Mercedes Cano

Effect of aging and oxidative stress on elongation factor-2 in hypothalamus and hypophysis

The hypothalamic-hypophysis system (HHS) secretes peptide hormones whose synthesis requires the integrity of the translation machinery. As the organisms age, a considerable diminution of the protein synthesis takes place in several tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). We studied whether the level of this protein was affected in the HHS in old animals. The effects of aging are compared to those of an oxidant compound (cumene hydroperoxide) administered to young rats. The results indicate that oxidative stress could be involved in the alterations of eEF-2, which forms adducts with malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The alterations of eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes could contribute to the suboptimal hormone production from these tissues during aging. Besides eEF-2, proteomic analysis shows that several other proteins are affected.

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex.

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose >> D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin >> D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.

Chloride transport in brush-border membrane vesicles from chick jejunum.

This study sought to characterize the mechanism(s) of Cl− transport across brush-border membrane vesicles (BBMV) isolated from chick jejunum. An inwardly directed proton gradient stimulated chloride (36C1−) uptake. This uptake was inhibited by SITS and H2-DIDS. pH-gradient-stimulated Cl− uptake was electroneutral, since it was only slightly decreased by voltage clamping the BBMV with K+ and valinomycin. An outwardly directed HCO3− gradient significantly increased chloride uptake in the presence of a pH gradient. pH-driven chloride uptake was reduced by the presence of several anions in the uptake buffer. The rank order of potency for inhibition of pH-driven Cl− uptake was Cl− > SCN− > HCO3− > I− > Glu− > HPO42−. In the absence of a pH gradient, chloride was less concentrated inside the vesicles than outside. Chloride uptake under these conditions was stimulated by a positive electrical potential inside the vesicles. This stimulation was inhibited by the addition of several anions outside the vesicles. The order of inhibitory potency was SCN− > I− > Cl− > HCO3− > Glu− > HPO42−. The results are consistent with the presence of a Cl−/base exchanger and a chloride conductance pathway in the brush-border membrane of chick small intestine.

In vitro and in vivo protection by melatonin against the decline of elongation factor-2 caused by lipid peroxidation: preservation of protein synthesis.

Abstract:  As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor‐2 (eEF‐2). eEF‐2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF‐2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF‐2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4‐hydroxynoenal (HNE). We have previously reported that hepatic eEF‐2 forms adducts with low‐molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF‐2 must be specifically carried out by a compound with lipoperoxyl radical‐scavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF‐2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF‐2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF‐2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH‐induced oxidative stress, which was partially or totally prevented by melatonin.

EXPERIENCIAS DE APRENDIZAJE EN INGENIERÍA QUÍMICA: DISEÑO, MONTAJE Y PUESTA EN MARCHA DE UNA UNIDAD DE DESTILACIÓN A ESCALA LABORATORIO MEDIANTE EL APRENDIZAJE BASADO EN PROBLEMAS

espanolSe ha propuesto y puesto en practica una nueva metodologia de aprendizaje basado en problemas en una asignatura de caracter experimental en la carrera de ingenieria quimica en la Escuela Tecnica Superior de Ingenieria de la Universidad de Sevilla (Espana). La metodologia se basa en el trabajo con grupos reducidos y aprendizaje basado en problemas, proponiendo nuevos desafios y oportunidades para el desarrollo de competencias de alumnos de ingenieria. Los alumnos han desarrollado tareas de diseno de procesos, seleccion de alternativas, toma de decisiones, diseno de ingenieria basica y gestion de compras, que han culminado con el montaje y puesta en marcha de una unidad de destilacion a escala laboratorio. La metodologia ha permitido trabajar todas las competencias adscritas a la asignatura con un alto grado de participacion y motivacion del alumnado. Por lo tanto, se considera una herramienta atractiva y util para su incorporacion en asignaturas de experimentacion en ingenieria quimica. EnglishA new problem solving methodology is proposed for a course which is essentially experimental for student of chemical engineer at the Higher Technical Engineering School of the University of Seville in Spain is proposed. The methodology is based on small group work and problem-based learning, proposing new challenges and opportunities to develop skills and competencies in engineering student. The students developed tasks regarding process design, selection of optimal alternative, decision making, basic engineering design and purchase management, which concluded with the assembly and starting-up of a laboratory scales distillation unit. The methodology allowed working all subject competences with high participation and motivation from the students. Therefore, it is considered to be a useful and attractive tool to be incorporated to experimental chemical engineering courses.Resumen Se ha propuesto y puesto en practica una nueva metodologia de aprendizaje basado en problemas en una asignatura de caracter experimental en la carrera de ingenieria quimica en la Escuela Tecnica Superior de Ingenieria de la Universidad de Sevilla (Espana). La metodologia se basa en el trabajo con grupos reducidos y aprendizaje basado en problemas, proponiendo nuevos desafios y oportunidades para el desarrollo de competencias de alumnos de ingenieria. Los alumnos han desarrollado tareas de diseno de procesos, seleccion de alternativas, toma de decisiones, diseno de ingenieria basica y gestion de compras, que han culminado con el montaje y puesta en marcha de una unidad de destilacion a escala laboratorio. La metodologia ha permitido trabajar todas las competencias adscritas a la asignatura con un alto grado de participacion y motivacion del alumnado. Por lo tanto, se considera una herramienta atractiva y util para su incorporacion en asignaturas de experimentacion en ingenieria quimica

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla.

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L‐[3H]‐carnitine in a Na+‐dependent and ‐independent manner. OCTN2 mediates the Na+/L‐carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na+/L‐carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na+‐independent L‐carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na+‐independent L‐carnitine uptake was inhibited by both, anti‐OCTN2 and anti‐OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high‐ and a low‐affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na+‐independent L‐carnitine uptake exhibited trans‐stimulation by intravesicular L‐carnitine or betaine. This trans‐stimulation was inhibited by anti‐OCTN3 antibody, but not by anti‐OCTN2 antibody, indicating that OCTN3 can function as an L‐carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla. J. Cell. Physiol. 223: 451–459, 2010.

CNS voltage-dependent Na+ channel expression and distribution in an undifferentiated and differentiated CNS cell line

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channels structural differences play a role in channel distribution.

Comparative Study of the in Vitro Protective Effects of Several Antioxidants on Elongation Factor 2 under Oxidative Stress Conditions

One of the biochemical pathways affected by aging in all organisms is protein synthesis. Previous reports from our laboratory have indicated that the elongation step is specially affected by aging as a consequence of alterations in elongation factor-2 (eEF-2). In the present work, we studied in vitro the effectiveness of several individual nutritional antioxidants in protecting the levels of hepatic eEF-2 subjected to oxidative stress induced by cumene hydroperoxide. The in vitro system employed consisted of rat liver homogenates treated with cumene hydroperoxide. The antioxidants used in this study were lipoic acid, coenzyme Q10, tethrahydrofolic acid, and N-tert-butyl-alpha-phenylnitrone. The results indicate that the antioxidants have different capacities to prevent eEF-2 loss, folic acid being the most effective. A comparison between the antioxidants used and their potential pro-oxidant activity is also discussed, on the basis of the oxidative stress parameters measured.

Role of Melatonin in the Inflammatory Process and its Therapeutic Potential

Melatonin is an indolamine synthesized and secreted by the pineal gland along with other extrapineal sources including immune system cells, the brain, skin and the gastrointestinal tract. Growing interest in this compound as a potential therapeutic agent in several diseases stems from its pleiotropic effects. Thus, melatonin plays a key role in various physiological activities that include regulation of circadian rhythms, immune responses, the oxidative process, apoptosis or mitochondrial homeostasis. Most of these processes are altered during inflammatory pathologies, among which neurodegenerative and bowel diseases stand out. Therapeutic assays with melatonin indicate that it has a beneficial therapeutic value in the treatment of several inflammatory diseases, such as Alzheimer, Amiotrophic Lateral, Multiple Sclerosis and Huntigton´s disease as well as ulcerative colitis. However, contradictory effects have been demonstrated in Parkinson´s and Chron´s diseases, which, in some cases, the reported effects were beneficial while in others the pathology was exacerbated. These various results may be related to several factors. In the first place, it should be taken into account that at the beginning of the inflammation phase there is a production of reactive oxygen species (ROS) that should not be blocked by exclusively antioxidant molecules, since, on the one hand, it would be interfering with the action of neutrophils and macrophages and, on the other, with the apoptotic signals activated by ROS. It is also important to keep in mind that the end result of an anti-inflammatory molecule will depend on the degree of inflammation or whether or not it has been resolved and has therefore become chronic. In this review we present the use of melatonin in the control of inflammation underlying the above mentioned diseases. These actions are mediated through their receptors but also with their direct antioxidant action and melatonins ability to break the vicious cycle of ROSinflammation. This review is aimed at evaluating the effect of melatonin on activity of the inflammatory process and at its immunomodulator effects.

Apical ouabain-sensitive K+-activated-ATPase activity in colon and caecum of the chick.

Abstract This study sought to investigate the presence and characteristics of K+-ATPase activity in chicken intestinal epithelia. A cytochemical method revealed Na+-independent, ouabain-sensitive, K+-ATPase activity in the apical, but not in the basolateral, membrane of chicken colonic and caecal epithelial cells. K+-ATPase activity was not observed in the small intestine. The measurement of K+-activated pNPPase activity was used to characterize the K+-ATPase activity evidenced by the cytochemical method. In addition, K+ and NH4+, but neither Na+ nor Li+, could activate pNPPase activity in chicken intestinal epithelia. Vanadate abolished ouabain-sensitive, K+-activated pNPPase activity in the three membrane preparations tested, whereas oligomycin and SCH 28080 were without effect. The Km for K+ and the ouabain IC50 values for the apical colonic and caecal K+-activated pNPPase activity were higher than those measured for K+-activated pNPPase activity measured in the basolateral membrane of chicken jejunal enterocytes. The results indicate that the apical membranes of chicken colon and caecum possess Na+-independent, ouabain-sensitive K+-activated-ATPase activity.