Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymp...Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.展开更多
AIM: To prove anthrax lethal toxin(Le Tx) blocks the mitogen activated protein kinases(MAPKs) activation by degrading the MAPK/ERK kinases(MEKs) to suppress vascular endothelial growth factor(VEGF) secretion.METHODS: ...AIM: To prove anthrax lethal toxin(Le Tx) blocks the mitogen activated protein kinases(MAPKs) activation by degrading the MAPK/ERK kinases(MEKs) to suppress vascular endothelial growth factor(VEGF) secretion.METHODS: Human adult retinal pigmented epithelium(ARPE) cells were cultured and treated with normal glucose, high glucose or high glucose with Le Tx for additional 24, 48 or 72 h for viable cell count. Total RNA from the ARPE was isolated for reverse transcription polymerase chain reaction(RT-PCR). The conditioned medium of ARPE cells treated in different group for 48 h was filtered and diluted to detect the concentration of VEGF by enzyme-linked immunosorbant assays.Evaluate the role of MEK/MAPK pathway in the secretion of VEGF by immunoblotting. RESULTS: In this study, we proved high glucose induced activation of the MAPK extracellular signal-regulated kinase(ERK1/2) and p38 in the ARPE cell line was blocked by anthrax Le Tx. Le Tx also inhibited high glucose induced ARPE cell over proliferation.CONCLUSION: Le Tx suppressed high glucose induced VEGF over secretion in the ARPE cells, mainly through a post-translational mechanism.展开更多
Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, w...Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 （Racl）, PI3K/Akt and p21- actived kinase （PAK1） along with cell migration. Ectopic expression of PAK1 K299R, a dominant negative PAK1 mutant, could largely abolish EGF-induced cell migration. Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration. Furthermore, expression of dominant-negative Racl （T17N） could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration. Interestingly, EGF could induce a significant production of ROS, and N-acetyl-L-cysteine, a scavenger of ROS which abolished the EGF-induced ROS generation, cell migration, as well as activation of PI3K/Akt and PAK, but not Racl. Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events, including activation of Racl, generation of ROS and subsequent activation of PI3K/Akt and PAK1.展开更多
基金supported by grants from the National "973" Basic Research Program of China (No.2012CB944703)the National Key Technology Research and Development Program of China (No.2011BAI17B00)the Shandong Provincial Science and Technology Development Projects (No.2009GG10002008 and No.2011GSF12103)
文摘Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.
基金Supported by National Natural Science Foundation of China (No.81170855)
文摘AIM: To prove anthrax lethal toxin(Le Tx) blocks the mitogen activated protein kinases(MAPKs) activation by degrading the MAPK/ERK kinases(MEKs) to suppress vascular endothelial growth factor(VEGF) secretion.METHODS: Human adult retinal pigmented epithelium(ARPE) cells were cultured and treated with normal glucose, high glucose or high glucose with Le Tx for additional 24, 48 or 72 h for viable cell count. Total RNA from the ARPE was isolated for reverse transcription polymerase chain reaction(RT-PCR). The conditioned medium of ARPE cells treated in different group for 48 h was filtered and diluted to detect the concentration of VEGF by enzyme-linked immunosorbant assays.Evaluate the role of MEK/MAPK pathway in the secretion of VEGF by immunoblotting. RESULTS: In this study, we proved high glucose induced activation of the MAPK extracellular signal-regulated kinase(ERK1/2) and p38 in the ARPE cell line was blocked by anthrax Le Tx. Le Tx also inhibited high glucose induced ARPE cell over proliferation.CONCLUSION: Le Tx suppressed high glucose induced VEGF over secretion in the ARPE cells, mainly through a post-translational mechanism.
基金supported by grants from the National Natural Science Foundation of China (No. 30872926)the Program for AdvancedTalents within Six Industries of Jiangsu Province (08-D) to Dr. Luo Gu+1 种基金the Science Development Foundation of Nanjing Medical University (No. 2010NJMUZ35)the Research Program funded by Schoolof Basic Medical Science, Nanjing Medical University to Dr. Jun Du
文摘Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 （Racl）, PI3K/Akt and p21- actived kinase （PAK1） along with cell migration. Ectopic expression of PAK1 K299R, a dominant negative PAK1 mutant, could largely abolish EGF-induced cell migration. Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration. Furthermore, expression of dominant-negative Racl （T17N） could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration. Interestingly, EGF could induce a significant production of ROS, and N-acetyl-L-cysteine, a scavenger of ROS which abolished the EGF-induced ROS generation, cell migration, as well as activation of PI3K/Akt and PAK, but not Racl. Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events, including activation of Racl, generation of ROS and subsequent activation of PI3K/Akt and PAK1.
