The following proposals were approved for funding in 2005:

Project 2-2005:

Project: A novel function of a translesion DNA polymerase against DNA-protein crosslinks
Principal Investigator: Jun Nakamura, D.V.M., Ph.D., Research Assistant Professor of Environmental Sciences and Engineering

Abstract

Cellular DNA is continuously exposed to endogenous and exogenous agents that produce DNA-protein crosslinks (DPCs). While the biological significance of DPCs have not been fully investigated, DPCs likely interrupt DNA replication, repair, recombination, transcription, and chromatin remodeling. Interestingly, epidemiological studies have reported a positive association between basal levels of DPCs and the incidence of breast cancer. Many low fidelity translesion DNA polymerases involved in the bypass of DNA lesions have been recently discovered in eukaryotic cells. The various insertive functions of these polymerases allow them to bypass DNA lesions but cause the main replicative polymerase to stall. Strikingly, our preliminary results showed that cells deficient in one of the translesion DNA polymerases are hypersensitive to physiological levels of endogenous bifunctional aldehydic compounds. Based on this evidence, we hypothesize that a single specific polymerase is involved in the removal of DPCs during the course of DNA repair or replicative bypass of these DNA lesions. This hypothesis will be extensively tested using a variety of genetic and functional approaches. The project will provide critical information regarding the mechanisms by which cells tolerate DPCs and the roles that translesion synthesis DNA polymerases have in counteracting DPCs induced under physiological conditions in order to maintain genomic integrity.

Project 3-2005:

Project: Correlating breast cancer and obesity: detection of biomarkers of susceptibility using an obese mouse model
Principal Investigator: Christoph H. Borchers, Ph.D., Assistant Professor of Biochemistry and Biophysics

Abstract

Breast cancer is a leading cause of cancer-related deaths among women, second only to lung cancer. Many epidemiological studies have uncovered a correlation between obesity and breast cancer. Not all obese women, however, will get breast cancer. Furthermore, the mitigating effects of exercise have also been postulated.

Our hypothesis is that there is a genetic basis for this difference in susceptibility and that it should be reflected in a difference in the proteome. The specific aim of this proposal is to discover protein biomarkers for breast cancer susceptibility in obese mice, which can be used to uncover the corresponding human biomarker. We will use quantitative proteomic approaches on blood from obese mice which did not develop breast cancer, vs. blood from mice which did develop breast cancer.

The results obtained will be used for a joint R01 between Dr. Borchers and Dr. Threadgill with the goal of using these biomarkers to study the mitigating effects of exercise in susceptible mice. Ultimately, we will develop a protein chip for screening human subjects in order to target prevention strategies to susceptible individuals.

Project 4-2005:

Project: Health effects of air pollution on lupus (HEAPL): a nested pilot study on a community-based panel of lupus patients and controls
Principal Investigator: Jiu-Chiuan (JC) Chen, MD, MPH, Sc.D., Assistant Professor of Epidemiology

Abstract

In the proposed pilot panel of 265 patients with systemic lupus erytheomatosus (SLE) and 302 community-based controls from the Carolina Lupus Study, we will use geocoded residential information, air pollution data recorded in EPA’s Aerometric Information Retrieval System, and other relevant spatiotemporal data to construct Bayesian Maximum Entropy (BME) models and estimate personal exposures to ambient PM10, PM2.5 (particulate matter with aerodynamic diameter =10 and 2.5µm respectively), and ozone. Inflammatory responses to air pollution (as assessed by increases in C-reactive protein [CRP]) will be compared between SLE patients and controls. Among SLE patients, clinical markers of disease severity and activity will be examined for their associations with exposures to air pollution. We will employ generalized linear models to estimate effects of air pollution on CRP and indicators of disease severity and activity, adjusting for relevant confounders. By demonstrating a sophisticated modeling approach applicable to studying other environmental determinants of health outcomes in North Carolina, our pilot study will generate preliminary but essential data to evaluate the susceptibility of SLE patients and the potential effects of air pollution on rheumatoid disorders.

Project 5-2005:

Project: Regulation of neuronal apoptosis: focus on identifying novel functions of MicroRNAs
Principal Investigator: Mohanish Deshmukh, Ph.D., Assistant Professor of Cell and Developmental Biology, School of Medicine and Neuroscience Center

Abstract

Exposure to environmental neurotoxins or other pathological situations can induce neurodevelopmental and neurological deficits by triggering excessive neuronal death by apoptosis. Therefore, understanding how neurons undergo apoptosis is essential for developing therapeutic strategies aimed at preventing neuronal death.

Recently, microRNAs (miRNAs) have been discovered as important regulators of many biological processes. MiRNAs are short, noncoding RNA molecules that negatively regulate protein expression. The focus of this pilot proposal is to determine whether miRNAs regulate apoptosis in mammalian neurons (using primary mouse sympathetic and cerebellar granule neurons). In Specific Aim 1, we will examine whether two known antiapoptotic Drosophila miRNAs and the cancer-associated mammalian miRNAs inhibit apoptosis in mammalian neurons. If these miRNAs are found to be neuroprotective, we will examine the mechanism by which they inhibit neuronal apoptosis. In Specific Aim 2 we will take an unbiased approach to discover novel miRNAs that regulate neuronal apoptosis by using a custom miRNA microarray to identify miRNAs that are transcriptionally induced in neurons undergoing apoptosis. Subsequent experiments will focus on identifying the mechanism by which these miRNAs promote neuronal apoptosis.

Project 6-2005:

Project: Effects of posttranslational modifications of P450 2E1 on butadiene metabolism in vitro
Principal Investigator: Gunnar Boysen, Ph.D., Postdoctoral Fellow, Environmental Sciences and Engineering

Abstract

Butadiene (BD) is a multi-species, multi-site carcinogen in rodents, with mice being a much more sensitive species than rats. This proposal evaluates whether phosphorylation or covalent binding of activated metabolites alter the catalytic activity of P450 2E1 and are responsible for the differences in BD metabolism and tumorigenesis. State of the art mass spectrometry approaches will be used to identify covalent binding sites of 1,2-epoxybutene (EB) and 1,2;3,4-diepoxybutane (DEB) and phosphorylation sites. Preliminary data have already demonstrated distinct binding sites of EB. After these posttranslational modifications have been identified, their effects on catalytic activity will be determined. Therefore, it is proposed to exam the kinetics for the conversions of BD to EB and EB to DEB. To normalize and verify these results, the catalytic activities will also be determined for p-Nitrophenol hydroxylation and Chlorzoxazone 6-hydroxylation. If posttranslational modifications alter the catalytic activity of P450 2E1 in vitro, these studies will establish a novel level of P450 2E1 regulation and it is proposed to extent this project to in vivo samples from tissues of BD treated mice and rats.

Project 7-2005:

Project: Commensal gut microflora, inflammation and obesity
Principal Investigator: P. Kay Lund, Ph. D, Professor of Cell and Molecular Physiology, Nutrition and Pediatrics

Abstract

In mice commensal gut microflora promote suppress intestinal fasting induced adipocyte factor (Fiaf) mRNA and promote adiposity. Gut microflora also induce cytokines implicated in obesity associated insulin resistance. We will use existing colon biopsy RNA from a human population characterized for BMI, waist: hip ratio, plasma insulin and glycated hemoglobin (Hb1Ac). We will test the hypothesis that in humans, low expression of colonic Fiaf, and elevated expression of pro-inflammatory cytokines predict obesity and early signs of insulin resistance. We also hypothesize that obesity and altered Fiaf or cytokines reflect increased load of total or specific colonic commensal bacteria. Aim 1 : real time PCR will quantify expression of Fiaf, cytokines and a panel of 16S rRNAs specific to common bacteria in 580 human colon RNA samples. Aim 2: logistic regression will test for associations between Fiaf, cytokine expression levels, or bacterial RNAs and BMI, waist to hip ratio, plasma insulin or Hb1Ac levels. Overall, we aim to define new biomarkers for bacterial colonization or responses to colonization in gut, and establish if these predict obesity or insulin resistance.