RESUMEN
Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15-30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m-2 s-1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m-2 s-1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.
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Resumo Este trabalho apresenta uma investigação sobre o desempenho dos estudantes brasileiros em Matemática no Pisa de 2003 e 2012. O objetivo foi verificar em qual conteúdo avaliado os estudantes apresentaram melhor desempenho e dispersão positiva na escala de níveis da avaliação. A estratégia empírica adotada foi descritiva e comparativa. Foram analisados relatórios do Inep (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira) e da OCDE/Pisa para compor a base de dados para análise comparativa das duas aplicações da prova. Os resultados apontam que os estudantes brasileiros apresentaram melhor desempenho e a mais significativa distribuição na escala Pisa em Indeterminação e Dados ou Probabilidade. Contudo o desempenho dos estudantes brasileiros, influenciado por uma multiplicidade de fatores, ainda está longe de atingir os níveis da OCDE.
Abstract This study presents an investigation on the performance of Brazilian students in Mathematics in Pisa 2003 and 2012. Our goal was to identify the assessed content in which the students had their best performance and most significant dispersion in the scale of assessment levels. Our empirical study consisted of descriptive and comparative analyses. Reports from Inep (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira) and OECD/Pisa were analyzed to make up the database for comparing the two editions of the test. The results show that Brazilian students had their best performance and most significant distribution on the Pisa scale in Indetermination and Data or Probability. However, the performance of Brazilian students, influenced by innumerous factors, is still far below OECD levels.
Résumé Cet article présente une recherche sur la performance des élèves brésiliens en mathématiques dans au Pisa 2003 et 2012. L'objectif était de vérifier quels étaient les contenus évalués dans lesquels les élèves avaient obtenu une meilleure performance et dispersion positive sur l'échelle des niveaux d'évaluation. La stratégie empirique adoptée a été d'ordre descriptif et comparatif. Des rapports de l'Inep (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira) et de l' OCDE/Pisa ont été analysés pour composer la base de données de l'analyse comparative des deux épreuves du test. Les résultats indiquent que, sur l'échelle Pisa, les élèves brésiliens ont présenté une meilleure performance et la distribution la plus marquée en Indétermination et en Données et Probabilité. Cependant, dû à une multitude de facteurs, le score des élèves brésiliens est encore loin d'atteindre les niveaux de l'OCDE.
Resumen Este trabajo presenta una investigación sobre el desempeño de los estudiantes brasileños en Matemática en el Pisa de 2003 y 2012. El objetivo fue verificar en cuál contenido evaluado los estudiantes presentaron mejor desempeño y dispersión positiva en la escala de niveles de evaluación. La estrategia empírica adoptada fue descriptiva y comparativa. Fueron analizados informes del Inep (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira) y de la OCDE/Pisa para componer la base de datos para análisis comparativa de las dos aplicaciones de la prueba. Los resultados apuntan que los estudiantes brasileños presentaron mejor desempeño y la más significativa distribución en la escala Pisa en Indeterminación y Datos o Probabilidad. Aun considerando esto, el desempeño de los estudiantes brasileños, influenciado por una multiplicidad de factores, todavía esta lejos de alcanzar los niveles de la OCDE.
RESUMEN
IMPACT, a highly conserved protein, is an inhibitor of the eIF2α kinase GCN2. In mammals, it is preferentially expressed in neurons. Knock-down of IMPACT expression in neuronal cells increases basal GCN2 activation and eIF2α phosphorylation and decreases translation initiation. In the mouse brain, IMPACT is particularly abundant in the hypothalamus. Here we describe that the lack of IMPACT in mice affects hypothalamic functions. Impact-/- mice (Imp-KO) are viable and have no apparent major phenotypic defect. The hypothalamus in these animals shows increased levels of eIF2α phosphorylation, as expected from the described role of IMPACT in inhibiting GCN2 and from its abundance in this brain region. When fed a normal chow, animals lacking IMPACT weight slightly less than wild-type mice. When fed a high-fat diet, Imp-KO animals gain substantially less weight due to lower food intake when compared to wild-type mice. STAT3 signaling was depressed in Imp-KO animals even though leptin levels were identical to the wild-type mice. This finding supports the observation that Imp-KO mice have defective thermoregulation upon fasting. This phenotype was partially dependent on GCN2, whereas the lean phenotype was independent of GCN2. Taken together, our results indicate that IMPACT contributes to GCN2-dependent and -independent mechanisms involved in the regulation of autonomic functions in response to energy availability.
Asunto(s)
Regulación de la Temperatura Corporal/efectos de los fármacos , Grasas de la Dieta/efectos adversos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Regulación de la Temperatura Corporal/genética , Grasas de la Dieta/farmacología , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Hipotálamo/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/patología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
O envelhecimento da população e a inversão da pirâmide demográfica é uma realidade bem conhecida pelos profissionais da saúde. Com o avanço da idade, o idoso tende a restringir sua vida social. Consideram-se políticas de saúde aquelas que existem para apoiar aos idosos dependentes para as atividades básicas ou instrumentais da vida diária na área social e da saúde. Através da revisão bibliográfica dessas políticas em Portugal, considera-se que o estado atual não consegue colmatar as necessidades.
The aging of the population and the inversion of the demographic pyramid is a reality well known to health professionals. With advancing age, the elderly tend to restrict their social life. Health policies are those that exist to support elderly dependents for basic or instrumental activities of daily living in the social and health area. Through the bibliographic review of these policies in Portugal, it is considered that the current state cannot meet the needs.
El envejecimiento de la población y la inversión de la pirámide demográfica es una realidad bien conocida por los profesionales de la salud. Con el avance de la edad, el anciano tiende a restringir su vida social. Se consideran políticas de salud aquellas que existen para apoyar a los ancianos dependientes para las actividades básicas o instrumentales de la vida diaria en el área social y de la salud. A través de revisión de la literatura de estas políticas en Portugal, se considera que el estado actual no puede satisfacer las necesidades.
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Humanos , Masculino , Femenino , Anciano , Anciano de 80 o más Años , Envejecimiento , Políticas , Política de Salud , Hogares para Ancianos , Anciano Frágil , Atención Domiciliaria de SaludRESUMEN
BACKGROUND: Historically, the primary sensory areas of the cerebral cortex have been exclusively associated with the processing of a single sensory modality. Yet the presence of tactile responses in the primary visual (V1) cortex has challenged this view, leading to the notion that primary sensory areas engage in cross-modal processing, and that the associated circuitry is modifiable by such activity. To explore this notion, here we assessed whether the exploration of novel objects in the dark induces the activation of plasticity markers in the V1 cortex of rats. METHODS: Adult rats were allowed to freely explore for 20 min a completely dark box with four novel objects of different shapes and textures. Animals were euthanized either 1 (n = 5) or 3 h (n = 5) after exploration. A control group (n = 5) was placed for 20 min in the same environment, but without the objects. Frontal sections of the brains were submitted to immunohistochemistry to measure protein levels of egr-1 and c-fos, and phosphorylated calcium-dependent kinase (pCaKMII) in V1 cortex. RESULTS: The amount of neurons labeled with monoclonal antibodies against c-fos, egr-1 or pCaKMII increased significantly in V1 cortex after one hour of exploration in the dark. Three hours after exploration, the number of labeled neurons decreased to basal levels. CONCLUSIONS: Our results suggest that non-visual exploration induces the activation of immediate-early genes in V1 cortex, which is suggestive of cross-modal processing in this area. Besides, the increase in the number of neurons labeled with pCaKMII may signal a condition promoting synaptic plasticity.
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ABSTRACT Tubulointerstitial nephritis and uveitis syndrome is a rare and probably underdiagnosed condition. Renal and ocular manifestations may not occur simultaneously, making the diagnosis more difficult. Nephritis may be asymptomatic; therefore, renal function evaluation is essential for diagnosis. Urinary β2-microglobulin levels may be particularly useful. Uveitis, mostly anterior, nongranulomatous and bilateral, occurs usually after the onset of nephritis. Treatment includes corticosteroids and, eventually, other immunosuppressant agents. Renal disease is usually benign and resolves spontaneously or after treatment with systemic corticosteroids. Uveitis, however, may be chronic or recurrent. The authors described the cases of three pediatric patients diagnosed with tubulointerstitial nephritis and uveitis syndrome. The goal of this paper was to warn the medical community over the need to screen patients with uveitis for renal disease.
RESUMO A síndrome nefrite tubulointersticial e uveíte é uma doença rara, provavelmente subdiagnosticada. As manifestações renais e oculares podem não ocorrer simultaneamente, tornando o diagnóstico mais difícil. A nefrite é geralmente assintomática, tornando fundamental a avaliação da função renal em doentes com uveíte. O doseamento da excreção urinária de β2-microglobulina é particularmente útil para o diagnóstico. A uveíte, tipicamente anterior, não granulomatosa e bilateral, manifesta-se após a nefrite na maioria dos casos. O tratamento inclui corticoides e, por vezes, outros imunossupressores. A doença renal tem evolução benigna, resolvendo-se espontaneamente ou com terapêutica com corticoides sistêmicos na maioria dos casos, no entanto, a uveíte pode ser crônica ou recorrente. Os autores descrevem três casos de síndrome nefrite tubulointersticial e uveíte, diagnosticados em idade pediátrica, e pretendem alertar para a necessidade de pesquisar sempre alterações renais nos doentes com uveíte.
Asunto(s)
Humanos , Femenino , Niño , Adolescente , Uveítis/diagnóstico , Uveítis/tratamiento farmacológico , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/tratamiento farmacológicoRESUMEN
Tubulointerstitial nephritis and uveitis syndrome is a rare and probably underdiagnosed condition. Renal and ocular manifestations may not occur simultaneously, making the diagnosis more difficult. Nephritis may be asymptomatic; therefore, renal function evaluation is essential for diagnosis. Urinary ß2-microglobulin levels may be particularly useful. Uveitis, mostly anterior, nongranulomatous and bilateral, occurs usually after the onset of nephritis. Treatment includes corticosteroids and, eventually, other immunosuppressant agents. Renal disease is usually benign and resolves spontaneously or after treatment with systemic corticosteroids. Uveitis, however, may be chronic or recurrent. The authors described the cases of three pediatric patients diagnosed with tubulointerstitial nephritis and uveitis syndrome. The goal of this paper was to warn the medical community over the need to screen patients with uveitis for renal disease.
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Nefritis Intersticial , Uveítis , Adolescente , Niño , Femenino , Humanos , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/tratamiento farmacológico , Uveítis/diagnóstico , Uveítis/tratamiento farmacológicoRESUMEN
(AU)Os problemas éticos, enfrentados pelos profissionais do nível primário, são complexos, porque surgem de situações únicas pela variedade de dimensões do cuidado. O acompanhamento de pacientes resistentes ao tratamento, difíceis de cuidar é uma dessas situações que necessita de um manejo diferenciado. Para discutir esta questão, será analisado um caso de difícil acompanhamento, relatado por uma equipe da atenção primária. O caso emergiu numa pesquisa qualitativa sobre problemas éticos realizada num município da região metropolitana de Porto Alegre. A coleta de dados foi por meio de discussões focais sobre problemas éticos, e seus cursos de solução. Para superar as dificuldades da relação terapêutica entre usuários e profissionais, os resultados apontaram para a necessidade de ampliar a compreensão das necessidades em saúde, de reconhecer o contexto relacional da aplicação dos procedimentos clínicos e a importância da experiência subjetiva do adoecimento.(AU)
Los problemas éticos enfrentados por los profesionales del nivel primario son complejos puesto que surgen de situaciones únicas por la variedad de dimensiones del cuidado. El acompañamiento de pacientes resistentes al tratamiento, difíciles de cuidar, es una de esas situaciones que precisa un manejo diferenciado. Para discutir tal cuestión se analizará un caso de difícil acompañamiento relatado por un equipo de atención primaria. El caso surgió durante una encuesta cualitativa sobre problemas éticos realizada en un municipio de la región metropolitana de Porto Alegre. La colecta de datos se realizó por medio de discusiones en grupo sobre problemas éticos y sus cursos de solución. Para superar las dificultades de la relación terapéutica entre usuarios y profesionales, los resultados mostraron una necesidad de ampliar la comprensión de las necesidades de salud, de reconocer el contexto relacional de la aplicación de los procedimientos clínicos y la importancia de la experiencia subjetiva de la enfermedad.(AU)
Ethical problems faced by professionals of primary care are complex, due to their emergence in unique situations, caused by the multiple dimension of care. The followup of patients resistant to treatment, which are difficult to care are cases that demand a differentiated handling. The article reports the analysis of a case of tough follow-up by a primary care team. The case emerged in a qualitative research about ethical problems, which took place in a municipality of the metropolitan region of Porto Alegre. The data collected by focus discussions regarding ethical problems and its possible ways of solution. To overcome the difficulties of the therapeutic relation between users and practitioners, the results pointed out to the need to enlarge the understanding of the health needs, recognizing the relational context of the application of clinic proceeds and the significance of the subjective experience of illness.(AU)
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Humanos , Femenino , Anciano , Atención Primaria de Salud , Barreras de Comunicación , Atención a la Salud , Necesidades y Demandas de Servicios de SaludRESUMEN
Genetically-modified mice without the dopamine transporter (DAT) are hyperdopaminergic, and serve as models for studies of addiction, mania and hyperactive disorders. Here we investigated the capacity for object recognition in mildly hyperdopaminergic mice heterozygous for DAT (DAT +/-), with synaptic dopaminergic levels situated between those shown by DAT -/- homozygous and wild-type (WT) mice. We used a classical dopamine D2 antagonist, haloperidol, to modulate the levels of dopaminergic transmission in a dose-dependent manner, before or after exploring novel objects. In comparison with WT mice, DAT +/- mice showed a deficit in object recognition upon subsequent testing 24h later. This deficit was compensated by a single 0.05mg/kg haloperidol injection 30min before training. In all mice, a 0.3mg/kg haloperidol injected immediately after training impaired object recognition. The results indicate that a mild enhancement of dopaminergic levels can be detrimental to object recognition, and that this deficit can be rescued by a low dose of a D2 dopamine receptor antagonist. This suggests that novel object recognition is optimal at intermediate levels of D2 receptor activity.
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Haloperidol/farmacología , Haloperidol/uso terapéutico , Trastornos de la Memoria/tratamiento farmacológico , Reconocimiento en Psicología/efectos de los fármacos , Animales , Antagonistas de Dopamina/farmacología , Antagonistas de Dopamina/uso terapéutico , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Relación Dosis-Respuesta a Droga , Trastornos de la Memoria/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estadísticas no ParamétricasRESUMEN
In response to a range of environmental stresses, phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) represses general protein synthesis coincident with increased translation of specific mRNAs, such as those encoding the transcription activators GCN4 and ATF4. The eIF2α kinase GCN2 is activated by amino acid starvation by a mechanism involving GCN2 binding to an activator protein GCN1, along with association with uncharged tRNA that accumulates during nutrient deprivation. We previously showed that mammalian IMPACT and its yeast ortholog YIH1 bind to GCN1, thereby preventing GCN1 association with GCN2 and stimulation of this eIF2α kinase during amino acid depletion. GCN2 activity is also enhanced by other stresses, including proteasome inhibition, UV irradiation and lack of glucose. Here, we provide evidence that IMPACT affects directly and specifically the activation of GCN2 under these stress conditions in mammalian cells. We show that activation of mammalian GCN2 requires its interaction with GCN1 and that IMPACT promotes the dissolution of the GCN2-GCN1 complex. To a similar extent as the overexpression of YIH1, overexpression of IMPACT in yeast cells inhibited growth under all stress conditions that require GCN2 and GCN1 for cell survival, including exposure to acetic acid, high levels of NaCl, H2O2 or benomyl. This study extends our understanding of the roles played by GCN1 in GCN2 activation induced by a variety of stress arrangements and suggests that IMPACT and YIH1 use similar mechanisms for regulating this eIF2α kinase.
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Proteínas Portadoras/metabolismo , Secuencia Conservada/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas/genética , Proteínas/metabolismo , Estrés Fisiológico/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Activación Enzimática , Evolución Molecular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Proteínas de Unión al ARN , TransactivadoresRESUMEN
IMPACT is an inhibitor of GCN2, a kinase that phosphorylates the alpha subunit of the translation initiation factor 2 (eIF2 alpha). GCN2 has been implicated in regulating feeding behavior and learning and memory in mice. IMPACT is highly abundant in the brain, suggesting its relevance in the control of GCN2 activation in the central nervous system. We describe here the distribution of IMPACT in the brain of rodents (mice and rats) and of a primate (marmoset) using highly specific antibodies raised against the mouse IMPACT protein. Neurons expressing high levels of IMPACT were found in most areas of the brain. In the hippocampal formation the lack of IMPACT in the dentate gyrus granule cells was striking. The hypothalamus is exceptionally rich in neurons expressing high levels of IMPACT, particularly in the suprachiasmatic nucleus. The only exception to this pattern was the ventromedial nucleus. The thalamic neurons are mostly devoid of IMPACT, with the exception of the paraventricular, reuniens and reticular nuclei, and intergeniculate leaf. The brainstem displayed high levels of IMPACT. For the marmoset, IMPACT expression in the brain is not as prominent when compared to other organs. In the marmoset brain the pattern of IMPACT expression was similar to rodents in most areas, except for the very strong labeling of the Purkinje cells, the lack of IMPACT-positive neurons in the nucleus reuniens, and weak labeling of interneurons in the hippocampus. GCN1, the activator of GCN2 to which IMPACT binds, is widely distributed in all neuronal populations, and all IMPACT-positive cells were also GCN1-positive. The data presented herein suggest that IMPACT may be involved in biochemical homeostatic mechanisms that would prevent GCN2 activation and therefore ATF4 (CREB-2) synthesis in neurons.
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Encéfalo/metabolismo , Callithrix/metabolismo , Ratones/metabolismo , Proteínas/metabolismo , Ratas/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Masculino , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Especificidad de la Especie , Médula Espinal/metabolismoRESUMEN
In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.
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Encéfalo/metabolismo , Muerte Celular , Biosíntesis de Proteínas , Estado Epiléptico/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Epilepsia del Lóbulo Temporal/fisiopatología , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Ratones Endogámicos C57BL , Mióticos , Fosforilación , PilocarpinaRESUMEN
Translational control directed by the eukaryotic translation initiation factor 2 alpha-subunit (eIF2alpha) kinase GCN2 is important for coordinating gene expression programs in response to nutritional deprivation. The GCN2 stress response, conserved from yeast to mammals, is critical for resistance to nutritional deficiencies and for the control of feeding behaviors in rodents. The mouse protein IMPACT has sequence similarities to the yeast YIH1 protein, an inhibitor of GCN2. YIH1 competes with GCN2 for binding to a positive regulator, GCN1. Here, we present evidence that IMPACT is the functional counterpart of YIH1. Overexpression of IMPACT in yeast lowered both basal and amino acid starvation-induced levels of phosphorylated eIF2alpha, as described for YIH1 (31). Overexpression of IMPACT in mouse embryonic fibroblasts inhibited phosphorylation of eIF2alpha by GCN2 under leucine starvation conditions, abolishing expression of its downstream target genes, ATF4 (CREB-2) and CHOP (GADD153). IMPACT bound to the minimal yeast GCN1 segment required for interaction with yeast GCN2 and YIH1 and to native mouse GCN1. At the protein level, IMPACT was detected mainly in the brain. IMPACT was found to be abundant in the majority of hypothalamic neurons. Scattered neurons expressing this protein at higher levels were detected in other regions such as the hippocampus and piriform cortex. The abundance of IMPACT correlated inversely with phosphorylated eIF2alpha levels in different brain areas. These results suggest that IMPACT ensures constant high levels of translation and low levels of ATF4 and CHOP in specific neuronal cells under amino acid starvation conditions.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Quinasas/metabolismo , Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/genética , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Factores de Elongación de Péptidos , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas/metabolismo , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae/genética , TransactivadoresRESUMEN
B13, one of the immunodominant antigens of Trypanosoma cruzi, is composed of repeats of a 12-amino-acid motif. Using synthetic peptides, the sequence FGQAAAGDK was previously shown to contain the B13 immunodominant epitope recognized by chagasic patients sera. To investigate the effects of neighboring sequences in the immunodominance, we tested serum recognition of two B13 sequences fused to LamB. GDKPSPFGQAAA-LamB and FGQAAAGDKPSP-LamB were recognized, respectively, by 15% and 80% of 80 sera reactive to B13 antigen. Recognition of FGQAAAGDKPSP-LamB was inhibited by AAAGDK-containing synthetic peptides. FGQAAAGDKPSP-LamB competed with a B13 recombinant protein containing 16.6 repeats for binding to chagasic antibodies. These results strengthen previous conclusions on the immunodominant epitope of B13 and provide a comparison of two methods for epitope mapping.
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Antígenos de Protozoos/inmunología , Mapeo Epitopo , Proteínas de Escherichia coli/inmunología , Proteínas Recombinantes de Fusión/inmunología , Secuencias Repetitivas de Ácidos Nucleicos/inmunología , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Linfocitos B/inmunología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Mapeo Epitopo/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
In this work, we show extensive phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha) occurring in the brain of mice subjected to 30 min of status epilepticus induced by pilocarpine. eIF2alpha(P) immunoreactivity was detected in the hippocampal pyramidal layer CA1 and CA3, cortex layer V, thalamus and amygdala. After 2 h of recovery, there was a marked decrease in total brain eIF2alpha(P), with the cortex layer V showing the most pronounced loss of anti-eIF2alpha(P) labeling, whereas the CA1 subregion had a significant increase in eIF2alpha(P). These results indicate that inhibition of protein synthesis in experimental models of epilepsy might be due to low levels of eIF2-GTP caused by the phosphorylation of eIF2alpha, and suggest that translational control may contribute to cell fate in the affected areas.
Asunto(s)
Encéfalo/metabolismo , Estado Epiléptico/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting/métodos , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Densitometría/métodos , Modelos Animales de Enfermedad , Inmunohistoquímica/métodos , Masculino , Ratones , Fosforilación/efectos de los fármacos , Pilocarpina , Estado Epiléptico/inducido químicamente , Factores de TiempoRESUMEN
The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strainscauses diarrhoea by altering the fluid secretion in intestinal epithelial cells.Here, the effectiveness of a flagellin fusion protein of Salmonella containing a19-amino-acid sequence derived from the ST sequence (FLAST) in generatingantibodies capable of neutralizing the toxic activity of ST was evaluated. Thisfusion protein, and an alternative construction where two cysteine residues inthe ST sequence were substituted by alanines (STmt), were delivered to theimmune system by three distinct strategies: (i) orally, using an attenuatedSalmonella strain expressing FLAST; (ii) intraperitoneally, by injection ofpurified FLAST; (iii) orally, using attenuated Salmonella carrying a eukaryoticexpression plasmid (pCDNA3) with the gene encoding FLAST. The resultsshowed that the flagellin system can be used as a carrier to generateST-neutralizing antibodies. However, it should be mentioned that humoralimmune response against ST was only obtained when the mutated ST sequencewas employed. FLAST was found to be non-immunogenic when delivered viathe oral route with attenuated Salmonella strains. However, a flagellinantibody response was obtained by immunizing mice with Salmonella carryingpCDNA3/FLA-STmt. Oral immunization with Salmonella carrying the eukaryoticexpression plasmid (pCDNA3/FLASTmt) seems to be a promising method toelicit an appropriate response against fusions to flagellin.