The gingiva was treated with 0.025% trypsin and 0.01% EDTA overnight at 4°C and human gingival epithelial cells (HGECs) were isolated as previously Selleckchem RAD001 described . The HGECs were seeded in 60-mm plastic tissue culture plates coated with type-I collagen (BD Biocoat, Franklin Lakes, NJ, USA) and incubated in 5% CO2 at 37°C using K-SFM
medium (Invitrogen, Carlsbad, CA, USA) containing 10 μg/ml of insulin, 5 μg/ml of transferrin, 10 μM of 2-mercaptoethanol, 10 μM of 2-aminoethanol, 10 mM of sodium selenite, 50 Quisinostat molecular weight μg/ml of bovine pituitary extract, 100 units/ml of penicillin/streptomycin and 50 ng/ml of fungizone (complete medium). When the cells reached sub-confluence, they were harvested and sub-cultured as previously selleck described . Bacterial strains and conditions P. gingivalis ATCC 33277 was purchased from the ATCC (Manassas, VA, USA) and the derivative KDP128, an RgpA/RgpB/Kgp triple mutant , was kindly provided by Dr. K. Nakayama (Nagasaki University Graduate School of Biomedical Sciences). P. gingivalis W50 (ATCC 53978), and the derivative mutants E8, an RgpA/RgpB double mutant, and K1A, a Kgp mutant ,
were kindly provided by Dr. M. Curtis (Barts and The London, Queen Mary’s School of Medicine and Dentistry). All P. gingivalis strains at low passage were grown in GAM media (Nissui Pharmaceutical, Tokyo, Japan) under anaerobic conditions (85% N2, 10% CO2 and 10% H2; Coy Laboratory) for 2 days. After cultivation, the bacteria were harvested by centrifugation, washed in PBS (pH 7.4) and used immediately for the live cell challenge or heat-inactivated for 1 h at Vasopressin Receptor 60°C. For the bacterial culture supernatant assays, the supernatant was filtered sterilized using a 0.22 μm pore PVDF membrane (Millipore, USA). The Rgp and Kgp activity of each strain was determined using the enzymatic substrate hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) (Sigma), for
Rgp activity, or acetyl-lysine-p-nitroanilide (ALNA) (Bachem), for Kgp activity. The Rgp and Kgp activity were negligible for the heat-killed bacteria. Purified gingipains and gingipain inhibitors Purified HRgpA, RgpB and Kgp were isolated as previously described [25–27]. The purified gingipains were used at a final concentration of 8 μg/ml for HRgpA, 5.2 μg/ml for RgpB and 3 μg/ml for Kgp (all equivalent to 113 units of Rgp activity/ml or 12.4 units of Kgp activity/ml) in the presence of 5 mM L-cysteine . For the gingipain inhibition assays, live P. gingivalis 33277 or its culture supernatant was incubated with gingipain inhibitors for 15 min at 37°C, just prior to the HGEC challenge. zFKck, a specific Kgp inhibitor , was used at a final concentration of 10 μM. Leupeptin (Sigma), a specific Rgp inhibitor, was used at a final concentration of 100 μM.