BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription fact...BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.展开更多
A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicam...A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.展开更多
Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which ...Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which activates unfolded protein response （UPR） to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α （xIRE1α）. The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptofic cells were measured by TUNNEL assays. Although both gain and loss of xlRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.展开更多
Conformation analysis of the model compounds for tunicamycin V and its natural donor substrate, UDP-N-acetylglucose, is performed to reveal the detail of the inhibition process.
为探究秦川牛宰后成熟期间基础免疫球蛋白(basigin,BSG)对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路及细胞凋亡的影响,利用4D-非标记定量组学技术分析BSG及其差异蛋白的变化情况。向宰后秦川牛肉的背最长肌...为探究秦川牛宰后成熟期间基础免疫球蛋白(basigin,BSG)对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路及细胞凋亡的影响,利用4D-非标记定量组学技术分析BSG及其差异蛋白的变化情况。向宰后秦川牛肉的背最长肌中注射BSG抑制剂衣霉素,通过蛋白质免疫印迹法测定秦川牛肉在4℃贮藏过程中MAPK通路关键蛋白质表达水平,并测定caspase-3的活力变化。研究表明:在秦川牛宰后贮藏期内,BSG的表达量总体呈先上升后下降趋势;利用京都基因与基因组百科全书通路分析发现BSG及其差异蛋白质显著注释于氧化磷酸化通路、钙信号通路、MAPK信号通路,说明BSG通过MAPK途径发挥作用。另外,衣霉素组MAPK通路关键蛋白质的相对表达量均显著下调,说明BSG抑制剂使得MAPK信号通路失活。这为研究BSG对MAPK信号通路影响奠定了很好的基础。在抑制BSG的表达后,衣霉素组caspase-3的活力明显上升,说明细胞凋亡是细胞损伤机制的重要环节,衣霉素作用于BSG的N端结构使蛋白质发生去糖基化作用。通过抑制细胞内蛋白质的折叠使其生物学活性受到抑制,从而诱导细胞凋亡。展开更多
Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called ...Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called endoplasmic reticulum stress （ERS）. Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer＇s disease （AD）, Parkinson＇s disease and so on. Therefore, the protection effect of the traditional Chinese drug Tiantai No. 1 （天泰1号） on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. Methods： Primarily cultured marrow mesenchymat stem cells （MSCs） of rats were treated by tunicamycin （TM） in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 （which would assist cells to resist cellular stress injury）, and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. Results： Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs （P〈0.05）. All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group （P〈0.05 or P〈0.01） after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group （P〈0.05 or P〈0.01）. This effect showed a dose dependent manner. Conclusion： Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.展开更多
Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic...Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.展开更多
基金Supported by:Scientific and Technological Foundation of the National Administration of Traditional Chinese Medicine of China,No.02-03LP41the Scientific and Technological Key Project of Guangdong Province,No. 2006B35630007
文摘BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.
基金supported by the National Natural Science Foundation of China,No. 30900802the Research Fund for the Doctoral Program of Higher Education,No. 20070001801+1 种基金the Leading Academic Discipline Project of Beijing Education Bureauthe Fund for Fostering Talents in Basic Science of the National Natural Science Foundation of China,No. J0630853/J0108
文摘A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.
文摘Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which activates unfolded protein response （UPR） to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α （xIRE1α）. The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptofic cells were measured by TUNNEL assays. Although both gain and loss of xlRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.
文摘Conformation analysis of the model compounds for tunicamycin V and its natural donor substrate, UDP-N-acetylglucose, is performed to reveal the detail of the inhibition process.
文摘为探究秦川牛宰后成熟期间基础免疫球蛋白(basigin,BSG)对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路及细胞凋亡的影响,利用4D-非标记定量组学技术分析BSG及其差异蛋白的变化情况。向宰后秦川牛肉的背最长肌中注射BSG抑制剂衣霉素,通过蛋白质免疫印迹法测定秦川牛肉在4℃贮藏过程中MAPK通路关键蛋白质表达水平,并测定caspase-3的活力变化。研究表明:在秦川牛宰后贮藏期内,BSG的表达量总体呈先上升后下降趋势;利用京都基因与基因组百科全书通路分析发现BSG及其差异蛋白质显著注释于氧化磷酸化通路、钙信号通路、MAPK信号通路,说明BSG通过MAPK途径发挥作用。另外,衣霉素组MAPK通路关键蛋白质的相对表达量均显著下调,说明BSG抑制剂使得MAPK信号通路失活。这为研究BSG对MAPK信号通路影响奠定了很好的基础。在抑制BSG的表达后,衣霉素组caspase-3的活力明显上升,说明细胞凋亡是细胞损伤机制的重要环节,衣霉素作用于BSG的N端结构使蛋白质发生去糖基化作用。通过抑制细胞内蛋白质的折叠使其生物学活性受到抑制,从而诱导细胞凋亡。
基金Supported by the National Foundation of Traditional Chinese Medical Science and Technology(No.02-03LP41)Key Project of the Foundation of Science and Technology of Guangdong Province(No.2006B35630007)
文摘Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called endoplasmic reticulum stress （ERS）. Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer＇s disease （AD）, Parkinson＇s disease and so on. Therefore, the protection effect of the traditional Chinese drug Tiantai No. 1 （天泰1号） on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. Methods： Primarily cultured marrow mesenchymat stem cells （MSCs） of rats were treated by tunicamycin （TM） in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 （which would assist cells to resist cellular stress injury）, and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. Results： Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs （P〈0.05）. All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group （P〈0.05 or P〈0.01） after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group （P〈0.05 or P〈0.01）. This effect showed a dose dependent manner. Conclusion： Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.
基金This work was supported by the National Basic Research Program(973 Program)the National Programs for High Technology Research Development Program(863 Program)from the Ministry of Science and Technology,the National Science Foundation of China,the Ministry of Education,the Science and Technology Commission of Shanghai Municipality,and Shanghai Leading Academic Discipline Project B203.
文摘Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.
