A sequential precipitation was done using 40% ammonium sulphate saturation with slow
stirring for 30 min, equilibrating for 30 min at 4° C and centrifuging Protein Tyrosine Kinase inhibitor at 39,200 g for 45 min. The supernatant was precipitated with 60% ammonium sulfate saturation and treated as previously described, but in this case the precipitate was recovered and the supernatant was discarded. The fraction was resuspended in a minimum volume of deionizer water, dialyzed through a 3-kDa pore size membrane and centrifuged, loading 4 mL aliquots on a Sephadex G-75 gel filtration 167 × 1.7 cm column (Pharmacia Biotech, Uppsala, Switzerland). The column was equilibrated with 0.01 M ammonium bicarbonate buffer pH 7.8. The experiment was performed at 4° C collecting 0.3 mL/min fractions with the same buffer. Protein was monitored at 280 nm in a Beckman DU-65 spectrophotometer. Agglutination activity [22] was determined by microscopic counting using glutaraldehyde-fixed type A+ human erythrocytes [23]. Specific activity was determined using protein concentration [24]. Electrophoretic profile was obtained by 10% polyacrylamide SDS-PAGE [25]. Glycoproteins were confirmed
by periodic acid-Schiff staining (PASS) [26]. Additionally, lectins were observed by western blot using an anti-phytohemagglutinin antibody from Phaseolus vulgaris (Vector Laboratories Inc. Burlingame, CA, USA. cat. N° AS-2300). The fraction was dialyzed against deionized water, lyophilized and stored at -20° C until use. Five-week old male SD rats Akt inhibitor in vivo were divided into 2 groups (n = 8 per group). After fasting for 24 h, the treated group received a single dose of the lyophilized 50 mg/kg TBLF dissolved in standard saline solution (0.9% NaCl in deionized water) using an intragastric cannula [20], while the control group received saline solution. Autoclaving (121° C for 15 min) was necessary
for denature food lectins. Feeding 4-Aminobutyrate aminotransferase was restarted with ad libitum water and autoclaved chow food (Rodent Laboratory Chow 5001. Saint Louis, MO, USA). Feces form 4 rats per group were collected at 0, 24, 48, 72, 96 and 120 h, fecal protein was extracted in PBS, filtered through a 0.22 mm membrane and agglutination specific activity was determined by microscopic counting [22]. Other 4 rats per group were sacrificed at 24 h in order to recover blood for CBC (CellDyn® 1600) and a commercial kit for differential blood cells staining was used for cell counting from blood smear (Hycel, Mexico; cat. number 548). Erythrocytes, neutrophils, eosinophils, basophils, lymphocytes, monocytes and platelets were counted using a 100X microscope objective. Results are expressed as absolute blood counts or percentage respect to control animals. Fifteen-week old male SD rats were randomly selected in 2 groups (n = 12 per group). Treated rats were dosed with 50 mg/kg TBLF dissolved in saline solution and control group was administered with saline solution by using an intragastric cannula.