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
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
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.
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.
