RESUMO
The immunogenicity of a truncated HCV core protein (Co.120) was studied in BALB/c and C57BL/6 mice, given three intramuscular injections of antigen, adjuvanted with either aluminum hydroxide or Freund's adjuvant. A rapid antibody response was noted after the first dose, with both strains of mice eventually exhibiting comparable levels of anti-core IgG (titers >1:100000), with a mixed IgG1/IgG2a subclass response. Spleen cells from Co.120-immunized mice gave a significant specific proliferative response. IFN-gamma gene expression was also detected after an ex-vivo specific stimulation of spleen cells in all immunized mice. This response was independent of dose, H-2 genetic background or type of adjuvant. The results indicated that immunization with the Co.120 protein elicits a potent anti-HCV humoral and cellular immune response.
Assuntos
Hepacivirus/imunologia , Antígenos da Hepatite C/imunologia , Proteínas do Core Viral/imunologia , Animais , Especificidade de Anticorpos , Feminino , Expressão Gênica , Hepacivirus/genética , Anticorpos Anti-Hepatite C/biossíntese , Antígenos da Hepatite C/química , Antígenos da Hepatite C/genética , Imunidade Celular , Imunoglobulina G/biossíntese , Técnicas In Vitro , Interferon gama/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Células Th1/imunologia , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.
Assuntos
Hepacivirus/fisiologia , Pichia , Proteínas do Core Viral/biossíntese , Proteínas do Envelope Viral/biossíntese , Montagem de Vírus , Expressão Gênica , Humanos , Microscopia Imunoeletrônica , Pichia/ultraestrutura , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/genética , Vírion/ultraestruturaRESUMO
A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.
Assuntos
Glicosídeo Hidrolases/metabolismo , Pichia/metabolismo , Oxirredutases do Álcool/genética , Compartimento Celular , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Vetores Genéticos , Glicerol/farmacologia , Glicosídeo Hidrolases/genética , Metanol/metabolismo , Pichia/efeitos dos fármacos , Pichia/genética , Pichia/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , beta-FrutofuranosidaseRESUMO
Sao descritos dois novos casos de sindrome dolorosa renal e pionefrose que envolve o grupo caliceal superior do rim, secundario a uma compressao extrinseca deste grupo caliceal. Patologia deste descrita por Elwin E. Fraley em 1966, pela primeira vez, a evolucao tem inicio com um simples retardo de esvaziamento do grupo caliceal superior do rim; tem um quadro clinico de simples dor lombar, ocorrendo no entanto o acometimento geral do orgao; o tratamento quase sempre cirurgico, dependendo da lesao, nao so em gravidade ou extensao, o procedimento consistira em infundibuloplastia simplesmente ou heminefrectomia