[ Objective ] The aim of this study was to investigate the infectivity of Nosema bombycis to drosophila, which offered a new vision for systematical studies on the infection mechanism of Nosema bombycis, and also prov...[ Objective ] The aim of this study was to investigate the infectivity of Nosema bombycis to drosophila, which offered a new vision for systematical studies on the infection mechanism of Nosema bombycis, and also provided reference for the bio-control effect of Nosema bombycis. [ Method ] Nosema bombycis was used to feed wild type and mutant drosophila, and the morphological observation of Nosema bombycis in drosophila body fluid was also analyzed by calcofluor white M2R fluorescent staining. [ Result] Nosema bombycis could infect drosophila, and the number of Nosema bombycis in the infected mutant drosophila was higher than that in wild type drosophila. [ Conclusion ] Nosema bombycis can infect drosophila, which provides primary reference for studies on the infectivity of Nosema bombycis to other hosts and also lays a foundation for further study on the infection mechanism of Nosema bombycis.展开更多
A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophilaatrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. C...A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophilaatrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. Comparativestudies on morphology, infectivity and spore surface protein were conducted. 24Nbh was short and wide, and had asignificant difference (P<0.01) over the Nb spores. The infectivity tests conducted on second instar silkworm larvaeshowed that IC50 of 24Nbh was 1.98104 spores mL-1 and of Nb was 1.72103 spores mL-1, thus indicating that the infectivityof Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The IC50 of spores from silkworm infected with24 Nbh was 6.9 times less than Nb, showing that the infectivity of 24Nbh spores rejuvenated very fast when reinfected tosilkworms, further more, the length and width of such spore was larger than 24Nbh (P<0.01) and smaller than Nb (P<0.05).The SDS-PAGE profiles of Nb and 24Nbh were generally the same, 4 distinct proteins of 12, 17, 30, 33 kDa were obtainedwith difference in quantity. When 120 g of protein was applied for 2D-PAGE, five suspected different proteins withdifference in quantity were observed. These results demonstrate that these differential proteins maybe associated withvariation in infectivity of the spores.展开更多
Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involve...Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involves its polar tube acting as a needle-syringe system. However, recent studies show microsporidian spores can also gain access to host cells by phagocytosis. The present study investigated the phagocytic uptake process of causative agent of the pebrine disease, Nosema bombycis, in several insect cell lines. We observed KOH-treated spores and cold-storaged spores can be easily uptaken by all the studied cell types 4 h post inoculation. In contrast, large numbers of freshly recovered spores remained in the culture medium. To further investigate the intracellular fates of KOH-treated spores and cold-storaged spores, electron and fluorescence microscopy were performed. No intracellular germination or subsequent parasite development were observed. Intracellular spores can be detected in host cells by polyclonal antibody 7 d post inoculation, suggesting phagocytized N. bombycis could not be digested by these non-professional phagocytes. Our results suggest that, phagocytic uptake of N. bombycis spores might represent a defense mechanism of the host cells and the intact spore wall barrier enable freshly recovered spores to keep resistance to this mechanism.展开更多
The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this st...The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBmswp26-egfp bacmid. The vBmswp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.展开更多
Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this ph...Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this phylum. We present here the mitochondrial pyruvate dehydrogenase El （PDH, including PDHα and PDHβ） of the microsporidian Nosema bombycis, the pathogen of silkworm pebrine. Compared with PDH of microsporidian Encephalitozoon cuniculi and Antonospora locustae, both subunits are eonscrced. The phylogeny indicated that both subunits are mitochondrial. The syntenic maps revealed the subunits organization of NbPDH is distributed in different scaffolds, similar to that of EcPDH but different with AIPDH, and the relationship between phylogeny tree and organization of PDH suggest that the AlPDH subunits organization is the ancestral style of microsporidia, and through the genome evolution, the reshuffling of the chromosome of microsporidia occurred, the adjacent style of ALPDHE1 organization changed, and the two subunits separated and located to different chromosomes in E. cuniculi. For N. bombycis and N. ceranae, they locate to different scaffolds. In order to determine NbPDH subcellular localizations, we prepared the polyclonal antibodies against NbPDH prokaryotic fusion proteins, and adopted the colloidal gold immunological electron microscopy, the expression signals of NbPDH were observed in spores however, the subcellular localization were not definited. In general, through comparison of three mierosporidian PDH molecular phylogeny, subunits organization in chromosomes, localization indicated that PDH is an interesting marker in microsporidia evolution展开更多
Objective To study the chemical constituents of Bombycis Feculae.Methods Chemical constituents were isolated by HPLC-ELSD.The structures of the isolated compounds were determined by spectral means.Results Two compound...Objective To study the chemical constituents of Bombycis Feculae.Methods Chemical constituents were isolated by HPLC-ELSD.The structures of the isolated compounds were determined by spectral means.Results Two compounds were isolated and identified as 1-deoxynojirimycin(1)and(2R,3R,5R)-2-(hydroxymethyl) piperidine-3,5-diol,named as 1,3-dideoxygalatonojirimycin(2).Conclusion Compound 2 is a new alkaloid.The extract of Bombycis Feculae,compound 1 and compound 2 show inhibitory activities againstα-glucosidase.展开更多
Objective To establish a simple and rapid method for the determination of 1,3-dideoxygalactonojirimycin in Bombycis Faeces,a potent glucosidase inihibitor,by HPLC.Methods A RP-HPLC method with fluorescence detection h...Objective To establish a simple and rapid method for the determination of 1,3-dideoxygalactonojirimycin in Bombycis Faeces,a potent glucosidase inihibitor,by HPLC.Methods A RP-HPLC method with fluorescence detection has been developed.Results The HPLC method developed in this research has a good reliability including accuracy and precision.The detection limit was less than 72 ng.Conclusion This method is sufficiently sensitive for determining 1,3-dideoxygalactonojirimycin in Bombycis Faeces and other related products.展开更多
A 24-membered ring macrolide compound,macrolactin A has potential applications in pharmaceuticals for its anti-infectious and antiviral activity.In this study,macrolactin A was produced by a marine bacterium,which was...A 24-membered ring macrolide compound,macrolactin A has potential applications in pharmaceuticals for its anti-infectious and antiviral activity.In this study,macrolactin A was produced by a marine bacterium,which was identified as Bacillus subtilis by 16S ribosomal RNA(rRNA) sequence analysis.Electrospray ionization mass spectrometry(ESI/MS) and nuclear magnetic resonance(NMR) spectroscopy analyses were used to characterize this compound.To improve the production,response surface methodology(RSM) involving Box-Behnken design(BBD) was employed.Faeces bombycis,the main by-product in sericulture,was used as a nitrogen source in fermentation.The interactions between three significant factors,F.bombycis,soluble starch,and(NH4)2SO4 were investigated.A quadratic model was constructed to fit the production and the factors.Optimum medium composition was obtained by analysis of the model.When cultivated in the optimum medium,the production of macrolactin A was increased to 851 mg/L,2.7 times as compared to the original.This study is also useful to find another way in utilizing F.bombycis.展开更多
基金Supported by Natural Science Foundation of Chongqing(2008BB1368)~~
文摘[ Objective ] The aim of this study was to investigate the infectivity of Nosema bombycis to drosophila, which offered a new vision for systematical studies on the infection mechanism of Nosema bombycis, and also provided reference for the bio-control effect of Nosema bombycis. [ Method ] Nosema bombycis was used to feed wild type and mutant drosophila, and the morphological observation of Nosema bombycis in drosophila body fluid was also analyzed by calcofluor white M2R fluorescent staining. [ Result] Nosema bombycis could infect drosophila, and the number of Nosema bombycis in the infected mutant drosophila was higher than that in wild type drosophila. [ Conclusion ] Nosema bombycis can infect drosophila, which provides primary reference for studies on the infectivity of Nosema bombycis to other hosts and also lays a foundation for further study on the infection mechanism of Nosema bombycis.
基金funded by the National Natural Science Foundation of China(30270898).
文摘A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophilaatrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. Comparativestudies on morphology, infectivity and spore surface protein were conducted. 24Nbh was short and wide, and had asignificant difference (P<0.01) over the Nb spores. The infectivity tests conducted on second instar silkworm larvaeshowed that IC50 of 24Nbh was 1.98104 spores mL-1 and of Nb was 1.72103 spores mL-1, thus indicating that the infectivityof Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The IC50 of spores from silkworm infected with24 Nbh was 6.9 times less than Nb, showing that the infectivity of 24Nbh spores rejuvenated very fast when reinfected tosilkworms, further more, the length and width of such spore was larger than 24Nbh (P<0.01) and smaller than Nb (P<0.05).The SDS-PAGE profiles of Nb and 24Nbh were generally the same, 4 distinct proteins of 12, 17, 30, 33 kDa were obtainedwith difference in quantity. When 120 g of protein was applied for 2D-PAGE, five suspected different proteins withdifference in quantity were observed. These results demonstrate that these differential proteins maybe associated withvariation in infectivity of the spores.
基金supported by the National Natural Science Foundation of China (30771456)
文摘Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involves its polar tube acting as a needle-syringe system. However, recent studies show microsporidian spores can also gain access to host cells by phagocytosis. The present study investigated the phagocytic uptake process of causative agent of the pebrine disease, Nosema bombycis, in several insect cell lines. We observed KOH-treated spores and cold-storaged spores can be easily uptaken by all the studied cell types 4 h post inoculation. In contrast, large numbers of freshly recovered spores remained in the culture medium. To further investigate the intracellular fates of KOH-treated spores and cold-storaged spores, electron and fluorescence microscopy were performed. No intracellular germination or subsequent parasite development were observed. Intracellular spores can be detected in host cells by polyclonal antibody 7 d post inoculation, suggesting phagocytized N. bombycis could not be digested by these non-professional phagocytes. Our results suggest that, phagocytic uptake of N. bombycis spores might represent a defense mechanism of the host cells and the intact spore wall barrier enable freshly recovered spores to keep resistance to this mechanism.
文摘The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBmswp26-egfp bacmid. The vBmswp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.
基金supported by the Project of Chongqing Science and Technology Commission(CSTC,2006AA5019)National Basic Research Program of China under the grant No.2005CB121000
文摘Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this phylum. We present here the mitochondrial pyruvate dehydrogenase El （PDH, including PDHα and PDHβ） of the microsporidian Nosema bombycis, the pathogen of silkworm pebrine. Compared with PDH of microsporidian Encephalitozoon cuniculi and Antonospora locustae, both subunits are eonscrced. The phylogeny indicated that both subunits are mitochondrial. The syntenic maps revealed the subunits organization of NbPDH is distributed in different scaffolds, similar to that of EcPDH but different with AIPDH, and the relationship between phylogeny tree and organization of PDH suggest that the AlPDH subunits organization is the ancestral style of microsporidia, and through the genome evolution, the reshuffling of the chromosome of microsporidia occurred, the adjacent style of ALPDHE1 organization changed, and the two subunits separated and located to different chromosomes in E. cuniculi. For N. bombycis and N. ceranae, they locate to different scaffolds. In order to determine NbPDH subcellular localizations, we prepared the polyclonal antibodies against NbPDH prokaryotic fusion proteins, and adopted the colloidal gold immunological electron microscopy, the expression signals of NbPDH were observed in spores however, the subcellular localization were not definited. In general, through comparison of three mierosporidian PDH molecular phylogeny, subunits organization in chromosomes, localization indicated that PDH is an interesting marker in microsporidia evolution
基金Key National Science & Technology Specific Projects(2009ZX09103-363)
文摘Objective To study the chemical constituents of Bombycis Feculae.Methods Chemical constituents were isolated by HPLC-ELSD.The structures of the isolated compounds were determined by spectral means.Results Two compounds were isolated and identified as 1-deoxynojirimycin(1)and(2R,3R,5R)-2-(hydroxymethyl) piperidine-3,5-diol,named as 1,3-dideoxygalatonojirimycin(2).Conclusion Compound 2 is a new alkaloid.The extract of Bombycis Feculae,compound 1 and compound 2 show inhibitory activities againstα-glucosidase.
文摘Objective To establish a simple and rapid method for the determination of 1,3-dideoxygalactonojirimycin in Bombycis Faeces,a potent glucosidase inihibitor,by HPLC.Methods A RP-HPLC method with fluorescence detection has been developed.Results The HPLC method developed in this research has a good reliability including accuracy and precision.The detection limit was less than 72 ng.Conclusion This method is sufficiently sensitive for determining 1,3-dideoxygalactonojirimycin in Bombycis Faeces and other related products.
基金Project supported by the Science and Technology Department of Zhejiang Province,China(No.2009C33019)the IndustryUniversity-Research Institution Alliance for Microbial Medicine Technology Innovation and New Drug Development of China (No.2010ZX090401-403)the National Science and Technology Major Project of New Drug of China(Nos.2011ZX09201-101 and 2012ZX09103101-075)
文摘A 24-membered ring macrolide compound,macrolactin A has potential applications in pharmaceuticals for its anti-infectious and antiviral activity.In this study,macrolactin A was produced by a marine bacterium,which was identified as Bacillus subtilis by 16S ribosomal RNA(rRNA) sequence analysis.Electrospray ionization mass spectrometry(ESI/MS) and nuclear magnetic resonance(NMR) spectroscopy analyses were used to characterize this compound.To improve the production,response surface methodology(RSM) involving Box-Behnken design(BBD) was employed.Faeces bombycis,the main by-product in sericulture,was used as a nitrogen source in fermentation.The interactions between three significant factors,F.bombycis,soluble starch,and(NH4)2SO4 were investigated.A quadratic model was constructed to fit the production and the factors.Optimum medium composition was obtained by analysis of the model.When cultivated in the optimum medium,the production of macrolactin A was increased to 851 mg/L,2.7 times as compared to the original.This study is also useful to find another way in utilizing F.bombycis.
