The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a mo...The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain （NICD）. A Disintegrin and Metalloprotease 10 （ADAM10） might be a physiological rate-limiting $2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is cdtical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAMIO mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 （E14.5） to postnatal day 1 （P1）. ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin （neural stem cell marker）, Tujl （mature neuron marker）, and S100β （gila marker） showed that ADAM10 expression highly matched that of S10013 and partially matched that of Tujl at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.展开更多
【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。...【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。【结果】(1)选择能为大肠杆菌稀有密码子提供额外tRNA的Escherichia coli.Rosetta(DE3)作为宿主菌,将质粒pGEX-ADAM15转化于其中在最佳诱导表达条件下获得298mg/L融合蛋白GST-ADAM15;(2)采用PCR体外定点突变技术将目标蛋白编码区稀有密码子GGA(Gly425)替换为GGC,使融合蛋白表达水平提高9.4%;(3)通过消除凝血酶识别序列附近的Pro-Glu-Phe残基,提高凝血酶酶切效率,使rhADAM15产量提高了35.7%;(4)在GGA替换为GGC基础上切除"Pro-Glu-Phe"残基,使rhADAM15产量提高到68mg/L,比分别切除"Pro-Glu-Phe"残基、GGA替换为GGC和野生型提高了19.2%、51.1%和61.9%。【结论】这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达。展开更多
We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More impor...We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.展开更多
目的在毕赤酵母中表达重组人ADAM15去整合素区域蛋白(recombinant human disintegrin of ADAM15,rhddADAM15)与人血清白蛋白融合蛋白(HSA-rhddADAM15-His),并测定其活性。方法以rhddADAM15基因为模板,PCR扩增rhddADAM15-His基因,克隆入p...目的在毕赤酵母中表达重组人ADAM15去整合素区域蛋白(recombinant human disintegrin of ADAM15,rhddADAM15)与人血清白蛋白融合蛋白(HSA-rhddADAM15-His),并测定其活性。方法以rhddADAM15基因为模板,PCR扩增rhddADAM15-His基因,克隆入pBluescript-HSA质粒中,经酶切后与pPIC9k载体连接,构建重组表达质粒pPIC9k-HSA-rhddADAM15-His,将重组表达质粒转化毕赤酵母GS115,甲醇诱导表达。表达产物经Blue-Sepharose、Ni2+螯合层析及DEAE阴离子交换层析纯化,纯化产物经SDS-PAGE和Western blot鉴定后,利用划痕及SRB法检测其活性。结果重组表达质粒经双酶切及测序鉴定证明构建正确;融合蛋白HSA-rhddADAM15-His相对分子质量约76 000,以可溶性分子形式存在于发酵液上清中;纯化的融合蛋白纯度约为75%,可与兔抗rhddADAM15多克隆抗体特异性结合;融合蛋白对小鼠黑色素瘤细胞B16的迁移具有明显的抑制作用,但对其增殖无明显抑制作用。结论成功在毕赤酵母GS115中表达并纯化了HSA-rhddADAM15-His蛋白,为其作用机理的研究奠定了基础。展开更多
基金supported by the National Natural Science Foundation of China,No.30800322Shanghai Pujiang Program,No.08PJ1401300+4 种基金Shanghai Leading Academic Discipline Project,No.B111Ministry of Education Research Fund for New Teachers in Doctoral Program of Higher Educational Institutes,No.200802461050National Basic Research Program of China(973 Program),No.2011CB503703Ministry of Education Start Fund to Returned Overseas ScholarsZhuo Xue Program of Fudan University
文摘The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain （NICD）. A Disintegrin and Metalloprotease 10 （ADAM10） might be a physiological rate-limiting $2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is cdtical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAMIO mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 （E14.5） to postnatal day 1 （P1）. ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin （neural stem cell marker）, Tujl （mature neuron marker）, and S100β （gila marker） showed that ADAM10 expression highly matched that of S10013 and partially matched that of Tujl at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.
文摘【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。【结果】(1)选择能为大肠杆菌稀有密码子提供额外tRNA的Escherichia coli.Rosetta(DE3)作为宿主菌,将质粒pGEX-ADAM15转化于其中在最佳诱导表达条件下获得298mg/L融合蛋白GST-ADAM15;(2)采用PCR体外定点突变技术将目标蛋白编码区稀有密码子GGA(Gly425)替换为GGC,使融合蛋白表达水平提高9.4%;(3)通过消除凝血酶识别序列附近的Pro-Glu-Phe残基,提高凝血酶酶切效率,使rhADAM15产量提高了35.7%;(4)在GGA替换为GGC基础上切除"Pro-Glu-Phe"残基,使rhADAM15产量提高到68mg/L,比分别切除"Pro-Glu-Phe"残基、GGA替换为GGC和野生型提高了19.2%、51.1%和61.9%。【结论】这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达。
基金supported by the National Natural Science Foundation of China,Nos. 82071304 (to QXZ), 81671149 (to QXZ),and 81971179 (to XML)the Natural Science Foundation of Jiangsu Province,Nos. BK20191463 (to XML) and BK20161167 (to QXZ)。
文摘We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.
文摘目的在毕赤酵母中表达重组人ADAM15去整合素区域蛋白(recombinant human disintegrin of ADAM15,rhddADAM15)与人血清白蛋白融合蛋白(HSA-rhddADAM15-His),并测定其活性。方法以rhddADAM15基因为模板,PCR扩增rhddADAM15-His基因,克隆入pBluescript-HSA质粒中,经酶切后与pPIC9k载体连接,构建重组表达质粒pPIC9k-HSA-rhddADAM15-His,将重组表达质粒转化毕赤酵母GS115,甲醇诱导表达。表达产物经Blue-Sepharose、Ni2+螯合层析及DEAE阴离子交换层析纯化,纯化产物经SDS-PAGE和Western blot鉴定后,利用划痕及SRB法检测其活性。结果重组表达质粒经双酶切及测序鉴定证明构建正确;融合蛋白HSA-rhddADAM15-His相对分子质量约76 000,以可溶性分子形式存在于发酵液上清中;纯化的融合蛋白纯度约为75%,可与兔抗rhddADAM15多克隆抗体特异性结合;融合蛋白对小鼠黑色素瘤细胞B16的迁移具有明显的抑制作用,但对其增殖无明显抑制作用。结论成功在毕赤酵母GS115中表达并纯化了HSA-rhddADAM15-His蛋白,为其作用机理的研究奠定了基础。
