RESUMEN
Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261--1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.
Asunto(s)
Antígenos Bacterianos/toxicidad , Proteínas de la Membrana Bacteriana Externa , Streptococcus pyogenes/patogenicidad , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/toxicidad , Proteínas Portadoras/toxicidad , Complemento C3/metabolismo , Reacciones Cruzadas , Humanos , Ratones , Datos de Secuencia Molecular , Fagocitosis , Conejos , Streptococcus pyogenes/inmunología , VirulenciaAsunto(s)
Genes de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Mutagénesis Sitio-Dirigida , Animales , Bacteriófago M13/genética , Células COS , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Vectores Genéticos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes , Mapeo Restrictivo , TransfecciónRESUMEN
Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.
Asunto(s)
Vectores Genéticos , Región Variable de Inmunoglobulina/genética , Reacción en Cadena de la Polimerasa , Animales , Secuencia de Bases , Clonación Molecular , Exones , Humanos , Regiones Constantes de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Intrones , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transfección , Células Tumorales CultivadasRESUMEN
Inhibition of rat liver microsomal glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) by orthophosphate and organophosphate esters was examined at pH 6.0 and 7.5 with and without enzyme pretreatment with 0.2% (w/v) deoxycholate. Inhibition by orthophosphate and monoethyl phosphate was competitive with respect to glucose-6-P while inhibition by mono- and di-phenyl phosphate was of the mixed type. Monoalkyl phosphates were more effective inhibitors than the analogous di- and tri-alkyl phosphates and deoxycholate potentiated the inhibitory effects. Mono- and di-phenyl phosphates were more effective inhibitors than triphenyl phosphate, and deoxycholate decreased these inhibitory effects. The results are interpreted in terms of inhibitor and deoxycholate interactions with the enzyme.
