目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中...目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中带;而MⅡ期表现为中、下带。结论:可以用电泳迁移率的变化作为观察p34cdc2磷酸化状态的一种方法。展开更多
Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after ...Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection; Western blotting analysis showed that the level of p3cdc2, and p33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (below 10 ℃). 25S-Met incorporation analysis showed that when the oocytes were incubated at 6℃, synthesis of about thirty defferent polypeptides was promoted or induced, including p34cdc2 and some other p13suc1-binding proteins. All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and store some cell cycle drivers and its regulators, and to gain the maturation competence. These results will also provide a new clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature and how to maintain the geographical distribution of some animals.展开更多
Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced...Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937 ASPI3K (ATM, negative) and U937 pZeosv2(+) (ATM, wild type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2 (Thr 161) or p34cdc2 (Thr 14,Tyr 15). RT PCR was used to estimate CDC25 transcript levels. U937 ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine threonine phosphatase inhibitor or cyclin dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937 pZeosv2 (+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937 ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In abstract, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.展开更多
Objective: To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods: We treated SGC-7901 cells with allitridi, and observed the proliferati...Objective: To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods: We treated SGC-7901 cells with allitridi, and observed the proliferation inhibitory rate with MTT colometric assay, changes of cell cycle using flow cytometry and Switzerland-Giemsa's staining, and morphologic changes of the microtubule structure and location changes of cyclin BI expression using immunofluorescence and confocal laser scanning microscope. Furthermore, the expression of cyclin B1 was analyzed quantitatively using Leica confocal software. Results: SGC-7901 cells were inhibited after exposure to allitridi and the IC50 was 7.2μg/ml for 24 h, 20μg/ml for 72 h. When the cells were treated with allitridi at concentrations of 3, 6, and 9μg/ml for 24 h respectively, there was a declining tendency in the percentage of G0/G1 cell but an increasing tendency in GE/M cell in the allitridi treated group compared with that of control (P〈0.01). When cells were treated allitridi at concentration of 6 μg/ml for 24 h, its mitotic index was much higher (P〈0.01) than that of control, suggesting that allitridi caused arrest of gastric cancer cells in M phase. The cells were treated with allitridi became more shrunken and nepheloid, in which the microtubule networks disappeared, while the control cell exhibited an intact microtubule network. Contrasting with normal existence mainly in the cytoplasm, the cyclin B1 was expressed more significantly and concentrated in the nucleus after exposure to allitridi. Fluorescence intensity of cyclin B 1 protein in cells treated with allitridi was much more higher than that of control (P〈0.001). Conclusion: Allitridican induce arrest of SGC-7901 cells in M phase, probably through enhancing microtubule depolymerization by elevating the expression of cyclin B1.展开更多
HL-60 cells were synchronized from G1 to S phase boundary with a double thymidine block. Samples of the cells were collected at scheduled time points after the release of the block. It was found with immunoblot analys...HL-60 cells were synchronized from G1 to S phase boundary with a double thymidine block. Samples of the cells were collected at scheduled time points after the release of the block. It was found with immunoblot analysis that the protein expression of cyclin E and D, fluctuated periodically. They both began to increase in G, phase,reached the peak in S phase and declined gradually in G, phase. The protein expression of cyclin-dependent kinase P34cdk2 showed no periodical changes- The antiproliferation effect of retinoic acid or arotinoidethylester on HL-60cells was manifested by a block in Go/G1 of most of the cells and resulted in a marked decrease of the protein expression of cyclin E and D1 and no significant change of P34cdk2. These findings suggest that retinoic acid or arotinoidethylester is able to suppress the proliferation of HL-cells or to induce their differentiation.展开更多
文摘目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中带;而MⅡ期表现为中、下带。结论:可以用电泳迁移率的变化作为观察p34cdc2磷酸化状态的一种方法。
文摘Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection; Western blotting analysis showed that the level of p3cdc2, and p33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (below 10 ℃). 25S-Met incorporation analysis showed that when the oocytes were incubated at 6℃, synthesis of about thirty defferent polypeptides was promoted or induced, including p34cdc2 and some other p13suc1-binding proteins. All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and store some cell cycle drivers and its regulators, and to gain the maturation competence. These results will also provide a new clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature and how to maintain the geographical distribution of some animals.
基金the National NaturalScience Foundation of China (Serial No.3 980 0 1 49)
文摘Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937 ASPI3K (ATM, negative) and U937 pZeosv2(+) (ATM, wild type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2 (Thr 161) or p34cdc2 (Thr 14,Tyr 15). RT PCR was used to estimate CDC25 transcript levels. U937 ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine threonine phosphatase inhibitor or cyclin dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937 pZeosv2 (+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937 ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In abstract, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.
基金the National 10th Five-year Plan Key Technologies R & D Program of China(No.2004BA703B04-02)
文摘Objective: To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods: We treated SGC-7901 cells with allitridi, and observed the proliferation inhibitory rate with MTT colometric assay, changes of cell cycle using flow cytometry and Switzerland-Giemsa's staining, and morphologic changes of the microtubule structure and location changes of cyclin BI expression using immunofluorescence and confocal laser scanning microscope. Furthermore, the expression of cyclin B1 was analyzed quantitatively using Leica confocal software. Results: SGC-7901 cells were inhibited after exposure to allitridi and the IC50 was 7.2μg/ml for 24 h, 20μg/ml for 72 h. When the cells were treated with allitridi at concentrations of 3, 6, and 9μg/ml for 24 h respectively, there was a declining tendency in the percentage of G0/G1 cell but an increasing tendency in GE/M cell in the allitridi treated group compared with that of control (P〈0.01). When cells were treated allitridi at concentration of 6 μg/ml for 24 h, its mitotic index was much higher (P〈0.01) than that of control, suggesting that allitridi caused arrest of gastric cancer cells in M phase. The cells were treated with allitridi became more shrunken and nepheloid, in which the microtubule networks disappeared, while the control cell exhibited an intact microtubule network. Contrasting with normal existence mainly in the cytoplasm, the cyclin B1 was expressed more significantly and concentrated in the nucleus after exposure to allitridi. Fluorescence intensity of cyclin B 1 protein in cells treated with allitridi was much more higher than that of control (P〈0.001). Conclusion: Allitridican induce arrest of SGC-7901 cells in M phase, probably through enhancing microtubule depolymerization by elevating the expression of cyclin B1.
文摘HL-60 cells were synchronized from G1 to S phase boundary with a double thymidine block. Samples of the cells were collected at scheduled time points after the release of the block. It was found with immunoblot analysis that the protein expression of cyclin E and D, fluctuated periodically. They both began to increase in G, phase,reached the peak in S phase and declined gradually in G, phase. The protein expression of cyclin-dependent kinase P34cdk2 showed no periodical changes- The antiproliferation effect of retinoic acid or arotinoidethylester on HL-60cells was manifested by a block in Go/G1 of most of the cells and resulted in a marked decrease of the protein expression of cyclin E and D1 and no significant change of P34cdk2. These findings suggest that retinoic acid or arotinoidethylester is able to suppress the proliferation of HL-cells or to induce their differentiation.