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.
