2e). Although the above studies ascertained the formation of free radicals during PCD in Xcg, it was not clear whether these radicals are the cause or the effect of PCD. To answer this question, the effects of the ROS scavengers DMSO, glutathione

(GSH), nPG, and catalase on PCD were tested. Cell survival almost doubled in the presence of DMSO (0.25–0.5%) compared with the control at the end of a selleck chemicals llc 96-h incubation period and the increase was found to be statistically significant (P≤0.05) (Fig. 3a). However, the increase in survival was not found to be significantly affected by an increase in the DMSO concentration (P≤0.05). When GSH was added to PIM, a concentration-dependent increase in cell survival was observed when assayed at 96 h of incubation and PCD was completely inhibited with 10 mM GSH (Fig. 3b). As for GSH, PCD was also significantly abolished with 100 μM nPG (Fig. 3c) and 500 U mL−1 of catalase (Fig. 3d). No growth was observed at higher concentrations of GSH or nPG and both were found to be more effective than DMSO in inhibiting PCD. Caspase-3 biosynthesis was also found to be lower in cells grown in the presence of these ROS scavengers (Fig. 3e). In comparison with PIM-grown Xcg cells, the caspase-3 band intensity was 14%, 25%, 53%,

and 57% in cells grown in PIM in the presence of GSH (10 mM), DMSO (0.5%), nPG (100 μM), and catalase (500 U mL−1), respectively. The inhibition of caspase-3 expression Pexidartinib by DMSO (0.5%) or GSH (10 mM) was quite prominent compared with nPG or catalase.

This effect may be due to a difference in the mechanism of action of different ROS scavengers. Caspase-3 activity decreased by 15%, 10%, and 20% in Xcg cells grown in PIM 4-Aminobutyrate aminotransferase in the presence of GSH (10 mM), nPG (100 μM), and catalase (100 U mL−1), respectively, as compared with Xcg cells grown in PIM alone (Fig. 3f). Caspase-3 activity in Xcg cells grown in PIM in the presence of DMSO (0.5%) was negligible (data not shown). When a PNIM-grown Xcg cell lysate was exposed to H2O2, the level of caspase activity increased in a concentration-dependent manner, as evidenced by the observed increase in the intensity of fluorescence (Fig. 4a). Therefore, these findings indicate that H2O2 is involved in both intercellular and intracellular communication of the PCD signal in Xcg. No H2O2 could be detected by scopoletin assay in the Xcg cells grown in PIM in the presence of 500 μM 2,4-dinitrophenol (DNP) (Fig. 4b). When Xcg cells were grown in PIM with a sublethal concentration of DNP, cell survival increased by one log cycle (Fig. 4c). Figure 4d shows the effect of the addition of nalidixic acid (DNA gyrase inhibitor) to Xcg culture in PIM. The minimum inhibitory concentration of nalidixic acid for Xanthomonas sp. has been reported to be around 8–16 μg mL−1 (Pruvost et al., 1998). The results show that the addition of nalidixic acid at sublethal concentrations (0.8 and 1.