Characterization of HPE
Gel filtration was used to estimate the molecular weight of the adhesion molecule-inducing factor in HPE from NCTC11637. Freeze-dried extract (10 mg) mixed with 1.5 mL of 150 mM NaCl was eluted through a HiPrep Sephacryl S-100 HR column (Pharmacia Fine Chemicals, USA), with collection of 2 mL fractions. Each fraction (10%) was estimated for adhesion molecule-induction activity. Data were expressed as a ratio of each sample OD to control OD (IL-1P).
To further characterize the adhesion molecule-inducing factor in HPE, the active fractions were heated (56°C for 30 min or 100°C for 10 min) in a water bath or incubated them with trypsin (250 mg/mL, final concentration) for 1 h at 37°C. Treated and untreated fractions were assayed for adhesion molecule-induction activity. Data were expressed as a ratio of each sample OD to control OD (IL-1P). To test for a relationship between induction of adhesion molecules and the presence of LPS, LPS concentrations in the five HPE were measured by a tur-bidimetric Limulus amebocyte lysate assay using the Limulus ES-J Test Wako (Wako Pure Chemical Industries Ltd, Japan) with a Toxinometer ET-301 BL (Wako Pure Chemical Industries Ltd, Japan).
All values were expressed as the mean ± SE. Data were compared using analysis of variance (ANOVA) followed by Scheffe’s test. P<0.05 was considered statistically significant.