This crude material was dialyzed in distilled water. The water solution was then lyophilized to obtain CMWS. Polysaccharides were completely hydrolyzed in 2.0 M CF3CO2H (115°C, 1.5 hr). The sugars were converted to alditol acetates by reduction followed by treatment with acetic anhydride in an equal volume of pyridine (100°C, 1 hr), and then analyzed by GLC using a GC-2014AF instrument (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a 30 m × 0.25 mm (0.25 mM) DB-225 capillary column (J&W Scientific, Folsom, CA,
USA). The total carbohydrate concentration was determined by the phenol-sulfuric acid method using a mixture of d-mannose and d-glucose as Alectinib a standard. Total protein was determined by using the BCA Protein Assay Regent kit (Pierce Biotechnology, Rockford, IL, USA), with BSA as a standard. Endotoxin content was determined by the Toxicolor LS-50M Set (Seikagaku Biobusiness, Tokyo, Japan). We used the DBA/2 mouse strain in this experiment because this strain shows the most serious coronary arteritis after treatment with the CAWS that is secreted into the culture supernatant by Candida albicans (11). In week1, CMWS (4 mg/mouse) was administered intraperitoneally for 5 consecutive days to each mouse. The hearts of the animals were fixed with 10% neutral formalin and embedded in paraffin blocks. Tissue sections were stained with HE. Preparation of paraffin blocks and HE staining was done by Japan SLC. The incidence
and severity of rapid buy Ulixertinib enough anaphylactoid shock was assessed within 1 hr of i.v. injection (0.1 mL/10 g body weight) of CMWS (8 mg/kg) into ICR mice. The subsequent mortality (in the first hour after injection) was recorded. The reactivity
of cell wall extracts to serum factors from Candida Check, which consists of rabbit polyclonal antibodies against Candida cell wall mannan (23–25), was assessed by ELISA. A solution of cell wall extracts in 50 mM carbonate buffer (pH 9.6) was coated onto Nunc immunoplates (Roskilde, Denmark), which were then incubated at 4°C overnight. The plates were washed extensively with PBST; unbound sites were blocked by the addition of BPBST to wells for 40 min at 37°C; and then the wells were washed six times with PBST. Candida serum factors serially diluted with BPBST were added and incubated for 60 min at 37°C. After six washes with PBST, the wells were treated with peroxidase-conjugated goat anti-rabbit IgG and the TMB microwell peroxidase substrate system (KPL, Gaithersburg, MD, USA). After 45 min, the reaction was stopped with 1 N H3PO4. The optical density of each well was then read at 450 nm on an automatic microplate reader. The reactions were evaluated as positive when the maximum optical density was over 1.0 at an 80-fold dilution ratio of Candida serum factor because Candida serum factors are polyclonal antibodies. Exchangeable protons were removed by dissolving cell wall extracts in D2O, and samples were then lyophilized. This exchange process was repeated three times.