Four new 9,19-cycloartane triterpenoids,cimilactone E(1),cimilactone F(2),20-O-(E)-butenoyl-23-epi-26-deoxyactein(3),and 20,12b-O-diacetylcimiracemonol-3-O-b-D-xylopyranoside(4),together with four known constituents(5...Four new 9,19-cycloartane triterpenoids,cimilactone E(1),cimilactone F(2),20-O-(E)-butenoyl-23-epi-26-deoxyactein(3),and 20,12b-O-diacetylcimiracemonol-3-O-b-D-xylopyranoside(4),together with four known constituents(5–8)were isolated from the roots of Cimicifuga foetida.The new structures were elucidated by extensive spectroscopic analysis.In addition,compounds 7 and 8 showed significant Wnt signaling pathway inhibitory activity,with IC50 values of 3.33 and 13.34 lM,respectively,using the luciferase reporter gene assay.展开更多
目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基...目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基因表达体系检测转录活性。结果 PCR产物经测序证实后,分别定向连接至pGL3-Promoter载体中报告基因启动子上游的Kpn I和Xho I酶切位点和报告基因下游的BamHI和Sal I酶切位点上,经酶切鉴定后,构建了6种荧光素酶报告基因表达体系。以pSV-β-Galactosidase质粒为内对照,瞬时转染HEK293细胞,培养48h后检测细胞裂解液中荧光素酶活性。当3个DNA片段位于报告基因启动子上游时,均不具有增强转录的能力。将它们分别连接于报告基因下游时,E1和E3所调控的荧光素酶活性分别是对照pGL3-Promoter的2.83倍(P<0.05)和1.59倍(P<0.05)。结论 LUNX基因的+3770～+3959bp和+14553～+14652bp序列具有增强转录的能力,为LUNX表达调控机制的深入研究奠定了基础。展开更多
Objective： We previously found that hUTP14a binds P53 and promotes P53 degradation. However, if hUTP14a is a downstream gene of P53 remains to be determined. This study aimed to identify the promoter of h UTP14a and ...Objective： We previously found that hUTP14a binds P53 and promotes P53 degradation. However, if hUTP14a is a downstream gene of P53 remains to be determined. This study aimed to identify the promoter of h UTP14a and investigate if h UTP14a is regulated by P53. Methods： The hUTPI4a promoter region was cloned into pGL3-Basic-luciferase reporter plasmid to get pGL3-hUTP14a-luc. The reporter plasmid was transfected into 293T cells and luciferase activity was evaluated by the Dual-Luciferase Reporter Assay System. Putative transcription factors were identified through searching Matlnspector Professional and Algorismica i Genetica databases. Either pGL3-hUTPI4aluc or p21 promoter reporter plasmid was co-transfected with increasing dose of p53 plasmid, and luciferase activity was evaluated. A series of deletion constructs of pGL3-hUTP14a-luc were constructed and minimal promoter region of hUTP14a was determined. Differences of the lnciferase activities between different groups were assessed by statistical analysis. Results： The hUTP14a gene promoter reporter construct was correctly cloned and was demonstrated to possess promoter activity. The transcription of hUTP14a was not regulated by P53. The minimal promoter region of h UTP14a gene is located between -203 to -100 of the transcription initiation site. Conclusion： Unlike other P53-interacting proteins such as MDM2, Pirh2 and Cop I which promote P53 degradation and whose transcriptions are regulated by P53, does not hUTP14a transcription form a regulation feedback loop with P 53.展开更多
基金Program for National Natural Science Foundation of China(Nos.U1132604 and 81302670)The Major Deployment Program of the CAS(No.KSZDEW-Z-004-01)Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China(P2008-ZZ05).
文摘Four new 9,19-cycloartane triterpenoids,cimilactone E(1),cimilactone F(2),20-O-(E)-butenoyl-23-epi-26-deoxyactein(3),and 20,12b-O-diacetylcimiracemonol-3-O-b-D-xylopyranoside(4),together with four known constituents(5–8)were isolated from the roots of Cimicifuga foetida.The new structures were elucidated by extensive spectroscopic analysis.In addition,compounds 7 and 8 showed significant Wnt signaling pathway inhibitory activity,with IC50 values of 3.33 and 13.34 lM,respectively,using the luciferase reporter gene assay.
文摘目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基因表达体系检测转录活性。结果 PCR产物经测序证实后,分别定向连接至pGL3-Promoter载体中报告基因启动子上游的Kpn I和Xho I酶切位点和报告基因下游的BamHI和Sal I酶切位点上,经酶切鉴定后,构建了6种荧光素酶报告基因表达体系。以pSV-β-Galactosidase质粒为内对照,瞬时转染HEK293细胞,培养48h后检测细胞裂解液中荧光素酶活性。当3个DNA片段位于报告基因启动子上游时,均不具有增强转录的能力。将它们分别连接于报告基因下游时,E1和E3所调控的荧光素酶活性分别是对照pGL3-Promoter的2.83倍(P<0.05)和1.59倍(P<0.05)。结论 LUNX基因的+3770～+3959bp和+14553～+14652bp序列具有增强转录的能力,为LUNX表达调控机制的深入研究奠定了基础。
基金supported by grants from Beijing Municipal Natural Science Foundation (Grant No. 7122032)the National Natural Science Foundation of China (Grant No. 81071672)+1 种基金the National Basic Research Program of China (973 program) (Grant No. 2010CB529303)National High Technology Research and Development Program of China (863 Program, Grant No. 2008AA02Z131)
文摘Objective： We previously found that hUTP14a binds P53 and promotes P53 degradation. However, if hUTP14a is a downstream gene of P53 remains to be determined. This study aimed to identify the promoter of h UTP14a and investigate if h UTP14a is regulated by P53. Methods： The hUTPI4a promoter region was cloned into pGL3-Basic-luciferase reporter plasmid to get pGL3-hUTP14a-luc. The reporter plasmid was transfected into 293T cells and luciferase activity was evaluated by the Dual-Luciferase Reporter Assay System. Putative transcription factors were identified through searching Matlnspector Professional and Algorismica i Genetica databases. Either pGL3-hUTPI4aluc or p21 promoter reporter plasmid was co-transfected with increasing dose of p53 plasmid, and luciferase activity was evaluated. A series of deletion constructs of pGL3-hUTP14a-luc were constructed and minimal promoter region of hUTP14a was determined. Differences of the lnciferase activities between different groups were assessed by statistical analysis. Results： The hUTP14a gene promoter reporter construct was correctly cloned and was demonstrated to possess promoter activity. The transcription of hUTP14a was not regulated by P53. The minimal promoter region of h UTP14a gene is located between -203 to -100 of the transcription initiation site. Conclusion： Unlike other P53-interacting proteins such as MDM2, Pirh2 and Cop I which promote P53 degradation and whose transcriptions are regulated by P53, does not hUTP14a transcription form a regulation feedback loop with P 53.
