2006 Highlights

Title: Concentration-Dependent Protection of Retinal Ganglion Cells from Pressure-Induced Apoptosis by Pre-treatment with a Flavonol Flavonoid
Investigator: David Calkins, M.D., Associate Professor, Department of Ophthalmology and Visual Sciences
Description: Loss of retinal ganglion cells (RGCs) in glaucoma is associated with sensitivity to intraocular pressure. A component of RGC loss involves oxidative stress, so this project sought to ascertain whether a naturally occurring antioxidant, the flavonol flavonoid quercetin, could reduce the susceptibility of RGCs to pressure-induced apoptosis. The immortalized RGC-5 cell line was used to test whether a brief pre-treatment with quercetin modulates their survival with elevated hydrostatic pressure in vitro. Samples of RGC-5 cells were treated to one of four concentrations of quercetin dihydrate, and cells were exposed to either ambient pressure or a uniform column of elevated hydrostatic pressure. All experiments were completed in triplicate, and the statistical difference between samples was determined using ANOVA and Mann-Whitney rank tests, which require no underlying assumption of sample distribution. While low concentrations of quercetin do not enhance survival of RGC-5 cells at ambient pressure, for RGC-5 cells challenged by elevated hydrostatic pressure, quercetin at 2.5 uM and 25 uM decreases the fraction of apoptotic cells. Surprisingly, the highest concentration tested induced a 10-fold increase in apoptosis at ambient pressure and a 4-fold increase at elevated pressure, indicating that higher doses of flavonols may actually be harmful.

Title: Complexities of Complex II: One Target, Many Toxins
Investigator: Tina Iverson, Ph.D., Assistant Professor, Department of Pharmacology
Description: Complex II is a mitochondrial respiratory protein that plays a central role in metabolism by linking the electron transport chain with the Krebs cycle. Inhibition, mutation, or downregulation of complex II can lead to several types of genetically linked cancer, reactive oxygen species generation and aging, and neurodegenerative disease. This project had two specific aims: 1) Characterization of the binding of the fungal toxin 3-nitropropionic acid to complex II and 2) determination of how the identity of catalytic residues affects 3-nitropropionic acid inhibition. Specific Aim 1 proposed a combination of x-ray crystallography and mass spectrometry to determine if 3-nitropropionic (3-NPA) had pH-dependent modes of inhibition. The crystal structure of 3-NPA bound to complex II was determined at 3.3 Å resolution and currently has an Rcryst of 0.245 and an Rfree of 0.288. Mass spectral analysis proceeded in collaboration with Drs. Leibler and Amy Ham in the Vanderbilt mass spectrometry facility. Wild type protein digested in-gel with trypsin, chymotrypsin, and Aspn was analyzed by LC-MS-MS. This indicated coverage of 96.5% by amino acid count and verified feasibility of the experiment. Specific Aim 2 proposed refinement of the crystal structure of an active site mutation, purification of new protein in-house, and the growth of crystal of active site mutants hypothesized to alter 3-NPA binding. The crystal structure of the FrdA T234A mutation at a resolution of 3.65 Å has an Rcryst of 0.263 and an Rfree of 0.299 after refinement with CNS, PHENIX, RAPPERMC, and REFMAC. The structure shows that active site residues move away from the 3-NPA binding site in this mutation.

Title: Novel Approach to Suppression of Anticholinesterase-Associated Neurodegeneration
Investigator: Dejan Milatovic, Ph.D., Research Assistant Professor, Department of Pediatrics
Description: The focus of this project was to utilize a variety of independent experimental strategies to test the hypothesis that suppression of biomarkers of oxidative damage can prevent neurodegeneration induced by anticholinesterase neurotoxicity. During the first 4 months of this project, this study focused on determination of anticholinesterase-induced changes in biomarkers of oxidative damage in the rat brain by quantifying lipid peroxides (F2-IsoPs and F4-NeuroPs), nitric oxide, and high-energy phosphates.  The most prominent biochemical changes observed at one hour following anticholinesterase exposure are correlated with quantitative determination of dendritic degeneration in the CA1 hippocampal area. Analysis of multiple pyramidal neurons evaluated by Neurolucida-assisted morphometry revealed significant decline in dendritic length and spine density. During the subsequent 4 months, the study focused on suppression of neuronal oxidative damage and dendritic degeneration by using neuroprotectants known for beneficial effects in neurodegenerative diseases. Pretreatment with memantine 1) suppressed alterations in biomarkers of oxidative damage, nitric oxide, and high-energy phosphates induced by anticholinesterase and 2) attenuated a decrease in dendrite length and spine density of pyramidal neurons from the CA1 area. In addition, antioxidant vitamin E and spin trapping agents PBN also attenuated an anticholinesterase-induced increase in biomarkers of oxidative damage. During the remaining period of the project, work focused on evaluating the effect of antioxidants on suppressing anticholinesterase-induced sinaptodendritic changes in affected hippocampal area, as well as on assessing the biochemical and morphometric manifestations of repeated, subthreshold exposures to cholinesterase inhibitor.

Title: Identification of Lipid Peroxidation Protein Adducts via MS/MS Sequence Tags
Investigator: David Tabb, Ph.D., Assistant Professor, Department of Biomedical Informatics
Description: Environmental stresses often lead to the formation of oxidants, giving rise to cytotoxic and genotoxic effects. Protein adducts associated with these species have been studied as part of a Program Project Grant from NIEHS under Dr. Ned Porter (NIH P01 ES013125). In project 4 of this grant, Dr. Dan Liebler is evaluating the effects of 4-hydroxyalkenals on subcellular fractions of proteins and then progressing to more complex mixtures of reactive oxygen species, identifying proteins that are most prone to oxidative damage by tandem mass spectrometry. This project planned the use of Sequest and P-Mod to identify protein adducts produced by these oxidants. Sequest, however, is limited to identifying small numbers of chemical modifications for which the mass and affected residues are known. P-Mod is capable of finding unanticipated modifications, but it scales poorly to large numbers of proteins. To remedy these shortcomings, this project developed new algorithms for “sequence tag” identification in which partial sequences are inferred directly from spectra. The first goal to be achieved through such software was to stratify spectra, removing those of low quality and revealing those of high quality for which identification failed. The ultimate aim for this tool, however, is to identify comprehensively the masses and locations for unanticipated chemical modifications. As such, this project 1) will increase the amount of information that can be derived from toxicological proteomics data sets; 2) will demonstrate the viability of sequence tagging for identifying and locating unspecified chemical modifications in complex protein mixtures; and 3) made use of continued improvement in the scoring discrimination of tag inference with the aim of outperforming existing tag inference tools.

Title: Effects of Omega-3 Fatty Acid Supplementation in a Murine Model of Asthma
Investigator: Huiyong Yin, Ph.D., Research Assistant Professor, Departments of Pharmacology and Chemistry
Description: The purpose of this project is to study the effects of fish oil supplementation on a mouse model of asthma with the hypothesis that oxidation products from omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil are responsible for the beneficial effects of fish oil consumption in asthmatic patients. Liquid chromatography-mass spectrometry (LC-MS) based methods have been developed to analyze the fatty acid composition and the oxidation products of polyunsaturated fatty acids in vitro and in mouse tissues and fluids. These methods are comparable with the conventional GC-MS based methods, but multiple compounds can be analyzed at the same time with minimal sample workups. Analysis of enzymatic products, such as prostaglandins and leukotrienes, by LC-MS methods are under investigation. EPA and DHA are successfully incorporated into lung tissue after ten weeks of feeding, and Ovalbumin (OVA) challenge causes the oxidative stress in mouse lung tissue. Levels of F2-IsoPs, the gold standard for oxidative stress, are elevated in lung tissue after OVA challenge. Fish oil feeding significantly attenuates the formation of these pro-inflammatory lipid mediators. However, some unexpected results were obtained in that the levels of inflammatory cytokines such as IL-5 and IL-13 are also elevated in the fish oil-fed group as compared to the control groups. Experiments have been designed and were subsequently performed to address this finding by feeding low doses of fish oil and changing the timing of challenge. It is known in the literature that PGE2 is an anti-inflammatory lipid mediator in this asthma model acting through EP3 receptors. The significant reduction of arachidonic acid, a substrate for PGE2, by fish oil feeding results in the reduction of PGE2 production. In previous experiments, 4% fish oil was used in the diet, which replaced 50% of the arachidonic acid by EPA. This project used 2% fish oil in the diet, and the mice were sensitized by OVA before fish oil feeding.