The group continued to implement and validate the technique called immuno-spin trapping, which combines the specific free radical reactivity of the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin trap with nitrone-antibody sensitivity. Anti-DMPO has proven itself to be a highly specific antibody with the no-DMPO control easily identifying any non specificity issues. This type of control is uncommon among antibody work, but is of great utility. Unlike electron spin resonance (ESR) detection of radical intermediates, anti-DMPO immuno-spin trapping is not dependent on transient free radical intermediates and is about 10,000 times more sensitive than ESR and 100 times more sensitive than MS. In years we have continued using the immuno-spin trapping approach to investigate the formation of protein-centered free radicals in vitro, in cells, and in vivo. The immunoassay has many advantages compared to ESR spin-trapping: 1) immuno-spin trapping requires much less material (micrograms of protein rather than milligrams); 2) the stability of the final oxidation product, the DMPO-nitrone adduct (a stable non-radical species), is much greater than that of the paramagnetic DMPO-radical adducts (t1/2 = sec-min) required for ESR, resulting in greatly enhanced sensitivity, and 3) immuno-spin trapping can be performed using standard ELISA, Western blotting techniques, and immuno-histological techniques, making immuno-spin trapping far more accessible and versatile than ESR spin trapping. This advance greatly expanded the utility of the spin-trapping technique, freeing it from the quantum mechanical complexity of ESR spectroscopy. We found that expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP) induces oxidative stress in cells. The increased formation of reactive oxygen species (ROS) such as superoxide (O2) and hydrogen peroxide (H2O2) can explain the cytotoxicity and tissue abnormalities reported previously in mammalian and bacterial cells and animals overexpressing fluorescent proteins. Both O2 and H2O2 induce redox signaling mechanisms, leading to altered gene expression of cell regulatory proteins involved in cell proliferation, cell differentiation, and cell death. Thus, these findings could have a major influence on the interpretation of results obtained from numerous studies that routinely use fluorescent protein tags. In the Maneb and the herbicide paraquat induced Parkinsons disease model, we found that the cytochrome c plays crucial roles in -synuclein radical formation and oligomerization. Extensive evidence were provided that -synuclein affects several biological pathways, which ultimately contribute to neuronal death in the Maneb and the herbicide paraquat induced mouse model of Parkinsons disease. The presence or absence of -synuclein (in wild-type or -synuclein knockout mice, respectively) determined all the difference in response to Maneb and paraquat co-exposure. These results suggest that experimental approaches which rely on scavenging -synuclein radical or decreasing -synuclein levels, can possibly lead to development of potential therapeutics for Parkinsons disease.
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