RESUMO
Antioxidants may play an important role in immune evasion by schistosome parasites. Previous studies have focused on the roles of superoxide dismutase and glutathione S-transferase. In the present study, glutathione peroxidase (GPX) activity was measured in different fractions of worm extracts from several developmental stages of Schistosoma mansoni. The enzyme activity was shown to be developmentally regulated, with higher specific activities being found in the tegument-enriched Nonidet P-40 extract of adult worms (the stage least susceptible to immune killing) than in the larval stages (which are most susceptible to immune elimination). In all extracts tested, the activity against cumene hydroperoxide, even when glutathione S-transferase activity was removed, was higher than that for hydrogen peroxide. The expression of GPX cDNA in pGEX-2T by bacteria produced a 50-kDa fusion protein and a 32-kDa truncated protein. The latter was due to termination at the internal UGA codon that codes for selenocysteine. GPX activity was detected in the recombinantly produced GPX but not with Sj26-glutathione S-transferase from the vector. Mutating the TGA codon to TGT produced a full-length product, GPXm (19 kDa), that was used to produce 19 monoclonal antibodies. Anti-GPXm monoclonal antibodies recognized a 19-kDa molecule in adult-worm extract which, upon removal by immunoprecipitation, resulted in the loss of over 90% of the GPX activity, suggesting that a single form of GPX exists in the schistosome.
Assuntos
Glutationa Peroxidase/metabolismo , Schistosoma mansoni/enzimologia , Animais , Anticorpos Monoclonais/imunologia , Glutationa Peroxidase/imunologia , Humanos , Testes de PrecipitinaRESUMO
A 1.4-kb cDNA clone encoding a 50-kDa homologue of p59, a heat shock binding immunophilin, has been isolated from a Schistosoma mansoni cercariae cDNA library. We have designated this clone S. mansoni p50 (Smp50). From the sequence comparison, we speculate that Smp50 is similar in structural organization to other p59 proteins. As with other p59s, Smp50 also shows homology to various FK binding proteins (FKBPs). The amino acids in human FKBP12 which are proposed to be important for FK506 interaction are conserved in the schistosome protein. We have expressed and purified the recombinant protein (rSmp50) from Escherichia coli. rSmp50 demonstrated peptidyl-prolyl cis-transisomerase (PPIase) activity, typical of immunophilins. Western blot analysis of extracts from S. mansoni adult worms probed with rabbit polyclonal antisera, generated against the recombinant polypeptide, has indicated the presence of Smp50 in the parasite. The antisera did not cross-react with proteins from E. coli or HeLa cell extracts. All of the p59s characterized to date have been from vertebrate species. Therefore, our finding represents the first identification of the protein in an invertebrate system.
Assuntos
Proteínas de Helminto/genética , Schistosoma mansoni/genética , Isomerases de Aminoácido/química , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclosporinas/metabolismo , Primers do DNA/química , DNA de Helmintos/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/imunologia , Genes de Helmintos , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Humanos , Soros Imunes/imunologia , Dados de Sequência Molecular , Peptidilprolil Isomerase , Proteínas Recombinantes/imunologia , Schistosoma mansoni/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Software , Tacrolimo/metabolismo , Proteínas de Ligação a TacrolimoRESUMO
This report describes a rapid method of detection of poliovirus from viral isolates of clinical specimens using a single set of primers selected from the conserved 5' noncoding region of the poliovirus genome. Of the 144 clinical viral isolates examined, 81 were positive for polioviruses and 50 were positive for nonpoliovirus enteroviruses by tissue culture neutralization and infectivity. All 81 (100%) of the viral isolates identified as poliovirus by tissue culture infectivity were also positive by polymerase chain reaction. Of 50 nonpoliovirus enterovirus isolates found to be negative for poliovirus by tissue culture neutralization and infectivity, 48 were also negative by polymerase chain reaction. The high sensitivity (100%) and specificity (96%) of the primer set indicate that this assay has potential clinical applicability in the diagnosis of nonpoliovirus enterovirus infection.
Assuntos
Enterovirus/genética , Poliovirus/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , DNA Viral/genética , Enterovirus/isolamento & purificação , Infecções por Enterovirus/diagnóstico , Estudos de Avaliação como Assunto , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Poliovirus/isolamento & purificação , Reação em Cadeia da Polimerase/estatística & dados numéricos , DNA Polimerase Dirigida por RNA , Sensibilidade e Especificidade , Virologia/métodosRESUMO
The genome of Schistosoma mansoni, a human blood fluke, contains a family of short repetitive DNA elements which we have named the SM alpha family. In this paper we report the sequences of two SM alpha family members which are derived from tandem arrangements and four family members which are dispersed copies. The two tandemly repeated copies are 331 and 335 bp, while the four dispersed copies range in size from 107 to 322 bp. Three dispersed copies are flanked by direct repeats and have AT-rich 3' ends. The tandem copies and one of the dispersed copies have regions of homology to RNA polymerase III promoters and arginine tRNA genes. In addition the repeated element is rearranged in two of the dispersed copies when compared with the other dispersed and two tandem copies. Localization studies show that SM alpha elements are distributed in the sex and autosomal chromosomes. These observations suggest that members of this family may have been dispersed throughout the genome via RNA intermediates.
