Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apopt...Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apoptosis in glaucoma.Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma.In this study,we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation.Following this,we treated Müller glial cells in vitro and in vivo with the m Glu R I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr^(9)Asp overexpression.We found that both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited activation of Müller glial cells.Subsequently,we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr^(9)Asp in the eye,and observed similar results in Müller cells in vivo as those seen in vitro.Both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited Müller cell activation,regulated the balance of Bax/Bcl-2,and reduced the m RNA and protein levels of pro-inflammatory factors,including interleukin-1βand tumor necrosis factor-α.Furthermore,we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia.In this co-culture system,we observed elevated adenosine triphosphate concentrations in activated Müller cells,increased levels of translocator protein(a marker of microglial activation),and elevated interleukin-1βm RNA and protein levels in microglia induced by activated Müller cells.These changes could be reversed by Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression in Müller cells.Kir4.1 overexpression,but not Kir4.1 Tyr^(9)Asp overexpression,reduced the number of proliferative and migratory microglia induced by activated Müller cells.Collectively,these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma.Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression attenuated Müller cell activation,reduced ATP/P2X receptor–mediated interactions between glial cells,inhibited microglial activation,and decreased the synthesis and release of pro-inflammatory factors,consequently ameliorating retinal ganglion cell apoptosis in glaucoma.展开更多
Gibberella stalk rot(GSR)caused by Fusarium graminearum is one of the most devastating diseases of maize,seriously impacting maize yield and quality,as well as the ability to use technology of mechanical harvesting.En...Gibberella stalk rot(GSR)caused by Fusarium graminearum is one of the most devastating diseases of maize,seriously impacting maize yield and quality,as well as the ability to use technology of mechanical harvesting.Environmental conditions including photoperiod affect crop disease resistance.However,the mechanism underlying photoperiod-regulated maize GSR resistance remains unexplored.We found in this study that GSR resistance is regulated by the ZmPIF4.1(Phytochrome-Interacting Factor4)-ZmPTI1c(Pto-Interacting 1)-ZmMYB31 module coupled with photoperiod.The functional analysis of zmpti1c mutant indicated that ZmPTI1c negatively regulates maize GSR resistance.Short day promoted the disease progression in both zmpti1c and wild-type plants.ZmPTI1c promoter contains multiple predicted cis-acting elements for light responses.Yeast one-hybrid assay(Y1H),Electrophoretic mobility shift analysis(EMSA),and Dual-luciferase(LUC)reporter assays demonstrated that ZmPIF4.1 binds to the G-box in ZmPTI1c promoter and activates its expression.Moreover,expression levels of ZmPIF4 and ZmPTI1c were significantly higher under short day than under long day.ZmPTI1c interacted with and phosphorylated ZmMYB31.GSR resistance in zmmyb31 mutant was significantly increased than in wild type,indicating that ZmMYB31 also negatively regulated GSR resistance.Furthermore,ZmMYB31 suppressed the transcriptional activation of ZmPTI1c by ZmPIF4.1.Overall,ZmPIF4.1-ZmPTI1c-ZmMYB31negatively regulates maize immunity to GSR,which is likely modulated by photoperiod.展开更多
目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常...目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常规培养基础上加入0.5μmol/L羟苯磺酸钙)和高糖+羟苯磺酸钙组(高糖基础上加入0.5μmol/L羟苯磺酸钙)。使用CCK-8评估细胞增殖,流式细胞术检测细胞凋亡,试剂盒检测氧化应激指标,蛋白印迹技术检测内向整流钾离子通道4.1(inwardly rectifying K channel 4.1,Kir4.1)和水通道蛋白4(aquaporin-4,AQP4)蛋白水平。结果与对照组比较,高糖组Müller细胞增殖活性降低且凋亡率升高,细胞发生氧化应激,AQP4蛋白表达水平升高而Kir4.1蛋白表达水平降低(P<0.05)。与高糖组比较,高糖+羟苯磺酸钙组细胞增殖活性增加且凋亡率降低,细胞氧化应激损伤减轻,AQP4蛋白表达水平降低而Kir4.1蛋白表达水平升高(P<0.05)。结论羟苯磺酸钙可能通过调节AQP4/Kir4.1轴抑制高糖诱导的Müller细胞氧化损伤。展开更多
基金supported by the National Natural Science Foundation of China,Nos.32271043(to ZW)and 82171047(to YM)the both Science and Technology Major Project of Shanghai,No.2018SHZDZX01 and ZJLabShanghai Center for Brain Science and Brain-Inspired Technology(to ZW)。
文摘Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apoptosis in glaucoma.Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma.In this study,we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation.Following this,we treated Müller glial cells in vitro and in vivo with the m Glu R I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr^(9)Asp overexpression.We found that both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited activation of Müller glial cells.Subsequently,we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr^(9)Asp in the eye,and observed similar results in Müller cells in vivo as those seen in vitro.Both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited Müller cell activation,regulated the balance of Bax/Bcl-2,and reduced the m RNA and protein levels of pro-inflammatory factors,including interleukin-1βand tumor necrosis factor-α.Furthermore,we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia.In this co-culture system,we observed elevated adenosine triphosphate concentrations in activated Müller cells,increased levels of translocator protein(a marker of microglial activation),and elevated interleukin-1βm RNA and protein levels in microglia induced by activated Müller cells.These changes could be reversed by Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression in Müller cells.Kir4.1 overexpression,but not Kir4.1 Tyr^(9)Asp overexpression,reduced the number of proliferative and migratory microglia induced by activated Müller cells.Collectively,these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma.Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression attenuated Müller cell activation,reduced ATP/P2X receptor–mediated interactions between glial cells,inhibited microglial activation,and decreased the synthesis and release of pro-inflammatory factors,consequently ameliorating retinal ganglion cell apoptosis in glaucoma.
基金supported financially by the grants from the JBGS[2021]002 project from the Jiangsu Governmentthe National Nature Science Foundation of China(32472095)+2 种基金the National Key Research and Development Program of China(2020YFE02029002)Collaborative Innovation Center for Modern Crop Production(CIC-MCP)to Xiquan Gaosupported in part by the high-performance computing platform of Bioinformatics Center,Nanjing Agricultural University。
文摘Gibberella stalk rot(GSR)caused by Fusarium graminearum is one of the most devastating diseases of maize,seriously impacting maize yield and quality,as well as the ability to use technology of mechanical harvesting.Environmental conditions including photoperiod affect crop disease resistance.However,the mechanism underlying photoperiod-regulated maize GSR resistance remains unexplored.We found in this study that GSR resistance is regulated by the ZmPIF4.1(Phytochrome-Interacting Factor4)-ZmPTI1c(Pto-Interacting 1)-ZmMYB31 module coupled with photoperiod.The functional analysis of zmpti1c mutant indicated that ZmPTI1c negatively regulates maize GSR resistance.Short day promoted the disease progression in both zmpti1c and wild-type plants.ZmPTI1c promoter contains multiple predicted cis-acting elements for light responses.Yeast one-hybrid assay(Y1H),Electrophoretic mobility shift analysis(EMSA),and Dual-luciferase(LUC)reporter assays demonstrated that ZmPIF4.1 binds to the G-box in ZmPTI1c promoter and activates its expression.Moreover,expression levels of ZmPIF4 and ZmPTI1c were significantly higher under short day than under long day.ZmPTI1c interacted with and phosphorylated ZmMYB31.GSR resistance in zmmyb31 mutant was significantly increased than in wild type,indicating that ZmMYB31 also negatively regulated GSR resistance.Furthermore,ZmMYB31 suppressed the transcriptional activation of ZmPTI1c by ZmPIF4.1.Overall,ZmPIF4.1-ZmPTI1c-ZmMYB31negatively regulates maize immunity to GSR,which is likely modulated by photoperiod.
文摘目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常规培养基础上加入0.5μmol/L羟苯磺酸钙)和高糖+羟苯磺酸钙组(高糖基础上加入0.5μmol/L羟苯磺酸钙)。使用CCK-8评估细胞增殖,流式细胞术检测细胞凋亡,试剂盒检测氧化应激指标,蛋白印迹技术检测内向整流钾离子通道4.1(inwardly rectifying K channel 4.1,Kir4.1)和水通道蛋白4(aquaporin-4,AQP4)蛋白水平。结果与对照组比较,高糖组Müller细胞增殖活性降低且凋亡率升高,细胞发生氧化应激,AQP4蛋白表达水平升高而Kir4.1蛋白表达水平降低(P<0.05)。与高糖组比较,高糖+羟苯磺酸钙组细胞增殖活性增加且凋亡率降低,细胞氧化应激损伤减轻,AQP4蛋白表达水平降低而Kir4.1蛋白表达水平升高(P<0.05)。结论羟苯磺酸钙可能通过调节AQP4/Kir4.1轴抑制高糖诱导的Müller细胞氧化损伤。
