AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture,...AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.展开更多
AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activ...AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in 展开更多
AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore th...AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor. METHODS: Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry. RESULTS: Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P 【 0.01) and the average growth rate of tumor in SCID mice (35.5 +/- 7.6 vs 65.8 +/- 7.6, P 【 0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 0.01). CONCLUSION: Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.展开更多
WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cD...WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.展开更多
The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves t...The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).展开更多
原子力显微镜(atomic force microscope,AFM)在研究DNA与蛋白质的相互作用方面具有重要的应用。本文对DNA结合蛋白的AFM像的分析方法进行了研究,通过分析DNA及其与蛋白质结合的AFM图像,我们可以计算出已知序列DNA的长度及其未知DNA结合...原子力显微镜(atomic force microscope,AFM)在研究DNA与蛋白质的相互作用方面具有重要的应用。本文对DNA结合蛋白的AFM像的分析方法进行了研究,通过分析DNA及其与蛋白质结合的AFM图像,我们可以计算出已知序列DNA的长度及其未知DNA结合蛋白质在DNA链上的位置信息,从而可以估计该蛋白质的DNA结合位点序列。结果证明对AFM图像的分析可以得到DNA结合蛋白的位置信息。该研究对于寻找新的DNA结合蛋白,研究生物体中遗传信息的复制、转录、修复和重组的分子生物学机制具有重要意义。展开更多
Objective To examine the effect of combined treatment with Bojungikgi-tang(BJIGT,Buzhong Yiqi Decoction)and riluzole(RZ)in transactive response DNA-binding protein 43(TDP-43)stress granule(SG)cells,a amyotrophic later...Objective To examine the effect of combined treatment with Bojungikgi-tang(BJIGT,Buzhong Yiqi Decoction)and riluzole(RZ)in transactive response DNA-binding protein 43(TDP-43)stress granule(SG)cells,a amyotrophic lateral sclerosis(ALS)cell line using transcriptomic and molecular techniques.Methods TDP-43 SG cells were pretreated with BJIGT(100µg/mL),RZ(50µmol/L),and combined BJIGT(100µg/mL)/RZ(50µmol/L)for 6 h before treatment with lipopolysaccharide(LPS,200µmol/L).Cell viability assay was performed to elucidate cell toxicity in TDP-43 SC cells using a cell-counting kit-8(CCK8)assay kit.The expression levels of cell death-related proteins,including Bax,caspase 1,cleaved caspase 3 and DJ1 in TDP-43 SG cells were examined by Western blot analysis.The autophagy-related proteins,including pmTOR/mTOR,LC3b,P62,ATG7 and Bcl-2-associated athanogene 3(Bag3)were investigated using immunofluorescence and immunoblotting assays.Results Cell viability assay and Western blot analysis showed that combined treatment with BJIGT and RZ suppressed LPS-induced cell death and expression of cell death-related proteins,including Bax,caspase 1,and DJ1(P<0.05 or P<0.01).Immunofluorescence and immunoblotting assays showed that combined treatment with BJIGT and RZ reduced LPS-induced formation of TDP-43 aggregates and regulated autophagy-related protein levels,including p62,light chain 3b,Bag3,and ATG7,in TDP-43-expressing cells(P<0.05 or P<0.01).Conclusion The combined treatment of BJIGT and RZ might reduce inflammation and regulate autophagy dysfunction in TDP-43-induced ALS.展开更多
Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*...Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*G93A transgenic(ALS mouse model) and wide-type mice.This involved intraperitoneal injections of either granisetron,piboserod,or ritanserin,which inhibit the 5-HT3,5-HT4,and 5-HT2 receptors,respectively.The transgenic mice were found to have fewer5-HT-positive cells in the spinal cord compared with wide-type mice.We found that the administration of granisetron reduced the body weight of the transgenic mice,while piboserod and ritanserin worsened the motor functioning,as assessed using a hanging wire test.However,none of the 5-HT receptor antagonists affected the disease progression.We analyzed the distribution and/or expression of TAR DNA binding protein 43(TDP-43) and superoxide dismutase 1 G93A(SOD1-G93A),which fo rm abnormal aggregates in ALS.We found that the expression of these proteins increased following the administration of all three 5-HT receptor antagonists.In addition,the disease-related mislocalization of TD P-43 to the cytoplasm increased markedly for all three drugs.In ce rtain anatomical regions,the 5-HT receptor antagonists also led to a marked increase in the number of astrocytes and microglia and a decrease in the number of neurons.These results indicate that 5-HT deficiency may play a role in the pathogenesis of amyotrophic lateral sclerosis by inducing the abnormal expression and/or distribution of TDP-43 and SOD1-G93A and by activating glial cells.5-HT co uld therefore be a potential therapeutic target for amyotrophic lateral sclerosis.展开更多
A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA ...A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.展开更多
基金National Natural Science Foundation of China,No.39770300,30070873the Overseas Chinese Affairs Office of the State Council Foundation,No.98-33
文摘AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.
基金the National Natural Scientific Foundation of China,No.39670298
文摘AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in
基金Supported by the National Natural Science Foundation of China,No.39670298.
文摘AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor. METHODS: Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry. RESULTS: Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P 【 0.01) and the average growth rate of tumor in SCID mice (35.5 +/- 7.6 vs 65.8 +/- 7.6, P 【 0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 0.01). CONCLUSION: Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.
基金This work was supported by the State Basic Research and Development Plan(G200001 6203)the National Natural Science Foundation of China(Grant Nos.30370139&30471122).
文摘WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.
基金in part supported by the National Natural Science Foundation of China,Nos.30560042,81160161,81360198,and 82160255Education Department of Jiangxi Province,Nos.GJJ13198 and GJJ170021+1 种基金Jiangxi Provincial Department of Science and Technology,No.20192BAB205043Health and Family Planning Commission of Jiangxi Province,Nos.20181019 and 202210002(all to RX)。
文摘The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).
文摘原子力显微镜(atomic force microscope,AFM)在研究DNA与蛋白质的相互作用方面具有重要的应用。本文对DNA结合蛋白的AFM像的分析方法进行了研究,通过分析DNA及其与蛋白质结合的AFM图像,我们可以计算出已知序列DNA的长度及其未知DNA结合蛋白质在DNA链上的位置信息,从而可以估计该蛋白质的DNA结合位点序列。结果证明对AFM图像的分析可以得到DNA结合蛋白的位置信息。该研究对于寻找新的DNA结合蛋白,研究生物体中遗传信息的复制、转录、修复和重组的分子生物学机制具有重要意义。
基金Supported by the Korea Institute of Oriental Medicine(KIOM)under Grant(No.C18040)。
文摘Objective To examine the effect of combined treatment with Bojungikgi-tang(BJIGT,Buzhong Yiqi Decoction)and riluzole(RZ)in transactive response DNA-binding protein 43(TDP-43)stress granule(SG)cells,a amyotrophic lateral sclerosis(ALS)cell line using transcriptomic and molecular techniques.Methods TDP-43 SG cells were pretreated with BJIGT(100µg/mL),RZ(50µmol/L),and combined BJIGT(100µg/mL)/RZ(50µmol/L)for 6 h before treatment with lipopolysaccharide(LPS,200µmol/L).Cell viability assay was performed to elucidate cell toxicity in TDP-43 SC cells using a cell-counting kit-8(CCK8)assay kit.The expression levels of cell death-related proteins,including Bax,caspase 1,cleaved caspase 3 and DJ1 in TDP-43 SG cells were examined by Western blot analysis.The autophagy-related proteins,including pmTOR/mTOR,LC3b,P62,ATG7 and Bcl-2-associated athanogene 3(Bag3)were investigated using immunofluorescence and immunoblotting assays.Results Cell viability assay and Western blot analysis showed that combined treatment with BJIGT and RZ suppressed LPS-induced cell death and expression of cell death-related proteins,including Bax,caspase 1,and DJ1(P<0.05 or P<0.01).Immunofluorescence and immunoblotting assays showed that combined treatment with BJIGT and RZ reduced LPS-induced formation of TDP-43 aggregates and regulated autophagy-related protein levels,including p62,light chain 3b,Bag3,and ATG7,in TDP-43-expressing cells(P<0.05 or P<0.01).Conclusion The combined treatment of BJIGT and RZ might reduce inflammation and regulate autophagy dysfunction in TDP-43-induced ALS.
基金supported by the National Natural Science Foundation of China,Nos.30560042,81160161,8136019882160255+2 种基金Education Department of Jiangxi Province,Nos.GJJ13198 and GJJ170021Jiangxi Provincial Department of Science and Technology,Nos.20142BBG70062,20171 BAB215022,20192BAB205043Health and Family Planning Commission of Jiangxi Province,No.20181019 (all to RSX)。
文摘Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*G93A transgenic(ALS mouse model) and wide-type mice.This involved intraperitoneal injections of either granisetron,piboserod,or ritanserin,which inhibit the 5-HT3,5-HT4,and 5-HT2 receptors,respectively.The transgenic mice were found to have fewer5-HT-positive cells in the spinal cord compared with wide-type mice.We found that the administration of granisetron reduced the body weight of the transgenic mice,while piboserod and ritanserin worsened the motor functioning,as assessed using a hanging wire test.However,none of the 5-HT receptor antagonists affected the disease progression.We analyzed the distribution and/or expression of TAR DNA binding protein 43(TDP-43) and superoxide dismutase 1 G93A(SOD1-G93A),which fo rm abnormal aggregates in ALS.We found that the expression of these proteins increased following the administration of all three 5-HT receptor antagonists.In addition,the disease-related mislocalization of TD P-43 to the cytoplasm increased markedly for all three drugs.In ce rtain anatomical regions,the 5-HT receptor antagonists also led to a marked increase in the number of astrocytes and microglia and a decrease in the number of neurons.These results indicate that 5-HT deficiency may play a role in the pathogenesis of amyotrophic lateral sclerosis by inducing the abnormal expression and/or distribution of TDP-43 and SOD1-G93A and by activating glial cells.5-HT co uld therefore be a potential therapeutic target for amyotrophic lateral sclerosis.
基金supported by the National Natural Science Foundation of China(Nos.60501010,60701008 and 60771024)
文摘A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.
