The 33 ku protein, prepared from NaCI-treated PSII particles, has shown an single band by SDS-PAGE. After being dialyzed against the low-osmotic medium at 4癈, it has been found that the 33 ku protein degraded into se...The 33 ku protein, prepared from NaCI-treated PSII particles, has shown an single band by SDS-PAGE. After being dialyzed against the low-osmotic medium at 4癈, it has been found that the 33 ku protein degraded into several small fragments. This result suggests that the preparations of 33 ku protein probably contain some latent proteinases. it has also been found, by the 2-D electrophoresis and IEF, that the preparations of 33 ku protein not dialyzed against the low-osmotic medium contain several polypeptides with nearly the same molecular weight but not the same isoelectric point as the 33 ku protein.展开更多
By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in p...By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic celis. BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level. The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation. in western-blotting analysis, the antibodies reacted with the 72 ku protein expressed in E.coli.展开更多
Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway....Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.展开更多
OBJECTIVE To clarify the role of translocator protein 18 ku(TSPO)on cecum liga⁃tion and puncture(CLP)induced sepsis associat⁃ed encephalopathy(SAE)mice,which consis⁃tently demonstrated astrocyte activation and neu⁃roi...OBJECTIVE To clarify the role of translocator protein 18 ku(TSPO)on cecum liga⁃tion and puncture(CLP)induced sepsis associat⁃ed encephalopathy(SAE)mice,which consis⁃tently demonstrated astrocyte activation and neu⁃roinflammation.Background SAE,a brain dys⁃function,caused by systemic infection without clinical or laboratory evidence of direct infection.Most patients have symptoms such as long-term cognitive dysfunction.As the pathogenesis of SAE is very complex,neuroinflammation for SAE is one of the causes of the disease.TSPO as a marker of neuroinflammation that has the poten⁃tial to regulate neuroinflammation and SAE.METHODS The animal model of SAE was in⁃duced by CLP.TSPO ligands and TSPO knock⁃out mice were used for behavioral and molecular biology research.Survival rate of mice within 120 h on CLP mice was observed.The changes of cog⁃nitive function in mice were observed by Morris water maze and open field test.The changes of proinflammatory factors(IL-1β,TNF-α,IL-6)in hippocampus were observed by ELISA;Astro⁃cyte activation,marked by GFAP,in hippocam⁃pal was analyzed by tissue immunofluorescence and Western blotting.RESULTS Pretreatment with the TSPO ligands,XBD173 or PK11195,sig⁃nificantly improved the survival rate of CLP mice.The results of Morris water maze showed that TSPO ligands significantly increased the number of crossing the platform and the target quadrant time on CLP mice,suggesting that TSPO ligands may improve the learning and memory ability of CLP mice.Subsequent experiments revealed that TSPO ligands can reduce the inflammatory factors(IL-1β,TNF-α,IL-6)and astrocyte activa⁃tion in hippocampus of CLP mice.Similar results were also confirmed in TSPO knockout CLP mice,suggesting intervention of TSPO can reduce neuroinflammatory response and play a protec⁃tive role on SAE mice.CONCLUSION TSPO may play a critical role on SAE mice.Targeting TSPO by pharmacological means may improve the survival rate and cognitive function on CLP mice,which may through inhibiting astrocyte acti⁃vation and neuroinflammation in hippocampal.展开更多
文摘The 33 ku protein, prepared from NaCI-treated PSII particles, has shown an single band by SDS-PAGE. After being dialyzed against the low-osmotic medium at 4癈, it has been found that the 33 ku protein degraded into several small fragments. This result suggests that the preparations of 33 ku protein probably contain some latent proteinases. it has also been found, by the 2-D electrophoresis and IEF, that the preparations of 33 ku protein not dialyzed against the low-osmotic medium contain several polypeptides with nearly the same molecular weight but not the same isoelectric point as the 33 ku protein.
文摘By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic celis. BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level. The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation. in western-blotting analysis, the antibodies reacted with the 72 ku protein expressed in E.coli.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.81672743 and 81974464)Beijing Tianjin Hebei Basic Research Cooperation Project(Grant No.19JCZDJC64500(Z))+4 种基金Shenzhen Basic Research Project(Grant No.JCYJ20160331114230843)Tianjin Municipal Health Commission(Grant Nos.2015KR11 and 2013KG134)Tianjin Municipal Science and Technology Bureau(Grant No.18JCYBJC27800)US NIH grant RO 1 CAI33093,the Alabama Innovation Fund of the United Statesthe Tianjin Medical University Cancer Institute and Hospital Innovation Fund(Grant No.1803)。
文摘Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.
基金Joint Funds for the Innovation of Science and Technology of Fujian Province(2019Y9009)and Natural Science Foundation of Fujian Province(2020J01618)。
文摘OBJECTIVE To clarify the role of translocator protein 18 ku(TSPO)on cecum liga⁃tion and puncture(CLP)induced sepsis associat⁃ed encephalopathy(SAE)mice,which consis⁃tently demonstrated astrocyte activation and neu⁃roinflammation.Background SAE,a brain dys⁃function,caused by systemic infection without clinical or laboratory evidence of direct infection.Most patients have symptoms such as long-term cognitive dysfunction.As the pathogenesis of SAE is very complex,neuroinflammation for SAE is one of the causes of the disease.TSPO as a marker of neuroinflammation that has the poten⁃tial to regulate neuroinflammation and SAE.METHODS The animal model of SAE was in⁃duced by CLP.TSPO ligands and TSPO knock⁃out mice were used for behavioral and molecular biology research.Survival rate of mice within 120 h on CLP mice was observed.The changes of cog⁃nitive function in mice were observed by Morris water maze and open field test.The changes of proinflammatory factors(IL-1β,TNF-α,IL-6)in hippocampus were observed by ELISA;Astro⁃cyte activation,marked by GFAP,in hippocam⁃pal was analyzed by tissue immunofluorescence and Western blotting.RESULTS Pretreatment with the TSPO ligands,XBD173 or PK11195,sig⁃nificantly improved the survival rate of CLP mice.The results of Morris water maze showed that TSPO ligands significantly increased the number of crossing the platform and the target quadrant time on CLP mice,suggesting that TSPO ligands may improve the learning and memory ability of CLP mice.Subsequent experiments revealed that TSPO ligands can reduce the inflammatory factors(IL-1β,TNF-α,IL-6)and astrocyte activa⁃tion in hippocampus of CLP mice.Similar results were also confirmed in TSPO knockout CLP mice,suggesting intervention of TSPO can reduce neuroinflammatory response and play a protec⁃tive role on SAE mice.CONCLUSION TSPO may play a critical role on SAE mice.Targeting TSPO by pharmacological means may improve the survival rate and cognitive function on CLP mice,which may through inhibiting astrocyte acti⁃vation and neuroinflammation in hippocampal.
