DNA and protein reactions of NO', ONOO-, and reactive species produced by phagocy

项目2.我们在这个项目中的工作假设是， 炎症部位代表与人类疾病的致病联系。在结肠炎的主题中，中性粒细胞 和没有阻力共享整个程序，项目2重点是化学介质的反应， 炎症与DNA，脂质，碳水化合物和蛋白质在上皮细胞产生有毒产品， 导致生理改变，细胞死亡和与癌症相关的突变。目的是探索 这种损伤的机制，开发不稳定炎症介质的替代标记物， 开发炎症的候选生物标志物。在上一个资助期，我们开发了分析方法， DNA、RNA和蛋白质损伤产物（具有核心1），并将这些方法应用于培养的细胞（具有核心1）。 项目3）和炎症动物模型（项目4，核心2），目的是将 项目1中开发的化学模型与项目3和项目4。在拟议的研究中，我们将扩大 生物标志物开发，包括脂质过氧化产生的DNA、RNA和蛋白质损伤产物 和嗜中性粒细胞，并定量这些标记物在发炎和癌性人体组织。具体目标 具体如下： 目标1.定义由NO、N2 O3、ONOO* 产生的DNA、RNA和蛋白质损伤谱 和HOCI在分离的核酸和培养的细胞（与项目1，3，4）。我们将开发分析 DNA、RNA和蛋白质损伤产物的测定方法：氯酪氨酸、硝基酪氨酸;卤化 核碱基、鸟嘌呤氧化产物和所有DNA损伤的RNA形式。这些方法将 应用于项目1、3和4的模型细胞系统。 目标2.在小鼠炎症模型中应用方法并定义病变谱（具有 项目1、3、4）。我们将建立生物标志物在炎症和结肠炎动物模型中的效用， 项目4和量化DNA，RNA和蛋白质病变组织协调与其他项目，以发展 活性氮和卤素类物质生产的预测模型。 目标3.将生物标志物方法应用于发炎和癌变的人体组织（项目1，3， 4）。将生物标志物转化为正常人结肠组织和来自患有结肠癌的患者的样品。 克罗恩病和溃疡性结肠炎，目的是比较人类和小鼠的概况 模型我们还将开发分析存档石蜡中NO和HOCI化学标记物的方法 黑色素瘤肿瘤块，目的是区分来自黑色素瘤的NO的作用， 细胞本身，从那些攻击肿瘤的先天免疫细胞。

Project 2. Our working hypothesis in this Program is that chemical species generated by phagocytes at sites of inflammation represent a causative link to human disease. Within the themes of colitis, neutrophils and NO resistance shared across the Program, Project 2 focuseson reactions of the chemical mediators of inflammation with DNA, lipids, carbohydratesand proteins in epithelial cells to generate toxic products that lead to altered physiology, cell death and mutations associatedwith cancer. The objectives are to explore the mechanisms of this damage, to develop surrogate markers of the labile inflammatory mediators, and to develop candidate biomarkers of inflammation. In the last grant period, we developed analytical methods for DNA, RNA and protein damage products (with Core 1) and applied these methods to cultured cells (with Project 3) and to animal models of inflammation (with Projects 4, Core 2), with the goal of linking the chemical models developed in Project 1with Projects 3 and 4. In the proposed studies, we will expand biomarker development to include DNA, RNA and protein damage products derived from lipid peroxidation and neutrophils, and quantify these markers in inflamed and cancerous human tissues. The Specific Aims are as follow: Aim 1. Define the spectrum of DNA, RNAand protein lesions produced by NO, N2O3,ONOO* and HOCI in isolated nucleic acids and cultured cells (with Projects 1, 3,4). Wewill develop analytical methods for DNA, RNA and protein damage products: chlorotyrosine, nitrotyrosine; halogenated nucleobases, guanine oxidation products; and RNA versions of all DNA lesions. These methods will then be applied to model cell systems with Projects 1, 3 and 4. Aim 2. Apply methods and define lesion spectra in mouse models of inflammation (with Projects 1, 3, 4). We will establish the utility of biomarkers in animal models of inflammation and colitis with Project 4 and quantify DNA, RNA and protein lesions in tissues in coordination with other projects to develop predictive models for production of reactive nitrogen and halogenspecies. Aim 3. Apply biomarker methods to inflamed and cancerous human tissues (with Projects 1,3, 4). The biomarkers will be translated to samples of normal human colon tissue and from patients with Crohn's disease and ulcerative colitis, with the goal of comparing the profiles from human and mouse models. We will also develop methods to analyze markers of NO and HOCI chemistry in archived paraffin blocks of melanoma tumors, with the goal of distinguishing the effects of NO derived from the melanoma cells per se, from those of the innate immune cells attacking the tumor.