Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were devel...Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.展开更多
OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 ...OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 and MDA- MB231） were cultured and treated with XHP. Cell viability was detected using the 3-（4, 5-Dimeth- ylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Flow cytometry was used to measure the cell cycle and apoptosis. The cell cycle was ana- lyzed with propidium iodide staining. Apoptosis was evaluated using the Annexin V-fluorescein iso- thiocyanate/propidium iodide method. Western blotting was used to analyze the expression of es- trogen receptor （ER）-α and ER-13.RESULTS： XHP had growth-inhibitory effects on MCF-7 and MDA-MB231 cells with a half-maximal inhibitory concentration （IC50） of 10.14 mg/mL （MCF-7） and 8.98 mg/mL （MDA-MB231）. Apoptosis was induced to some extent. Certain changes in the ER were caused. Upregulation of ER-a protein was found in MCF-7 cells. ER-β expression in MDA-MB231 cells was increased. Cell-cycle arrest was not observed in the two BC cell lines. ER-β ex- pression in MCF-7 cells was unchanged. No ER-a ex- pression was shown in MDA-MB231 cells. CONCLUSION： These data suggest that XHP can af- fect cell viability and cause apoptosis, but that the cell cycle is not blocked. XHP has a certain impact on ER expression, but its mechanisms of action of anti-13C effects may not be due to regulation of ER expression.展开更多
基金Supported by the National Natural Science Foundation of China (Nos. 30800853 and 30901107)the National Key Projects, National Science and Technology Pillar Program during the 12th Five-Year-Plan (No. 2011BAD13B03)
文摘Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.
基金Supported by the Beijing Traditional Chinese Medicine Science and Technology Project(QN2010-3)National Natural Science Foundation of China(No.81001564)
文摘OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 and MDA- MB231） were cultured and treated with XHP. Cell viability was detected using the 3-（4, 5-Dimeth- ylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Flow cytometry was used to measure the cell cycle and apoptosis. The cell cycle was ana- lyzed with propidium iodide staining. Apoptosis was evaluated using the Annexin V-fluorescein iso- thiocyanate/propidium iodide method. Western blotting was used to analyze the expression of es- trogen receptor （ER）-α and ER-13.RESULTS： XHP had growth-inhibitory effects on MCF-7 and MDA-MB231 cells with a half-maximal inhibitory concentration （IC50） of 10.14 mg/mL （MCF-7） and 8.98 mg/mL （MDA-MB231）. Apoptosis was induced to some extent. Certain changes in the ER were caused. Upregulation of ER-a protein was found in MCF-7 cells. ER-β expression in MDA-MB231 cells was increased. Cell-cycle arrest was not observed in the two BC cell lines. ER-β ex- pression in MCF-7 cells was unchanged. No ER-a ex- pression was shown in MDA-MB231 cells. CONCLUSION： These data suggest that XHP can af- fect cell viability and cause apoptosis, but that the cell cycle is not blocked. XHP has a certain impact on ER expression, but its mechanisms of action of anti-13C effects may not be due to regulation of ER expression.
