RESUMEN
The benefits of physical exercise (PE) on memory consolidation have been well-documented in both healthy and memory-impaired animals. However, the underlying mechanisms through which PE exerts these effects are still unclear. In this study, we aimed to investigate the role of hippocampal protein synthesis in memory modulation by acute PE in rats. After novel object recognition (NOR) training, rats were subjected to a 30-min moderate-intensity acute PE on the treadmill, while control animals did not undergo any procedures. Using anisomycin (ANI) and rapamycin (RAPA), compounds that inhibit protein synthesis through different mechanisms, we manipulated protein synthesis in the CA1 region of the hippocampus to examine its contribution to memory consolidation. Memory was assessed on days 1, 7, and 14 post-training. Our results showed that inhibiting protein synthesis by ANI or RAPA impaired NOR memory consolidation in control animals. However, acute PE prevented this impairment without affecting memory persistence. We also evaluated brain-derived neurotrophic factor (BDNF) levels after acute PE at 0.5h, 2h, and 12h afterward and found no differences in levels compared to animals that did not engage in acute PE or were only habituated to the treadmill. Therefore, our findings suggest that acute PE could serve as a non-pharmacological intervention to enhance memory consolidation and prevent memory loss in conditions associated with hippocampal protein synthesis inhibition. This mechanism appears not to depend on BDNF synthesis in the early hours after exercise.
Asunto(s)
Amnesia , Anisomicina , Factor Neurotrófico Derivado del Encéfalo , Hipocampo , Condicionamiento Físico Animal , Ratas Wistar , Animales , Masculino , Condicionamiento Físico Animal/fisiología , Ratas , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Anisomicina/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Amnesia/metabolismo , Amnesia/prevención & control , Inhibidores de la Síntesis de la Proteína/farmacología , Sirolimus/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , Consolidación de la Memoria/efectos de los fármacos , Consolidación de la Memoria/fisiología , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiologíaRESUMEN
The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.
Asunto(s)
Factor 1 Eucariótico de Iniciación , Higromicina B , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Núcleo Celular/metabolismo , Núcleo Celular/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Higromicina B/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
INTRODUCTION: The function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. AIMS: In this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. METHODS: Mechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 µM), comparing its effects with those of the chaperones TUDCA (30 µM) and 4-PBA (3 mM). RESULTS: Emetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. CONCLUSION: Blocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.
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Calcio/metabolismo , Emetina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Contracción Miocárdica , Aturdimiento Miocárdico , Canales de Translocación SEC/antagonistas & inhibidores , Respuesta de Proteína Desplegada , Animales , Señalización del Calcio , Ratones , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Aturdimiento Miocárdico/tratamiento farmacológico , Aturdimiento Miocárdico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
KEY MESSAGE: The moss Pseudocrossidium replicatum is a desiccation-tolerant species that uses an inducible system to withstand severe abiotic stress in both protonemal and gametophore tissues. Desiccation tolerance (DT) is the ability of cells to recover from an air-dried state. Here, the moss Pseudocrossidium replicatum was identified as a fully desiccation-tolerant (FDT) species. Its gametophores rapidly lost more than 90% of their water content when exposed to a low-humidity atmosphere [23% relative humidity (RH)], but abscisic acid (ABA) pretreatment diminished the final water loss after equilibrium was reached. P. replicatum gametophores maintained good maximum photosystem II (PSII) efficiency (Fv/Fm) for up to two hours during slow dehydration; however, ABA pretreatment induced a faster decrease in the Fv/Fm. ABA also induced a faster recovery of the Fv/Fm after rehydration. Protein synthesis inhibitor treatment before dehydration hampered the recovery of the Fv/Fm when the gametophores were rehydrated after desiccation, suggesting the presence of an inducible protective mechanism that is activated in response to abiotic stress. This observation was also supported by accumulation of soluble sugars in gametophores exposed to ABA or NaCl. Exogenous ABA treatment delayed the germination of P. replicatum spores and induced morphological changes in protonemal cells that resembled brachycytes. Transcriptome analyses revealed the presence of an inducible molecular mechanism in P. replicatum protonemata that was activated in response to dehydration. This study is the first RNA-Seq study of the protonemal tissues of an FDT moss. Our results suggest that P. replicatum is an FDT moss equipped with an inducible molecular response that prepares this species for severe abiotic stress and that ABA plays an important role in this response.
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Adaptación Fisiológica/genética , Bryopsida/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Ácido Abscísico/farmacología , Adaptación Fisiológica/efectos de los fármacos , Alfa-Amanitina/farmacología , Bryopsida/metabolismo , Cicloheximida/farmacología , Deshidratación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Geografía , México , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , RNA-Seq/métodos , Estrés Fisiológico , Factores de TiempoRESUMEN
The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.
Asunto(s)
Bovinos/metabolismo , Oocitos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Femenino , Oocitos/efectos de los fármacosRESUMEN
Memory formation depends upon several parametric training conditions. Among them, trial number and inter-trial interval (ITI) are key factors to induce long-term retention. However, it is still unclear how individual training trials contribute to mechanisms underlying memory formation and stabilization. Contextual conditioning in Neohelice granulata has traditionally elicited associative long-term memory (LTM) after 15 spaced (ITI = 3 min) trials. Here, we show that LTM in crabs can be induced after only two training trials by increasing the ITI to 45 min (2t-LTM) and maintaining the same training duration as in traditional protocols. This newly observed LTM was preserved for at least 96 h, exhibiting protein synthesis dependence during consolidation and reconsolidation as well as context-specificity. Moreover, we demonstrate that 2t-LTM depends on inter-trial and post-training ERK activation showing a faster phosphorylation after the second trial compared to the first one. In summary, we present a new training protocol in crabs through a reduced number of trials showing associative features similar to traditional spaced training. This novel protocol allows for intra-training manipulation and the assessment of individual trial contribution to LTM formation.
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Conducta Animal/fisiología , Braquiuros/fisiología , Consolidación de la Memoria/fisiología , Memoria a Largo Plazo/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Práctica Psicológica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Cicloheximida/farmacología , Dimetilsulfóxido/administración & dosificación , Flavonoides/farmacología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de la Síntesis de la Proteína/administración & dosificaciónRESUMEN
Fully consolidated associative memories may be altered by alternative retrieval dependent memory processes. While a brief exposure to the conditioned stimulus (CS) can trigger reconsolidation of the original memory, a prolonged CS exposure will trigger memory extinction. The conditioned response is maintained after reconsolidation, but is inhibited after extinction, presumably by the formation of a new inhibitory memory trace. In rats and humans, it has been shown that CS exposure of intermediate duration leave the memory in an insensitive or limbo state. Limbo is characterised by the absence of reconsolidation or extinction. Here we investigated the evolutionary conserved nature of limbo using a contextual Pavlovian conditioning (CPC) memory paradigm in the crab Neohelice granulata. In animals with fully consolidated CPC memory, systemic administration of the protein synthesis inhibitor cycloheximide after 1 CS presentation disrupted the memory, presumably by interfering with memory reconsolidation. The same intervention given after 320 CSs prevented CPC memory extinction. Cycloheximide had no behavioural effect when administered after 80 CS presentations, a protocol that failed to extinguish CPC memory. Also, we observed that a stronger CPC memory engaged reconsolidation after 80 CS instead of limbo, indicating that memory strength affects the parametrical conditions to engage either reconsolidation or limbo. Altogether, these results indicate that limbo is an evolutionary conserved memory process segregating reconsolidation from extinction in the number of CSs space. Limbo appears as an intrinsic component of retrieval dependent memory processing, with a key function in the transition from memory maintenance to inhibition.
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Braquiuros , Extinción Psicológica , Animales , Condicionamiento Clásico , Memoria , Inhibidores de la Síntesis de la Proteína/farmacología , RatasRESUMEN
Memory reconsolidation occurs when a retrieving event destabilizes transiently a consolidated memory, triggering thereby a new process of restabilization that ensures memory persistence. Although this phenomenon has received wide attention, the effect of new information cooccurring with the reconsolidation process has been less explored. Here we demonstrate that a memory-retrieving event sets a neural tag, which enables the reconsolidation of memory after binding proteins provided by the original or a different contiguous experience. We characterized the specific temporal window during which this association is effective and identified the protein kinase A (PKA) and the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) pathways as the mechanisms related to the setting of the reconsolidation tag and the synthesis of proteins. Our results show, therefore, that memory reconsolidation is mediated by a "behavioral tagging" process, which is common to different memory forms. They represent a significant advance in understanding the fate of memories reconsolidated while being adjacent to other events, and provide a tool for designing noninvasive strategies to attenuate (pathological/traumatic) or improve (education-related) memories.
Asunto(s)
Conducta , Consolidación de la Memoria/fisiología , Memoria/fisiología , Animales , Biomarcadores , Masculino , Memoria/efectos de los fármacos , Consolidación de la Memoria/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , RatasRESUMEN
Findings of several experiments indicate that many treatments that typically interfere with memory consolidation are ineffective in preventing or attenuating memory induced by intense training. As extensive evidence suggests that the consolidation of newly acquired memories requires gene expression and de novo protein synthesis the present study investigated whether intense training prevents consolidation impairment induced by blockers of mRNA and protein synthesis. Rats were given a single inhibitory training trial using a moderate (1.0â¯mA) or a relatively intense (2.0â¯mA) foot-shock. Bilateral hippocampal infusions of the mRNA synthesis blocker DRB (10, 40 or 80â¯ng/0.5⯵L/hemisphere) or the protein synthesis inhibitor anisomycin (ANI), an inhibitor de novo protein synthesis (15.62, 31.25, or 62.50⯵g/0.5⯵L/hemisphere) were administered 15â¯min prior to training. Retention was measured at 30â¯min or 48â¯h following training. DRB and ANI impaired memory of moderate training in a dose-dependent manner without affecting short-term memory. In contrast, memory consolidation was not impaired in the groups trained with 2.0â¯mA. The findings showed that: (1) inhibitors of transcription and translation in the hippocampus impair the consolidation of memory of inhibitory avoidance learning induced by moderate levels of aversive stimulation and (2) blocking of mRNA and protein synthesis does not prevent the consolidation of memory induced by relatively high levels of aversive stimulation. These findings do not support the hypothesis that gene expression and de novo protein synthesis are necessary steps for long-term memory formation as memory was not impaired if intense foot-shock was used in training.
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Reacción de Prevención/efectos de los fármacos , Hipocampo/efectos de los fármacos , Consolidación de la Memoria/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Anisomicina/farmacología , Reacción de Prevención/fisiología , Diclororribofuranosil Benzoimidazol/farmacología , Electrochoque , Hipocampo/fisiología , Masculino , Consolidación de la Memoria/fisiología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas WistarRESUMEN
Fear generalization is defined as the transferring of fear experienced during a traumatic event to safe conditions resembling or not the traumatic event. It has been related to several psychological disorders. Here we set out to determine whether novelty exposure can be effective to avoid fear generalization. We evaluated the effect of a novelty exposure on fear memory generalization using an aversive memory task, the inhibitory avoidance (IA). Male Wistar rats were trained in IA (day 1) and 24 h after (day 2) they were exposed to a new context similar to the original (modified IA - MIA), with some rats being exposed to a novelty just before the exposure to the MIA, while others were not (controls). On day 3, retention tests for IA and MIA contexts were performed. The control rats generalized the memory, expressing aversive behavioral in both contexts whereas rats exposed to novelty only expressed aversion on IA. Furthermore, both anisomycin, an inhibitor of ribosomal protein synthesis, and rapamycin, an inhibitor of mTOR-mediated protein synthesis, injected in the CA1 region of dorsal hippocampus blocked the novelty effect, promoting memory generalization. We conclude that novelty exposure hinders aversive memory generalization depending on hippocampal protein synthesis.
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Reacción de Prevención/fisiología , Generalización Psicológica/fisiología , Hipocampo/metabolismo , Memoria/fisiología , Biosíntesis de Proteínas , Animales , Anisomicina/farmacología , Reacción de Prevención/efectos de los fármacos , Generalización Psicológica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Pruebas Neuropsicológicas , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas Wistar , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Abnormal fibrillary aggregation of tau protein is a pathological condition observed in Alzheimer's disease and other tauopathies; however, the presence and pathological significance of early non-fibrillary aggregates of tau remain under investigation. In cell and animal models expressing normal or modified tau, toxic effects altering the structure and function of several membranous organelles have also been reported in the absence of fibrillary structures; however, how these abnormalities are produced is an issue yet to be addressed. In order to obtain more insights into the mechanisms by which tau may disturb intracellular membranous elements, we transiently overexpressed human full-length tau and several truncated tau variants in cultured neuroblastoma cells. After 48âh of transfection, either full-length or truncated tau forms produced significant fragmentation of the Golgi apparatus (GA) with no changes in cell viability. Noteworthy is that in the majority of cells exhibiting dispersion of the GA, a ring-shaped array of cortical or perinuclear microtubule (Mt) bundles was also generated under the expression of either variant of tau. In contrast, Taxol treatment of non-transfected cells increased the amount of Mt bundles but not sufficiently to produce fragmentation of the GA. Tau-induced ring-shaped Mt bundles appeared to be well-organized and stable structures because they were resistant to Nocodazole post-treatment and displayed a high level of tubulin acetylation. These results further indicate that a mechanical force generated by tau-induced Mt-bundling may be responsible for Golgi fragmentation and that the repeated domain region of tau may be the main promoter of this effect.
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Citoesqueleto/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Microtúbulos/metabolismo , Neuroblastoma/ultraestructura , Proteínas tau/metabolismo , Brefeldino A/farmacología , Metabolismo de los Hidratos de Carbono/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Glicoproteínas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mutación/genética , Neuroblastoma/patología , Nocodazol/farmacología , Compuestos Orgánicos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Transfección , Proteínas tau/genéticaRESUMEN
Under laboratory conditions, crayfish establish hierarchical orders through agonistic encounters whose outcome defines the dominant one and one, or more, submissive animals. These agonistic encounters are ritualistic, based on threats, pushes, attacks, grabs, and avoidance behaviors that include retreats and escape responses. Agonistic behavior in a triad of unfamiliar, size-matched animals is intense on the first day of social interaction and the intensity fades on daily repetitions. The dominant animal keeps its status for long periods, and the submissive ones seem to remember 'who the boss is'. It has been assumed that animals remember and recognize their hierarchical status by urine signals, but the putative substance mediating this recognition has not been reported. The aim of this work was to characterize this hierarchical recognition memory. Triads of unfamiliar crayfish (male animals, size and weight-matched) were faced during standardized agonistic protocols for five consecutive days to analyze memory acquisition dynamics (Experiment 1). In Experiment 2, dominant crayfish were shifted among triads to disclose whether hierarchy depended upon individual recognition memory or recognition of status. The maintenance of the hierarchical structure without behavioral reinforcement was assessed by immobilizing the dominant animal during eleven daily agonistic encounters, and considering any shift in the dominance order (Experiment 3). Standard amnesic treatments (anisomycin, scopolamine or cold-anesthesia) were given to all members of the triads immediately after the first interaction session to prevent individual recognition memory consolidation and evaluate its effect on the hierarchical order (Experiment 4). Acquisition of hierarchical recognition occurs at the first agonistic encounter and agonistic behavior gradually diminishes in the following days; animals keep their hierarchical order despite the inability of the dominant crayfish to attack the submissive ones. Finally, blocking of protein synthesis or muscarinic receptors and cold anesthesia impair memory consolidation. These findings suggest that agonistic encounters induces the acquisition of a robust and lasting social recognition memory in crayfish.
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Conducta Animal/fisiología , Crioanestesia , Jerarquia Social , Consolidación de la Memoria/fisiología , Antagonistas Muscarínicos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Reconocimiento en Psicología/fisiología , Conducta Social , Percepción Social , Animales , Anisomicina/farmacología , Astacoidea , Conducta Animal/efectos de los fármacos , Masculino , Consolidación de la Memoria/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacos , Escopolamina/farmacologíaRESUMEN
Extinction is a process that involves new learning that inhibits the expression of previously acquired memories. Although temporarily effective, extinction does not erase an original fear association. Since the extinction trace tends to fade over time, the original memory can resurge. On the other hand, strengthening effects have been described in several reconsolidation studies using different behavioral and pharmacological manipulations. In order to know whether an extinction memory can be strengthened by reactivation-based interventions in the contextual fear conditioning task, we began by replicating the classic phenomenon of spontaneous recovery to show that brief reexposure sessions can prevent the decay of the extinction trace over time in a long-lasting way. This fear attenuation was shown to depend both on L-type calcium channels and protein synthesis, which suggests a reconsolidation process behind the reactivation-induced strengthening effect. The extinction trace was also susceptible to enhancement by a post-reactivation infusion of a memory-enhancing drug (NaB), which was also able to prevent rapid fear reacquisition (savings). These findings point to new reactivation-based approaches able to strengthen an extinction memory to promote its persistence. The constructive interactions between extinction and reconsolidation may represent a promising novel approach in the realm of fear-related disorder treatments.
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Condicionamiento Clásico , Extinción Psicológica/efectos de los fármacos , Memoria/efectos de los fármacos , Animales , Ácido Butírico/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cicloheximida/farmacología , Extinción Psicológica/fisiología , Miedo , Inhibidores de Histona Desacetilasas/farmacología , Masculino , Memoria/fisiología , Nimodipina/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas WistarRESUMEN
Drug dependence seems to involve a learning and memory process. Since learning and memory depend on protein synthesis, drug dependence may depend on protein synthesis, too. Drug-induced reward is a crucial effect for the development of drug-dependence. We used chloramphenicol (CAP, a protein synthesis inhibitor), to evaluate its effects on amphetamine (amph)-seeking behavior, on CB1R expression and on protein synthesis in general, in specific areas of the brain. Two groups of Wistar adult male rats were subjected to amph-induced conditioned place preference (CPP). Rats in group 1 received amph and were kept in the chamber for 30min. Once this period elapsed, they received a subcutaneous injection of saline (veh) and were returned to their home-cage. Rats in group 2 were also treated with amph but received CAP (150mg/kgsc) instead of saline. Once CPP was evaluated rats were sacrificed and the prefrontal cortex (PFC), the nucleus accumbens (NAcc) and the hippocampus (Hipp) were isolated and prepared for CB1R Western blot analysis. A vivarium reared group of rats was added as a non-experimentally manipulated control group. Results indicate that group 1 developed CPP while increasing CB1R expression in the NAcc. Group 2 did not develop CPP, had lower CB1R expression in the PFC and lacked the CB1R increase in the NAcc observed in the amph+veh group. These results support the notion that among the underlying mechanisms for amph-seeking reward is an increase in CB1R, further supporting an interaction between dopamine/endocannabinoids in CPP learning.
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Anfetamina/antagonistas & inhibidores , Anfetamina/farmacología , Estimulantes del Sistema Nervioso Central/antagonistas & inhibidores , Estimulantes del Sistema Nervioso Central/farmacología , Cloranfenicol/farmacología , Condicionamiento Operante/efectos de los fármacos , Núcleo Accumbens/metabolismo , Corteza Prefrontal/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Receptor Cannabinoide CB1/biosíntesis , Receptor Cannabinoide CB1/efectos de los fármacos , Animales , Masculino , Memoria/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.
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Aminoácidos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Insuficiencia Cardíaca/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Piperidinas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Quinazolinonas/farmacología , Estrés Fisiológico , Aminoácidos/deficiencia , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
Reconsolidation restabilizes memory after reactivation. Previously, we reported that the hippocampus is engaged in object recognition memory reconsolidation to allow incorporation of new information into the original engram. Here we show that BDNF is sufficient for this process, and that blockade of BDNF function in dorsal CA1 impairs updating of the reactivated recognition memory trace.
Asunto(s)
Anticuerpos/farmacología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Hipocampo/metabolismo , Consolidación de la Memoria/fisiología , Reconocimiento en Psicología/fisiología , Animales , Anisomicina/farmacología , Factor Neurotrófico Derivado del Encéfalo/inmunología , Hipocampo/efectos de los fármacos , Masculino , Consolidación de la Memoria/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Wistar , Reconocimiento en Psicología/efectos de los fármacosRESUMEN
The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Brefeldino A/farmacología , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Células HeLa , Humanos , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de ProteínasRESUMEN
Increasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that of in vitro fertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%, P 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.
Asunto(s)
Anisomicina/farmacología , Blastocisto/efectos de los fármacos , Ploidias , Inhibidores de la Síntesis de la Proteína/farmacología , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Adenina/análogos & derivados , Adenina/farmacología , Animales , Blastocisto/citología , Blastocisto/fisiología , Bovinos , Cicloheximida/farmacología , Técnicas de Cultivo de Embriones , Femenino , Masculino , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data-the sequencing of short ribosome-bound fragments of mRNA-is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.
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ARN Mensajero/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN/métodos , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter.