RUI: Isotopic Tracing of Nitric Oxide (NO)-Related Cell Signaling Pathways

一氧化氮（NO）在多种生物系统中合成，发挥着广泛的信号作用。尽管其合成的生物学后果已被广泛记载，但其在体内的化学作用模式和功能仍存在争议，而且在很大程度上不清楚。精确定位NO生物化学的一个困难源于缺乏能够(1)从其来源，通过化学中间体和其蛋白质靶点追踪NO的仪器，同时(2)提供足够的灵敏度来检测大多数生物样品中发现的这些产物的水平，这些产物通常位于低皮摩尔范围内。该项目的第一个目标是完善罗德里格斯实验室目前正在开发的一项实验技术，利用稳定同位素标记的NO形式来跟踪生物系统中NO的命运。该技术基于现有的分析分析方法，用于定量no相关产品，包括最初为no -臭氧化学发光检测而开发的no修饰蛋白。该项目纳入了在这些分析中使用质谱法的步骤，以便能够以同位素特异性检测低皮摩尔水平的no相关产品。该项目的第二个目标是验证这种新开发的技术在生物流体和基质中的应用。为此，该技术将应用于一种具有良好特征的生物模型，即巨噬细胞免疫细胞，该模型能够以高通量产生NO。作为这一过程的一部分，我们将研究这些细胞应对一氧化氮相关产物亚硝酸盐潜在毒性水平的机制，以及它们如何利用这种物质来实现其免疫作用。初步结果表明，这些细胞可能具有一种机制，当它们的免疫机制被激活时，可以防止细胞内亚硝酸盐水平达到过高水平。在15n标记的亚硝酸盐和NO前体l -精氨酸的帮助下，亚硝酸盐在细胞内和细胞外浓度的时间变化，以及它的产生、代谢和进出这些细胞的运输速度将被量化。这些数量的知识，结合数学建模，将允许阐明哪些过程积极参与调控亚硝酸盐。同样的模型将允许确定这些过程是否允许有毒水平的亚硝酸盐在吞噬体中积累，吞噬体是吞噬入侵病原体的细胞内空间。更广泛的影响。教育：该项目将允许具有不同数学和科学背景的本科生在生物学研究中合作，将数学、化学、物理和工程结合起来。在这个过程中，他们会发现，并希望将这些学科的训练有助于促进对现代生物学的理解传递给其他人。改善研究基础设施：本项目开发的硬件设计和技术将通过会议和出版物传播。此外，该仪器将提供给其他研究人员，特别是路易斯安那州西北部的研究人员，他们中的许多人对了解NO在哺乳动物和植物系统中的作用感兴趣。该技术还将提高植物科学家研究植物氮吸收和代谢的能力。

Nitric oxide (NO) is synthesized in a variety of biological systems, fulfilling a wide range of signaling roles. Although the biological consequences of its synthesis are widely documented, its chemical modes of action and function, in vivo, remain controversial and largely unclear. One difficulty in pinpointing the biochemistry of NO stems from the lack of instrumentation capable of (1) tracing NO from its sources, through chemical intermediates, and to its protein targets, while (2) affording sufficient sensitivity to detect these products at levels found in most biological samples, which typically lie in the low picomole range. The first goal of this project is to perfect an experimental technique currently under development in the Rodriguez laboratory for tracking the fate of NO in biological systems, using forms of NO labeled with stable isotopes. The technique is based on existing analytical assays for the quantification of NO-related products, including NO-modified proteins, which were originally developed for NO-ozone chemiluminescence detection. Steps are incorporated in the project for use of mass spectrometry in these assays, in a way that will enable detection of low picomole levels of NO-related products with isotope specificity.The second goal of the project is to validate the use of this newly-developed technique in biological fluids and matrices. To this end, the technique will be applied to a well-characterized biological model capable of producing NO at high fluxes, namely the macrophage immune cell. As part of this process, the mechanism that allows these cells to cope with potentially toxic levels of the NO related product, nitrite, and how they may use this substance to fulfill their immunological role will be investigated. Preliminary results suggest these cells may possess a mechanism that prevents intracellular levels of nitrite from reaching excessive levels when their immune machinery is activated. With the aid of 15N-labelled nitrite and NO precursor L-arginine, the temporal changes in intra- and extra-cellular concentrations of nitrite, as well as its rate of production, metabolism, and transport in and out of these cells will be quantitated. Knowledge of these quantities, combined with mathematical modeling, will permit elucidation of which processes are actively involved in the regulation of nitrite. The same modeling will allow determination of whether these processes allow toxic levels of nitrite to accumulate within phagosomes, the intracellular spaces that engulf invading pathogens.Broader impact. Education: This project will allow undergraduate students with diverse mathematical and scientific backgrounds to collaborate in biological research that interfaces mathematics, chemistry, physics, and engineering. In the process they will discover, and hopefully pass on to others, the realization that training in these disciplines helps to advance the understanding of modern biology. Improving research infrastructure: The hardware design, and techniques developed in this project will be disseminated through meetings and publications. In addition, the instrumentation will be made available to other investigators, particularly those in Northwest Louisiana, many of whom have an interest in understanding the role of NO in mammalian and plant systems. The technique will also provide plant scientists with improved capability to study nitrogen uptake and metabolism in plants.