IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injec...IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injection, 24 hours and one month after the first injection.METHODS 25AS in cell extracts of PBMCs from 10 normal persons and 15 chronic hepatitis C patients were determined with PEI cellulose thinlayer chromatography.RESULTS The assay of 25AS in human PBMC was found to be rapid, sensitive, specific and reliable. The 25AS activity of PBMC in normal persons was in a quite low level (20%), and it was increased about tenfolds after stimulation of IFN (197%), (P<001). In 15 chronic hepatitic C patients, the basal levels of 25AS before IFN treatment were higher than those of normal persons, being much higher in the group showing poor response to IFN treatment, but 24h after the first injection of IFNα the 25AS level showed a more rapid and much greater rise in those patients with a good response.CONCLUSION 25AS may be a useful parameter of biological response during the IFN therapy..展开更多
AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected wit...AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.展开更多
The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype...The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another dotlblestranded RNA-dependent enzyme,the 40kDa form of 2'5'oligoadenylate synthetase. Inhibition of both wile-type PKR or 2'5' oligoadenylate synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between PKR K296R and wild-type PKR or 2'5' oligoadenylate synthetase for limiting amounts of dublestranded RNA. Moreover,the data imply that the tumourigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-strandeed RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.展开更多
目的研究慢性丙型病毒性肝炎患者外周血单核细胞(PBMC)中2’,5’寡腺苷酸合成酶(2’,5’-Oligoadenylate synthetase,2-5AS)的表达及其传导与转录激活因子(Signal transducer and activator of transcription,Stat)Stat1和Stat2等的水...目的研究慢性丙型病毒性肝炎患者外周血单核细胞(PBMC)中2’,5’寡腺苷酸合成酶(2’,5’-Oligoadenylate synthetase,2-5AS)的表达及其传导与转录激活因子(Signal transducer and activator of transcription,Stat)Stat1和Stat2等的水平相关性分析。方法选择60例慢性丙肝患者,抽取外周血分离PBMC。首先测定每份标本的2-5AS活性。然后将所有丙肝患者的PBMC标本按按编号单双数分为对照组和实验组,进行体外培养。其中,对照组PBMC不添加干扰素α(interferon alpha,IFN-α),而实验组PBMC添加1000IU/ml IFN-α。体外培养大约24小时后,测定PBMC中2-5AS活性、Stat1和Stat2等的表达量。结果和对照组相比,实验组PBMC的2-5AS活性明显升高(P<0.05)。实验组PBMC中,在2-5AS活性增高的同时,Stat1和Stat2等表达量也呈现明显增多的趋势(P<0.05)。结论基础的2-5AS活性与加入IFNα刺激培养后Stat1的改变幅度呈负相关(P<0.05);2-5AS基础水平和IFNα刺激后的Stat2表达的变化没有相关性(P>0.05)。展开更多
(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular laten...(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular latent endonuclease (RNase L ), leading to the degradation of viral mRNA and cellular rRNA, and inhibiting viral replicatio and cellular proliferation, The level of 2-5 A synthetase in human leukocytes could be enhanced during virus infection or展开更多
文摘IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injection, 24 hours and one month after the first injection.METHODS 25AS in cell extracts of PBMCs from 10 normal persons and 15 chronic hepatitis C patients were determined with PEI cellulose thinlayer chromatography.RESULTS The assay of 25AS in human PBMC was found to be rapid, sensitive, specific and reliable. The 25AS activity of PBMC in normal persons was in a quite low level (20%), and it was increased about tenfolds after stimulation of IFN (197%), (P<001). In 15 chronic hepatitic C patients, the basal levels of 25AS before IFN treatment were higher than those of normal persons, being much higher in the group showing poor response to IFN treatment, but 24h after the first injection of IFNα the 25AS level showed a more rapid and much greater rise in those patients with a good response.CONCLUSION 25AS may be a useful parameter of biological response during the IFN therapy..
基金Supported by National Natural Science Foundation of China,No.30671846
文摘AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.
文摘The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another dotlblestranded RNA-dependent enzyme,the 40kDa form of 2'5'oligoadenylate synthetase. Inhibition of both wile-type PKR or 2'5' oligoadenylate synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between PKR K296R and wild-type PKR or 2'5' oligoadenylate synthetase for limiting amounts of dublestranded RNA. Moreover,the data imply that the tumourigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-strandeed RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.
文摘目的研究慢性丙型病毒性肝炎患者外周血单核细胞(PBMC)中2’,5’寡腺苷酸合成酶(2’,5’-Oligoadenylate synthetase,2-5AS)的表达及其传导与转录激活因子(Signal transducer and activator of transcription,Stat)Stat1和Stat2等的水平相关性分析。方法选择60例慢性丙肝患者,抽取外周血分离PBMC。首先测定每份标本的2-5AS活性。然后将所有丙肝患者的PBMC标本按按编号单双数分为对照组和实验组,进行体外培养。其中,对照组PBMC不添加干扰素α(interferon alpha,IFN-α),而实验组PBMC添加1000IU/ml IFN-α。体外培养大约24小时后,测定PBMC中2-5AS活性、Stat1和Stat2等的表达量。结果和对照组相比,实验组PBMC的2-5AS活性明显升高(P<0.05)。实验组PBMC中,在2-5AS活性增高的同时,Stat1和Stat2等表达量也呈现明显增多的趋势(P<0.05)。结论基础的2-5AS活性与加入IFNα刺激培养后Stat1的改变幅度呈负相关(P<0.05);2-5AS基础水平和IFNα刺激后的Stat2表达的变化没有相关性(P>0.05)。
基金Project supported by the National Natural Science Foundation of China
文摘(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular latent endonuclease (RNase L ), leading to the degradation of viral mRNA and cellular rRNA, and inhibiting viral replicatio and cellular proliferation, The level of 2-5 A synthetase in human leukocytes could be enhanced during virus infection or
