5% Triton X-100, rinsed with water, and incubated overnight at 37 °C in 50 mM Tris–HCl (pH 8) containing 5 mM calcium chloride and 2 nM zinc chloride. Gels were stained with Coomassie blue and destained with 25% ethanol and 10% acetic acid solution. Areas associated with gelatinolytic activity appeared as clear bands on a BMS-777607 supplier blue background. The molecular weights of lung tissue proteins present in the clear bands were estimated by comparison with those of

the placental sample. Gelatinolytic activity was densitometrically quantified as the intensity of the negative bands in relation to those determined in the positive control (Niu et al., 2000). For such purpose Scion Image 4.03 software (Scion Corporation, Frederick, MD, USA) was used. Aliquots of lung homogenates, each containing 30 μg of protein,

were denatured in 50 mM Tris–HCl (pH 6.8) containing 1% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.001% bromophenol blue, and heated in boiling water for 3 min. AZD5363 mw Samples, together with Rainbow molecular weight markers (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), were submitted to 12% SDS polyacrylamide gel electrophoresis and the separated lung tissue proteins transferred to nitrocellulose membranes. Membranes were blocked with Tween-TBS [20 mM Tris–HCl (pH 7.5) containing 500 mM sodium chloride and 0.5% Tween-20] supplemented with 2% BSA, and probed (1:1000) with the specific primary antibodies Liothyronine Sodium goat anti-mouse MMP-12 and goat anti-mouse

HMGB-1. After extensive washing in Tween-TBS, the membranes were incubated with biotinylated secondary antibody and ABP for 1 h and then visualized by DAB staining. The intensities of the bands were densitometrically quantified using Scion Image 4.03 software (Scion Corporation, Frederick, MD, USA) after ponceau staining of the membrane. All data were expressed as mean ± S.E.M. or as median and percentiles (10 and 90%), and analyzed using GraphPad Prism 5 data analysis software (GraphPad Software, CA, USA). Normally distributed continuous data (i.e. BALF counts, antioxidant enzyme activities and pulmonary mechanics) were analyzed using Student t-test with Welch’s correction, while discrete data (Vvair, Vvef and densitometric measurements) were treated using the Mann–Whitney test. In all cases, the level of significance was set at 5%. The mean (±S.E.M.) COHb level in air-exposed mice was 1.1 ± 0.2%, while that in CS-exposed mice was 13.4 ± 1.3%. Photomicrographs of lung sections in control animals presented normal alveoli with thin alveolar septa and few alveolar macrophages (Fig. 1a) and elastic fibers displaying fine branching in the alveolar septa (Fig. 1c). On the other hand, mice exposed to CS exhibited enlarged airspaces and thickened alveolar septa (Fig. 1b), a large amount of alveolar macrophages and rupture of elastic fibers in the alveolar septa (Fig. 1d). Lung static elastance and functional residual capacity were significantly higher (p < 0.