Fig. 9.

The heterologous expression of the Pbaox gene decreases the generation of intracellular ROS in S. cerevisiae cells. Cell culture, induction of Pbaox expression, spheroplast generation, and ROS measurement procedures were performed as described in Materials and Methods. The spheroplasts of S. cerevisiae/pYES-Pbaox and S. cerevisiae/pYES cells (5 × 10⁶ cells/ml) were loaded with the CM-H₂DCFDA (5 μM) fluorescent probe for 30 min at 37°C. The oxidation of the probe by ROS was monitored using a Hitachi F-4500 fluorescence spectrophotometer at 30°C under agitation. The excitation and emission wavelengths were 503 and 529 nm, respectively. The concentrations of the inhibitors in each addition were as follows: for KCN, 1 mM, and for antimycin A, 1.8 μM. The values represent increases in fluorescence at 10, 30, and 50 min in comparison to that at time zero (before the addition of inhibitors), and these values are shown as the averages ± SEMs of results from three independent assays. Asterisks indicate P values of <0.05 between S. cerevisiae/pYES-Pbaox and S. cerevisiae/pYES cells. The statistical analyses are described in Materials and Methods.