Furthermore, control samples, not exposed to labelled insulin, did not give PLX4032 a positive reaction when developed with DAB. The initial binding experiments used a concentration of insulin that was much higher than the physiological concentration, but in-line with what previous workers had used (Christopher & Sundermann, 1996; Souza & López, 2004). These experiments were repeated with insulin-binding positive bacteria using insulin at a normal physiological concentration.

The insulin-binding assay was repeated on B. multivorans and A salmonicida using 80 pM of insulin peroxidase at different exposure times 2, 5, 10, 20, 40 and 80 min. These experiments showed that A salmonicida produced a positive reaction after 5 min, and this grew stronger with time up to 80 min. However, the B. multivorans showed no reaction at exposure times of 2, 5 and 10 min, and the first positive reaction was seen at 20 min and grew stronger at 40 and 80 min. Also included is a microscopic image of cells of A. salmonicida CM30 showing binding of FITC-labelled insulin (Fig. 1b). Variation in the intensity of staining of individual cells may be attributable to the method of fixation, different planes of focus and/or the possibility that some labelled insulin may have entered

cells. Both wild-type A. salmonicida and B. multivorans showed significant insulin binding at all the time points tested; however, the amount of insulin binding to the fish pathogen A. salmonicida was about 105 ng per 109 cells after 15 min incubation time,

which was much higher compared JQ1 research buy to 28.3 and 21.1 ng per 109 cells binding to B. multiv-orans and the A. salmonicida A-layer mutant, respectively (Fig. 2). Furthermore, wild-type A. salmonicida and B. multivorans showed significant binding relatively early (after 1 min) compared to the mutant A. salmonicida MT004, which showed significant FITC-insulin binding only after 10 min. Cyclooxygenase (COX) The amount of nonspecific insulin that bound to the P. aeruginosa and Escherchia coli was about 0.08 and 0.03 ng per 109 cells, respectively. Insulin binding to wild-type A. salmonicida increased steadily with time; however, B. multivorans showed no significant increase in insulin binding up to 5 min (13.1 ng per 109 cells) but produced strong binding of 19.1 and 23.8 ng per 109 cells after 10 and 15 min, respectively. Whereas the mutant A. salmonicida MT004 showed significant binding of 15.5 and 21.1 ng per 109 cells only after 10 and 15 min, respectively, with no significant binding at earlier times. Various protocols were applied during this work to separate bacterial proteins on different gels using native, SDS-PAGE (Laemmli, 1970), blue native (BN-PAGE; Nijtmans et al., 2002) and agarose gel electrophoresis (Henderson et al., 2000) and both Burkholderia and A. salmonicida samples initially showed no IBP bands on Western ligand blotting.