Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ...Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.展开更多
Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1—335) and CK2β (rCK2β). The overall topolo...Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1—335) and CK2β (rCK2β). The overall topology of rCK2α-△C and rCK2β are very similar to the human enzyme, although large structural differences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi- cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conformational change. The major region of interaction within the interface of CK2α/CK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.展开更多
目的系统评价CDKAL1基因rs7756992位点A>G多态性与2型糖尿病(T2DM)易感性的关系。方法制定原始文献的纳入、排除标准及检索策略,通过检索学术期刊全文数据库(CNKI)、万方数据库及EMBASE、PubMed、ScienceDirect等数据库,收集有关CDK...目的系统评价CDKAL1基因rs7756992位点A>G多态性与2型糖尿病(T2DM)易感性的关系。方法制定原始文献的纳入、排除标准及检索策略,通过检索学术期刊全文数据库(CNKI)、万方数据库及EMBASE、PubMed、ScienceDirect等数据库,收集有关CDKAL1基因rs7756992位点A>G多态性与T2DM易感性的病例对照研究,以病例组与对照组CDKAL1基因rs7756992位点各种基因模型的比值比(OR)及其95%置信区间(CI)为效应指标进行meta分析,并根据研究人群种族不同进行亚组分析。结果本研究共纳入15篇文献,T2DM组和对照组病例数分别为24 315例和35 132例。Meta分析显示,CDKAL1基因rs7756992位点A>G多态性与T2DM易感性有关联[等位基因模式(G vs A):OR=1.171,95%CI1.122~1.223,P<0.001;共显性模式(GG vs AA):OR=1.380,95%CI1.258~1.515,P<0.001;共显性模式(AG vs AA):OR=1.131,95%CI 1.089~1.176,P<0.001;显性模式(AG+GG vs AA):OR=1.168,95%CI 1.101~1.240,P<0.001;隐性模式(GG vs AA+AG):OR=1.343,95%CI 1.282~1.405,P<0.001]。亚组分析显示,亚洲人群和白种人群中携带CDKAL1基因rs7756992位点G等位基因的人群发生T2DM的风险增加(P<0.05);而非洲人群中携带CDKAL1基因rs7756992位点G等位基因与A等位基因的人群发生T2DM风险的差异无统计学意义。结论在亚洲人群及白种人群中CDKAL1基因rs7756992位点A>G等位基因的突变可能是T2DM发病的危险因素之一。展开更多
】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H i...】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c展开更多
目的探讨CDC28蛋白激酶调节亚基2(CKS2)对A2780细胞丝足形成的影响及其可能机制。方法采用慢病毒介导的sh RNA敲除A2780细胞的CKS2基因,显微镜下观察细胞丝足的变化;采用细胞划痕实验检测A2780细胞迁移能力的变化;采用real time PCR检测...目的探讨CDC28蛋白激酶调节亚基2(CKS2)对A2780细胞丝足形成的影响及其可能机制。方法采用慢病毒介导的sh RNA敲除A2780细胞的CKS2基因,显微镜下观察细胞丝足的变化;采用细胞划痕实验检测A2780细胞迁移能力的变化;采用real time PCR检测CKS2敲除对CDC42两种剪接变异体(CDC42-V1和CDC42-V2)表达的影响,以及不同卵巢癌标本中CKS2、CDC42-V1和CDC42-V2剪接变异体的表达情况。结果 CKS2表达抑制后,A2780细胞丝足明显减少,细胞迁移能力明显减弱(P<0.05),CDC42-V1 m RNA表达降低,而CDC42-V2 m RNA表达升高(P<0.05)。Real time PCR结果显示,卵巢癌组织标本中CKS2及CDC42-V1的表达高于对应的正常卵巢组织,而CDC42-V2的表达低于对应的正常卵巢组织(P<0.05)。结论 CKS2通过调控CDC42的选择性剪接而影响细胞丝足的形成,进而影响卵巢癌细胞A2780的迁移能力。展开更多
AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27...AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.展开更多
文摘Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.
基金Supported by Tianjin Basic Research Program (Grant No. 07JCYBJC15600)National Natural Science Foundation of China (Grant No. 30770428)+2 种基金National Key Basic Research and Development Program of China (Grant Nos. 2007CB914301, 2006CB910200)National High-Technology Research and Development Program of China (Grant No. 2006AA02A319)National Institute of Health (Grant No. MH07752)
文摘Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1—335) and CK2β (rCK2β). The overall topology of rCK2α-△C and rCK2β are very similar to the human enzyme, although large structural differences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi- cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conformational change. The major region of interaction within the interface of CK2α/CK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.
文摘目的系统评价CDKAL1基因rs7756992位点A>G多态性与2型糖尿病(T2DM)易感性的关系。方法制定原始文献的纳入、排除标准及检索策略,通过检索学术期刊全文数据库(CNKI)、万方数据库及EMBASE、PubMed、ScienceDirect等数据库,收集有关CDKAL1基因rs7756992位点A>G多态性与T2DM易感性的病例对照研究,以病例组与对照组CDKAL1基因rs7756992位点各种基因模型的比值比(OR)及其95%置信区间(CI)为效应指标进行meta分析,并根据研究人群种族不同进行亚组分析。结果本研究共纳入15篇文献,T2DM组和对照组病例数分别为24 315例和35 132例。Meta分析显示,CDKAL1基因rs7756992位点A>G多态性与T2DM易感性有关联[等位基因模式(G vs A):OR=1.171,95%CI1.122~1.223,P<0.001;共显性模式(GG vs AA):OR=1.380,95%CI1.258~1.515,P<0.001;共显性模式(AG vs AA):OR=1.131,95%CI 1.089~1.176,P<0.001;显性模式(AG+GG vs AA):OR=1.168,95%CI 1.101~1.240,P<0.001;隐性模式(GG vs AA+AG):OR=1.343,95%CI 1.282~1.405,P<0.001]。亚组分析显示,亚洲人群和白种人群中携带CDKAL1基因rs7756992位点G等位基因的人群发生T2DM的风险增加(P<0.05);而非洲人群中携带CDKAL1基因rs7756992位点G等位基因与A等位基因的人群发生T2DM风险的差异无统计学意义。结论在亚洲人群及白种人群中CDKAL1基因rs7756992位点A>G等位基因的突变可能是T2DM发病的危险因素之一。
文摘】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c
文摘目的探讨CDC28蛋白激酶调节亚基2(CKS2)对A2780细胞丝足形成的影响及其可能机制。方法采用慢病毒介导的sh RNA敲除A2780细胞的CKS2基因,显微镜下观察细胞丝足的变化;采用细胞划痕实验检测A2780细胞迁移能力的变化;采用real time PCR检测CKS2敲除对CDC42两种剪接变异体(CDC42-V1和CDC42-V2)表达的影响,以及不同卵巢癌标本中CKS2、CDC42-V1和CDC42-V2剪接变异体的表达情况。结果 CKS2表达抑制后,A2780细胞丝足明显减少,细胞迁移能力明显减弱(P<0.05),CDC42-V1 m RNA表达降低,而CDC42-V2 m RNA表达升高(P<0.05)。Real time PCR结果显示,卵巢癌组织标本中CKS2及CDC42-V1的表达高于对应的正常卵巢组织,而CDC42-V2的表达低于对应的正常卵巢组织(P<0.05)。结论 CKS2通过调控CDC42的选择性剪接而影响细胞丝足的形成,进而影响卵巢癌细胞A2780的迁移能力。
基金Supported by A grant from Samsung Biomedical Research Institute,No.C-A9-210-1
文摘AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.
