RESUMO
Based on the hyperthermostable family 11 xylanase (EvXyn11(TS)) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5-Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11(C5T)-encoding gene, Syxyn11(C5T), was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11(C5T), were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11(C5T) decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.
Assuntos
Dissulfetos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Sequência de Aminoácidos , Dissulfetos/química , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pichia/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is lost or decreased in most human tumors. The role of WWOX in human lung carcinoma invasion is still not clear. This study aimed to elucidate the potential role of WWOX in lung cancer cell invasion. WWOX mRNA levels in human lung cancers and lung cancer cell lines were assayed by quantitative real-time PCR. WWOX in lung cancer cell lines was manipulated by transfection of expression vector or small interfering RNA. Cell migration and invasion were assessed by wound healing and/or transwell migration and invasion assays. The protein levels of WWOX, E-cadherin and RUNX2 were analyzed by western blot or immunofluorescence. WWOX expression is inversely correlated to invasiveness of lung cancer. WWOX overexpression in highly invasive H1299 cells reduced cell motility and invasiveness, and inhibited the expression of RUNX2 and its target gene matrix metalloproteinase-9 (MMP-9). Silencing WWOX in less invasive NL9980 cells resulted in opposite effects. Overexpressing RUNX2 in H1299 or silencing RUNX2 in NL9980 cells reversed the effects of WWOX. These results suggested that WWOX inhibited the invasive phenotype of lung cancer through downregulating the expression of RUNX2.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica , Humanos , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genéticaRESUMO
This article reports the detection of small double capsid virus and results of preliminary studies on its morphology, serology and relationship to gastroenteritis. A kind of small virus particles with morphological characteristics was found in fecal samples of 3 of 23 infants with autumn gastroenteritis. The average diameter of this virus particles is 34.1 +/- 1.1 nm, possessing core, double capsids and spoke-like capsomers. It can be differentiated from other viruses in morphology. Correlated antigenicity exists among 3 strains of the virus but not among other studied viruses and this virus. The titer of the antibody to small double capsid virus in convalescent phase-sera of 2 patients was more than 4-fold as high as that in control sera. This virus was absent in fecal samples of recovered patients as well as in those of normal infants. These show that small "double layer like" capsid virus might be one of the etiological agents of acute gastroenteritis.