Objective To investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G-bacteria, E. coli K12 anti Pse.FH2,...Objective To investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G-bacteria, E. coli K12 anti Pse.FH2, and one G^+ bactemm, B. subtilis. Methods The SOD, CAT, and ATPase specific activities of cell lysates were determined spectrophotometrically at 550 nm, 240 nm, and 660 nm, respectively, with kits A001, A016, and A007. SOD isozyme patterns were detected by native PAGE analysis. Results SOD and CAT activities in the tested bacteria increased significantly in a concentration-dependent manner after different concentrations of acetamiprid were applied. The activity of SOD in B. subtilis and Pse.FH2 was stimulated and reached the highest level after treatment with 100 mg/L acetamiprid for 0.5 h. For Pse.FH2, there was another stimulation of SOD activity after acetamiprid application for about 8.0 h and the second stimulation was stronger than the first. The stimulation by acetamiprid showed a relative lag for E. coli K12. Acetamiprid seemed to exhibit a similar effect on CAT activity of the two G bacteria and had an evident influence on ATPase activity in the three bacteria within a relatively short period. Only one SOD isozyme was detectable in Pse.FH2 and B. subtilis, while different isozyme compositions in E. coli could be detected by native PAGE analysis. Conclusion Acetamiprid causes a certain oxidative stress on the three bacteria which may not only elevate SOD and CAT activities but also generate new SOD isozymes to antagonize oxidative stress. However, this oxidative stress lasts for a relatively short time and does not cause a long-term damage.展开更多
By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue sample...By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.展开更多
The oligomers of Aβ1-40 peptide in PBS buffer solution were analyzed by SEC and native PAGE, and the trimer of Aβ1-40 was also isolated by SEC. In addition, the effects of the soluble Aβ1-40 trimer on intracellular...The oligomers of Aβ1-40 peptide in PBS buffer solution were analyzed by SEC and native PAGE, and the trimer of Aβ1-40 was also isolated by SEC. In addition, the effects of the soluble Aβ1-40 trimer on intracellular free calcium (Ca2+) balance of hippocampal neurons of postnatal rats were investi- gated by fluorescence microscopy. The experimental results indicated that Aβ1-40 peptide existed in the form of low molecular weight oligomers in 0.231 mmol/L fresh Aβ1-40 solution (20 mmol/L sodium phosphate buffer, pH 7.4, 0.02% sodium azide) within 24 h and the soluble trimer was the most abundant species. Both the trimeric and the fibrillar Aβ1-40 were able to increase the intracellular Ca2+ concentration, but the Aβ1-40 trimer caused a gradual rise and the potential was also stronger than that of the fibrils at the same concentration. In addition there were dif- ferent response modes for trimeric and fibrillar Aβ1-40, meaning that there are different mechanisms of in- crease in intracellular Ca2+ caused by Aβ1-40.展开更多
基金The project was supported by National Natural Science Foundation of China (No. 30370048).
文摘Objective To investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G-bacteria, E. coli K12 anti Pse.FH2, and one G^+ bactemm, B. subtilis. Methods The SOD, CAT, and ATPase specific activities of cell lysates were determined spectrophotometrically at 550 nm, 240 nm, and 660 nm, respectively, with kits A001, A016, and A007. SOD isozyme patterns were detected by native PAGE analysis. Results SOD and CAT activities in the tested bacteria increased significantly in a concentration-dependent manner after different concentrations of acetamiprid were applied. The activity of SOD in B. subtilis and Pse.FH2 was stimulated and reached the highest level after treatment with 100 mg/L acetamiprid for 0.5 h. For Pse.FH2, there was another stimulation of SOD activity after acetamiprid application for about 8.0 h and the second stimulation was stronger than the first. The stimulation by acetamiprid showed a relative lag for E. coli K12. Acetamiprid seemed to exhibit a similar effect on CAT activity of the two G bacteria and had an evident influence on ATPase activity in the three bacteria within a relatively short period. Only one SOD isozyme was detectable in Pse.FH2 and B. subtilis, while different isozyme compositions in E. coli could be detected by native PAGE analysis. Conclusion Acetamiprid causes a certain oxidative stress on the three bacteria which may not only elevate SOD and CAT activities but also generate new SOD isozymes to antagonize oxidative stress. However, this oxidative stress lasts for a relatively short time and does not cause a long-term damage.
文摘By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.
基金supported by the National Natural Science Foundation of China(Grant Nos.20273002&20103001).
文摘The oligomers of Aβ1-40 peptide in PBS buffer solution were analyzed by SEC and native PAGE, and the trimer of Aβ1-40 was also isolated by SEC. In addition, the effects of the soluble Aβ1-40 trimer on intracellular free calcium (Ca2+) balance of hippocampal neurons of postnatal rats were investi- gated by fluorescence microscopy. The experimental results indicated that Aβ1-40 peptide existed in the form of low molecular weight oligomers in 0.231 mmol/L fresh Aβ1-40 solution (20 mmol/L sodium phosphate buffer, pH 7.4, 0.02% sodium azide) within 24 h and the soluble trimer was the most abundant species. Both the trimeric and the fibrillar Aβ1-40 were able to increase the intracellular Ca2+ concentration, but the Aβ1-40 trimer caused a gradual rise and the potential was also stronger than that of the fibrils at the same concentration. In addition there were dif- ferent response modes for trimeric and fibrillar Aβ1-40, meaning that there are different mechanisms of in- crease in intracellular Ca2+ caused by Aβ1-40.