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Huang L.,Zhejiang Agriculture And forestry University | Fang W.,Zhejiang Agriculture And forestry University | Fang W.,Institute of Preventive Veterinary Medicine | Yu Y.,Institute of Preventive Veterinary Medicine | Song H.,Zhejiang Agriculture And forestry University
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2012

The on-site labeling and localization tracking of membrane proteins in pathogenic bacteria are tedious work. In order to develop a novel protein labeling technology at super resolution level (nanometer scale) using the photoactivatable localization microscopy (PALM), the chimeric protein of the outer membrane protein A (OmpA) of Mycobacterium tuberculosis and the photoactivatable mEos2m protein were expressed in the non-pathogenic Mycobacterium smegmatis. The recombinant bacteria were fixed on slide, activated by 405 nm laser and subject to PALM imaging to capture photons released by the fusion protein. Meanwhile, colony and cell morphology were visualized under regular fluorescent stereomicroscope and upright fluorescent microscope to characterize fluorescence conversion and protein localization. The fusion proteins formed a "belt"-like structure on cell membrane of M. smegmatis under PALM, providing direct evidence of on-site imaging of membrane proteins. Expression of fusion protein did not compromise the localization properties of OmpA. Thus, mEos2m could be used as a labeling probe to track localizations of non-oligomer oriented membrane proteins. This indicates non-pathogenic M. smegmatis could be served as a model strain to characterize the function and localization of the proteins derived from pathogenic M. tuberculosis. This is the first report using PALM to characterize localization of membrane proteins. © 2012 Chin J Biotech, All rights reserved. Source

Li L.,Institute of Preventive Veterinary Medicine | Li L.,Sichuan Agricultural University | Zhu D.K.,Sichuan Agricultural University | Zhou Y.,Sichuan Agricultural University | And 13 more authors.
Poultry Science | Year: 2012

Here, we investigated adhesion and invasion of Riemerella anatipestifer (RA) to primary duck embryo fibroblast (DEF) cells. The ability of RA to adhere to, and more importantly, to invade DEF cells was demonstrated by using a gentamicin invasion assay and was confirmed by transmission electron microscopy (TEM). Adhesion of RA could be found by TEM after 1 h of inoculation. Both apoptosis and necrocytosis of DEF were indicated by TEM after 10 h of incubation, which suggested a complex mechanism of DEF cell death induced by RA. Our results showed that internalized RA had the ability to leave the DEF cells. Inhibition studies indicated that RA proteins play a role in adhesion. Moreover, invasion of RA to DEF cells was shown to require rearrangement of action microfilaments and microtubular cytoskeletal elements. Because the adhesion and invasion ability of RA to DEF cells could be demonstrated in vitro, similar processes might occur in vivo, where DEF cells play a crucial role in the diffusion of RA in ducks. © 2012 Poultry Science Association Inc. Source

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