Aims: To investigate the hypothesis that commonly occurring bacterial toxins cause sudden infant death syndrome (SIDS) by (1), determining in which tissues bacterial toxins are concentrated after intravenous injection in rats; and (2), seeing if the same tissues contain detectable toxins in cases of SIDS. Methods: The tissue distribution of intravenously injected staphylococcal enterotoxin A (SEA), enterotoxin B (SEB), enterotoxin C (SEC), enterotoxin D (SED), toxic shock syndrome toxin (TSST-1), and a-haemolysin was studied in rats using immunohistology and polyacrylamide gel electrophoresis with immunoblotting. Immunostaining was also carried out on formalin fixed kidneys from cases of SIDS and a comparison series of necropsy cases using anti-SEA, anti-SEB, anti-SEC2 and anti-SED. Results: Immunohistology showed that SEB, SEC, SED and TSST-1 were all concentrated in the proximal convoluted tubular cells ofthe kidney. The Bead cultures stored at -70'C were grown on blood agar overnight at 37'C, then transferred to bijou bottles containing 5 ml brainheart infusion (BHI) medium, and again left overnight at 37'C. Two drops of the culture were spread on dialysis membrane overlying BHI-1 5% agar plates and incubated for three to four days at 37'C in 5% CO2. Phosphate buffered saline (PBS) (2 ml) was used to wash the growth from the membrane and the suspension was centrifuged at 4000 x g for 15 minutes. The supernatant was sterilised by passing through a 0 2 ,um Minisart disposable filter and stored at -20°C. ELECTROPHORESIS AND IMMUNOBLOTIINGToxin preparations were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using a 1-5 mm slab gel containing 12% (WY) acrylamide for the separating gel and a 4% acrylamide for the stacking gel. The buffer system was a slightly modified version of Laemmli. 12 The sample buffer contained 2% SDS, 10% sucrose, 002 M TRIS-HCI (pH 8 0), 0-002 M EDTA and 2 mg Pyronin Y as marker dye. (Mercapto-ethanol was added to the sample buffer for most toxins but was found to break down SEB into small fractions.) Equal quantities of sample and sample buffer were used at 25 ,um/well.Electrophoresis was carried out in "The Sturdier" vertical slab unit, SE 400 (Hoefer Scientific Instruments).Immediately after electrophoresis the separating gels were soaked in transfer buffer (0-25 MTRIS, 0-192 M glycine in 20% methanol) for 15 minutes before blotting. The separated proteins were transferred to nitrocellulose membrane for one hour at 100 mA, 716 on 10 May 2018 by guest. Protected by copyright.
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