There were no significant changes in these enzymes in the cells e

There were no significant changes in these enzymes in the cells exposed to H2O2 (Fig. 3). Hence, these data point to the channeling of substrates towards the formation of KG and NADPH with the subsequent decrease in the synthesis of NADH. This strategy ensures that during oxidative stress, sufficient NADPH, a potent reductive fuel, and KG, a powerful scavenger of ROS, are available.

The decrease in the KU-60019 purchase generation of NADH will further help decrease the oxidative burden as this moiety drives the production of ROS via the electron transport chain (ETC). Furthermore, it is critical that during oxidative stress, the effectors mediating ROS production be attenuated. Oxidative phosphorylation is a major generator of ROS (Ludwig et al., 2001; Murphy, 2009). Hence, it is quite conceivable that the complexes mediating this process are downgraded. These Fe-containing complexes are susceptible to H2O2 (Touati, 2000; Middaugh et al., 2005). Indeed, sharp reduction was observed in the activities of Complexes I, II, and IV (Fig. 4). The nature of Complexes I and IV was further confirmed by

the inclusion of rotenone mTOR inhibitor and KCN in the assay mixture. The former is a specific inhibitor for Complex I, while Complex IV is inhibited by KCN. The activity band was not detected in the control CFE in the presence of these inhibitors, respectively (data not included). This strategy of limiting the formation of NADH, coupled with decreased activities of the enzymes involved in its oxidation, provides an effective tool to mitigate H2O2 insult. Pseudomonas fluorescens appears to adopt this tactic in an effort to survive in the oxidative environment induced by H2O2. Numerous SDHB organisms do indeed resort to decreased oxidative phosphorylation and anaerobiosis with the goal of coping with a ROS challenge (Chen et al., 2003; Chenier et al., 2008). In eukaryotic systems, the promotion of the hypoxia-inducible factor (HIF-1α), an activator of anaerobic respiration, is favored (Mailloux et al., 2009a, b). As the catabolism of histidine was

providing glutamate, a moiety involved in the generation of the antioxidant KG, it was important to ascertain whether the enzymes involved in the formation and utilization of KG were modulated by H2O2. When control cells were exposed to H2O2 stress, the decrease in KGDH activity was coupled with the increase in GDH activity. However, when H2O2-stressed cells were introduced into control media, the reverse trend was observed i.e. the activity of KGDH was recovered while the activity of GDH was decreased. Western blot analyses revealed that the latter enzyme was more abundant in the H2O2-treated cells and was affected by this oxidative modulator (Fig. 5). Hence, it is clear that H2O2 was indeed controlling the status of KGDH, GDH, and ICDH and subsequently the levels of KG and NADPH.

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