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ABSTRACT: Bacillus sphaericus produces a mosquito-larvicidal binary toxin composed of BinB and BinA subunits. BinA is important for toxicity, whereas BinB acts as a specific receptor-binding component. To study the functional significance of two regions that are only present in BinB, four block mutations and two single mutations were initially introduced: (111)YLD(113)-->(111)AAA(113), (115)NNH(117)-->(115)AAA(117), (143)GEQ(145)-->(143)AAA(145), (147)FQFY(150)-->(147)AAAA(150), N114A and F146A. Only the replacements at (147)FQFY(150) resulted in a total loss of toxicity to Culex quinquefasciatus larvae. Further single alanine substitutions in this region, F147A, Q148A, F149A and Y150A, were introduced to identify residues playing a critical role in mosquito-larvicidal activity. Larvicidal activity assays revealed that only F149A and Y150A mutants exhibited a total loss of toxicity. The in vitro interaction assays demonstrated that all BinB mutants are able to interact with BinA. Immunohistochemistry analysis revealed that only the Y150A mutant was unable to bind to the larval midgut, suggesting an important role of this residue in receptor binding of the BinB subunit. Conservative aromatic substitutions at F149 and Y150 resulted in full recovery of larvicidal activity, indicating that the aromaticity of F149 and Y150 is a key determinant of larvicidal activity, possibly playing a key role in the membrane interaction and receptor binding.
FEMS Microbiology Letters 11/2009; 303(1):84-91. · 2.04 Impact Factor
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ABSTRACT: Dysfunction of endothelial cells (EC) to produce endothelial nitric oxide synthase (eNOS) by tumor necrosis factor-alpha (TNF-alpha) causes critical features of vascular inflammation associated with several disease states (eg, atherosclerosis including increased platelet aggregation and adhesion on EC, elevated adhesion molecules and enhanced inflammatory cells binding to EC). 17-beta estradiol (E2) can stimulate eNOS production and improve the critical features of atherosclerosis. Using TNF-alpha and E2, we attempted to develop an in vitro vascular model for studying atherosclerosis.
Human umbilical vein endothelial cells (HUVEC) grown in transwells were cocultured with smooth muscle cells in a 24-well plate to mimic the major components of the vascular wall. The model was incubated with TNF-alpha (10 ng/ml) for 12 h, prior exposed to E2 (100 pg/ml) for 6-12 h, then investigated by transmission and scanning electron microscopes. The result indicated recovered morphology with good tight junction, and decreased platelet adhesion was noted in defective HUVEC after E2 treatment.
17-beta estradiol was represented as an antiatherosclerogenic agent to demonstrate feasibility of the model. Although our finding focused only on the endothelium, this would be the basis for our future studies to develop ex vivo continuous perfusion of human vessel segments for a further atherosclerosis study.
Circulation Journal 07/2005; 69(6):730-8. · 3.77 Impact Factor
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ABSTRACT: Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.
The Journal of General and Applied Microbiology 07/1999; 45(3):105-113. · 0.98 Impact Factor
