James Travis - US grants

No summary has been provided to the Smithsonian Science Information Exchange.

No summary has been provided to the Smithsonian Science Information Exchange.

emphysema; alpha 1 antitrypsin deficiency; alpha 1 antitrypsin; enzyme structure; liver cells; glucose analog; genetic transcription; genetic manipulation; conformation; secretion; Golgi apparatus; microsomes; inclusion body; molecular pathology; isozymes; gene therapy; molecular cloning; neoplastic cell culture for noncancer research; tissue /cell culture; human tissue;

The objectives of this proposal are to continue investigations into the mechanisms by which inflammatory reactions occurring in the lung result in connective tissue damage. These studies primarily involve an examination of the proteinase-proteinase inhibitor balance within the lung, mechanisms by which this balance can be perturbed in favor of free enzyme, and the function of proteinase:proteinase inhibitor complexes in signalling for synthesis of acute phase proteins to offset this imbalance. In particular, the binding of both inhibitor:enzyme complexes and proteolytically inactivated inhibitors to specific receptors on cell surfaces will be followed to determine whether this induces cytokine synthesis and, indirectly, both protein synthesis and chemotaxis. Since the three-dimensional structure of the native form of inhibitors involved in the regulation of endogenous proteinases involved in tissue destruction is unknown (members of the Serpin family), it is also planned to complete the sequence and determine the crystal structure of one of these which has been isolated from equine leukocyte cytosol. In a separate study an investigation of the role of neutrophil proteinases in cell migration will also be carried out to determine whether such enzymes have alternate roles in addition to those involving the degradation of foreign or denatured proteins during phagocytosis. Finally, a study of the potential role of proteinases from pollens in lung disease will be initiated in order to determine whether such enzymes present in these gametes might be involved in the development of allergies or asthma.

We are requesting funds for an ultrasensitive protein sequencer with a supporting high pressure liquid chromatography apparatus to be placed in a central facility along with a gas-phase sequenator already installed. Nine faculty, eight from the Department of Biochemistry, have submitted proposals which require use of this equipment. All the projects have substantial funding by NIH and in some cases other Federal agencies and are diverse in their substance: structures of cystic fibrosis mucin and other glycoproteins, sequences of proteins involved in reproduction, gene expression and development, structures of proteins involved in cell communication, structures including active sites of enzymes implicated in diseased states, bacterial redox proteins, enzymes important in bioluminescence biotechnology and vitamin Bl2 synthesis. All these projects require the enhanced sequencing sensitivity provided by the apparatus, which though primarily for the Biochemistry Department will be made available to a very active faculty in the Biological Sciences Division as well. Although a similar apparatus is already operational on campus, the usage of that instrument is so intense that a four-month waiting list currently exists, a waiting period that grows steadily longer. A full-time highly effective staff member operates the available instrument and would be in charge of the requested apparatus as well.

[unreadable] DESCRIPTION (provided by applicant): A primary organism involved in the development and progression of periodontal disease is the periodontopathogen, Porphyromonas gingivalis. This bacterium utilizes an arsenal of virulence factors to avoid host defense, allowing for its growth and proliferation at infected sites. Among these factors are proteolytic enzymes, which appear to be involved in the deregulation of cascade pathways, the inactivation of plasma proteinase inhibitors, and the disruption of chemotactic processes, all to the benefit of the invading organism. Many of these enzymes have been isolated and characterized in this laboratory, and it seems clear that inhibitors developed against them would significantly reduce the pathological symptoms associated with periodontitis. Nevertheless, in some types of periodontal disease, especially its early phase, other bacterial pathogens are likely to be involved. For these reasons, the Specific Aims of this project are as follows: 1) to expand the model we have developed which directly points to major roles for P. gingivalis proteinases in the pathological hallmarks of periodontal disease (bleeding on probing, swelling, tissue resorption, bone loss), 2), to initiate experiments to determine if other bacterial pathogens which are found at the early stages of periodontal disease also utilize proteolytic enzymes for host defense evasion, and 3) to screen for and purify low and high molecular weight proteinase inhibitors which may be synthesized by other oral organisms for the possible control of P. gingivalis growth and proliferation. Our long-term goals are to determine whether the mechanisms used by P. gingivalis to avoid host defense are a general process that other pathogens have adopted and to develop inhibitors against such enzymes in order to control or eradicate bacterial infections.

emphysema; protein biosynthesis; enzyme structure; alpha 1 antitrypsin deficiency; alpha 1 antitrypsin; liver cells; glucose analog; genetic transcription; genetic manipulation; secretion; Golgi apparatus; microsomes; inclusion body; conformation; molecular pathology; isozymes; gene therapy; molecular cloning; glycosylation; neoplastic cell culture for noncancer research; tissue /cell culture; human tissue;

emphysema; protein biosynthesis; enzyme structure; alpha 1 antitrypsin deficiency; alpha 1 antitrypsin; liver cells; glucose analog; genetic transcription; genetic manipulation; secretion; Golgi apparatus; microsomes; inclusion body; conformation; molecular pathology; isozymes; gene therapy; molecular cloning; glycosylation; neoplastic cell culture for noncancer research; tissue /cell culture; human tissue;

The regulation of proteolysis is considered to be a pivotal requirement in the maintenance of homeostasis in all living organisms. This delicate balance, however, is continually being challenged by a triad of factors which may cause an increase in proteolysis, of primary importance being those effects exerted by proteinases from non-host systems. Thus, there is enormous interest in examining not only the mechanisms by which host enzymes and inhibitors interact but also how proteinases from viruses, pathogenic bacteria, and parasites deregulate tightly controlled biochemical pathways, such as the coagulation, complement, kallikrein/kinin, and cytokine systems, during inflammatory episodes. Significantly, one can now extend such studies into the plant field where it is now possible to understand not only haw proteinase-proteinase inhibitor synthesis is induced but also haw it may be enhanced in order to aid in both plant defense and food production. This symposium has been designed to bring together a broadly-based group of scientists whose primary interests involve an understanding of how proteinases and inhibitors from all living organisms and viruses function in normal and pathological states, and how the latter may be rectified by the use of either natural or synthetic inhibitors. It has also been organized in order to attract both graduate students and young M.D. and Ph.D. research scientists to a meeting place where they may become more familiar with a rapidly expanding area within the biosciences which is already of premier importance in pathobiochemistry, nutritional biochemistry, plant biochemistry, and virology.

The goals of the current proposal are to continue investigations into the role of proteinase inhibitors and their target enzymes in the development of lung disease. Focus will be made on determining the contribution of lung epithelial cells in synthesizing and secreting functional acute phase proteinase inhibitors for defense against tissue proteolysis in this by organ by neutrophil-derived proteinases. In this context, the specific aims of the grant involve a) an investigation of the properties of the epithelial cell-derived inhibitors and b) assessment of the effect on epithelial cells of various cytokines which are known to stimulate acute phase protein synthesis. Because the lung can easily be infected by a variety of bacterial and fungal organisms, many of which can release their own, uncontrolled proteolytic activities, experiments are also planned c) to examine the stability of cytokines involved in either acute phase protein synthesis or in neutrophil chemotaxis, after- treatment with non- host proteinases and d) to determine whether such enzymes can also degrade receptors for cytokines and chemotaxins on specific target cells. All of these results should give significant information as to whether locally synthesized proteinase inhibitors do contribute to lung defense against uncontrolled proteolysis by host-derived enzymes. In addition, important data will be obtained regarding the ability of host and non-host proteinases to dysregulate cell to cell communication through either cytokine or receptor degradation.

high performance liquid chromatography; aminoacid analyzer; biomedical equipment purchase;

Apple trees do not reproduce true-to-type from seed. Instead, desirable cultivars are propagated by grafting vegetative material onto suitable rootstocks. Rootstocks can alter scion traits of importance to agriculture, including fruit size, flowering timing, the number of flowers and fruits produced, drought tolerance, disease resistance, cold tolerance, and tree stature. In effect, the traits of genetically identical apple cultivar scions can be very different depending on the rootstock to which they are grafted. This project harnesses this phenomenon to identify genes in apple trees that are involved in controlling agriculturally important traits. The central hypothesis driving this research is that scions grafted to different rootstocks have different overall patterns of gene activity, and that these differences in gene activity play a role in determining the trait differences between the trees. Global gene expression patterns in "Gala" cultivar scions grafted to a series of different rootstocks will be compared. Genes whose expression level is rootstock-dependent will be identified (at least 500 unique genes). A DNA microarray comprised of the differentially expressed genes will be developed and used to survey a large number of scion/rootstock combinations in order to associate the expression of particular genes with particular traits.

This project will result in the identification of genes whose activity depends on the identity of the rootstock. The sequences of these genes will be made public though databases including GenBank (http://www.ncbi.nlm.nih.gov/) and the Genome Database for Rosaceae (http://www.genome.clemson.edu/gdr/). The results of the microarray experiments will be made available to the public though GEO database (http://www.ncbi.nlm.nih.gov/geo/). The sequence and microarray data will be made publicly available through the GEO database as soon as the first manuscript describing the results is accepted for journal publication, and will remain accessible after the completion of the project into the foreseeable future. The microarrays themselves will be made available at cost to other non-profit research groups. The broader impacts of this work include minority undergraduate student summer training through the SROP program (http://www.cic.uiuc.edu/programs/SROP/). Each summer, two or three underrepresented undergraduates will be given the opportunity to participate in the project. In addition, one or more graduate students will receive training in genomics research and undergraduates will be engaged in research related to this project during the academic year. The results of this project may also be useful for the development of gene activity markers that could be used in apple breeding programs to try to predict the traits of a tree early on in its development.