Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role...Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.展开更多
Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral sy...Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.展开更多
PP2Cs(2C type protein phosphatases)是一种单体丝氨酸/苏氨酸蛋白磷酸酶,在真核生物中,PP2Cs在脱落酸(ABA)、茉莉酸(JA)、水杨酸(SA)等激素信号传导途径中起着重要的调控作用。本研究通过序列比对,从谷子基因组中筛选出80个PP2C候选基...PP2Cs(2C type protein phosphatases)是一种单体丝氨酸/苏氨酸蛋白磷酸酶,在真核生物中,PP2Cs在脱落酸(ABA)、茉莉酸(JA)、水杨酸(SA)等激素信号传导途径中起着重要的调控作用。本研究通过序列比对,从谷子基因组中筛选出80个PP2C候选基因,聚类分析将其分为12个亚族(A、B、C、D、E1、E2、F1、F2、G、H、I、J)。与拟南芥PP2C基因家族比对表明,A^I为2个物种共有的亚族,J亚族只存在于谷子基因组中,L亚族只存在于拟南芥中。将谷子A亚族的10个成员命名为SiPP2CA1-10。基因表达谱分析表明,A亚族基因不同程度受ABA、干旱、高盐、低温和低氮诱导表达,其中,SiPP2CA6、SiPP2CA8在5种处理下诱导表达量都高。对10个A亚族成员的启动子分析发现,在这些基因的启动子序列中含有多种参与逆境胁迫应答的顺式作用元件,其中,SiPP2CA5、SiPP2CA6、SiPP2CA7、SiPP2CA8的启动子中含有参与低氮胁迫响应的元件。进一步研究发现,SiPP2CA8主要在根部表达,且在低氮胁迫下一直有较高的表达水平。亚细胞定位结果显示SiPP2CA8定位在细胞膜、细胞质、细胞核中;双分子荧光互补试验(BiFC)结果表明,SiPP2CA8与一个ABA受体类似蛋白SiRCAR3(基因号为Si018317m.g)在细胞膜、细胞质及细胞核上互作,表明SiPP2CA8在谷子中可能参与ABA信号传导过程。展开更多
基金Major State BasicResearch (973) Program of China, (G1999053905).
文摘Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.
基金supported by the Medical Research Fund of Guangdong Province of China,No.B2014449a grant from the Science and Technology Project of Zhongshan City of China,No.2014A1FC137
文摘Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.
文摘PP2Cs(2C type protein phosphatases)是一种单体丝氨酸/苏氨酸蛋白磷酸酶,在真核生物中,PP2Cs在脱落酸(ABA)、茉莉酸(JA)、水杨酸(SA)等激素信号传导途径中起着重要的调控作用。本研究通过序列比对,从谷子基因组中筛选出80个PP2C候选基因,聚类分析将其分为12个亚族(A、B、C、D、E1、E2、F1、F2、G、H、I、J)。与拟南芥PP2C基因家族比对表明,A^I为2个物种共有的亚族,J亚族只存在于谷子基因组中,L亚族只存在于拟南芥中。将谷子A亚族的10个成员命名为SiPP2CA1-10。基因表达谱分析表明,A亚族基因不同程度受ABA、干旱、高盐、低温和低氮诱导表达,其中,SiPP2CA6、SiPP2CA8在5种处理下诱导表达量都高。对10个A亚族成员的启动子分析发现,在这些基因的启动子序列中含有多种参与逆境胁迫应答的顺式作用元件,其中,SiPP2CA5、SiPP2CA6、SiPP2CA7、SiPP2CA8的启动子中含有参与低氮胁迫响应的元件。进一步研究发现,SiPP2CA8主要在根部表达,且在低氮胁迫下一直有较高的表达水平。亚细胞定位结果显示SiPP2CA8定位在细胞膜、细胞质、细胞核中;双分子荧光互补试验(BiFC)结果表明,SiPP2CA8与一个ABA受体类似蛋白SiRCAR3(基因号为Si018317m.g)在细胞膜、细胞质及细胞核上互作,表明SiPP2CA8在谷子中可能参与ABA信号传导过程。
文摘蛋白磷酸酶2C(protein phosphatase 2C,PP2Cs)家族是植物细胞去除磷酸化蛋白中磷酸基团的重要蛋白磷酸酶。拟南芥含有80多个编码PP2Cs的基因,然而大部分成员在植物抗逆反应中的功能仍有待研究。本文发现拟南芥PP2C31(At2g40860)基因的表达受高盐抑制,其T-DNA插入突变体(pp2c31-1突变体和pp2c31-2突变体)表现出对高盐胁迫的耐受性且Na+含量较低。实时定量PCR(quantitative real time-PCR,qRT-PCR)分析发现,PP2C31基因在植物体各组织均有表达;亚细胞定位结果显示PP2C31蛋白定位于细胞质和细胞核中。利用脱落酸合成抑制剂和外源脱落酸处理发现,突变体植株的高盐耐受性和PP2C31基因的表达均不受脱落酸的影响。研究结果表明:PP2C31蛋白是拟南芥响应高盐胁迫的一个非脱落酸依赖的负调控组分。
