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
Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein k...Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.展开更多
Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. Th...Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.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.
文摘Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.
基金the National Natural Science Foundation of China (No. 30370904 , 30671258) the National High Technology Research and Development Program (863 Project) of China (No. 2006AA10Z121) the Program for New Century Excellent Talents in University (No. NCET-07-0712).
文摘Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.
