Mouse-Liver Glutathione Reductase: Inactivation by NADPH sf Two Allelic Variants

Mouse-liver glutathione reductase has been purified to homogeneity from strain SWR/J by ammonium sulfate precipitation (40-80 %) and two additional steps of affinity chromatography in ATPR-Sepharose and 2', 5'-ADP-Sepharose from which it was specifically eluted by using NADP+ gradients. After 2032-fold purification the pure enzyme has a specific activity of 146 Uimg. The SWRiJ protein is slightly more basic than the other allelic variant from strain DBA/2J, with PI 7.0 and 6.5 respectively. Both pure proteins are immunologically identical, either by immunodiffusion or by quantitative imrnunoprecipitation. They can however be distinguished by their rate of inactivation in the presence of NADPH, their reduced cofactor. The SWR/J protein is much more resistant to that inactivation ( t x = 14 min) than the DBA/2J enzyme (t % = 5 min).La elizlma glutation reductasa de higado de raton ha sido purificada hasta homogeneidad a partir de la estirpe SWR/J mediante su precipitadbn fraccionada con sulfato am6nico (40-80 %) y dos pasos adicionales de cromatograiia de afinidad en geles de ATPR-Sefarosa y 2',5'-ADP-Sefarosa de 10s que fue eluida especificamente mediante sendos gradientes de NADP+. Tras una purificaci6n de 2.032 veces la enzima pura muestra una actividad especifica de 146 p/mg. La proteina de la estirpe SWR/J es ligeramente mas bhsica que la procedente de la otra variante alelica DBA/2J, con PI 7,O y 6,5, respectivamente. Ambm proteinas puras son inmunologicamente identicas, sea en inmunodifusi6n o en inmunoprecipitaci6n cuantitativa. Se distinguen, sin embargo, por su diferente velocidad de inactivacibn co

Amplification and cloning of cDNAS of cytochrome P4501A1 and metallothionein genes from Sparus aurata Linnaeus, 1758 and Liza aurata (Risso, 1810) by Race-PCR

The biotransformation of xenobiotics found in marine ecosystems is catalysed by inducible systems, a property that makes them useful as early-warning biomarkers of environmental pollution. We have focused our study on cytochrome P4501A1 (CYP 1A1) as a phase I response against organic aromatic xenobiotics, and metallothionein (MT), which reflects pollution by transition metals. The high homology existing between the sequences of both genes, already cloned in different fish species, enabled us to design degenerate oligos to amplify by RT-PCR specific sequences of CYP 1A1 and MT genes in two teleost fish species of the Spanish South Atlantic littoral, the gilthead seabream, Sparus aurata Linnaeus, 1758, and the grey mullet, Liza aurata (Risso 1810). To this end, the expression of both genes was previously induced by intraperitoneal injection with Aroclor 1254 (CYP 1A1) and CdCl2 (MT). The specific sequences for both genes amplified by RT-PCR with degenerate oligos were subsequently used to design new, specific oligos to obtain by Race-PCR (Rapid Amplification of cDNA Ends) the complete cDNAs coding for both genes in both fish species, which were subsequently cloned and sequenced. The coding sequences and the corresponding proteins were compared with those already obtained in other fish species. We are currently developing homologous probes for mRNA quantification using molecular biology techniques in both fish species, to be employed as molecular biomarkers of pollution in the Spanish South Atlantic littoralLa biotransformación de xenobióticos presentes en ecosistemas marinos está catalizada por varios sistemas inducibles, lo que permite su uso como biomarcadores de alerta temprana de contaminación ambiental. Este estudio se ha centrado en el citocromo P4501A1 (CYP 1A1), como respuesta de fase I contra xenobióticos orgánicos aromáticos, y la metalotioneína (MT), que refleja la contaminación por metales de transición. La alta homología existente entre las secuencias de ambos genes previamente clonados en diferentes especies de peces permitió diseñar oligos degenerados para amplificar por RT-PCR secuencias específicas de los genes CYP 1A1 y MT en dos especies de peces teleósteos del litoral suratlántico español, la dorada Sparus aurata Linnaeus, 1758 y la lisa Liza aurata (Risso, 1810). Para ello, la expresión de ambos genes se indujo por inyección intraperitoneal con Aroclor 1254 (CYP 1A1) y CdCl2 (MT). Las secuencias específicas de ambos genes, amplificadas por RT-PCR con tales oligos, se usaron posteriormente para diseñar nuevos oligos específicos; éstos se usaron para amplificar por Race-PCR (Rapid Amplification of cDNA Ends) los cDNAs completos que codifican tales genes en ambas especies de peces, que fueron posteriormente clonados y secuenciados. Las secuencias codificantes y las proteínas correspondientes se han comparado con las obtenidas en otras especies de peces. Actualmente se desarrollan sondas homólogas para cuantificar por métodos de biología molecular los mRNAs específicos en ambos peces, para su uso como biomarcadores moleculares de contaminación en el litoral suratlántico españo

Research on Etiological aspects of Dual Pathology

Introducción: Es importante evaluar la interacción entre los antecedentes psicopatológicos familiares (AF), la dinámica familiar (DF), los trastornos de conducta, los trastornos por uso de sustancias y los trastornos de personalidad (TP). Metodología: Estudio transversal con 350 drogodependientes evaluados los AF psiquiátricos, de alcoholismo y de drogodependencias; la DF; consumo de sustancias; y la presencia de TP, los problemas de conducta (PC) en la adolescencia y el trastorno disocial (TD). Las variables correlacionadas fueron incluidas en varios modelos de regresión logística. Resultados: Una edad de inicio en el consumo más temprana se relaciona con AF de drogodependencia y peor DF. Los AF de alcoholismo, drogodependencias y psiquiátricos se relacionan con peor DF. Edad de inicio en el consumo más temprana, los AF y una peor DF se relacionan con los PC y el TD. Edad de inicio en el consumo más temprana, tener AF, una peor DF, los PC y el TD se relacionan con la presencia de algún TP. Permiten predecir la presencia de algún TP la edad de inicio en el consumo de metadona (IC(95):1,005/3,222; p=0,048) y de otros opiáceos (IC(95):0,233/0,894; p=0,022). La puntuación en AF de alcoholismo permite predecir la presencia de TP límite (IC(95):1,137-2,942; p=0,013), y la edad de inicio en el consumo de cocaína permite predecir la presencia de TP antisocial (IC(95):0,864/0,992; p=0,028). Conclusiones: Los AF de consumo de sustancias y el consumo propio, predicen la presencia de algunos TP.Introduction: It is important to assess the interaction between family psychopathologic history (FH), family dynamics (FD), behavior disorders, substance-use disorders and personality disorders (PD). Methodology: Cross-sectional design. The sample was made up of 350 subjects with substance-use disorders who were assessed for FH including alcoholism and substance-use disorders through an interview; for substance use via a questionnaire; for FD; for PD using the International Personality Disorder Examination (IPDE); for behavior problems in adolescence; and for disocial disorder. Correlated variables were included in logistic regression models. Results: Early age of onset for substance use is related to FH of substanceuse disorders and poorer FD. FH of alcoholism, substance-use disorders and psychiatric disorders are related to poorer FD. Early age of onset for substance use, FH and a disruptive FD are related to behavior problems and disocial disorder. Early age of onset for substance use, FH, disruptive FD, behavior problems and disocial disorder are related to presence of PD. Logistic regression predicted the presence of PD by age of onset for use of methadone (CI(95):1.005/3.222; p=0.048) and of other opiates (CI(95):0.864/0.992;p=0.028). FH score in alcoholism predicted Borderline Personality Disorder (CI(95):1.137- 2.942; p=0.013), and age of onset of cocaine use predicted Antisocial Personality Disorder (CI(95):0.864/0.992; p=0.028). Conclusions: FH of substance use and own use predict the presence of some PDs

Direct Determination of Glutathione S-transferase and Glucose-6-phosphate Dehydrogenase Activities in Cells Cultured in Microtitre Plates as Biomarkers for Oxidative Stress

The enzymes glutathione S,-transferase (GST) and glucose-6-phosphate dehydrogenase (G-6PDH) are implicated in the defence against oxidative stress. GST is mainly involved in the conjugation of electrophilic compounds with glutathione (GSH), although some of its isoenzymes display peroxidase activity. G-6PDH and glutathione reductase regenerate NADPH and GSH, respectively, to restore the reduced intracellular redox .status following oxidative stress. Enzymatic assays for GST and G-6PDH were adapted and optimised to permit the direct in vitro determination of the effects of toxicants which induce oxidative stress in cells on microtitre plates, thereby avoiding the need to prepare cell-free extracts. To optimise the conditions of the enzymatic assays, GST activity was measured at substrate concentrations of 1-3mM GSH and 1-3mM 1-chloro-2,4-dinitrobenzenew, hile G-6PDH activity was measured at 7.5-37.5mM glucose-6-phosphate and 55-275mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 x 105 cellsiwell. The effects on GST and G-6PDH activities of three toxicants which induce oxidative stress - paraquat, iron (11) chloride and iron (111) chloride - were compared in cultured Vero cells to validate the new assays. Specific GST activity increased to 145% and 171% compared to the controls in cells treated with 5mM paraquat and 5mM iron (11) chloride, respectively, but was inhibited after exposure to 25mM iron (111) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5mM paraquat, but was inhibited in cells exposed to 5mM iron (11) chloride and 25mM iron (111) chlorid

Direct Determination of Glutathione Reductase in Cells Cultured in Microtitre Plates as a Biomarker for Oxidative Stress

A new method was developed for the direct determination of glutathione reductase (GOR) activity in Vero cells cultured in microtitre plates, avoiding cell-free extract preparation. The cells in each well were washed twice with phosphate-buffered saline, lysed with Triton X-100, and assayed in 0.1M potassium phosphate, pH 7.0. After subtracting oxidase activity, which increased with NADPH concentration, the net GOR activity was similar at different oxidised glutathione (GSSG) and NADPH concentrations, thus confirming enzyme saturation. The optimised GOR assay used 2.5mM GSSG and 0.12mM NADPH; 5mM EDTA was also added to prevent the enzyme from redox inactivation. The GOR activity was directly proportional to the number of cells per well for a wide range of cell densities, thus supporting the assay's validity for use with cultured cells. The effects on GORactivity of three chemicals which induce oxidative stress, namely, paraquat, iron (11) chloride and iron (111) chloride, were examined to validate the assay under experimental conditions. The specific enzymatic activity increased to 357% of untreated control activity in 5mM paraquat-treated cells, and to 407% of control activity in cells exposed to 7.5mM iron (11) chloride. By contrast, activity decreased to 56% of control activity in cells exposed to 5mM iron (111) chloride. In conclusion, the changes in GOR activity detected in Vero cells confirm that the new assay is suitable for routine in vitro screening of toxicants capable of inducing oxidative stress

Integration of proteomics with transcriptomics and metallomics for the study of environmental issues

Comunicaciones a congreso

Triplication of the photocurrent in dye solar cells by increasing the elongation of the π-conjugation in Zn-porphyrin sensitizers

Porphyrins are promising sensitizers for dye solar cells (DSCs) but narrow absorption bands at 400-450 and 500-650 nm limit their light-harvesting properties. Increasing elongation of the π-conjugation and loss of symmetry causes broadening and a red-shift of the absorption bands, which considerably improves the performance of the DSC. Herein we use an oligothienylenevinylene to bridge a Zn-porphyrin system and the anchoring group of the sensitizer. We separately study the performance of the two basic units: oligothienylenevinylene and Zn-porphyrin. The combined system provides a three-fold enhancement of the photocurrent with respect to parent dyes. This is caused by an additional strong absorption in the region 400-650 nm that leads to flat IPCE of 60%. Theoretical calculations support that the addition of the oligothienylenevinylene unit as a linking bridge creates a charge transfer band that transforms a Zn-porphyrin dye into a push-pull type system with highly efficient charge injection propertie

Voluminous plexiform neurofibromas of the neck region in neurofibromatosis 1

Objetivo. Presentar las características clínicas, de imagen y evolutivas de una serie de pacientes con neurofibromatosis tipo 1 que desarrollaron durante la infancia neurofibromas plexiformes voluminosos en el cuello (NFPVC). Pacientes y métodos. Nueve pacientes (cinco mujeres y cuatro varones) con edades entre 3 y 15 años en el momento del diagnóstico de los tumores, que podían extenderse también a la fosa posterior y a la zona torácica superior. El diagnóstico estuvo basado fundamentalmente en la clínica, la imagen y la histología. Resultados. Un tumor era intralaríngeo y causaba problemas respiratorios. Los otros ocho casos tenían su origen en varias raíces espinales de uno o de ambos lados y podían crecer también hacia el interior de la fosa posterior y de la región torácica en algunos pacientes, y desplazaban a las estructuras anatómicas vecinas, especialmente en tres casos, todos niñas, en las que el tumor creció hasta alcanzar gran volumen, especialmente por un lado, parándose el crecimiento entre los 11 y 12 años y no volviendo a crecer más tarde. Conclusiones. Los NFPVC son tumores histológicamente benignos. La extirpación es necesaria cuando están localizados en la laringe por los problemas respiratorios que causan, pero no en los de las otras regiones, pese a que el voluminoso tamaño que alcanzan en algunos casos puede causar exagerados desplazamientos de las estructuras vecinas. El estudio de nuestra serie parece indicar que al menos los tumores extralaríngeos sólo crecen hasta los 11-12 años. Puede ser recomendable retrasar la cirugía tanto como sea posible si no existe sintomatología aguda que la haga necesariaAim. To present the clinic, imaging and evolutive characteristics of a series of patients with neurofibromatosis 1 with voluminous plexiform neurofibromas in the neck (VPNFN) during childhood. Patients and methods. Nine patients (five females and four males) who were diagnosed as VPNFN at ages between 3 and 15 years. The VPNFN widespread to the posterior fossa or the upper thoracic region in some cases. The diagnosis was based on the clinical, imaging and histological findings. Results. One of the tumors was intralaryngeal and caused respiratory difficulties. The other eight patients had the origin of the tumor in several spinal roots of one or both sides and could growth to the posterior fossa and to the upper thoracic region in some cases with displacement of the surrounding organs, especially in three patients, all girls, in whom the tumor reached a voluminous size on one side, that was observed only until 10 to 11 years when the growth ceased. Conclusions. The VPNFN are histologically benign tumors. Those located in the larynx must be removed because of the respiratory problems, but it is not necessary in cases with other locations despite the voluminous size that can reach in some patients with great displacement of the surrounding organs. The analysis of the results of our series may demonstrate that al least the extralaryngeal tumors only grow to 11-12 years of age. This possibility may make recommendable to retard the surgical treatment as much as possible in cases that it is not necessar