Index        Introduction        Methods        Results        Discussion        References        Individual Project - Karl

Introduction:

     "Oxidative stress" may be referred to as “A disturbance in the pro-oxidant-antioxidant balance in favor of the former (ie. Pro-oxidant), leading to potential damage” (Sies, 1991).  Oxidative stress is responsible for substantial cellular damage to DNA, proteins, carbohydrates, lipids, and ion regulatory systems.  The massive cell damage can result when the available supply of the body's antioxidants are insufficient to handle and neutralize free radicals of different types (Brynes, 2003).  A significant amount of studies have been carried out on oxidative stress especially with respect to multiple sclerosis, ALS, Chronic Fatigue Syndrome and neurological diseases such as Parkinson’s disease, Alzheimer's disease, and AIDS dementia amongst others, are accelerated by oxidative stress which plays a major role in the breakdown of tissues.

         Quite a number of efforts have gone into developing a potential antioxidant therapy considering the implication of the condition. Blumberg (2004) emphasizes the need for research studies to be directed towards determining whether bio-markers reflect short –or-long term exposure to an antioxidant status or oxidative stress.  Of particular interest is the work being carried out by Dr. Slekar, here at James Madison University, who is focusing on studying anti-oxidant factors in yeast cells (Saccharomyces cerevisiae) using a genetic approach. Dr. Slekar and her team have shown that over expression of the yeast ZMS1 and ZMS2 genes suppress the mutant phenotypes of a zwf1.  The zwf1 gene in S. cerevisiae codes for enzymes responsible for catalyzing the NADPH production stage in the Pentose Phosphate pathway a metabolic process in yeast cells.  Glutathione, an electron donor and antioxidant that provides oxidative stress protection, is kept in its reduced and active form by NADPH

    We are seeking to investigate the sensitivity of the ZMS1 gene mutant to oxidative stress and compare it to the wild type strain.   Dr. Kim Slekar provided us with a  ZMS1 knockout strain of S. cerevisiae,  our model organism for observing possible phenotypic signs of sensitivity to oxidative stress.  Microarray techniques will be used to show the ZMS1 gene expression profile.  This investigation will employ statistical tools such as the MAGIC TOOL software to analyze the microarray data and reduce sources of error.

    S. cerevisiae is considered the an ideal model system for eukaryotic studies in which to study the metabolism of oxygen free radicals, because it is simple single-celled eukaryote.  Slekar et. al (1996) suggested that “yeast contain the same oxidative stress defense mechanisms present in higher eukaryotes and are extremely amenable to genetic, biochemical, and molecular biological manipulation model.”

    The objective of this project, is aimed at investigating which genes are turned on or off or are up regulated or down regulated. Blumberg (2004) agrees that this facet of new research is important in determining which individuals are most likely to respond to antioxidant interventions.  This will set up a basis for a breakthrough in treatment against the condition of “oxidative stress.”

Hypothesis:  It is hypothesized that there will be significant expression level increase in genes associated with resisting cellular oxidative stress in the ΔZMS1 yeast strain.

Noble Egekwu - egekwuni@jmu.edu    Karl Gorzelnik - gorzelkv@jmu.edu    Jonathan Baugher - baughejl@jmu.edu