[目的]基于连接蛋白43(Cx43)半通道探讨蛋白激酶基因表达在大鼠脑缺血再灌注过程中的动态变化规律以及针刺干预的影响。[方法]根据随机数字表将Wistar大鼠随机分为正常组、模型组、非穴位针刺组及针刺组。采用改良的线栓法制备大鼠大脑...[目的]基于连接蛋白43(Cx43)半通道探讨蛋白激酶基因表达在大鼠脑缺血再灌注过程中的动态变化规律以及针刺干预的影响。[方法]根据随机数字表将Wistar大鼠随机分为正常组、模型组、非穴位针刺组及针刺组。采用改良的线栓法制备大鼠大脑中动脉局灶性脑缺血再灌注模型,模型组及两个针刺组内分设4个亚组:缺血再灌注30 min组、缺血再灌注60 min组、缺血再灌注180 min组、缺血再灌注360 min组,每组10只大鼠。穴位组电针刺激大鼠的"顶中线"、"顶旁线";非穴位组取患侧肋下髂嵴上10 mm的固定点作为针刺点;模型组不予任何处理。采用荧光定量聚合酶链反应(PCR)技术从基因方面检测海马区蛋白激酶C(PKC)、酪蛋白激酶1(CK1)、蛋白磷酸酶(PP2B)的表达。[结果]与正常组相比,模型组PKC m RNA、CK1 m RNA及PP2B m RNA时间的推移表达量呈增高趋势;与模型组同一时间点比较,非穴位针刺组PKCm RNA表达无显著变化(P>0.05);调神通络针刺组可显著降低其在各时间点表达,与模型组及非穴位针刺组比较,差异具有统计学意义(P<0.05)。[结论]调神通络针刺组可抑制大鼠脑缺血再灌注过程中PKC m RNA、CK1 m RNA及PP2B m RNA表达,通过调节Cx43磷酸化或去磷酸化途径中的关键因子,来调控半通道的开放,在缺血性脑损伤中发挥神经保护作用。展开更多
The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphata...The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1–PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.展开更多
文摘[目的]基于连接蛋白43(Cx43)半通道探讨蛋白激酶基因表达在大鼠脑缺血再灌注过程中的动态变化规律以及针刺干预的影响。[方法]根据随机数字表将Wistar大鼠随机分为正常组、模型组、非穴位针刺组及针刺组。采用改良的线栓法制备大鼠大脑中动脉局灶性脑缺血再灌注模型,模型组及两个针刺组内分设4个亚组:缺血再灌注30 min组、缺血再灌注60 min组、缺血再灌注180 min组、缺血再灌注360 min组,每组10只大鼠。穴位组电针刺激大鼠的"顶中线"、"顶旁线";非穴位组取患侧肋下髂嵴上10 mm的固定点作为针刺点;模型组不予任何处理。采用荧光定量聚合酶链反应(PCR)技术从基因方面检测海马区蛋白激酶C(PKC)、酪蛋白激酶1(CK1)、蛋白磷酸酶(PP2B)的表达。[结果]与正常组相比,模型组PKC m RNA、CK1 m RNA及PP2B m RNA时间的推移表达量呈增高趋势;与模型组同一时间点比较,非穴位针刺组PKCm RNA表达无显著变化(P>0.05);调神通络针刺组可显著降低其在各时间点表达,与模型组及非穴位针刺组比较,差异具有统计学意义(P<0.05)。[结论]调神通络针刺组可抑制大鼠脑缺血再灌注过程中PKC m RNA、CK1 m RNA及PP2B m RNA表达,通过调节Cx43磷酸化或去磷酸化途径中的关键因子,来调控半通道的开放,在缺血性脑损伤中发挥神经保护作用。
基金supported by the National Natural Science Foundation of China(31970666)Taishan Scholar Project(tsqn201812054)from Shandong,China+3 种基金Work at the Novo Nordisk Foundation Center for Protein Research was supported by the grant NNF14CC0001J.N.is supported by grants from the Danish Cancer Society(R269-A15586-B17)Independent Research Fund Denmark(8021-00101B and 0134-00199B)Novo Nordisk Foundation(NNF20OC0065098).
文摘The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1–PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.
