RESUMEN
AIM: To examine the role of placental protein tyrosine nitration and p38-Mitogen-Activated Protein Kinase α (p38-MAPKα), Extra Cellular-Signal Regulated Kinase (ERK) and c-Jun NH2-Terminal Kinase (JNK) activity, in the pathogenesis of type 1 diabetic pre-eclampsia, and the putative modulation of these indices by maternal vitamin C and E supplementation. METHODS: Placental samples were obtained from a sub-cohort of the DAPIT trial: a randomised placebo-controlled trial of antioxidant supplementation to reduce pre-eclampsia in type 1 diabetic pregnancy. Placenta from placebo-treated: normotensive (NT) [n=17], gestational hypertension (GH) [n=7] and pre-eclampsia (PE) [n=6] and vitamin-treated: NT (n=20), GH (n=4) and PE (n=3) was analysed. Protein tyrosine nitration was assessed by immunohistochemistry in paraffin-embedded tissue. Catalytic activities of placental p38-MAPKα, ERK and JNK were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Nitrotyrosine immunostaining was present in placebo-treated NT, GH and PE placentae, with no significant difference observed between the groups. There was a non-significant trend towards decreased p38-MAPKα activity in PE vs NT control placentae. ERK and JNK were similar among the three outcome placebo groups and vitamin supplementation did not significantly alter their activity. CONCLUSION: Nitrotyrosine immunopositivity in normotensive diabetic placentae indicates some degree of tyrosine nitration in uncomplicated diabetic pregnancy, possibly due to inherent oxidative stress and peroxynitrite production. Our results suggest that p38-MAPKα, ERK and JNK are not directly involved in the pathogenesis of type 1 diabetic pre-eclampsia and are not modulated by vitamin-supplementation.
Asunto(s)
Antioxidantes/farmacología , Diabetes Mellitus Tipo 1/complicaciones , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Nitratos/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Proteínas Gestacionales/metabolismo , Vitaminas/farmacología , Adulto , Antioxidantes/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Suplementos Dietéticos , Femenino , Humanos , Placebos , Preeclampsia/tratamiento farmacológico , Embarazo , Embarazo en Diabéticas/tratamiento farmacológico , Embarazo en Diabéticas/metabolismo , Procesamiento Proteico-Postraduccional , Tirosina/metabolismo , Vitaminas/uso terapéuticoRESUMEN
Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.
Asunto(s)
Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Humanos , Cinética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transferasas (Grupos de Otros Fosfatos Sustitutos)/químicaRESUMEN
This study investigates the relationship between dietary fat and ion transport in rat intestine. Animals were fed isocaloric diets with high fat content as corn oil, evening primrose oil (Efamol), fish oil, Efamol/fish oil, olive oil, coconut oil, and butter. A low-fat (corn oil) diet was used in a control group. Biopsies of the small intestine from these animals were mounted in an Ussing chamber, and the intestinal mucosal to serosal ion transport was measured as short-circuit current (SCC). The SCC was stimulated in rats fed on diets high in polyunsaturated fatty acids such as corn oil, Efamol, fish oil, and Efamol/fish oil. SCC was also stimulated in rats fed on diets high in monosaturated fatty acids such as olive oil. Animals fed on diets high in saturated fatty acids such as coconut oil and butter, on the other hand, showed an inhibition in the SCC. To determine whether the effect of membrane lipids was mediated by a differential effect on membrane receptor proteins, the response to secretogogue challenge was studied. Biochemical agents and secretogogues including acetylcholine, noradrenaline, dibutyryl-cAMP, calcium ionophore A2318, and prostaglandin E2 were analysed and compared. These agents were able to influence the SCC, showing responses with marked differences between diet groups.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Mucosa Intestinal/metabolismo , Iones , Animales , Transporte Biológico , Bucladesina/farmacología , Mantequilla , Calcimicina/farmacología , Aceite de Coco , Aceite de Maíz/administración & dosificación , Dinoprostona/farmacología , Estimulación Eléctrica , Ácidos Grasos/administración & dosificación , Ácidos Grasos/metabolismo , Ácidos Grasos Esenciales/administración & dosificación , Aceites de Pescado/administración & dosificación , Intestinos/efectos de los fármacos , Ácidos Linoleicos , Masculino , Microvellosidades/metabolismo , Oenothera biennis , Aceite de Oliva , Aceites de Plantas/administración & dosificación , Ratas , Ratas Wistar , Ácido gammalinolénicoRESUMEN
Multiple mechanisms of regulation in the CDP-choline pathway for phosphatidylcholine (PC) synthesis were revealed by exploring the effects of choline and inositol on this pathway in Saccharomyces cerevisiae. At exogenous choline concentrations below 100 microM, phosphocholine cytidylyltransferase was rate-limiting; at higher choline concentrations the conversion of choline to phosphocholine by choline kinase became rate-limiting. Choline and inositol were found to regulate choline uptake; this established another regulatory mechanism by which PC synthesis is regulated in yeast. Inositol addition did not immediately affect labeled choline uptake or its incorporation into PC in actively dividing cells; however, preculturing the cells in the presence of choline decreased the rate of choline uptake, and this effect was amplified by the concomitant addition of inositol and choline. Additionally, a growth phase dependent effect of inositol supplementation was observed. Inositol addition to stationary phase cells resulted in an increase in choline uptake and subsequent PC production in these cells. This increase was shown to be due to an increase in the rate of choline transport into the cell. In the presence of inositol, choline transport is the main regulatory mechanism controlling flux through the CDP-choline pathway in S. cerevisiae. Inositol supplementation resulted in changes in the levels of enzyme activity detected in vitro. However, the effects observed in vivo correlated exclusively with changes in choline uptake. Choline transporter assays were consistent with these results. Since both the CPT1 and EPT1 gene products catalyze the cholinephosphotransferase reaction in vitro (Hjelmstad, R. H., and Bell, R. M. (1991) J. Biol. Chem. 266, 4357-4365), the effect of inositol on these two separate routes for PC biosynthesis was investigated. The data revealed that only cells harboring a functional CPT1 gene synthesized PC in vivo. These cells (ept1-delta 1::URA3) also displayed an identical mode of regulation in response to inositol as did cells containing an intact EPT1 gene (wild type) indicating there is no requirement for an alternate functional CDP-amino-alcohol pathway for inositol to regulate PC synthesis via the CDP-choline pathway.