Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mech...Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.展开更多
Mitogen-activated protein kinase （MAPK） cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multipl...Mitogen-activated protein kinase （MAPK） cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekkl that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complemen- tation （BiFC） analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases, mkkl and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkkl mkk2 double mutants were generated. The mkkl mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekkl mutants, the mkkl mkk2 double mutant seedlings accumulate high levels of H202, display spontaneous cell death, constitutively express Pathogenesis Related （PR） genes and exhibit pathogen resistance. In addition, activation of MPK4 by fig22 is impaired in the mkkl mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.展开更多
AIM： To study the protective effect of non-mitogenic human acidic fibroblast growth factor （FGF） on cardiac oxidative injury in vivo. METHODS： Ventricular cardiomyocytes were isolated from 1- to 3-d-old neonatal S...AIM： To study the protective effect of non-mitogenic human acidic fibroblast growth factor （FGF） on cardiac oxidative injury in vivo. METHODS： Ventricular cardiomyocytes were isolated from 1- to 3-d-old neonatal SD mice and cultured in Dulbecco＇s minimum essential medium supplemented with 15% fetal bovine serum under an atmosphere of 50 mL/L CO2-95% air at 37℃, as well as assessed by immunooltochemical assay. We constructed the cardiomyoolte injury model by exposure to a certain concentration of H2O2. Cellular viability, superoxide dismutase （SOD） activity, leakage of maleic dialdehyde and anti-apoptosis effect were included to evaluate the cardiac protective effect of non-mitogenic human acidic FGF. RESULTS： Over 50% of the cardiomyocytes beat spontaneously on the 2nd d of culture and synchronously beat after being cultured for 3 d. Forty-eight hours after plating was completed, the purity of such cultures was 95% myocytes, assessed by an immunocytochemical assay. Cellular viability dramatically decreased with the increasing of the concentration of H2O2. Non-mitogenic human acidic FGF showed significant resistance to thetoxic effect of H2O2, significantly increased the cellular viability as well as the activity of SOD, and dramatically decreased the leakage of maleic dialdehyde as well as the cellular apoptosis rate. CONCLUSION： Hydrogen peroxide shows strong cytotoxicity to the cultured cardiac myocytes, and non-mitogenic human acidic FGF shows strong cardio-protective effect when exposed to a certain concentration of H2O2.展开更多
The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off...The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramati- cally inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.展开更多
Objective To investigate whether the effect of E. coli on U937 cell fines apoptosis is mediated via p38 mitogen-activated protein kinase （MAPK） activation. Methods The U937 cell lines were treated with E. coli at d...Objective To investigate whether the effect of E. coli on U937 cell fines apoptosis is mediated via p38 mitogen-activated protein kinase （MAPK） activation. Methods The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry, p38 activities were detected by Western blotting. Results E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. Conclusion The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.展开更多
Optimum conditions for in vitro production of interleukin 2 （IL 2） like activity from the Peripheral blood lymphocytes （PBL） of an Indian major carp, Labeo rohita were studied. Culture supernatants were generated ...Optimum conditions for in vitro production of interleukin 2 （IL 2） like activity from the Peripheral blood lymphocytes （PBL） of an Indian major carp, Labeo rohita were studied. Culture supernatants were generated by culturing the PBL in RPMI-1640 media supplemented with Glutamine and 10% fetal bovine serum （FBS） and stimulating with two different mitogens： concanavalin A （Con A） and Phytohaemagglutinin （PHA） at different concentrations separately. Significantly （P 〈 0.01） higher proliferation response was obtained from the culture supematant stimulated with concanavalin A （Con A） at a concentration of 10 lag mLl. The effect of phorbol myristate acetate （PMA） was also studied by co-stimulating PBL with Con A and PHA separately and it was found to synergistically enhancing the stimulation index with Con A whereas the stimulation index remain unchanged with PHA. The Con A （10 μg mLl） stimulated PBL were also cultured at different cell density, incubation period and incubation temperature in order to optimize the in vitro L. rohita IL2 production. The IL2 like activity was studied by lymphocyte proliferation assay on 72 h Con A blasts using WST based assay technique. Significantly （P 〈 0.01） higher stimulation indices were obtained when the PBL were cultured at a cell density of 1 × 10^6 cells mL^-1 for 30-36 h at an incubation temperature of 30 ℃. The IL2 like activity was purified by DEAE-Sepharose anion exchange chromatography and recorded between 70-130 mM NaCI with peak activity at 110 Mm NaCI. The molecular weight of the factor responsible for IL2 like activity was found to be 15-17 KD.展开更多
基金the National Natural Science Foundation of China (No. 30570627)
文摘Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.
文摘Mitogen-activated protein kinase （MAPK） cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekkl that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complemen- tation （BiFC） analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases, mkkl and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkkl mkk2 double mutants were generated. The mkkl mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekkl mutants, the mkkl mkk2 double mutant seedlings accumulate high levels of H202, display spontaneous cell death, constitutively express Pathogenesis Related （PR） genes and exhibit pathogen resistance. In addition, activation of MPK4 by fig22 is impaired in the mkkl mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.
基金Supported by the National 863 Project, No. 2001AA215131 and No. 2002AA2Z3318
文摘AIM： To study the protective effect of non-mitogenic human acidic fibroblast growth factor （FGF） on cardiac oxidative injury in vivo. METHODS： Ventricular cardiomyocytes were isolated from 1- to 3-d-old neonatal SD mice and cultured in Dulbecco＇s minimum essential medium supplemented with 15% fetal bovine serum under an atmosphere of 50 mL/L CO2-95% air at 37℃, as well as assessed by immunooltochemical assay. We constructed the cardiomyoolte injury model by exposure to a certain concentration of H2O2. Cellular viability, superoxide dismutase （SOD） activity, leakage of maleic dialdehyde and anti-apoptosis effect were included to evaluate the cardiac protective effect of non-mitogenic human acidic FGF. RESULTS： Over 50% of the cardiomyocytes beat spontaneously on the 2nd d of culture and synchronously beat after being cultured for 3 d. Forty-eight hours after plating was completed, the purity of such cultures was 95% myocytes, assessed by an immunocytochemical assay. Cellular viability dramatically decreased with the increasing of the concentration of H2O2. Non-mitogenic human acidic FGF showed significant resistance to thetoxic effect of H2O2, significantly increased the cellular viability as well as the activity of SOD, and dramatically decreased the leakage of maleic dialdehyde as well as the cellular apoptosis rate. CONCLUSION： Hydrogen peroxide shows strong cytotoxicity to the cultured cardiac myocytes, and non-mitogenic human acidic FGF shows strong cardio-protective effect when exposed to a certain concentration of H2O2.
基金supported by National Natural Science Foundation of China(No.30270556)The National Basic Research Program(No.2002CB513004).
文摘The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramati- cally inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.
文摘Objective To investigate whether the effect of E. coli on U937 cell fines apoptosis is mediated via p38 mitogen-activated protein kinase （MAPK） activation. Methods The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry, p38 activities were detected by Western blotting. Results E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. Conclusion The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.
文摘Optimum conditions for in vitro production of interleukin 2 （IL 2） like activity from the Peripheral blood lymphocytes （PBL） of an Indian major carp, Labeo rohita were studied. Culture supernatants were generated by culturing the PBL in RPMI-1640 media supplemented with Glutamine and 10% fetal bovine serum （FBS） and stimulating with two different mitogens： concanavalin A （Con A） and Phytohaemagglutinin （PHA） at different concentrations separately. Significantly （P 〈 0.01） higher proliferation response was obtained from the culture supematant stimulated with concanavalin A （Con A） at a concentration of 10 lag mLl. The effect of phorbol myristate acetate （PMA） was also studied by co-stimulating PBL with Con A and PHA separately and it was found to synergistically enhancing the stimulation index with Con A whereas the stimulation index remain unchanged with PHA. The Con A （10 μg mLl） stimulated PBL were also cultured at different cell density, incubation period and incubation temperature in order to optimize the in vitro L. rohita IL2 production. The IL2 like activity was studied by lymphocyte proliferation assay on 72 h Con A blasts using WST based assay technique. Significantly （P 〈 0.01） higher stimulation indices were obtained when the PBL were cultured at a cell density of 1 × 10^6 cells mL^-1 for 30-36 h at an incubation temperature of 30 ℃. The IL2 like activity was purified by DEAE-Sepharose anion exchange chromatography and recorded between 70-130 mM NaCI with peak activity at 110 Mm NaCI. The molecular weight of the factor responsible for IL2 like activity was found to be 15-17 KD.
