During the past several years, my lab is working in the field of cellular responses to oxidative stress. Free radicals and ROS are involved in a variety of cellular processes ranging from cell proliferation to apoptosis and necrosis. Identification of how ROS might specifically regulate signaling pathways by altering the activity of direct target molecules is a major goal of my lab. In order to study the role of reactive oxygen species in pathological conditions in vivo we are using transgenic mice which overexpress human antioxidant enzymes. The research carried out by my laboratory and collaborators clearly demonstrates that transgenic mice in general, and those with antioxidant enzymes in particular, provide unique models for investigating the mechanisms of numerous pathological processes resulting from the actions of reactive oxygen species, such as neurotoxicity, ischemia/reperfusion injury, septic shock, tumorigenesis etc.

Successful development of therapeutic agents for the treatment of CNS trauma and stroke requires understanding of the basic molecular mechanisms underlying the permanent loss of neural tissue and function. This project is devoted to elucidation of the mechanisms of protection effect of the elevated level of antioxidant enzymes (glutathione peroxidases in particular) in a well-established model of focal cerebral ischemia, temporally MCA occlusion. Increased level of antioxidant enzyme (GPx1 in particular) was able to protect against I/R damage by decreasing the sensitivity of brain cells to induced cell death and by modulating inflammatory response. The ability of ROS and antioxidant enzymes to modulate specific signaling pathways as well as execution mechanisms of apoptosis are currently under investigation.

It is clear that other early-activated regulatory proteins induced during pathological process significantly influence development of tissue injury. We use targeted differential display method to carry out systematical analysis of these proteins. Among new discovered proteins, is a mammalian homolog of Sua5/YrdC family that is a family of highly conserved and uncharacterized proteins. We are using variety of biochemical approaches as well as two-hybrid yeast system to elucidate function and to search for the interacting targets of this new protein. We were able to demonstrate that this protein can regulate activity of mammalian transporters, including dopamine transporter. A number of approaches in several species are undertaken to characterize exact function of the new proteins, such as micRNA and antisense RNA in C. elegans and human cell cultures, transgenic and knockout mice models etc.

Another project we are working on currently is Parkinson's disease model induced by combination of environmental toxicants. Data from human postmortem tissue as well as cell culture studies suggest that ROS are important in the pathogenesis of sporadic Parkinson disease. Relative lack of in vivo evidence has contributed to this controversy surrounding the exact role of ROS in the pathophysiology of this disease. Therefore, it is extremely important to characterize recently introduced early environmental PD model based on combined exposure to low dosages of herbicides and fungicides. In the course of the proposed studies we are following signaling pathways leading to neurodegeneration in several cell culture and transgenic animals. The future studies will attempt to address the issue of possible sources of the ROS as well as immediate sensitive molecules mediating cell-death inducing signals.

	Glutathione peroxidase inhibits cell death and glial activation following experimental stroke. Ishibashi N, Prokopenko O, Weisbrot-Lefkowitz M, Reuhl KR, Mirochnitchenko O. Brain Res Mol Brain Res. 2002 Dec 30;109(1-2):34-44.
	Chemokine expression in transgenic mice overproducing human glutathione peroxidases. Ishibashi N, Mirochnitchenko O. Methods Enzymol. 2002;353:460-76.
	Inflammatory response and glutathione peroxidase in a model of stroke. Ishibashi N, Prokopenko O, Reuhl KR, Mirochnitchenko O. J Immunol. 2002 Feb 15;168(4):1926-33.
	Endotoxemia in transgenic mice overexpressing human glutathione peroxidases. Mirochnitchenko O, Prokopenko O, Palnitkar U, Kister I, Powell WS, Inouye M. Circ Res. 2000 Aug 18;87(4):289-95.