1), streptococcal culture JNK inhibitor supernatants were harvested
by centrifugation exactly at the mid log phase (A600 = 0.6–0.7) (Svensson et al., 2002). In addition, to ensure the absence of the zymogene (40 kDa) and active form (28 kDa) of the SpeB protease, the proteins of mid log phase culture supernatants were precipitated using 100% trichloroacetic acid and were evaluated by standard 12% SDS-PAGE and Coomassie blue staining (Svensson et al., 2002). The culture supernatants were subsequently filtered through 0.22-μm filter (Whatman, Germany) and stored at −70 °C until use. For each colorimetric assay, 50 μL of the culture supernatant was incubated at 37 °C with 100 μL 50 mM Tris–HCl, pH 7.4 in microplate containing 50 μg mL−1 of human Plg (Sigma) for 15 min. The S-2251 substrate (50 μL of 2.5 mM) was added, and absorbance was measured at 405 nm every 5 min for 60 min. Each assay was performed in duplicate, both in the presence and absence of Plg/S-2251. Assays containing the intact (unused) THB media and culture supernatants of the reference strains were also employed as negative and positive controls, respectively. Serial dilutions of Streptase® (CSL, Behring, Germany), a commercial SK, were used to prepare the standard curve for
SK Ribociclib datasheet activity. Optical densities were plotted against time, and activity rates were determined from linear portion of the curve. The level of SK activity in each bacterial culture supernatant was converted to IU mL−1 using the standard curve. The PCR product of a representative of each digestion pattern (Fig. 1) was selected for nucleotide sequencing. DNA sequencing data were used for alignment studies and restriction site mapping via application Rutecarpine of the Molecular Evolutionary Genetic Analysis (mega 4) analytical package (Tamura et al., 2007). Difference in SK activities among GAS and GCS/GGS groups was determined using the one-way
anova test. The Kruskal–Wallis analysis of variance was employed to calculate the level of significance of SK activity among variants. All statistical analyses were carried out using spss version 16.0 (SPSS, Inc., Chicago, IL). A value of P < 0.05 was considered significant. PCR-based amplification of the sk-V1 region produced the expected 339-bp fragments (Fig. 1). Digestion of the PCR products (sk-V1) by MluI, PvuII, DraI and DdeI restriction enzymes also provided exactly the earlier described restriction patterns of the DNA fragments (Tewodros et al., 1996) (Fig. 1, Supporting Information, Table S1). The reference strains GCS/GGS and NZ131 were classified as sk5 and sk1, respectively, as expected. In total, 21 sk allelic variants were detected among strains investigated in our study (Fig. 2). Besides the several previously reported sk allelic variants of GAS and GCS/GGS isolates (Tewodros et al.