1,721,021 research outputs found
Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity
Get PDFRod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.Fil: Castagnet, Paula Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
Phospholipase D signaling during oxidative stress
No full textCompared to other organs, the brain contains a high amount of phospholipids with a structural role in maintaining cellular membrane fluidity and properties; however, their specific function in the pathogenesis of the nervous system is still poorly understood. Oxidative stress is one common factor underlying neurodegenerative disorders and triggering numerous signaling cascades during neurodegeneration processes. Evidence suggests that phospholipid-derived signaling might play an important role in oxidative stress-induced neuronal degeneration and death. In this review, we will summarize the link between phospholipase D, a phosphatidic acid-synthesizing enzyme, with neuronal damage induced by oxidative stress. We will focus on the specific role of phospholipase D isoforms in signaling mechanisms, paying particular attention to the characterization of downstream ironinduced synaptic signaling.Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina
Get PDFWe have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-3H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-3H]DAG generated by LPP action on [2-3H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-3H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.Fil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
Involvement of Lysophosphatidic Acid, Sphingosine 1-Phosphate and Ceramide 1-Phosphate in the Metabolization of Phosphatidic Acid by Lipid Phosphate Phosphatases in Bovine Rod Outer Segments
Get PDFThe aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark–light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.Fil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
Age-related changes in the metabolization of phosphatidic acid in cerebral cortex synaptosomes
Get PDFIn this study, phosphatidic acid (PA) metabolization is found to generate diacylglycerol (DAG), monoacylglycerol (MAG) and glycerol by the sequential action of lipid phosphate phosphatase (LPP), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) in cerebral cortex (CC) synaptosomes. It is also demonstrated that PA is metabolized by phospholipases A (PLA)/lysophosphatidic acid phosphohydrolase (LPAPase) in synaptic endings. Age-related changes in the metabolization of PA have been observed in rat cerebral cortex synaptosomes in the presence of the alternative substrates for LPP, namely LPA, sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P). In addition, LPA and C1P up to concentrations of about 50 μM favor the metabolism in the direction of MAG and glycerol in aged and adult synaptosomes, respectively. At equimolecular concentrations with PA, LPA decreases DAG formation in adult and aged synaptosomes, whereas S1P decreases it and C1P increases it only in aged synaptosomes. Sphingosine (50 μM) or ceramide (100 μM) increase PA metabolism by the pathway that involves LPP/DAGL/MAGL action in aged membranes. Using RHC-80267, a DAGL inhibitor, we could observe that 50% and 33% of MAG are produced as a result of DAGL action in adult and aged synaptosomes, respectively. Taken together, our findings indicate that the ageing modifies the different enzymatic pathways involved in PA metabolization.Fil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Gaveglio, Virginia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
GSK3 beta and Erk are activated downstream of PI3K in synaptic endings during oxidative injury
No full textIron induced oxidative injury is comparable to that of ß-amyloid peptide on the brain of Alzheimer’s disease patients. Our purpose was to evaluate the state of PI3K pathway and its downstream effectors Akt and GSK3ß in cerebral cortex synaptosomes exposed 2+ to Fe (50 µM) for different periods of time (5, 30 and 60 min). The increase in Akt phosphorylation in serine 473 and threonine 308 was temporally coincident with PI3K activation (5 min). GSK3ß, the downstream effector of Akt, was also phosphorylated after 5 and 30 min of iron exposure and this phosphorylation was inhibited by LY294002. Additionally, Erk activation was also observed after 5 and 30 min of insult exposure, and this activation was PI3Kdependent. Immunoprecipitations carried out with anti-cSrc demonstrated a strong association between activated Akt and this tyrosine kinase induced by oxidative stress. Our results demonstrate that oxidative stress triggers the activation of different synaptic signaling pathways that operate downstream PI3K/AktFil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaXLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología MolecularVilla Carlos PazArgentinaSociedad Argentina de Investigación en Bioquímica y Biología Molecula
Retinoic acid modifies lipid metabolism in isolated adult and aged rat cerebellum nuclei
No full textNuclear lipid metabolism gives rise to several lipid second messengers that seem to be involved in the regulation of nuclear structure and gene expression. The purpose of the present research was therefore to study the metabolic pathways involved in the metabolism of phosphatidic acid (PA) and its regulation in isolated nuclei from the central nervous system. This study was also conducted in isolated nuclei from aged animals allowing us to analyze the effects of neurodegeneration processes on PA metabolism as a result of ageing. Adult (4 mo) and aged (28 mo) rat cerebellums were homogenized and highly purified nuclei were isolated by sucrose-density ultracentrifugation. Using radiolabelled substrates we demonstrated lipid phosphate phosphatases (LPPs), diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), lysophosphatidate phosphohydrolase (LPAPase) and phospholipase (LPAase) as well as PA-phospholipase type A (PLA) activities. We further studied their regulation by the nuclear agonist retinoic acid (RA), which was observed to decrease DAGL and MAGL activities. Significant aged-related changes in the above-mentioned enzymatic activities as well as in its regulation by RA were observed. Taken together, our results demonstrate a RA-regulated PA metabolism in rat cerebellum nuclei which could be involved in neurodegeneration processes.Fil: Gaveglio, Virginia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaXLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología MolecularCiudad de MendozaArgentinaSociedad Argentina de Investigación en Bioquímica y Biología Molecula
Blancos moleculares de la vía PI3K/Akt en neuronas de hipocampo sometidas a estrés oxidativo
No full textLa vía PI3K/Akt tiene un reconocido rol en la señalización in- tracelular involucrada en la plasticidad sináptica y la supervivencia neuronal. Los objetivos de este trabajo fueron: i) caracterizar un modelo de neurodegeneración inducido por Fe2+, y ii) estudiar la participación de la vía PI3K/Akt y su relación con distintos blancos moleculares tales como GSK3β y los factores de transcripción FoxO en los eventos disparados durante la injuria neuronal oxida- tiva. En este trabajo se utilizó la línea neuronal de hipocampo de ratón HT22, la cual fue expuesta a diferentes concentraciones de Fe2+ durante 24 h. Los niveles de especies reactivas de oxígeno (medidos con la sonda DCF) se vieron incrementados por la expo- sición al metal, de igual manera que los productos de peroxidación lipídica (cuantificados por TBARS). La morfología celular mostró alteraciones características del daño: cuerpo celular redondeado con disminución del número de proyecciones neuronales. Las enzi- mas antioxidantes presentaron un perfil diferencial frente al estrés oxidativo: los niveles de SOD1 disminuyeron, los de catalasa no mostraron cambio, y los de SOD2 y HO1 aumentaron. Simultá- neamente, Akt y GSK3β exhibieron un incremento en sus niveles de fosforilación. Además, se estudió en las fracciones nuclear y citosólica la presencia de las formas fosforiladas (inactivas) de FoxO3a y FoxO1, las cuales aumentaron de manera dependiente de PI3K en la fracción citosólica de las células tratadas con Fe2+. Coincidentemente, el contenido de FoxO3a total disminuyó en la fracción nuclear. Asimismo, se observó la translocación nuclear de PI3K y Akt en respuesta al Fe2+. Estos resultados demuestran que nuestras condiciones experimentales reproducen un modelo de estrés oxidativo incipiente inducido por Fe2+ en neuronas de hipocampo, y que en este modelo, la vía PI3K/Akt se activa, transloca al núcleo, y promueve la fosforilación, inactivación y exportación nuclear de FoxO3a/FoxO1Fil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaLVII Reunión Anual de la Sociedad Argentina de Investigación Clínica y LX Reunión Científica Anual de la Sociedad Argentina de InmunologíaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de Inmunologí
Aged-related changes in 2-AG metabolism enzymes expression and activity in rat cerebral cortex
No full textEndocannabinoid 2-arachidonoylglycerol (2-AG) is synthesized by the enzymes diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAase). Its hydrolysis is carried out by monoacylglycerol lipase (MAGL) although other enzymes, such as fatty acid amide hydrolase (FAAH) and serine hydrolase ABHD may also be involved. The aim of this study was to analyze 2-AG synthesis and hydrolysis during physiological aging. Cerebral cortex membrane and soluble fractions; and synaptosomes from adult (3 mo) and aged (28 mo) rats were isolated by differential centrifugation and the synaptosomes were purified in ficoll gradients. LPAase, DAGL, and MAGLactivities were assayed using radiolabeled substrates, and their products were quantified from aqueous or lipid phase, previously separated by TLC. The expression of DAGL, MAGL and FAAH was analyzed by Western Blot. Both DAGL and ß were expressed in membranes while the second was only expressed in synaptosomes. The expression and activitiy of DAGL changed during aging. 2-AG hydrolysis showed no changes in the membrane fraction during aging in coincidence with the absence of changes in MAGL expression. 2-AG hydrolysis was observed to be higher in synaptosomes whereas MAGL expression decreased during aging. Our results show that the expression and activity of the enzymes involved in 2-AG metabolism are differently modulated by aging.Fil: Pascual, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Pasquaré, Susana Juana. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaXLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología MolecularMendozaArgentinaSociedad Argentina de Investigaciones en Bioquímica y Biología Molecula
Insulin modifies aging-related inhibition of 1-stearoyl, 2-achidonoylglycerol phosphorylation in rat synaptic terminals
Get PDFThe purpose of the present study was to analyze diacylglycerol kinase (DAGK) activity in synaptic terminals from cerebral cortex (CC) and hippocampus (Hp) from adult (3-4 month-old) and aged (26-28 month-old) rats. The effect of insulin through DAGK activity on synaptosomes from adult and aged rats was also analyzed under conditions favoring saturated or unsaturated phosphatidic acid (PA) formation, using exogenous di-palmitoil glycerol (DPG) or 1-stearoyl-2-arachidonoylglycerol (SAG) as substrates. Results showed that the enzymatic activity preferentially uses 1-stearoyl-2-arachidonoylglycerol (SAG) as substrate, thus indicating the presence of -type DAGK. A significant decrease in DAGK activity transforming SAG into PA was also observed in both tissues from aged rats. Western Blot detection of DAGK showed that enzyme content undergoes no changes with aging. [3H]inositol incorporation into phosphoinosites was also analyzed to evaluate the role of DAGK in their synthesis. Data obtained from 3H-inositol incorporation into phosphoinositides revealed that in synaptosomes from aged rats phosphatidylinositol (PI) synthesis is lower than in adult animals. Interestingly, in the presence of SAG, PI synthesis was restored to adult values. DAGK activity over SAG was more highly stimulated by insulin in CC and Hp synaptosomes of aged rats with respect to adult rats. On the other hand, insulin exerted a stimulatory effect on PI and phosphatidylinositol 4 phosphate (PI(4)P) synthesis in synaptosomal CC from aged rats. Taken together, our findings indicate that in aged rats insulin triggers a stimulatory mechanism that reverts the diminished synaptosomal ability to synthesize arachidonoyl phosphatidic acid (20:4 PA). The recovery of this PA species indicates that insulin positively regulates phosphoinositide synthesis.Fil: Zulian, Sandra Edith. Consejo Nacional de Investigaciones Cientificas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Bahia Blanca. Instituto de Investigaciones Bioqu__itilde__micas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Ilincheta, Monica Graciela. Consejo Nacional de Investigaciones Cientificas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Bahia Blanca. Instituto de Investigaciones Bioqu__itilde__micas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Cientificas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Bahia Blanca. Instituto de Investigaciones Bioqu__itilde__micas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; Argentin
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