PubMed · 14206430
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J G STEMPAK, R T WARD. 1964. AN IMPROVED STAINING METHOD FOR ELECTRON MICROSCOPY.. https://doi.org/10.1083/jcb.22.3.697
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A sensitive agarose-gel electrophoresis method has been developed for the visualization of lipopolysaccharide (LPS) samples from several Escherichia coli serotypes, such as 026:B6, 055:B5, 0128:B12, 0111:B4, 0127:B8, and K235. This method can detect as little as 0.5-6 microg of LPS depending on the serotype by treatment of the plates with toluidine blue, and it is able to measure sub-microgram amounts of samples, approx. 0.05-0.5 microg, when the staining with toluidine blue followed by Stains-All procedure is adopted. Treatment of LPS with alkali under anhydrous conditions removes the ester-linked fatty acids and the phosphate groups of the Lipid A component, producing the detoxified LPS. The carbohydrate neutral moiety obtained from the hydrolysate does not migrate during electrophoresis. This was utilized to monitor quantitatively the removal process of the Lipid A component for the formation of the detoxified LPS.
The bactericidal activity of copper-deposited titanium dioxide thin film (Cu/TiO2) was investigated under very weak ultraviolet (UV) light illumination. To elucidate the roles of the film photocatalyst and the deposited copper in the bactericidal activity, cells from a copper-resistant Escherichia coli (E. coli) strain were utilized. A decrease in survival rate was not observed with the copper-resistant cells under dark conditions, but when illuminated with a very weak UV intensity of 1 microW/cm2, the survival rate decreased, suggesting photocatalytic bactericidal activity. The decay curve of survival on the Cu/TiO2 film under very weak UV light illumination consisted of two steps, similar to the survival change of normal E. coli on TiO2 films under rather strong UV illumination. The first step is due to the partial decomposition of the outer membrane in the cell envelope by a photocatalytic process, followed by permeation of the copper ions into the cytoplasmic membrane. The second step is due to a disorder of the cytoplasmic membrane caused by the copper ions, which results in a loss of the cell's integrity. These processes explain why the Cu/TiO2 film system shows an effective bactericidal activity even under very weak UV light illumination.
Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.