RESUMEN
To investigate structure and biological properties of the nucleocapsid (N) protein of respiratory syncytial virus (RSV), we have generated a panel of 16 monoclonal antibodies, raised against recombinant N protein, and epitope mapped seven of these to three antigenic sites (Site I aa 16-30; Site II aa 341-350; Site III aa 351-365). Characterization by immunofluorescence and by immunoprecipitation assay demonstrated that a monoclonal antibody to antigenic site I can detect N protein complexed with phospho (P) protein. Antibodies to antigenic sites II and III, which are adjacent to each other near the carboxyl terminus of the N protein, have distinct properties. A site III monoclonal antibody detected N protein in cytoplasmic inclusion bodies and in the cytosol, but not when N was complexed to P protein, while the site II antibody reacted with N protein in the nucleocapsid fraction but did not detect cytosolic N protein. Further investigation into the reactivities of the antibodies after binding of P to N in vitro demonstrated that antigenic sites II and III were blocked by the interaction, indicating an involvement for the carboxy domain of N in the N-P interaction. This was confirmed by the ability of peptides from the carboxy terminus of N to inhibit the N-P interaction in vitro.
Asunto(s)
Proteínas de la Nucleocápside/inmunología , Fosfoproteínas/metabolismo , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Sitios de Unión , Epítopos/genética , Epítopos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/genética , Conejos , Proteínas Recombinantes/inmunología , Relación Estructura-Actividad , Proteínas Virales/genéticaRESUMEN
We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992).
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Viral/genética , Simplexvirus/genética , Animales , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Cinética , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas Recombinantes/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [32P]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of Gs alpha. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD+ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of Gs alpha demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for Gs alpha as neither the alpha-subunits of the pertussis toxin-sensitive G-proteins Gi2 and Gi3, nor the beta subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of Gs alpha nor of the alpha-subunits of Gi2 or Gi3 although it did cause ADP ribosylation of the entire complement of both Gi2 and Gi3 in the membranes. Despite the reduction in levels of immunoreactive Gs alpha from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted.
Asunto(s)
Toxina del Cólera/farmacología , Proteínas de Unión al GTP/metabolismo , Glioma/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Immunoblotting , NAD/metabolismo , Toxina del Pertussis , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Histological studies of the sweat glands of anhidrotic horses in the Hong Kong summer and under conditions of reduced thermal stress, both natural and controlled, were undertaken to determine if glandular regeneration occurs. Clinical data were collected for comparison with the histological results in each instance. Horses were assigned to one of three categories on the basis of the resulting change in the number of thin glandular profiles in a cooler environment. Group 1, which was classed as normal, had a low initial value, which was maintained. Group 2, typical of mild and moderately affected animals, had a high initial value, which fell markedly after as little as six weeks in the cool environment. Animals in Group 3, classed as severely affected, had a high initial value which remained high even after prolonged exposure to the cool environment. Light microscopical examination of the sweat glands in the heat, and after six weeks in a cool environment, provided a means of predicting the degree of anhidrotic severity and the potential for recovery in a cool climate. This was superior to clinical observation, although a diagnostic test based on glandular function is still required.