Abstract:

El CCl4 afectó a proteínas por distintos mecanismos. In vivo disminuyó transitoriamente la incorporación de (14)C-Leucina a proteínas nucleares (totales y distintas fracciones proteicas) y microsomales hepáticas de rata, siendo el efecto máximo a la hora. La cicloheximida causó un efecto similar. Los metabolitos reactivos del CCl4 (tridonometilo ('CCl3) y triclorometilperoxilo (CCl3O2')) formados por activacióncon luz ultravioleta C (UVC)causaron el aumento de grupos carbonilos (↑CO)y la disminución del contenido de grupos sulfhidrilo (↓SH) en albúmina de huevo (alb) y el ↑CO en 20 aminoácidos. El ↑CO en alb fue parcialmente inhibido por Trolox y EDTA pero no α-tocoferol. Los metabolitos del CCl4;formaron HCCl3 por reacción con alb (parcialmente inhibido por Trolox) y cisteína, posiblemente por abstracción de hidrógeno, la luz UVC per se ↑CO,abrió el puente disulfuro, pero casi no afectó los SH de la alb. El 'CCl3 y el CCl3O2 ↑CO y ↓SH en proteínas microsomales in vitro, por acción directa o vía productos de peroxidación de lípidos (PL) ya que fue parcialmente inhibido por antioxidantes y EDTA. No afectaron los CO y SH de proteínas nucleares, mitocondriales y citosólicas in vitro. El NADPH in vitro (probablemente vía PL) ↑CO y ↓SH de proteínas microsomales y nucleares, pero no mitocondriales Produjo ↑SH pero no de CO de compustos citosólicos. El CCl4 in vivo aumentó transitoriamente el contenido de CO de proteínas microsomales pero no de nucleares hepáticas de rata. Las alteraciones causadas por los metabolitos reactivos del CCl4 en proteínas permitirían explicar algunas de las alteraciones observadas en la intoxicación por CCl4(pérdida de funciones celulares, daño a la bomba de calcio del retículo endoplásmico),que podrían causar la muerte o desencadenar mecanismos de protección celulares.

CCI4 affected proteins through different mechanisms. It temporarily decreased 14C-Leucine incorporation into both, nuclear proteins (total and different protein fractions) and microsomal proteins in vivo. The main effect was observed at 1-hour poisoning. The effect of cicloheximide on 14C-Leu incorporation was similar to that of CCl4. Trichloromethyl (CCl3) and trichloromethylperoxyl (CCl3O2') free radicals generated by ultraviolet C light (UVC) activation of CCl4 promoted both, carbonyl formation (COT) and sulfhydryl decrease (SH↓) in chicken egg albumin (alb) and CO↑ in the common 20 amino acids. They also led to HCCb formation from alb (partially inhibited by Trolox)and cysteine, probably by hydrogen abstraction. The CO↑ in alb was partially prevented by Trolox and EDTA,but not by α-tocopherol. UVC light by itself caused CO↑,opened disulfide bonds, but it did not affect SH in alb. 'CCl3 and CCl3O2 CO↑ and SH↓ in microsomal proteins in vitro. These effects were partially prevented by antioxidants and EDTA,which indicates these could be due to both, CCl4-free radical direct action or lipid peroxidation products (PL). CCl4-free radicals did not affect CO and SH in nuclear, mitochondrial and citosolic proteins in vitro.NADPH in vitro (probably through PL) caused CO↑ and ↓SH in microsomal and nuclear but not in mitochondrial proteins.It also produced SH increase but not CO↑ in citosolic substances. CCl4 in vivo caused a transitory CO↑ in rat liver microsomal but not in nuclear proteins. These CCl4-induced alterations might explain some of the effects of this hepatotoxin (for example, cellular function loss or damage to microsomal calcium pump) that might induce cellular death or trigger cellular protection mechanisms.

Author affiliation: Galelli, Mirta Esther. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

La reconsolidación y la extinción de la memoria son dos procesos mnésicos funcionalmente relacionados, ya que ambos están involucrados en el procesamiento y almacenamiento de nueva información relacionada con un aprendizaje anterior. Sin embargo, ambos procesos están basados en mecanismos muy distintos. Mientras que la reconsolidación involucra una desestabilización y reestabilización de la traza del aprendizaje original, la extinción genera una nueva traza que compite con la anterior. Trabajos previos de nuestro laboratorio revelaron por primera vez una relación mecanística entre reconsolidación y extinción, mostrando que la presentación de un estímulo condicionado (CS) puede inducir uno u otro proceso dependiendo de su duración. En este trabajo revelamos nuevas características paramétricas que determinan la inducción de la reconsolidación y la extinción, la cinética con la que estos procesos ocurren, y un hipotético mecanismo que los vincula, que tiene lugar tras el fin del CS. Encontramos que (a) durante toda la presentación del CS la memoria original permanece intacta y consolidada, mostrando que ni reconsolidación ni extinción son inducidas hasta la terminación del CS, independientemente de su duración, (b) la presentación de un único refuerzo (US) durante la exposición al CS tiene la capacidad de prevenir la inducción tanto de la extinción como de la reconsolidación, (c) la extinción de la memoria ocurre pocos segundos después del fin del CS, y (d) reconsolidación y extinción son inducidos de forma mutuamente excluyente tras un único CS, pero ambos procesos pueden dispararse por sendos CSs y desarrollarse simultáneamente. Como resultado, presentamos un modelo que integra estos hallazgos, reflejando la dinámica e interrelación entre los procesos de reconsolidación y extinción de la memoria.

Memory reconsolidation and extinction are two functionally related memory processes, since they are both involved in the processing and storage of new information related to a previous learning. However, both processes are based on very different mechanisms. Reconsolidation involves a destabilization and a restabilization of the original memory trace, whereas extinction generates a new memory trace that competes with the former. Previous work from our laboratory showed for the first time that there is a mechanistic relationship between reconsolidation and extinction, since a single conditioned stimulus (CS) exposure could trigger one or the other process depending on its duration. Here we disclose new parametric conditions for the triggering of reconsolidation and extinction, the kinetics of these processes, and an hypothetic mechanism operating after CS offset that links both mnesic process. We have found that (a) during the entire CS presentation the original memory remains intact and consolidated, showing that neither reconsolidation nor extinction are triggered before CS termination, regardless of its duration, (b) a single reinforcement (US) presentation during CS exposure is able to prevent both reconsolidation and extinction triggering, (c) memory extinction is triggered a few seconds after CS offset, and (d) triggering of reconsolidation and extinction is mutually exclusive when a single CS is presented, but both processes can be serially triggered by respective CS presentations, and develop simultaneously. As result, we present a model integrating these findings, showing the dynamics and interrelationship of memory reconsolidation and extinction.

Author affiliation: Pérez-Cuesta, Luis María. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

The medial prefrontal cortex (mPFC) is known for its role in decision making and memory processing, including the participation in the formation of extinction memories. However, little is known regarding its contribution to aversive memory consolidation. Here we demonstrate that neural activity and protein synthesis are required in the dorsal mPFC for memory formation of a conditioned taste aversion (CTA) task and that this region is involved in the retrieval of recent and remote long-term CTA memory. In addition, both NMDA receptor and CaMKII activity in dorsal mPFC are needed for CTA memory consolidation, highlighting the complexity of mPFC functions.

Author affiliation: González, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina

Author affiliation: Villar, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina

Author affiliation: Igaz, Lionel M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Author affiliation: Viola, Haydee Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina

Author affiliation: Medina, Jorge Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina

Abstract:

Memory storage is a temporally graded process involving different phases and different structures in the mammalian brain. Cortical plasticity is essential to store stable memories, but little is known regarding its involvement in memory processing. Here we show that fear memory consolidation requires early post-training macromolecular synthesis in the anterior part of the retrosplenial cortex (aRSC), and that reversible pharmacological inactivation of this cortical region impairs recall of recent as well as of remote memories. These results challenge the generally accepted idea that neocortical areas are slow encoding systems that participate in the retrieval of remote memories only.

Author affiliation: Katche, Cynthia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina

Author affiliation: Dorman, Guido. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina

Author affiliation: Slipczuk, Leandro. Einstein Medical Center; Estados Unidos

Author affiliation: Cammarota, Martin Pablo. Pontificia Universidade Catolica Do Rio Grande Do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Medina, Jorge Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina

Abstract:

Regulation of protein synthesis contributes to maintenance of homeostasis and adaptation to environmental changes. mRNA translation is controlled at various levels including initiation, elongation and termination, through post-transcriptional/translational modifications of components of the protein synthesis machinery. Recently, protein and RNA hydroxylation have emerged as important enzymatic modifications of tRNAs, elongation and termination factors, as well as ribosomal proteins. These modifications enable a correct STOP codon recognition, ensuring translational fidelity. Recent studies are starting to show that STOP codon read-through is related to the ability of the cell to cope with different types of stress, such as oxidative and chemical insults, while correlations between defects in hydroxylation of protein synthesis components and STOP codon read-through are beginning to emerge. In this review we will discuss our current knowledge of protein synthesis regulation through hydroxylation of components of the translation machinery, with special focus on STOP codon recognition. We speculate on the possibility that programmed STOP codon read-through, modulated by hydroxylation of components of the protein synthesis machinery, is part of a concerted cellular response to stress.

Author affiliation: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Gándara, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: de Lella Ezcurra, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

Abstract:

Leptin, the LEP gene product, is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy. Thus, we have recently described the antiapoptotic and trophic effect of leptin on choriocarcinoma cell line JEG-3, stimulating DNA and protein synthesis. We have also demonstrated the presence of leptin receptor and leptin signaling in normal human trophoblastic cells, activating JAK-STAT, PI3K and MAPK pathways. In the present work we have employed dominant negative forms of MAPK and PKB constructs to find out the signaling pathways that specifically mediates the effect of leptin on protein synthesis. As previously shown, leptin stimulates protein synthesis as assessed by 3H-leucine incorporation. However, both dominant negative forms of MAPK and PKB inhibited protein synthesis in JEG-3 choriocarcinoma cells. The inhibition of PKB and MAPK activity by transfection with the dominant negative kinases prevented the leptin stimulation of p70 S6K, which is known to be an important kinase in the regulation of protein synthesis. Moreover, leptin stimulation of phosphorylation of EIF4EBP1 and EIF4E, which allows the initiation of translation was also prevented by MAPK and PI3K dominant negative constructs. Therefore, these results demonstrate that both PI3K and MAPK are necessary to observe the effect of leptin signaling that mediates protein synthesis in choriocarcinoma cells JEG-3. © 2010 Elsevier Inc. All rights reserved.

Author affiliation: Pérez-Pérez, Antonio. Universidad de Sevilla; España

Author affiliation: Gambino, Yésica Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Author affiliation: Maymo, Julieta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Author affiliation: Goberna, Raimundo. Universidad de Sevilla; España

Author affiliation: Fabiani, Fernando. Universidad de Sevilla; España

Author affiliation: Varone, Cecilia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Author affiliation: Sánchez Margalet, Víctor. Universidad de Sevilla; España

Abstract:

Neuronal activity can alter the phosphorylation state of eukaryotic elongation factor 2 (eEF2) and thereby regulates protein synthesis. This is thought to be the underlying mechanism for a form of synaptic plasticity that involves changes in the expression of synaptic AMPA type glutamate receptors. Phosphorylation of eEF2 by Ca/calmodulin-dependent eEF2 kinase reduces the activity of eEF2, and this is prevented by a commonly used eEF2 kinase inhibitor, NH125. Here we show that 10 μM NH125 increased the expression of synaptic GluA2-containing receptors in mouse cerebellar stellate cells and this was prevented by a protein synthesis inhibitor. However NH125 at 10 μM also reduced the level of CPEB3, a protein that is known to bind to GluA2 mRNA and suppress GluA2 (also known as GluR2) synthesis. In contrast, a low concentration of NH125 lowered the peEF2 level, but did not alter CPEB3 expression and also failed to increase synaptic GluA2 receptors. A selective eEF2 kinase inhibitor, A-484954, decreased the level of peEF2, without changing the expression of CPEB3. This suggests that reducing peEF2 does not lead to a decrease in CPEB3 levels and is not sufficient to increase GluA2 synthesis. Thus NH125 at 10 μM reduced the level of CPEB3, and promoted GluA2 translation via a mechanism independent of inhibition of eEF2 kinase. Therefore NH125 does not always alter protein synthesis via selective inhibition of eEF2 kinase and the effects of NH125 on translation of mRNAs should be interpreted with caution.

Author affiliation: Bender, Crhistian Luis. Louisiana State University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Yang, Qian. Louisiana State University; Estados Unidos

Author affiliation: Sun, Lu. Louisiana State University; Estados Unidos

Author affiliation: Liu, Siqiong June. Louisiana State University; Estados Unidos

Abstract:

The retrosplenial cortex (RSC) is involved in a range of cognitive functions. However, its precise involvement in memory processing is unknown. Pharmacological and behavioral experiments demonstrate that protein synthesis and c-Fos expression in the anterior part of RSC (aRSC) are necessary late after training to maintain for many days a fear-motivated memory. Long-lasting memory storage is regulated by D1/D5 dopamine receptors in aRSC and depends on the functional interplay between dorsal hippocampus and aRSC. These results suggest that the RSC recapitulates some of the molecular events that occur in the hippocampus to maintain memory trace over time.

Author affiliation: Katche, Cynthia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina

Author affiliation: Dorman, Guido. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina

Author affiliation: González, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina

Author affiliation: Kramar, Cecilia Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina

Author affiliation: Slipczuk, Leandro. Einstein Medical Center; Estados Unidos

Author affiliation: Rossato, Janine Inez. Pontificia Universidade Catolica Do Rio Grande Do Sul; Brasil

Author affiliation: Cammarota, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidade Catolica Do Rio Grande Do Sul; Brasil

Author affiliation: Medina, Jorge Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina

Abstract:

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A51 residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T. cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.

Author affiliation: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Smulski, Cristian Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Ma, Kit Wan. The Chinese University of Hong Kong; China

Author affiliation: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Institut Cochin; Francia

Author affiliation: Shaw, Pang Chui. The Chinese University of Hong Kong; China

Author affiliation: Wong, Kam Bo. The Chinese University of Hong Kong; China