Five different isoforms （IrlVHA-c1-c5） of V-ATPase subunit c （VHA-c） were cloned from a Japanese iris （Iris lactea Pall. var. chinensis Fisch. Koidz） cDNA library using degenerate primers PCR and the 5＇-RACE te...Five different isoforms （IrlVHA-c1-c5） of V-ATPase subunit c （VHA-c） were cloned from a Japanese iris （Iris lactea Pall. var. chinensis Fisch. Koidz） cDNA library using degenerate primers PCR and the 5＇-RACE technique. The sequence analysis showed the open reading frame （ORF） of the IrlVHA-c1 c5 to be 495 bp, corresponding to a protein of 164 amino acids. Among the five isoforms, IrlVHA-c1 and IrlVHA-c2 are completely homologous. The IrlVHA-c protein is localized at the vacuolar membrane as indicated by a green fluorescent protein （GFP） marker. Its over-expression in yeast could enhance yeast tolerance to NaCl stress. These results show that there are at least five genes encoding different isoforms of IrlVHA-c in Japanese iris and IrlVHA-c is important for the function of V-ATPase.展开更多
The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-dow...The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.展开更多
基金supported by the Heilongjiang Province Postdoctoral Science Foundation(LBH-Q10144)
文摘Five different isoforms （IrlVHA-c1-c5） of V-ATPase subunit c （VHA-c） were cloned from a Japanese iris （Iris lactea Pall. var. chinensis Fisch. Koidz） cDNA library using degenerate primers PCR and the 5＇-RACE technique. The sequence analysis showed the open reading frame （ORF） of the IrlVHA-c1 c5 to be 495 bp, corresponding to a protein of 164 amino acids. Among the five isoforms, IrlVHA-c1 and IrlVHA-c2 are completely homologous. The IrlVHA-c protein is localized at the vacuolar membrane as indicated by a green fluorescent protein （GFP） marker. Its over-expression in yeast could enhance yeast tolerance to NaCl stress. These results show that there are at least five genes encoding different isoforms of IrlVHA-c in Japanese iris and IrlVHA-c is important for the function of V-ATPase.
基金supported by China Postdoctoral Science Foundation(LRB06-262)Funds of Northeast Forestry University
文摘The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.
