placenta (1a), ovary (1c) and testis (1d) were over-expressed in arteries of leiomyomas.

Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.

Putrescine, spermidine and spermine are natural compounds found in up to millimolar concentrations in eukaryotic and prokaryotic cells. At physiologic pH, the polyamines are protonated (+2, +3 and +4 charges), their polycationic properties lead to the assumption that they could affect physiological systems by binding to anionic sites of the cellular membrane and/or by modulating ion channels. At the cardiovascular level, their effects are not completely understood. However, these compounds may be able to exert the induction of synthesis and release of cellular mediators. In an attempt to explore this possibility, we used the isolated and perfused rat heart, Langendorff, model in order to evaluate the inotropic effects of these polyamines, putrescine, spermidine and spermine. Dose-response curves (0.1-0.6 mM) for putrescine, spermidine and spermine were constructed; with the finding that spermine had the largest negative effect. The obtained effects were not blocked by nitric oxide synthesis inhibitors (L-NAME), H(1) and H(2) receptor antagonists (Brompheniramine and Cimetidine) or by Glibenclamide, an antagonist of ATP-sensitive K(+) channels. We found that spermine-induced and increased ATP concentration in cardiac effluents. Reactive Blue, a P(2y) purinoreceptor antagonist and Aminophylline, an unspecific adenosine receptor antagonist, blocked the spermine-induced effects. These results showed that ATP, at least in part, is responsible of the spermine cardiovascular effects. Adenosine was shown to also play an important role on those effects.