AIM:To investigate the role of P115 in the proliferation of gastric cancer cells and the mechanism involved.METHODS:The RNA and protein level of P115 and macrophage migration inhibitory factor(MIF)in gastric cancer an...AIM:To investigate the role of P115 in the proliferation of gastric cancer cells and the mechanism involved.METHODS:The RNA and protein level of P115 and macrophage migration inhibitory factor(MIF)in gastric cancer and normal gastric tissue/cells were measured and the effect of P115 on cell proliferation was assessed.The role of P115 in cell cycle checkpoints was investigated and the related proteins and signaling pathways,such as cyclin D1,Mcm2,p53,PCNA as well as the MAPK signaling pathway were determined.The interaction between P115 and MIF and the effect of P115 on MIF secretion were examined.The data were analyzed via one-way ANOVA comparisons between groups and P<0.05 was considered significant.RESULTS:P115 and MIF were both specifically expressed in gastric cancer tissues compared with normal gastric mucosa(both P<0.01).The mRNA and protein levels of P115 and MIF in gastric cancer cell lines MKN-28 and BGC-823 were higher than in the human gastric epithelial cell line GES-1(both P<0.01).In MKN-28 and BGC-823 cell lines,P115 promoted cell proliferation and G0-G1to S phase transition.In addition,several cell cycle-related regulators,including cyclin D1,Mcm2,PCNA,pERK1/2 and p53 were up-regulated by P115.Furthermore,the interaction between P115 and MIF was confirmed by co-immunoprecipitation assay.ELISA showed that P115 stimulated the secretion of MIF into the culture supernatant(P<0.01)and the compensative expression of MIF in cells was observed by Western blotting.CONCLUSION:P115 promotes proliferation of gastric cancer cells through an interaction with MIF.展开更多
Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterifica...Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications, was previously mostly expressed intracellularly as inclusion bodies in Escherichia coli. Denaturation and renaturation of inclusion bodies had a significant influence on the lipase activity. Thereupon, our present work described the secretion expression of gene encoding of this lipase in Pichia pastoris GS 115 and characterization of the recombinant enzyme. Firstly, the obtained lipA gene fragment was introduced into P. pastoris expression vector pPIC9K, the lipA gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOXI promoter, and the recombinant plasmid pPIC9K-lipA was transformed into P. pastoris strain GS115 by electroporation, and this recombinant P. pastoris were identified by PCR. Then lipase activity was detected on BMMY-tributyrin and olive oil agar plates containing Rhodamine B. Transformants with lipase activity by screening were induced 6 days by methanol, one band of 77 kDa protein could be observed by 10% SDS-PAGE. p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The pH and temperature optimum of lipase were pH 8.5 and 40℃ respectively. The stability and effects of metal ions and other reagents were also determined. However, the recombinant fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 4 U of lipase activity per milliliter of culture media supernatant.展开更多
文摘AIM:To investigate the role of P115 in the proliferation of gastric cancer cells and the mechanism involved.METHODS:The RNA and protein level of P115 and macrophage migration inhibitory factor(MIF)in gastric cancer and normal gastric tissue/cells were measured and the effect of P115 on cell proliferation was assessed.The role of P115 in cell cycle checkpoints was investigated and the related proteins and signaling pathways,such as cyclin D1,Mcm2,p53,PCNA as well as the MAPK signaling pathway were determined.The interaction between P115 and MIF and the effect of P115 on MIF secretion were examined.The data were analyzed via one-way ANOVA comparisons between groups and P<0.05 was considered significant.RESULTS:P115 and MIF were both specifically expressed in gastric cancer tissues compared with normal gastric mucosa(both P<0.01).The mRNA and protein levels of P115 and MIF in gastric cancer cell lines MKN-28 and BGC-823 were higher than in the human gastric epithelial cell line GES-1(both P<0.01).In MKN-28 and BGC-823 cell lines,P115 promoted cell proliferation and G0-G1to S phase transition.In addition,several cell cycle-related regulators,including cyclin D1,Mcm2,PCNA,pERK1/2 and p53 were up-regulated by P115.Furthermore,the interaction between P115 and MIF was confirmed by co-immunoprecipitation assay.ELISA showed that P115 stimulated the secretion of MIF into the culture supernatant(P<0.01)and the compensative expression of MIF in cells was observed by Western blotting.CONCLUSION:P115 promotes proliferation of gastric cancer cells through an interaction with MIF.
文摘Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications, was previously mostly expressed intracellularly as inclusion bodies in Escherichia coli. Denaturation and renaturation of inclusion bodies had a significant influence on the lipase activity. Thereupon, our present work described the secretion expression of gene encoding of this lipase in Pichia pastoris GS 115 and characterization of the recombinant enzyme. Firstly, the obtained lipA gene fragment was introduced into P. pastoris expression vector pPIC9K, the lipA gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOXI promoter, and the recombinant plasmid pPIC9K-lipA was transformed into P. pastoris strain GS115 by electroporation, and this recombinant P. pastoris were identified by PCR. Then lipase activity was detected on BMMY-tributyrin and olive oil agar plates containing Rhodamine B. Transformants with lipase activity by screening were induced 6 days by methanol, one band of 77 kDa protein could be observed by 10% SDS-PAGE. p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The pH and temperature optimum of lipase were pH 8.5 and 40℃ respectively. The stability and effects of metal ions and other reagents were also determined. However, the recombinant fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 4 U of lipase activity per milliliter of culture media supernatant.
