RESUMEN
The majority of cases with early onset familial Alzheimer's disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight membrane-bound complex that also contains beta-catenin. The physiological relevance of the association between PS1 and beta-catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3beta consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser(353) and Ser(357) are glycogen synthase kinase-3beta targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with beta-catenin. By disrupting this interaction, we demonstrate that the association between PS1 and beta-catenin has no effect on Abeta peptide production, beta-catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/beta-catenin association, we found no alteration in beta-catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or beta-catenin and a Tcf/Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and beta-catenin in familial Alzheimer's disease.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas del Citoesqueleto/química , Proteínas de la Membrana/química , Transducción de Señal , Transactivadores , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Apoptosis , Sitios de Unión , Western Blotting , Muerte Celular , Línea Celular , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Citosol/metabolismo , ADN Complementario/metabolismo , Vectores Genéticos , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Humanos , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fosforilación , Pruebas de Precipitina , Presenilina-1 , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Serina/química , Transfección , beta CateninaRESUMEN
T cells with CD4-CD8- (double negative, DN) phenotype in MRL-lpr/lpr mouse serve as a model to establish the correlation between the extremely low IL-2 gene expression and the specific signaling inactivation. The extent of nonresponsiveness in lpr DN cells was distinctive in several unusual defects. First, the poor IL-2 production in lpr DN cells could not be restored by supplement of signals known to augment IL-2 response in normal T cells. Second, the activations of both mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) were attenuated in lpr DN cells upon direct activation by TPA/A23187. Third, IL-2 mRNA was degraded much faster in lpr DN cells than that in normal T cells. Fourth, of the four major transcriptional elements on IL-2 promoter, only AP-1 and nuclear factor of activated T cells (NFAT)-binding activities were suppressed in lpr DN T cells. Altogether, these results suggest that an extremely low level of IL-2 production in lpr DN T cells was due to both the increased instability of mRNA and the reduced activation of IL-2 gene promoter, the latter defect could be attributed to the inactivation of AP-1 and NF-AT as well as the poor activation of the upstream MAP kinase and JNK.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-2/biosíntesis , Lupus Eritematoso Sistémico/inmunología , Trastornos Linfoproliferativos/inmunología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Nucleares , Animales , Calcimicina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos , Lupus Eritematoso Sistémico/genética , Trastornos Linfoproliferativos/genética , Ratones , Ratones Mutantes , Factores de Transcripción NFATC , ARN Mensajero/metabolismo , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A simple method for drying virus on inanimate objects (cover slips) under vacuum in the cold is described. Following this procedure virus maintains high titers (10(6-7)) for periods of 1-3 wk at -70 degrees C depending on the virus. For virucidal assay of disinfectants, cover slips are exposed to medium simulating the disinfectant (virus control) or disinfectant in an upright position in an Ultra-Vu cuvette. Cover slips are readily removed and placed in tissue culture medium for dilution of virus and determination of virus titer. Cytotoxicity of disinfectant is determined by exposing cover slip without virus to disinfectant, then placing it in medium, diluting the medium and incubating with the indicator cells. The use of this technique results in high titers of virus on cover slips, which are inanimate objects requiring minimal manipulation. The titration of virus or cytotoxicity in microplates is cell, medium, serum, and labware economical.
Asunto(s)
Antivirales , Desinfectantes , Virus/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Técnicas Microbiológicas , VacioRESUMEN
Turnover rates, as estimated from the accumulation of the intermediates, 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) following decarboxylase inhibition, were used to investigate the relationship between central catecholaminergic and serotonergic neurons and the development of hypertension in the one-kidney, one-clip renal hypertensive rats. Results indicated that at one week following clipping, 5-HTP accumulation was decreased in the posterior hypothalamus. At 5 weeks no changes were observed. At 20 weeks higher accumulations of both DOPA and 5-HTP were observed in the medulla oblongata while in the anterior hypothalamus DOPA accumulation was increased.