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，发挥广泛的信号作用。虽然其合成的生物学后果被广泛记录，但其在体内的化学作用和功能模式仍然存在争议，并且在很大程度上不清楚。 确定NO的生物化学的一个困难源于缺乏能够（1）通过化学中间体从其来源追踪NO并追踪其蛋白质靶点的仪器，同时（2）提供足够的灵敏度以检测在大多数生物样品中发现的水平的这些产物，其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实验技术，用于使用稳定同位素标记的NO形式跟踪生物系统中NO的命运。该技术是基于现有的分析测定NO相关产品的定量，包括NO修饰的蛋白质，这最初是为NO-臭氧化学发光检测开发的。该项目包括在这些分析中使用质谱法的步骤，其方式将能够检测具有同位素特异性的低皮摩尔水平的NO相关产物。该项目的第二个目标是验证这种新开发的技术在生物液体和基质中的使用。为此，该技术将被应用到一个良好的特点，能够产生NO在高通量的生物模型，即巨噬细胞免疫细胞。作为该过程的一部分，将研究允许这些细胞科普NO相关产物亚硝酸盐的潜在毒性水平的机制，以及它们如何使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的水平。借助15 N标记的亚硝酸盐和NO前体L-精氨酸，将定量测定亚硝酸盐的细胞内和细胞外浓度的时间变化，以及其生产、代谢和运输进出这些细胞的速率。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亚硝酸盐的调节。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内积累，吞噬入侵病原体的细胞内空间。学历：该项目将允许具有不同数学和科学背景的本科生在数学，化学，物理和工程接口的生物研究中进行合作。 在这个过程中，他们将发现，并希望传递给其他人，认识到这些学科的培训有助于促进对现代生物学的理解。改善研究基础设施：将通过会议和出版物传播这一项目中开发的硬件设计和技术。此外，仪器将提供给其他研究人员，特别是那些在路易斯安那州西北部，其中许多人有兴趣了解一氧化氮在哺乳动物和植物系统中的作用。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.