DEVELOPMENT OF A CD-ROM OF CHROMATOGRAPHIC IMAGES

在这个项目中，我们建议建立一个包含图像的CD-ROM数据库 高度核实的化学物质的色谱图，特别是那些 癌症/健康研究。对这些图像进行适当的索引将使 快速检索，我们将研究如何使光盘用户能够 将这些数据下载到现有的色谱分析软件中进行 自动分析。这张光盘的设计目的是解决两个问题 与色谱学研究活动相关：访问 快速、适当和可靠的色谱图，并在使用这些 参考色谱图进行计算。一般说来，层析 是用于分离/纯化组件的一系列技术 利用不同运输方式的化学混合物 这些化学物质在某些介质中的性质。的应用 色谱学包括分析研究和 提纯/生产。色谱学与其说是一门艺术不如说是一门艺术 科学在某种意义上说，选择一种“媒介”和相关的 条件(温度、压力、柱长等)通常基于 基于先例，而不是基于第一原则的计算。 然而，在期刊中找到可靠和完整的先例并不是 容易，而且，即使一个人有一个完整的先例说明，一个 调查员通常必须从这种情况推断出他/她的情况 可以做到--因为要分离的混合物与 公布的案例，或者是因为现有的分离设备 有些不同，或者是因为所需的性能不同。 这种从已发表的案例中推断出来的方法通常是通过“目测”来完成的。 现有的色谱图，这是一种允许改进的技术。 格兰特-R01AI32097 这项提议的主要焦点是对致命的 败血梭菌毒素(α毒素)。来自这些的数据 实验将为进一步研究该毒素奠定基础 以及它在败血症中的作用。我们的长期目标是获得 更好地了解影响经济增长的因素 败血链霉菌的致病性及潜在治疗药物的开发 这将降低非创伤性败血症的高死亡率。 毒气坏疽。败血症是非创伤性气体的主要原因。 结肠癌或癌症患者的坏疽(远端肌坏死) 转移到结肠、白血病和循环性中性粒细胞减少症。 感染败血症的死亡率通常为50%-100%，这一数字 可能是由于感染的暴发性和 生产有毒物质。相对较少的是 关于因素对疾病过程的贡献的信息 由败血链霉菌产生。然而，现有的证据和临床上的 观察表明，致命因素在几个层面上都有影响。 疾病的进程。败血症梭菌产生的唯一致死因子 到目前为止已经被确认的是阿尔法毒素。一种基本的方法来处理 由于α毒素似乎是一种新的毒素，因此对它的研究是必要的。 这个建议的具体目的是：1)克隆和测序该基因 2)继续研究其膜机制。 裂解；3)纯化能进行蛋白水解性的霉菌蛋白水解酶 激活α毒素；4)阐明蛋白水解物上的裂解部位 负责激活原毒素的原毒素。几个 败血症杆菌染色体DNA基因库已经建立，这些将 用平板法或溶血法筛选毒素基因 硝酸纤维素上的菌斑升高和对α的亲和纯化抗体 毒素。或者，一种涉及到生成一个 或更多基于氨基末端的冗余寡核苷酸探针 α毒素序列可用于毒素基因的筛选。Alpha 我们已经证明毒素是一种聚集的、形成毛孔的溶血素。 第二个目标是继续检查渠道的性质 在平面双层中的形成，确定通道的孔径和 检测原毒素的膜结合特性和 活化型毒素与放射性标记的经典结合分析 毒素。将使用特定的任一特定的方法来纯化败血症梭菌的蛋白酶。 亲和方法或经典的高分辨率层析(某些步骤 都已经制定出来了)。α毒素的蛋白水解性激活 通过在羧基末端区域的裂解进行。所在的地点 胰酶和败血症梭菌蛋白酶裂解并激活毒素将 通过获得活化形式的精确分子质量来阐明 用电子喷雾质谱仪(EMS)分析阿尔法毒素。分子 通过EMS分析获得的质量将与 用于鉴定这些酶的裂解位点的初级序列。

In this project we propose to build a CD-ROM database containing images of highly verified chromatograms of chemicals, especially those pertinent to cancer/health research. Suitable indexing of these images will make retrieval rapid, and we will research ways to enable CD-ROM users to download these data to existing chromatogram analysis software to perform automated analyses. This CD-ROM will be designed to address 2 problems associated with research activities in chromatography: accessing appropriate and reliable chromatograms quickly, and in using those reference chromatograms to make computations. In general, chromatography is a family of techniques used to separate/purify the components of chemical mixtures by taking advantage of the different transport properties of those chemicals in some medium. Applications of chromatography include analytical studies as well as purification/production. Chromatography is more like an art than a science in the sense that the choice of a "medium" and the associated conditions (temperature, pressure, column length, etc.) is usually based on precedent rather than on a calculation from first principles. However, locating reliable and complete precedents in journals is not easy, and, even when one has a complete specification of precedent, an investigator must usually extrapolate from that condition to what he/she can do - because the mixture to be separated is somewhat different from the published case, or because the separation equipment that is available is somewhat different, or because the performance required is different. This extrapolation from published cases is usually done by "eyeballing" the available chromatograms, a technique allowing room for improvement. GRANT-R01AI32097 The primary focus of this proposal is the characterization of the lethal toxin (alpha toxin) from Clostridium septicum. The data from these experiments will lay a foundation for the continued study of the toxin and its role in the C. septicum disease. Our long term goals are to gain a better understanding of the factors that contribute to the pathogenicity of C. septicum and to develop potential therapeutic agents that will decrease the high mortality rate from C. septicum nontraumatic gas gangrene. C. septicum is the primary cause of nontraumatic gas gangrene (distal myonecrosis) in patients with colonic cancers or cancers which have metastasized to the colon, leukemia and cyclic neutropenia. Mortality from C. septicum infection is usually 50-100%, a figure which is probably the result of the fulminant nature of the infection and the production of toxic substances. There is comparatively little information on the contribution to the disease process of factors produced by C. septicum. However, the available evidence and clinical observations suggest the involvement of a lethal factor at several levels of the disease process. The only lethal factor produced by C. septicum that has been identified to date is alpha toxin. A basic approach to the study of alpha toxin is necessary since it appears to be a novel toxin. The specific aims of this proposal are: 1) clone and sequence the gene for alpha toxin; 2) to continue to characterize its mechanism of membrane lysis; 3) purify the C. septicum protease that can proteolytically activate alpha toxin; 4) elucidate the proteolytic cleavage site on the protoxin that is responsible for activation of the protoxin. Several gene banks of C. septicum chromosomal DNA have been generated, these will be screened for the toxin gene using plate hemolytic activity assays or plaque lifts onto nitrocellulose and affinity purified antibody to alpha toxin. Alternatively, a procedure that involves the generation of one or more redundant oligonucleotide probes based on the amino terminal sequence of alpha toxin can be used to screen for the toxin gene. Alpha toxin has been shown by us to be an aggregating, pore forming hemolysin. The second aim will be to continue to examine the nature of the channel formation in planar bilayers, determine the pore size of the channel and to examine the membrane binding characteristics of the protoxin and activated toxin using classical binding analysis with radiolabelled toxin. The C. septicum protease will be purified using either specific affinity methods or classical high resolution chromatography (some steps have already been worked out). The proteolytic activation of alpha toxin proceeds by a cleavage in the carboxy-terminal region. The site at which trypsin and the C. septicum protease cleave and activate the toxin will be elucidated by obtaining precise molecular mass for the activated form of alpha toxin by electron spray mass spectroscopy (EMS). The molecular mass obtained by EMS analysis will be used in conjunction with the primary sequence to identify the cleavage site of these proteases.