Using an isotig encoding a putative polypeptide with high similarity to Arabidopsis LEA14 as a query, a 613 bp long cDNA was in silico cloned from the transeriptome data of rubber tree. The sequence nominated as HbLEA...Using an isotig encoding a putative polypeptide with high similarity to Arabidopsis LEA14 as a query, a 613 bp long cDNA was in silico cloned from the transeriptome data of rubber tree. The sequence nominated as HbLEA14L2 contains an ORF of 456 bp with 3 bp 5＇ UTR and 154 bp 3＇ UTR. Subsequently, a 464 bp eDNA and an 834 bp genome sequence containing this ORF was amplified and sequenced. Sequence analysis suggested that HbLEA14L2 has one intron and encodes 151 amino acids with a theoretical molecular weight of 16.55 kDa, isoleetric point of 4.93 and GRAVY value of -0.022, indicating a cytoplasmle localization pattern; HbLEA14L2 protein contains a conserved LEA_2 domain and belongs to LEA_2 subfamily, sharing 91%, 76%, 75%, 72% and 63% similarity with the homologous proteins in castor bean, leafy spurge, poplar, cotton, and Arabidopsis, respectively. Swiss-Model indicated that the tertiary structure of HbLEA14L2 is consisted of one α-helix and seven β-sheets, which was proposed to serve as a regulatory protein to prevent cellular desiccation.展开更多
OBJECTIVE： To investigate the antioxidant activities as well as phytochemical constituents of Antidesma thwaitesianum Mull. Arg. leaf extracts. METHODS： The leaves of A. thwaitesianum were extracted using three diff...OBJECTIVE： To investigate the antioxidant activities as well as phytochemical constituents of Antidesma thwaitesianum Mull. Arg. leaf extracts. METHODS： The leaves of A. thwaitesianum were extracted using three different methods： blending with distilled water, maceration with ethanol and decoction. The chemical antioxidant activity of the plant leaf extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl （DPPH） radical and 2,2＇-azinobis（3- ethylbenzo-thiazoline-6-sulfonic acid） diammonium salt （ABTS＊） radical scavenging assays, as well as the ferric reducing antioxidant power assay. Cellular antioxidant activity was determined by superoxide and nitric oxide scavenging assays. The cytotoxicity of the leaf extracts in RAW 264.7 and differentiated HL-60 cells was tested in parallel using 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide and 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium assays, respectively. The total phenolic and flavonoid contents were also assessed by spectrophotometric analysis Phytochemical constituents of the most potent extract were investigated by liquid chromatography with an electrospray ionization quadrupole time-of-flight mass spectrometer （LC-ESI-QTOF-MS/MS）. RESULTS： The ethanolic （ME） and decoction （LW） extracts of dried leaves had the highest chemical scavenging activity against DPPH and ABTS＋ free radicals with half maximal effective concentration （EC50） values ranging from 3.54 to 6.44 μg/mL. ME and LW exerted moderate ferric reducing activity, with ferric reducing antioxidant power values of 847.41 and 941.26 mg Fe2＋/g extract, respectively. Similarly, ME showed potent cellular scavenging activity against superoxide and nitric oxide radicals with EC50 values of 58.12 and 71.90 μg/mL, respectively. However, LW exhibited only strong nitric oxide scavenging activity with an EC50 value of 91.20 μg/mL. The cell viability of RAW 264.7 and HL-60 cells was greater than 70% in all tested concentrations of both extracts, thus confirming the absence of their cytotoxicity. ME and LW contained high total phenolic contents of 231.14 and 274.42 mg gallic acid equivalents per gram, respectively, as well as high total flavonoid contents of 18.82 and 22.17 mg quercetin equivalents per gram, respectively. LC-ESI-QTOF- MS/MS analysis revealed the presence of 52 structurally characterized compounds in ME, 43 of whichwere tentatively identified. Hydroxycinnamic acids such as caffeic acid and its derivatives were the predominant phenolic compounds. CONCLUSION： This is the first report describing potent chemical and cellular antioxidant effects of the ethanolic leaf extract of A. thwaitesianum. The extract contained high total phenolic and flavonoid contents LC-ESI-QTOF-MS/MS analysis further revealed an abundance of caffeic acid derivatives and flavonoids. These data support its potential use as dietary supplements in oxidative stress prevention.展开更多
目的:分析tsRNA-Arg-5-0022在口腔癌中的异常表达,对口腔癌细胞恶性增殖的影响及其潜在的分子机制。方法:通过tsRFun数据库分析头颈部鳞状细胞癌中差异表达的tsRNAs,并通过RT-PCR在口腔癌组织中进行验证;CCK8和克隆形成实验检测tsRNA-Ar...目的:分析tsRNA-Arg-5-0022在口腔癌中的异常表达,对口腔癌细胞恶性增殖的影响及其潜在的分子机制。方法:通过tsRFun数据库分析头颈部鳞状细胞癌中差异表达的tsRNAs,并通过RT-PCR在口腔癌组织中进行验证;CCK8和克隆形成实验检测tsRNA-Arg-5-0022对口腔癌细胞增殖能力的影响;OncotRF网站预测tsRNA-Arg-5-0022下游靶基因及潜在的结合位点;Western Blot检测tsRNA-Arg-5-0022对口腔癌细胞中DNA结合抑制因子4(inhibitor of DNA binding 4,ID4)表达水平的影响;双荧光素酶报告基因实验检测tsRNA-Arg-5-0022与ID4的3'UTR区的结合能力;UALCAN数据库分析ID4在头颈鳞癌中的表达水平,并通过RT-PCR在口腔癌组织中进行验证,分析其与tsRNA-Arg-5-0022表达水平的相关性;CCK8和克隆形成实验检测tsRNA-Arg-5-0022通过靶向抑制ID4对口腔癌细胞增殖能力的影响。结果:tsRFun数据库分析结果显示tsRNA-Arg-5-0022在头颈鳞癌中显著上调(P<0.05);RT-PCR结果显示tsRNA-Arg-5-0022在口腔癌中显著上调(P<0.05);tsRNA-Arg-5-0022抑制物能够显著抑制tsRNA-Arg-5-0022表达(P<0.05);tsRNA-Arg-5-0022抑制物能够显著抑制口腔癌细胞的增殖能力;OncotRF网站预测结果显示tsRNA-Arg-5-0022与ID4的3'UTR区存在结合位点;Western Blot检测结果显示tsRNA-Arg-5-0022抑制物能够显著升高口腔癌细胞中ID4表达水平(P<0.05);双荧光素酶报告基因检测结果显示,tsRNA-Arg-5-0022能够与ID4的3'UTR区结合;UALCAN数据库分析结果显示ID4在头颈鳞癌中表达水平下调(P<0.05);RT-PCR结果显示ID4在口腔癌中表达水平下调,且与tsRNA-Arg-5-0022表达水平呈负相关关系;CCK8和克隆形成实验结果显示tsRNA-Arg-5-0022能够通过靶向抑制ID4促进口腔癌细胞增殖。结论:tsRNA-Arg-5-0022在口腔癌中高表达,能够通过直接靶向ID4促进口腔癌细胞恶性增殖,是口腔癌潜在的分子标志物。展开更多
基金Supported by National Natural Science Foundation of China(31100460)Key Science and Technology Project of Hainan Province(90107)+1 种基金Natural Science Foundation of Hainan Province(312026)Fundamental Research Fund for the Rubber Research Institute in Chinese Academy of Tropical Agricultural Sciences(1630022011014)
文摘Using an isotig encoding a putative polypeptide with high similarity to Arabidopsis LEA14 as a query, a 613 bp long cDNA was in silico cloned from the transeriptome data of rubber tree. The sequence nominated as HbLEA14L2 contains an ORF of 456 bp with 3 bp 5＇ UTR and 154 bp 3＇ UTR. Subsequently, a 464 bp eDNA and an 834 bp genome sequence containing this ORF was amplified and sequenced. Sequence analysis suggested that HbLEA14L2 has one intron and encodes 151 amino acids with a theoretical molecular weight of 16.55 kDa, isoleetric point of 4.93 and GRAVY value of -0.022, indicating a cytoplasmle localization pattern; HbLEA14L2 protein contains a conserved LEA_2 domain and belongs to LEA_2 subfamily, sharing 91%, 76%, 75%, 72% and 63% similarity with the homologous proteins in castor bean, leafy spurge, poplar, cotton, and Arabidopsis, respectively. Swiss-Model indicated that the tertiary structure of HbLEA14L2 is consisted of one α-helix and seven β-sheets, which was proposed to serve as a regulatory protein to prevent cellular desiccation.
基金supported by the National Research University Project of Thailand Office of Higher Education Commission,Faculty of Medicine,Thammasat University
文摘OBJECTIVE： To investigate the antioxidant activities as well as phytochemical constituents of Antidesma thwaitesianum Mull. Arg. leaf extracts. METHODS： The leaves of A. thwaitesianum were extracted using three different methods： blending with distilled water, maceration with ethanol and decoction. The chemical antioxidant activity of the plant leaf extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl （DPPH） radical and 2,2＇-azinobis（3- ethylbenzo-thiazoline-6-sulfonic acid） diammonium salt （ABTS＊） radical scavenging assays, as well as the ferric reducing antioxidant power assay. Cellular antioxidant activity was determined by superoxide and nitric oxide scavenging assays. The cytotoxicity of the leaf extracts in RAW 264.7 and differentiated HL-60 cells was tested in parallel using 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide and 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium assays, respectively. The total phenolic and flavonoid contents were also assessed by spectrophotometric analysis Phytochemical constituents of the most potent extract were investigated by liquid chromatography with an electrospray ionization quadrupole time-of-flight mass spectrometer （LC-ESI-QTOF-MS/MS）. RESULTS： The ethanolic （ME） and decoction （LW） extracts of dried leaves had the highest chemical scavenging activity against DPPH and ABTS＋ free radicals with half maximal effective concentration （EC50） values ranging from 3.54 to 6.44 μg/mL. ME and LW exerted moderate ferric reducing activity, with ferric reducing antioxidant power values of 847.41 and 941.26 mg Fe2＋/g extract, respectively. Similarly, ME showed potent cellular scavenging activity against superoxide and nitric oxide radicals with EC50 values of 58.12 and 71.90 μg/mL, respectively. However, LW exhibited only strong nitric oxide scavenging activity with an EC50 value of 91.20 μg/mL. The cell viability of RAW 264.7 and HL-60 cells was greater than 70% in all tested concentrations of both extracts, thus confirming the absence of their cytotoxicity. ME and LW contained high total phenolic contents of 231.14 and 274.42 mg gallic acid equivalents per gram, respectively, as well as high total flavonoid contents of 18.82 and 22.17 mg quercetin equivalents per gram, respectively. LC-ESI-QTOF- MS/MS analysis revealed the presence of 52 structurally characterized compounds in ME, 43 of whichwere tentatively identified. Hydroxycinnamic acids such as caffeic acid and its derivatives were the predominant phenolic compounds. CONCLUSION： This is the first report describing potent chemical and cellular antioxidant effects of the ethanolic leaf extract of A. thwaitesianum. The extract contained high total phenolic and flavonoid contents LC-ESI-QTOF-MS/MS analysis further revealed an abundance of caffeic acid derivatives and flavonoids. These data support its potential use as dietary supplements in oxidative stress prevention.
文摘目的:分析tsRNA-Arg-5-0022在口腔癌中的异常表达,对口腔癌细胞恶性增殖的影响及其潜在的分子机制。方法:通过tsRFun数据库分析头颈部鳞状细胞癌中差异表达的tsRNAs,并通过RT-PCR在口腔癌组织中进行验证;CCK8和克隆形成实验检测tsRNA-Arg-5-0022对口腔癌细胞增殖能力的影响;OncotRF网站预测tsRNA-Arg-5-0022下游靶基因及潜在的结合位点;Western Blot检测tsRNA-Arg-5-0022对口腔癌细胞中DNA结合抑制因子4(inhibitor of DNA binding 4,ID4)表达水平的影响;双荧光素酶报告基因实验检测tsRNA-Arg-5-0022与ID4的3'UTR区的结合能力;UALCAN数据库分析ID4在头颈鳞癌中的表达水平,并通过RT-PCR在口腔癌组织中进行验证,分析其与tsRNA-Arg-5-0022表达水平的相关性;CCK8和克隆形成实验检测tsRNA-Arg-5-0022通过靶向抑制ID4对口腔癌细胞增殖能力的影响。结果:tsRFun数据库分析结果显示tsRNA-Arg-5-0022在头颈鳞癌中显著上调(P<0.05);RT-PCR结果显示tsRNA-Arg-5-0022在口腔癌中显著上调(P<0.05);tsRNA-Arg-5-0022抑制物能够显著抑制tsRNA-Arg-5-0022表达(P<0.05);tsRNA-Arg-5-0022抑制物能够显著抑制口腔癌细胞的增殖能力;OncotRF网站预测结果显示tsRNA-Arg-5-0022与ID4的3'UTR区存在结合位点;Western Blot检测结果显示tsRNA-Arg-5-0022抑制物能够显著升高口腔癌细胞中ID4表达水平(P<0.05);双荧光素酶报告基因检测结果显示,tsRNA-Arg-5-0022能够与ID4的3'UTR区结合;UALCAN数据库分析结果显示ID4在头颈鳞癌中表达水平下调(P<0.05);RT-PCR结果显示ID4在口腔癌中表达水平下调,且与tsRNA-Arg-5-0022表达水平呈负相关关系;CCK8和克隆形成实验结果显示tsRNA-Arg-5-0022能够通过靶向抑制ID4促进口腔癌细胞增殖。结论:tsRNA-Arg-5-0022在口腔癌中高表达,能够通过直接靶向ID4促进口腔癌细胞恶性增殖,是口腔癌潜在的分子标志物。
