Isolation of Staphylococcus aureus from sputum in cystic fibrosis.
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The success in the isolation of Staphylococcus aureus of different methods of sputum processing was investigated in 60 specimens collected from 14 patients with cystic fibrosis during a seven-month period. Fifty specimens (83%) from 11 patients yielded Staph. aureus by one or more methods. Direct plating of purulent portions of sputum on to media designed for general use in respiratory infections gave unsatisfactory results (35% yield of Staph. aureus). Some increase in isolations was obtained with preliminary liquefaction of sputum; but the best results were given by the addition of a medium selective for staphylococci (mannitol salt agar, BBL) or by initial sonication of sputum (each 83% yield). Seven of the 11 strains of Staph. aureus were thymidine-dependent and otherwise atypical in laboratory characteristics; these were isolated from patients who had received co-trimoxazole. (+info)
Partial characterization of a major autolysin from Mycobacterium phlei.
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Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene. (+info)
Involvement of outer-membrane proteins in the aggregation of Azospirillum brasilense.
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A bioassay was developed to investigate biological factors involved in the aggregation of Azospirillum brasilense strain Cd. Cells were grown for 24 h under aggregation-inducing and non-aggregation-inducing conditions (high and low C:N, respectively) and sonicated for 20 s. The cells were washed by centrifugation and resuspended in potassium phosphate buffer containing the two types of sonication extract. A greater extent of aggregation and higher flocculation were observed after 2-3 h incubation in the presence of sonicates from cells grown at high C:N (H-cells) compared to cells grown at low C:N. Flocculation did not occur after incubation of these cells in phosphate buffer. Boiled or proteinase K-treated sonicates originating from H-cells had lower aggregation-inducing capacity. After fractionation of the crude sonicate, both the outer-membrane protein (OMP) and the total membrane (mostly OMP) fractions possessed relatively high aggregation specific activities. The aggregation-inducing capacity of the OMP fraction strongly correlated with its protein concentration in the bioassay. Treatment of this fraction with proteinase K also decreased its aggregation-inducing activity. These findings suggest that OMPs are involved in the aggregation process of cells of A. brasilense. (+info)
Physical and biological properties of cationic triesters of phosphatidylcholine.
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The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective. (+info)
Alamethicin-mediated fusion of lecithin vesicles.
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It was recently shown that alamethicin greatly facilitates the fusion of small, sonicated, lecithin bilayer vesicles. In the present work the details of this fusion process have been followed by monitoring the inner and outer choline methyl signals separately by proton magnetic resonance spectroscopy. It is shown that during the alamethicine-induced fusion some of the antibiotic molecules become translocated from the extravesicular aqueous medium into the enclosed intravesicular space, and these alamethicine molecules were found to affect the choline methyl signals from the inner half of the bilayer only. No evidence was obtained for transmembrane coupling of the two halves of the bilayer in the presence of alamethicin or for any effects that might be construed as due to incorporation of alamethicin molecules into the hydrophobic core of the bilayer. (+info)
Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates.
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The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum. (+info)
Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.
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A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes rather than binding to the reconstituted liposomes. (+info)
Sonicated diagnostic immunoblot for bartonellosis.
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Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction. Both methods were then tested for sensitivity and specificity. Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In a healthy group, it is 100% specific. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity. (+info)