Chemicals. Trypsin-chymotrypsin inhibitor (T9777), protein L (P3101), and phenylmethylsulfonyl fluoride (PMSF P7626) were purchased from Sigma-Aldrich (St. Louis, MO). MacConkey agar (DF0113-17-5) and blood agar were purchased from Fisher Scientific (Pittsburgh, MEK162 molecular weight PA) and Ward’s Natural Science (Rochester, NY), respectively. Protein block buffer (X0909) was from DAKO (Carpinteria, CA). For tight junction protein Western blotting experiments, the primary antibodies [rabbit anti-zonula occludens-1 (ZO-1) (61-7300) and mouse anti-occludin (33-1500)] were from Zymed. Anti-��-actin was from Sigma Chemical (St. Louis, MO).

Secondary antibodies Alexa Fluor 680 goat anti-mouse (“type”:”entrez-nucleotide”,”attrs”:”text”:”A31562″,”term_id”:”1567162″A31562) and donkey anti-goat (“type”:”entrez-nucleotide”,”attrs”:”text”:”A21084″,”term_id”:”583471″A21084) were from Molecular Probes (Carlsbad, CA), and IRdye 800 anti-rabbit IgG (KFB001) was from Rockland (Gilbertsville, PA). Protease inhibitor Complete Mini solution was from Roche (Indianapolis, IN). To detect total and anti-LPS IgG by ELISA, the secondary antibody used was goat anti-rat IgG [horseradish peroxidase (HRP) conjugated, sc-2006] from Santa Cruz Biotechnology (Santa Cruz, CA). To detect fecal total, anti-flagellin, and anti-LPS IgG by ELISA, the secondary antibody was goat anti-rat IgA (Serotec, Raleigh, NC). The same antibody was also used for gut lamina propria IgA cell staining. Purified flagellin and LPS were provided by Dr. Andrew Gewirtz. Experimental diets. All rats were fed standard pelleted rat chow prior to operation.

After operation, the rats were fed semipurified diets custom prepared by Harlan Teklad (Madison, WI), with or without l-GLN supplementation (Table 1). The GLN content of GLN-supplemented diet was 4% of total diet weight, approximately fivefold higher than the GLN content derived from casein in the control diet [AIN-93G (29)]. The protein (casein) content in the GLN diet was reduced such that the GLN-supplemented and control diets were isocaloric and isonitrogenous (Table 1). Table 1. Composition of semipurified diets Operative procedures and pair feeding. Rats were randomized by body weight and fasted overnight before operation. The following day (day 1), rats underwent laparotomy between 9:00 AM and 1:00 PM, as previously described (23).

Briefly, animals were anesthetized with intraperitoneal ketamine (100 g/l) and xylazine (20 g/l), and a ventral abdominal midline incision was performed. The locations for bowel transection (TX, operative control) and partial small bowel-colonic resection (RX) were identified by using defined landmarks. The resected rats underwent removal of the distal 60% of the small intestine, the entire cecum, and 1 cm of the proximal colon. The transected rats had small AV-951 bowel transection at 60 cm proximal to the ileal-cecal valve (23).