RESUMO
The hyperphosphorylation of tau is a central mechanism in the pathogenesis of Alzheimer's disease (AD). Lithium is a potent inhibitor of glycogen synthase kinase-3beta (GSK3ß), the most important tau kinase in neurons, and may also affect tau phosphorylation by modifying the expression and/or activity of other kinases, such as protein kinase A (PKA), Akt (PKB), and calcium calmodulin kinase-II (CaMKII). The aim of the present study is to determine the effect of chronic lithium treatment on the protein expression of tau and its major kinases in cortical and hippocampal neurons, at distinct working concentrations. Primary cultures of cortical and hippocampal neurons were treated with sub-therapeutic (0.02 mM and 0.2 mM) and therapeutic (2 mM) concentrations of lithium for 7 days. Protein expression of tau and tau-kinases was determined by immunoblotting. An indirect estimate of GSK3ß activity was determined by the GSK3ß ratio (rGSKß). Statistically significant increments in the protein expression of tau and CaMKII were observed both in cortical and hippocampal neurons treated with subtherapeutic doses of lithium. GSK3ß activity was increased in cortical, but decreased in hippocampal neurons. Distinct patterns of changes in the expression of the remaining tau tau-kinases were observed: in cortical neurons, lithium treatment was associated with consistent decrements in Akt and PKA, whereas hippocampal neurons displayed increased protein expression of Akt and decreased PKA. Our results suggest that chronic lithium treatment may yield distinct biological effects depending on the concentration range, with regional specificity. We further suggest that hippocampal neurons may be more sensitive to the effect of lithium, presenting with changes in the expression of tau-related proteins at subtherapeutic doses, which may not be mirrored by the effects observed in cortical neurons.
Assuntos
Hipocampo/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Neurônios/efeitos dos fármacos , Proteínas tau/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/metabolismo , Cloreto de Lítio/administração & dosagem , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos WistarRESUMO
Objective: To evaluate the evolution of mammographic image quality in the state of Rio de Janeiro on the basis of parameters measured and analyzed during health surveillance inspections in the period from 2006 to 2011. Materials and Methods: Descriptive study analyzing parameters connected with imaging quality of 52 mammography apparatuses inspected at least twice with a one-year interval. Results: Amongst the 16 analyzed parameters, 7 presented more than 70% of conformity, namely: compression paddle pressure intensity (85.1%), films development (72.7%), film response (72.7%), low contrast fine detail (92.2%), tumor mass visualization (76.5%), absence of image artifacts (94.1%), mammography-specific developers availability (88.2%). On the other hand, relevant parameters were below 50% conformity, namely: monthly image quality control testing (28.8%) and high contrast details with respect to microcalcifications visualization (47.1%). Conclusion: The analysis revealed critical situations in terms of compliance with the health surveillance standards. Priority should be given to those mammography apparatuses that remained non-compliant at the second inspection performed within the one-year interval. .
Objetivo: Avaliar a evolução da qualidade da imagem de mamógrafos localizados no Estado do Rio de Janeiro, de 2006 a 2011, com base em parâmetros medidos e observados durante inspeções sanitárias. Materiais e Métodos: Estudo descritivo sobre a evolução de parâmetros que condicionam a qualidade da imagem focalizou 52 mamógrafos, inspecionados no mínimo duas vezes, com intervalo de um ano. Resultados: Dos 16 parâmetros avaliados, 7 apresentaram mais de 70% de conformidade: força do dispositivo de compressão (85,1%), processamento dos filmes (72,7%), resposta do filme do serviço (72,7%), detalhes lineares de baixo contraste (92,2%), visualização de massas tumorais (76,5%), ausência de artefatos de imagem (94,1%), existência de processadoras específicas para mamografia (88,2%). Importantes parâmetros apresentaram-se abaixo de 50% de conformidade: realização de testes mensais da qualidade de imagem pelo estabelecimento (28,8%) e detalhes de alto contraste, que dizem respeito à visualização de microcalcificações (47,1%). Conclusão: A análise revelou situações críticas da atuação da vigilância sanitária, cuja prioridade deveria ser dirigida aos estacionários, ou seja, os mamógrafos que permaneceram na situação de não conformidade nas inspeções realizadas com intervalo de um ano. .
Assuntos
Animais , Coelhos , Canais de Cálcio Tipo L/metabolismo , Células Musculares/metabolismo , Sequência de Aminoácidos , Agonistas dos Canais de Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Eletrofisiologia , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Ativação do Canal Iônico/fisiologia , Ligantes , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Peptídeos/farmacologiaRESUMO
Objetivo. Analizar la percepción que el prestador de servicios de salud y el adulto mayor (AM) tienen sobre el maltrato al AM en los servicios públicos de salud, en ciudades seleccionadas de México. Material y métodos. De 2009 a 2012 se realizó un estudio con diseño cualitativo y estrategia de triangulación de fuentes de datos; se efectuaron entrevistas semiestructuradas a 13 prestadores y a 12 ancianos para recuperar su experiencia en el tema. El análisis utilizó procedimientos de la Teoría Fundamentada. Resultados. El maltrato contra el AM es una práctica naturalizada por el personal y por el anciano, la cual se manifiesta de formas diversas. Conclusiones. La institucionalización, profesionalización histórica y falta de conciencia sobre las necesidades de los AM demandan cambios de planeación, organización y supervisión del Sistema de Salud. El personal requiere intervenciones de formación, capacitación y cambio de actitudes/comportamiento, para otorgar atención integral, digna, humana y de respeto a los Derechos Humanos de los AM.
Objective. To analyze the health care providers (HCP) and elderly patients' perceptions about abuse of the elderly by health personnel of public health services, in selected cities in Mexico. Materials and methods. A qualitative study and a strategy of data triangulation were performed during 2009 and 2012; 13 HCPs and 12 elders were interviewed, in order to obtain their experience regarding elder abuse. Grounded Theory proceedings were used for the analysis. Results. Elder abuse is a naturalized practice, from HCP and elderly people's point of view; these perceptions are showed in different ways. Conclusion. Institutionalization, historical professionalization and lack of consciousness about needs of the elderly (sociocultural and economic), require changes in planning, organization and monitoring process in the Health System; training and educational interventions on staff and exchange attitudes and behavior are necessary in order to offer a health care that is comprehensive, decent, human and with respect for the human rights.
Assuntos
Animais , Feminino , Humanos , Camundongos , Antimetabólitos Antineoplásicos/farmacologia , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Fenilacetatos/farmacologia , Elementos Antissenso (Genética) , Neoplasias da Mama , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Regulação para Cima/efeitos dos fármacosRESUMO
AIM: To verify the methylation status of CDH1, DAPK, COX2, hMLH1 and CDKN2A genes and to evaluate their association with Helicobacter pylori (H. pylori)-cagA(+) and Epstein Barr virus (EBV) infections in gastric adenocarcinomas. METHODS: Methylation-specific PCR (MSP) assay was performed in 89 primary gastric carcinomas (intestinal and diffuse types). Microsatellite instability (MSI) analysis was performed using the BAT26 primer set and PCR products were analyzed with the ABI PRISM 3100 Genetic Analyzer using Genescan 3.7 software (Applied Biosystems). Detection of H. pylori and genotyping were performed by PCR, using specific primers for ureaseC and cagA genes. The presence of EBV was assessed by in situ hybridization. Statistical analyses were performed using the chi(2) or Fisher's exact test. RESULTS: The most frequent hypermethylated gene was COX-2 (63.5%) followed by DAPK (55.7%), CDH1 (51%), CDKN2A (36%) and hMLH1 (30.3%). Intestinal and diffuse adenocarcinomas showed different methylation profiles and there was an association between methylation of E-CDH1 and H. pylori-cagA(+) in the intestinal adenocarcinoma type. MSI was correlated with hMLH1 methylation. There was an inverse correlation between DAPK hypermethylation and MSI. CONCLUSION: We found a strong association between CDH1 methylation and H. pylori-cagA(+) in intestinal-type gastric cancer, association of MSI and better prognosis and an heterogeneous COX-2 overexpression.
Assuntos
Adenocarcinoma/genética , Metilação de DNA , DNA de Neoplasias/metabolismo , Helicobacter pylori/isolamento & purificação , Herpesvirus Humano 4/isolamento & purificação , Instabilidade de Microssatélites , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/microbiologia , Adenocarcinoma/virologia , Antígenos CD , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caderinas/genética , Caderinas/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas Quinases Associadas com Morte Celular , Infecções por Vírus Epstein-Barr/complicações , Feminino , Infecções por Helicobacter/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estudos Retrospectivos , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/virologiaRESUMO
OBJECTIVE: Evaluation of promoter methylation of the death-associated protein kinase (DAPK) gene and HPV and EBV infections in cervical cells from patients with normal cytology and colposcopy. STUDY DESIGN: Twenty women, who had been patients at the Institute of Gynecology of the Federal University of Rio de Janeiro (UFRJ) for routine examinations and who showed normal cytology and colposcopy, were selected for this work. Cervical brushings were used for DNA extraction, and the analysis of methylation patterns of the DAPK gene was done through chemical modification with sodium bisulfite. Analysis of viral infection was done using polymerase chain reaction (PCR). RESULTS: Of the 20 patients studied, six (30%) presented methylation of the DAPK gene, five (25%) presented infection with EBV and three (15%) presented coinfection with HPV/EBV. Associating methylation with viral infection, we found methylated DAPK in one patient (16%) with EBV, in two patients (33%) with co-infection and in three patients (50%) with no viral infection. CONCLUSIONS: In the present study, we verified, for the first time, the methylation pattern of the DAPK gene in cervical smears from patients with normal cytology and colposcopy. The results also showed the presence of viral infections in these patients. EBV infection, irrespective of whether associated with HPV or not, may contribute to cervical carcinogenesis as a cofactor. Methylation of the DAPK gene is associated with cell transformation, suggesting that DAPK methylation might be an important marker for the development of cervical epithelial neoplasias.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Colo do Útero/metabolismo , Colo do Útero/patologia , Metilação de DNA , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Papillomavirus/metabolismo , Adulto , Proteínas Reguladoras de Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Estudos de Casos e Controles , Colposcopia , Proteínas Quinases Associadas com Morte Celular , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Feminino , Humanos , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Regiões Promotoras Genéticas/fisiologiaRESUMO
The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Epitélio Pigmentado Ocular/citologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases , Receptores de Glutamato/metabolismo , Receptores de Glutamato/fisiologia , Transdução de SinaisRESUMO
AIM: To present a panorama of the main features and possible identity of the synaptic tag, such as to discuss some of its functional implications. DEVELOPMENT: Long-term potentiation (LTP) constitutes a very attractive synaptic/cellular memory model. LTP, like memory, can manifest itself early (essentially depending on the modification of pre-existing proteins at synapse) and late (depending on new protein synthesis). As LTP is a highly specific phenomenon, a dilemma arises: how can the proteins, required to plastic change stabilization, that are synthesized at the soma of a neuron containing thousands of synaptic contacts--all depending of the same nucleus--go to the appropriate synapses? In this review, we present some of the models that intend to explain this question, making emphasis on synaptic tagging hypothesis. Some of the main findings that have contributed to tagging hypothesis are exposed. The local protein synthesis and the activation of protein kinases are analyzed as candidates to be the synaptic tag. Additionally, some of the functional implications of synaptic tagging are discussed. CONCLUSIONS: The synaptic tagging hypothesis offers a very flexible and reasonable solution to the specificity of long-lasting synaptic changes. Although some of the tagging features are known, the synaptic tag identity has not yet been elucidated. It seems that there is not a unique synaptic tag, but there are rather multiple molecular synaptic tags involved. Each of them might function as a synaptic tag under particular circumstances. Each might be differentially recruited by specific stimuli and mediate plasticity over different time domains.
Assuntos
Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Sinapses/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Neurológicos , Plasticidade Neuronal/fisiologia , Sinapses/ultraestruturaRESUMO
We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Benzilaminas/farmacologia , Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico , Relação Dose-Resposta a Droga , Isoproterenol/farmacologia , Isoquinolinas/farmacologia , Cinética , Masculino , Nifedipino/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologiaRESUMO
Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Prolactina/farmacologia , Proteínas SNARE/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Cinesinas/genética , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Fatores de TempoRESUMO
Long-term potentiation (LTP) is an activity-dependent strengthening of synapses that is thought to underlie memory storage. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been a leading candidate as a memory molecule because it is persistently activated after LTP induction and can enhance transmission. Furthermore, a mutation that blocks persistent activation blocks LTP and forms of learning. However, direct evidence for a role of the kinase in maintaining synaptic strength has been lacking. Here, we show that a newly developed noncompetitive inhibitor of CaMKII strongly reduces synaptic transmission in the CA1 region of the hippocampal slice. This occurs through both presynaptic and postsynaptic action. To study the role of CaMKII in the maintenance of LTP, inhibitor was applied after LTP induction and then removed. Inhibition occurred in both LTP and control pathways but only partially recovered. The nonrecovering component was attributable primarily to a postsynaptic change. To test whether nonrecovery was attributable to a persistent reversal of LTP, we first saturated LTP and then transiently applied inhibitor. This procedure allowed additional LTP to be induced, indicating a reversal of an LTP maintenance mechanism. This is the first procedure that can reverse LTP by chemical means and suggests that a component of synaptic memory is attributable to CaMKII. The procedure also enhanced the LTP that could be induced in the control pathway, consistent with the idea that CaMKII is involved in controlling basal synaptic strength, perhaps as a result of LTP that occurred in vivo.