Previous studies have confirmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying ...Previous studies have confirmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains un- clear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunopre- cipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II （LC3Ⅱ） and stimulated the conversion of EGFP-LC3Ⅰ to EGFP-LC3Ⅱ, but had little influence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during auto- phagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase I G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.展开更多
Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize.In the present study,a novel maize endosperm mutant A0178 of natural variation w...Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize.In the present study,a novel maize endosperm mutant A0178 of natural variation was used as the experimental material and identified and then characterized.Through phenotypic identification,genetic analysis,main ingredients measurement and embryo rescue,development of genetic mapping population from A0178,the endosperm mutant gene was located.The results showed that the mutant exhibited extremely low germination ability as attributed to the inhibited embryo development,and amounts of sugars were accumulated in the mutant seeds and more sugars content was detected at 23 days after pollination(DAP)in A0178 than B73.Employing genetic linkage analysis,the mutant trait was mapped in the bin 5.04 on chromosome 5.Sequence analysis showed that two sites of base transversion and insertion presented in the protein coding region and non-coding region of the mutant brittle-1(bt1),the adenylate translocator encoding gene involved in the starch synthesis.The single base insertion in the coding region cause frameshift mutation,early termination and lose of function of Brittle-1(BT1).All results suggested that bt1 is a novel allelic gene and the causal gene of this endosperm mutant,providing insights on the mechanism of endosperm formation in maize.展开更多
AIM: To investigate whether IDH1R132 C mutant in combination with loss of p53 and activated Notch signaling promotes intrahepatic cholangiocarcinoma(ICC) development.METHODS: We applied hydrodynamic injection and slee...AIM: To investigate whether IDH1R132 C mutant in combination with loss of p53 and activated Notch signaling promotes intrahepatic cholangiocarcinoma(ICC) development.METHODS: We applied hydrodynamic injection and sleeping beauty mediated somatic integration to induce loss of p53(via sh P53), activation of Notch [via intracellular domain of Notch1(NICD)] and/or overexpression of IDH1R132 C mutant together with the sleeping beauty transposase into the mouse liver. Specifically, we co-expressed sh P53 and NICD(sh P53/NICD, n = 4), sh P53 and IDH1R132C(sh P53/IDH1R132 C, n = 3), NICD and IDH1R132C(NICD/IDH1R132 C, n = 4), as well as NICD, sh P53 and IDH1R132C(NICD/sh P53/IDH1R132 C, n = 9) in mice. Mice were monitored for liver tumor development and euthanized at various time points. Liver histology was analyzed by hematoxylin and eosin staining. Molecular features of NICD/sh P53/IDH1R132 C ICC tumor cells were characterized by Myc tag, Flag tag, Ki-67, p-Erk and p-AKT immunohistochemical staining. Desmoplastic reaction in tumor tissues was studied by Picro-Sirius red staining.RESULTS: We found that co-expression of sh P53/NICD, sh P53/IDH1R132 C or NICD/IDH1R132 C did not lead to liver tumor formation. In striking contrast, coexpression of NICD/sh P53/IDH1R132 C resulted in ICC development in mice(P < 0.01). The tumors could be identified as early as 12 wk post hydrodynamic injection. Tumors rapidly progressed, and by 18 wk post hydrodynamic injection, multiple cystic lesions could be identified on the liver surface. NICD/sh P53/IDH1R132 C liver tumors shared multiple histological features of human ICCs, including hyperplasia of irregular glands. Importantly, all tumor cells were positive for the biliary epithelial cell marker cytokeratin 19. Extensive collagen fibers could be visualized in tumor tissues using Sirus red staining, duplicating the desmoplastic reaction observed in human ICC. Tumors were highly proliferative and expressed ectopically injected genes. Together these studies supported that NICD/sh P53/IDH1R132 C liver tumors were indeed ICCs. Finally, no p-AKT or p-ERK positive staining was observed, suggesting that NICD/sh P53/IDH1R132 C driven ICC development was independent of AKT/m TOR and Ras/MAPK signaling cascades. CONCLUSION: We have generated a simple, nongermline murine ICC model with activated Notch, loss of p53 and IDH1R132 C mutant. The study supported the oncogenic potential of IDH1R132 C.展开更多
As a kind of mutagen, ion beam irradiation can create abundant biological mutations. A population of about 2000 lines was generated by irradiating dry wheat seeds of XiaoYan 81 with low-energy nitrogen ion beams. The ...As a kind of mutagen, ion beam irradiation can create abundant biological mutations. A population of about 2000 lines was generated by irradiating dry wheat seeds of XiaoYan 81 with low-energy nitrogen ion beams. The traits of the plant, such as height, spike type, fertility, stem color and awn length, were investigated. The mutation rate in terms of the plant height in M2 was 2.9%. Eighteen deletion mutants of TaGA2ox1 were obtained. Associate analysis showed that TaGA2ox1 was closely related to the plant height. Most of the TaGA2ox1-deleted mutants were higher than the control, suggesting that the biological function of TaGA2ox1 is similar to its homologues in other plants. These results demonstrate that ion beam irradiation is an efficient tool in the construction of a mutant library for wheat.展开更多
For over one hundred years, viruses have been recognized as capable of killing tumor cells. At present, people are still researching and constructing more suitable oncolytic viruses for treating different malignant tu...For over one hundred years, viruses have been recognized as capable of killing tumor cells. At present, people are still researching and constructing more suitable oncolytic viruses for treating different malignant tumors. Although extensive studies have demonstrated that herpes simplex virus type 1 (HSV-1) is the most potential oncolytic virus, therapies based on herpes simplex virus type 1 vectors still arouse bio-safety and risk management issues. Researchers have therefore introduced the new idea of treating cancer with HSV-1 mutants labeled with radionuclides, combining radionuclide and oncolytic virus therapies. This overview briefly summarizes the status and mechanisms by which oncolytic viruses kill tumor cells, discusses the application of HSV-1 and HSV-1 derived vectors for tumor therapy, and demonstrates the feasibility and prospect of HSV-1 mutants labeled with radionuclides for treating tumors.展开更多
Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This co...Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .展开更多
The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which ...The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins （protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain）, and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.展开更多
基金supported in part by an Oversea Study Fellowship from the China Scholarship Council,No.2008630089
文摘Previous studies have confirmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains un- clear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunopre- cipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II （LC3Ⅱ） and stimulated the conversion of EGFP-LC3Ⅰ to EGFP-LC3Ⅱ, but had little influence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during auto- phagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase I G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.
基金This work was financially supported in part by grants from National Science and Technology Support Project of China(2016YFD0101205)the Natural Science Foundation of Jiangsu Province,China(BK20160586)+1 种基金National Transgenic Major Project of China(2019ZX08010-004)as well as Six Talent Peaks Project of Jiangsu Province,China(NY-020).
文摘Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize.In the present study,a novel maize endosperm mutant A0178 of natural variation was used as the experimental material and identified and then characterized.Through phenotypic identification,genetic analysis,main ingredients measurement and embryo rescue,development of genetic mapping population from A0178,the endosperm mutant gene was located.The results showed that the mutant exhibited extremely low germination ability as attributed to the inhibited embryo development,and amounts of sugars were accumulated in the mutant seeds and more sugars content was detected at 23 days after pollination(DAP)in A0178 than B73.Employing genetic linkage analysis,the mutant trait was mapped in the bin 5.04 on chromosome 5.Sequence analysis showed that two sites of base transversion and insertion presented in the protein coding region and non-coding region of the mutant brittle-1(bt1),the adenylate translocator encoding gene involved in the starch synthesis.The single base insertion in the coding region cause frameshift mutation,early termination and lose of function of Brittle-1(BT1).All results suggested that bt1 is a novel allelic gene and the causal gene of this endosperm mutant,providing insights on the mechanism of endosperm formation in maize.
基金Supported by Grants from National Institutes of HealthNo.R01CA136606(in part+5 种基金to Chen X)UCSF Liver CenterNo.P30DK026743China Scholarship CouncilcontractNo.201206010086(to Ding N)and No.201306590021(to Li XL)
文摘AIM: To investigate whether IDH1R132 C mutant in combination with loss of p53 and activated Notch signaling promotes intrahepatic cholangiocarcinoma(ICC) development.METHODS: We applied hydrodynamic injection and sleeping beauty mediated somatic integration to induce loss of p53(via sh P53), activation of Notch [via intracellular domain of Notch1(NICD)] and/or overexpression of IDH1R132 C mutant together with the sleeping beauty transposase into the mouse liver. Specifically, we co-expressed sh P53 and NICD(sh P53/NICD, n = 4), sh P53 and IDH1R132C(sh P53/IDH1R132 C, n = 3), NICD and IDH1R132C(NICD/IDH1R132 C, n = 4), as well as NICD, sh P53 and IDH1R132C(NICD/sh P53/IDH1R132 C, n = 9) in mice. Mice were monitored for liver tumor development and euthanized at various time points. Liver histology was analyzed by hematoxylin and eosin staining. Molecular features of NICD/sh P53/IDH1R132 C ICC tumor cells were characterized by Myc tag, Flag tag, Ki-67, p-Erk and p-AKT immunohistochemical staining. Desmoplastic reaction in tumor tissues was studied by Picro-Sirius red staining.RESULTS: We found that co-expression of sh P53/NICD, sh P53/IDH1R132 C or NICD/IDH1R132 C did not lead to liver tumor formation. In striking contrast, coexpression of NICD/sh P53/IDH1R132 C resulted in ICC development in mice(P < 0.01). The tumors could be identified as early as 12 wk post hydrodynamic injection. Tumors rapidly progressed, and by 18 wk post hydrodynamic injection, multiple cystic lesions could be identified on the liver surface. NICD/sh P53/IDH1R132 C liver tumors shared multiple histological features of human ICCs, including hyperplasia of irregular glands. Importantly, all tumor cells were positive for the biliary epithelial cell marker cytokeratin 19. Extensive collagen fibers could be visualized in tumor tissues using Sirus red staining, duplicating the desmoplastic reaction observed in human ICC. Tumors were highly proliferative and expressed ectopically injected genes. Together these studies supported that NICD/sh P53/IDH1R132 C liver tumors were indeed ICCs. Finally, no p-AKT or p-ERK positive staining was observed, suggesting that NICD/sh P53/IDH1R132 C driven ICC development was independent of AKT/m TOR and Ras/MAPK signaling cascades. CONCLUSION: We have generated a simple, nongermline murine ICC model with activated Notch, loss of p53 and IDH1R132 C mutant. The study supported the oncogenic potential of IDH1R132 C.
基金supported by National Natural Science Foundation of China (No.30800204)Chinese Ministry of Agriculture supported by the Open Project of the Chinese Academy of Sciences
文摘As a kind of mutagen, ion beam irradiation can create abundant biological mutations. A population of about 2000 lines was generated by irradiating dry wheat seeds of XiaoYan 81 with low-energy nitrogen ion beams. The traits of the plant, such as height, spike type, fertility, stem color and awn length, were investigated. The mutation rate in terms of the plant height in M2 was 2.9%. Eighteen deletion mutants of TaGA2ox1 were obtained. Associate analysis showed that TaGA2ox1 was closely related to the plant height. Most of the TaGA2ox1-deleted mutants were higher than the control, suggesting that the biological function of TaGA2ox1 is similar to its homologues in other plants. These results demonstrate that ion beam irradiation is an efficient tool in the construction of a mutant library for wheat.
基金National Natural Science Foundation of China, No. 30770604
文摘For over one hundred years, viruses have been recognized as capable of killing tumor cells. At present, people are still researching and constructing more suitable oncolytic viruses for treating different malignant tumors. Although extensive studies have demonstrated that herpes simplex virus type 1 (HSV-1) is the most potential oncolytic virus, therapies based on herpes simplex virus type 1 vectors still arouse bio-safety and risk management issues. Researchers have therefore introduced the new idea of treating cancer with HSV-1 mutants labeled with radionuclides, combining radionuclide and oncolytic virus therapies. This overview briefly summarizes the status and mechanisms by which oncolytic viruses kill tumor cells, discusses the application of HSV-1 and HSV-1 derived vectors for tumor therapy, and demonstrates the feasibility and prospect of HSV-1 mutants labeled with radionuclides for treating tumors.
基金This work was supported by grants from the National Natural Science Foundation of China(30170270)
文摘Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .
基金the National Natural Science Foundation of China, No. 30300116
文摘The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins （protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain）, and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.
