背景与目的:不育α基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋白1(sterile alpha motif and histidine/aspartic acid domain-containing protein 1,SAMHD1)具有抑制多种肿瘤细胞生长的作用,但其调节肝细胞癌(hepatocellular c...背景与目的:不育α基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋白1(sterile alpha motif and histidine/aspartic acid domain-containing protein 1,SAMHD1)具有抑制多种肿瘤细胞生长的作用,但其调节肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖的作用及机制尚未见报道。探究SAMHD1调控p27的表达对HCC细胞Huh7的增殖、凋亡和细胞周期的影响。方法:首先通过蛋白质印迹法(Western blot)检测正常肝细胞和不同类型HCC细胞中SAMHD1的表达情况。利用基因修饰技术构建过表达SAMHD1、dNTP酶活性位点突变体(SAMHD1-D207N)和磷酸化位点突变体(SAMHD1-T592E)的重组质粒,然后利用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测过表达SAMHD1及其突变体或siRNA干扰沉默SAMHD1对HCC细胞增殖的影响,采用流式细胞术检测细胞周期与细胞凋亡情况。在癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中分析SAMHD1和p27表达的相关性。结果:HCC细胞中SAMHD1表达上调,过表达SAMHD1、SAMHD1-D207N和SAMHD1-T592E可以抑制Huh7细胞增殖,细胞周期停滞在G1/G0期;相反,干扰SAMHD1后细胞增殖加快,细胞周期停滞在G2/M期。机制研究表明,SAMHD1上调细胞周期蛋白激酶抑制因子p27的表达。在HCC组织中,p27的表达与SAMHD1表达呈正相关。结论:过表达SAMHD1可以上调p27的表达,导致细胞周期停滞在G1/G0期,从而抑制HCC细胞增殖,这种抑制作用不依赖于其dNTP酶活性和磷酸化修饰。展开更多
Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment ...Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. Scs Est01 was successfully co-expressed in E scherichia coli BL21(DE3) with chaperones(dnaK-dna J-grp E) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2 +. The enzyme was characterized using p-nitrophenol butyrate as a substrate. Scs Est01 had the highest lipolytic activity at 35℃ and p H 8.0, indicative of a meso-thermophilic alkaline esterase. Scs Est01 was thermostable at 20℃. The lipolytic activity of scs Est01 was strongly increased by Fe2 +, Mn 2+ and 1% Tween 80 or Tween 20.展开更多
Ecdysis is a comm on phe nomenon that happe ns throughout the life phase of the giant freshwater prawn Macrobrachium rosenbergii.It is vital to better understand the correlation between cannibalism and biochemical com...Ecdysis is a comm on phe nomenon that happe ns throughout the life phase of the giant freshwater prawn Macrobrachium rosenbergii.It is vital to better understand the correlation between cannibalism and biochemical compound that exists during the moulting process.The objective of the present study was to determine the amino acid profile released by M.rosenbergii during the ecdysis process that promotes cannibalism.To accomplish this,changes in amino acid levels(total amino acid(TAA)and free amino acid(FAA))of tissue muscle,exoskeleton,and sample water of culture medium from the moulting(E-stage)and non-moulting(C-stage)prawns were analysed using highperformanee liquid chromatography(HPLC).Comparison study revealed that among the TAA compounds,proline and sarcosine of tissues from moulting prawn were found at the highest levels.The level of FAA from water that contains moulting prawns(E-stage)was dominated by tryptophan and proline.Significant values obtained in the present study suggested that these amino acid compounds act as a chemical cue to promote cannibalism in M.rosenbergii during ecdysis.The knowledge of compositions and compounds that were released during the moulting process should be helpful for better understanding of the mechanism and chemical cues that play roles on triggering cannibalism,and also for future dietary man ipulati on to improve feeding efficie ncies and feeding man agement,which in directly impacts productivity and profitability.展开更多
基金Supported by the Strategic Priority Research Program of Chinese Academy of Sciences(No.XDA11030404)the National High Technology Research and Development Program of China(863 Program)(Nos.2012AA092103,2014AA093501,2014AA093505)+1 种基金the Knowledge Innovation Program of Chinese Academy of Sciences(No.KZCX2-YW-JC201)the Open Project Program of Key Laboratory of Marine Bio-Resources Sustainable Utilization,South China Sea Institute of Oceanology,Chinese Academy of Sciences(No.LMB121006)
文摘Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. Scs Est01 was successfully co-expressed in E scherichia coli BL21(DE3) with chaperones(dnaK-dna J-grp E) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2 +. The enzyme was characterized using p-nitrophenol butyrate as a substrate. Scs Est01 had the highest lipolytic activity at 35℃ and p H 8.0, indicative of a meso-thermophilic alkaline esterase. Scs Est01 was thermostable at 20℃. The lipolytic activity of scs Est01 was strongly increased by Fe2 +, Mn 2+ and 1% Tween 80 or Tween 20.
基金Fundamental Research Grant Scheme from the Ministry of Higher Education,Malaysia(No.59324)。
文摘Ecdysis is a comm on phe nomenon that happe ns throughout the life phase of the giant freshwater prawn Macrobrachium rosenbergii.It is vital to better understand the correlation between cannibalism and biochemical compound that exists during the moulting process.The objective of the present study was to determine the amino acid profile released by M.rosenbergii during the ecdysis process that promotes cannibalism.To accomplish this,changes in amino acid levels(total amino acid(TAA)and free amino acid(FAA))of tissue muscle,exoskeleton,and sample water of culture medium from the moulting(E-stage)and non-moulting(C-stage)prawns were analysed using highperformanee liquid chromatography(HPLC).Comparison study revealed that among the TAA compounds,proline and sarcosine of tissues from moulting prawn were found at the highest levels.The level of FAA from water that contains moulting prawns(E-stage)was dominated by tryptophan and proline.Significant values obtained in the present study suggested that these amino acid compounds act as a chemical cue to promote cannibalism in M.rosenbergii during ecdysis.The knowledge of compositions and compounds that were released during the moulting process should be helpful for better understanding of the mechanism and chemical cues that play roles on triggering cannibalism,and also for future dietary man ipulati on to improve feeding efficie ncies and feeding man agement,which in directly impacts productivity and profitability.
