RESUMEN
Sequences front a cDNA of dengue virus type 4 were cloned into transcription vectors. These sequences included the E, NS1, NS2A, NS2B, NS3 genes. RNA transcripts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. With these RNA-probes we have been able to detect molecules of serotype-specific dengue 4 viral RNA. Moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might be useful to differentiate serotypes.
Asunto(s)
ADN Viral/genética , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Animales , Células Cultivadas , Clonación Molecular , Culicidae/microbiología , Genes ViralesRESUMEN
OBJECTIVE: This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model. MATERIAL AND METHODS: Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-beta1 (TGF-beta1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted. RESULTS AND CONCLUSIONS: Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-beta1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Condrocitos/citología , Alginatos , Animales , Cartílago Articular/citología , Cartílago Articular/patología , Desdiferenciación Celular , Proliferación Celular/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Técnicas de Cocultivo/métodos , Colágeno Tipo II/metabolismo , Medios de Cultivo Condicionados/farmacología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/genética , Ácido Glucurónico , Ácidos Hexurónicos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Mitosis , Osteoartritis/patología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The type II voltage-dependent sodium channel is present in neuronal cells, where it mediates the propagation of nerve impulses. Restricted expression of the type II sodium channel gene to neurons is due, at least in part, to binding of the repressor protein REST (also termed NRSF or XBR) to the RE1 (also called NRSE) sequence in the type II sodium channel gene. Previous studies have shown that a domain in REST containing eight GL1-Krüppel zinc finger motifs mediates DNA binding. Deletional and GAL4-fusion gene analyses now reveal repressor domains that lie outside of the DNA-binding domain in both the amino and carboxyl termini of REST. Mutational analysis further identifies a single zinc finger motif in the carboxyl-terminal domain as being essential for repressing type II sodium channel reporter genes. These studies reveal two domains in REST that may mediate interactions with other proteins involved in restricting expression of a large set of genes to the vertebrate nervous system.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genética , Canales de Sodio/genética , Factores de Transcripción , Dedos de Zinc/genética , Análisis Mutacional de ADN , Proteínas del Tejido Nervioso/biosíntesis , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión , Eliminación de Secuencia , Canales de Sodio/biosíntesis , Relación Estructura-ActividadRESUMEN
Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.