The Deltanif E MoFe protein (Deltanif E Av1) was obtained by a chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinela...The Deltanif E MoFe protein (Deltanif E Av1) was obtained by a chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE showed that the Delta nif EAv1 was similar to OP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (alpha and beta subunit). When complemented with nitrogenase Fe protein (M), the A nif EAv1 had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1. After the Delta nif E Av1 was treated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under atmosphere of Ar, Delta nif E Av1(C) was obtained. In the presence of both Av2 and MgATP regeneration system, the Delta nif EAv1(C), rather than A nif EAv1, was significantly activated in vitro by a reconstituent solution containing Mn which composed of KMnO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation of Delta nifE Av1(C) did not happen. It indicates that activation of Delta nif EAv1 by RS-Mn requires the pretreatment with o-phen and the simultaneous presence of Av2 and MgATP.展开更多
Objective： To detect the expressions of brain-derived neurotrophic factor （BDNF） and nerve growth factor （NGF） in purified rat choroid plexus epithelial cells in vitro. Methods： Primary and passage choroid plexu...Objective： To detect the expressions of brain-derived neurotrophic factor （BDNF） and nerve growth factor （NGF） in purified rat choroid plexus epithelial cells in vitro. Methods： Primary and passage choroid plexus epithelial cells were obtained from newborn, one-day Spragne-Dawley rats. The expressions of BDNF and NGF were measured by qRT-PCR and Western blottingting. The secretions of BDNF and NGF were detected by ELISA. Cell supematants of primary cells, purified cells and passage 1 cells were harvested. Results： The expression of BDNF in the purified cells was significantly lower than that in the primary cells （P〈0.05）, and it in the primary cells and the purified cells was significantly higher than that in the passage 1 cells （P〈0.05）. The expression of NGF was significantly higher in the purified cells than in the primary cells and the passage 1 cells （P〈0.05）. It in the passage 1 cells was significantly higher than that in the primary cells （P〈0.05）. Conclusion： The time of CPECs transplantation for central nervous system diseases should be selected based on their secretory function and features,which could lead to better and more effective treatment.展开更多
A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold pur...A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.展开更多
文摘The Deltanif E MoFe protein (Deltanif E Av1) was obtained by a chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE showed that the Delta nif EAv1 was similar to OP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (alpha and beta subunit). When complemented with nitrogenase Fe protein (M), the A nif EAv1 had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1. After the Delta nif E Av1 was treated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under atmosphere of Ar, Delta nif E Av1(C) was obtained. In the presence of both Av2 and MgATP regeneration system, the Delta nif EAv1(C), rather than A nif EAv1, was significantly activated in vitro by a reconstituent solution containing Mn which composed of KMnO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation of Delta nifE Av1(C) did not happen. It indicates that activation of Delta nif EAv1 by RS-Mn requires the pretreatment with o-phen and the simultaneous presence of Av2 and MgATP.
基金Supported by grants from the National Natural Science Foundation of China(No.30973099 and No.81271341)
文摘Objective： To detect the expressions of brain-derived neurotrophic factor （BDNF） and nerve growth factor （NGF） in purified rat choroid plexus epithelial cells in vitro. Methods： Primary and passage choroid plexus epithelial cells were obtained from newborn, one-day Spragne-Dawley rats. The expressions of BDNF and NGF were measured by qRT-PCR and Western blottingting. The secretions of BDNF and NGF were detected by ELISA. Cell supematants of primary cells, purified cells and passage 1 cells were harvested. Results： The expression of BDNF in the purified cells was significantly lower than that in the primary cells （P〈0.05）, and it in the primary cells and the purified cells was significantly higher than that in the passage 1 cells （P〈0.05）. The expression of NGF was significantly higher in the purified cells than in the primary cells and the passage 1 cells （P〈0.05）. It in the passage 1 cells was significantly higher than that in the primary cells （P〈0.05）. Conclusion： The time of CPECs transplantation for central nervous system diseases should be selected based on their secretory function and features,which could lead to better and more effective treatment.
基金Supported by the National High Technology Research and Development Program of China（2007AA021506） the Natural Science Foundation of Zhejiang Province（R207609）
文摘A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.
