Grant: Research Tools and Instruments - Category 1 (<$150,000)

Oxidative stress in a biological system can be caused by the overproduction of harmful reactive oxygen molecules or the inability to detoxify these molecules once formed, and can result in cellular damage.  The struggle between the reducing and oxidizing conditions that naturally arise in cells is often referred to as the "redox" state.  Disturbances in the normal redox state can cause toxic effects through the increased production of reactive oxygen species (ROS), such as peroxides and oxygen free radicals, leading to damage of essential components of the cell including DNA, membranes and proteins.  Although the prevailing opinion has been that ROS cause non-specific damage to cellular components, new evidence suggests that ROS can target specific proteins leading to a change in their activity.  Low levels of ROS, produced at the cell membrane and in specialized organelles called mitochondria, can trigger redox signaling events.  These events can then lead to increased gene expression in the nucleus and either activation of a protective anti-oxidant response or, if the levels of ROS are high, the initiation of programmed cell death.  Disulfide bonds are a type of chemical linkage formed between proteins during oxidative stress.  Recent studies in yeast and bacteria have shown that the formation of disulfide bonds within specific oxidant sensing proteins leads to their activation.  New evidence suggests that a similar disulfide-bond controlled activation system may also occur in mammalian cells.  Cell culture models are key to understanding how oxidative stress can activate redox signaling processes.  By culturing animal-derived cells in the presence of either high or low oxygen levels, it is possible to mimic the physiological fluctuations of ROS that occur in tissues.  In addition, addition of inflammatory factors called cytokines can also cause the production of ROS within cells and trigger cell death.  The development of cell culture models of oxidative stress and the isolation of disulfide bonded proteins from these cells can provide insight into how the cellular redox state influences both normal and disease-associated signaling processes.