scholarly journals The Immunologic Response of Dogs to Bartonella Vinsonii Subspecies Berkhoffii Antigens: as Assessed by Western Immunoblot Analysis

2003 ◽  
Vol 15 (4) ◽  
pp. 349-354 ◽  
Author(s):  
Edward B. Breitschwerdt ◽  
Jiraporn Suksawat ◽  
Bruno Chomel ◽  
Barbara C. Hegarty
Keyword(s):  
Cell Culture ◽  
Immunoblot Analysis ◽  
Progressive Increase ◽  
Protein Recognition ◽  
Molecular Testing ◽  
Serum Samples ◽  
Tissue Samples ◽  
Antigenic Protein ◽  
Western Immunoblot ◽  
Western Immunoblot Analysis

Bartonella vinsonii subspecies berkhoffii is a recently recognized zoonotic pathogen that causes endocarditis, granulomatous rhinitis, and granulomatous lymphadenitis in dogs. Isolation of B. vinsonii ( berkhoffii) from blood or tissue samples is frequently unsuccessful; therefore, diagnosis is primarily dependent on serologic or molecular testing modalities. Because previous canine serologic studies have used an indirect immunofluorescence assay (IFA), without Western immunoblot (WI) confirmation, the overall objective of this study was to examine the diagnostic use of WI for confirmation of B. vinsonii ( berkhoffii) infection in dogs. To confirm that agar-grown and cell culture–grown organisms yielded similar patterns of WI antigenic protein recognition, the 2 preparations were compared using IFA-reactive sera obtained from dogs experimentally infected with B. vinsonii ( berkhoffii). Temporal changes in the pattern of antigenic protein recognition were characterized using sera obtained from dogs at various time points after experimental B. vinsonii ( berkhoffii) infection. The specificity of B. vinsonii ( berkhoffii) WI was examined by testing canine sera that were reactive to B. henselae, B. clarridgeiae, Ehrlichia canis, Rickettsia rickettsii, Babesia canis, Anaplasma phagocytophilum (previously E. equi), or Brucella canis antigens. Clinical accessions including serum samples obtained from B. vinsonii ( berkhoffii) culture–positive dogs and B. vinsonii ( berkhoffii) culture–negative dogs that were IFA seroreactive to B. vinsonii ( berkhoffii) antigens were examined by WI. The results of this study indicate that WI using agar-grown or cell culture–grown B. vinsonii ( berkhoffii) antigens produce identical patterns of antigenic protein recognition. After experimental infection, there is a progressive increase in the number of antigenic proteins that are recognized by WI, with the 33-kD antigen representing the first and the most persistent antigen recognized by B. vinsonii ( berkhoffii)–infected dogs. Regarding specificity, sera from dogs that were reactive to various heterologous antigens did not recognize B. vinsonii ( berkhoffii) antigens by IFA or WI, and sera from dogs experimentally infected with B. henselae did not recognize B. vinsonii ( berkhoffii) antigens by WI. Regarding clinical accessions, there was good agreement between B. vinsonii ( berkhoffii) IFA test results and WI analysis. Western immunoblot analysis can be used to detect or confirm exposure to B. vinsonii ( berkhoffii) in dogs.

scholarly journals Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii

1993 ◽  
Vol 122 (4) ◽  
pp. 877-886 ◽  
Author(s):  
JD Harper ◽  
MA Sanders ◽  
JL Salisbury
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transcriptional Activation ◽  
Kinase Inhibitor ◽  
Nuclear Periphery ◽  
Immunoblot Analysis ◽  
Basal Region ◽  
Western Immunoblot ◽  
Flagellar Regeneration ◽  
Basal Apparatus ◽  
Western Immunoblot Analysis

The antiphosphoprotein monoclonal antibody MPM-2 was used to investigate protein phosphorylation during flagellar regeneration in Chlamydomonas reinhardtii. MPM-2 recognizes a phosphorylated epitope and detects several Chlamydomonas proteins by Western immunoblot analysis. Two MPM-2 reactive proteins (34 and 90 kD) increase in Western immunoblot intensity after flagellar excision and decrease in intensity during flagellar regeneration. Immunofluorescence and immunogold labeling revealed MPM-2 staining within the nucleus, especially towards the nuclear periphery, the flagellar basal apparatus, and the nucleus-basal body connector after flagellar excision. Comparison of MPM-2 reactivity in wild-type cells and in the mutant bald-2, which lacks functional basal bodies, demonstrates that the 34-kD protein is localized in the nucleus and the 90-kD protein is localized in the flagellar basal region. MPM-2 reactivity is observed in cells competent for flagellar regeneration. However, when cells were treated with the kinase inhibitor, staurosporine, MPM-2 reactivity did not increase after flagellar excision and flagellar regeneration was impaired. These observations suggest that phosphorylation of the 34- and 90-kD proteins may be important for flagellar regrowth. Possible roles for phosphorylation in flagellar regeneration include transcriptional activation and transport of flagellar precursors to the base of the growing flagella.

scholarly journals Secretion of Proteases by Pseudomonas aeruginosa Biofilms Exposed to Ciprofloxacin

2005 ◽  
Vol 49 (8) ◽  
pp. 3281-3288 ◽  
Author(s):  
Ewa Ołdak ◽  
Elżbieta A. Trafny
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Defense Mechanisms ◽  
Immunoblot Analysis ◽  
Fresh Medium ◽  
Western Immunoblot ◽  
Unexposed Control ◽  
Proteolytic Activities ◽  
Western Immunoblot Analysis ◽  
Host Tissues

ABSTRACT Pseudomonas aeruginosa proteases are considered important virulence factors which damage host tissues and interfere with host antibacterial defense mechanisms. P. aeruginosa biofilm cells are not completely killed by antibacterials, and therefore this study addresses the question whether ciprofloxacin attenuates the virulence of biofilm communities by abolishing their secretion of proteases. The surviving cells of the colony biofilms studied, despite their cyclical exposure to four doses of ciprofloxacin at bactericidal concentrations (one dose a day), still secreted active proteases to the environment surrounding the biofilms. The biofilm cells secreted elastase B (LasB) over the duration of the experiments as confirmed by Western immunoblot analysis. The colony biofilms did not secrete LasA—a protease with staphylolytic activity. The same profiles on zymogram gels with gelatin were observed for the proteases secreted by both ciprofloxacin-exposed and unexposed (control) biofilms. Total proteolytic activities of the colony biofilms studied were significantly reduced after exposure to ciprofloxacin at bactericidal concentrations—after 96 h of exposure they dropped to 38% for the strain intermediate resistant to ciprofloxacin and to 65% for the strain highly resistant to the antibiotic, relative to the control biofilms. The surviving cells of the colony biofilms after their release into a fresh medium displayed transient increased resistance to ciprofloxacin compared to their planktonic counterparts.

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