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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. NIFTMORADII.)UM U J ~ ~ T0: Dr. S.C. Scmers and Staff FRC]M: Dr. D.H Ford and D. Stone Re: Site visit with Dr. H.P. Jones, University of South Alabama, Nbbile, AL April 26, 1984 Grant No. 1568 entitled "Calcium-dependent regulatory proteins and neutrophil activation. " Dr. Jones is an energetic young man with an excellent grasp of his subject area who at one time trained with Dr. J. Foster. He appears to have developed the same degree of intensity of interest for. his research as demonstrated by Dr. Foster. His laboratory is well equipped and he see3ns to have established close working relationships with a ntunber of other investigators. Goals: Zb understand the role of Ca++ dependent regulatory proteins (calmodulin and a protease)+0 regulating the activation of NADPH - oxidase systen in PMNs. Ca binds to the protein causing a structural change. The altered protein then binds to and activates the-NADPH - oxidase, which catalyzes superoxide formation. NP,DPH + 2 02 ~ NADP + H+ + 202 the Superoxide then is converted to a peroxide: 2 02 + 2H+ .- .)PH202 + O 2 myeloperoxidase then converts the peroxide to the hydrochlorous ion H202 + Cl > OCL- +H20 or, alternativelv forms the extremely cytotoxic hydroxyl radical Fe+~- 02 + H2O2 --* 02 + OH •+ OH ' or some of each Both OH• and OCl'"* are extremely cytotoxic in killing trapped bacteria. Further the superoxide produced by the PMNs appear to activate, a latent chemotactic factor in the plasma which recruits other PMNs to the infected area. The "flip side" is that the oxidants which kill the bacteria may also mutate, transform or age the host tissue and, therefore, may do as much harm as good. Progress: Due to several conflicting observations in the literature, Dr. Jones first had to demonstrate the calmodulin was really present in PNIlVs. His studies showed tWt it is present and his current data verifies that calmcdulin is Ca dependent in turning on NADPH -

2 oxidase activity. However, he also observed tint the enzyme in resting PNIlJs can not be activated by calmodulin and Ca . The cell has to be activated first, and the question is, what are the natural compounds which serve to activate the PMN for this function? He also still has to determine that the interaction between calmodulin and the oxidase is allostearic. Further, he has had to rule out that xanthine plays a role in the production of superoxide in this system. The data he has collected so far has lead him to conclude that it is not involved, or if it is, only to a minor degree. A further problem is to determine the factors which influence the avaliability of NADPH produced via the pentose phosphate shunt and the factors regulating the the levels of available precursor nuc-l.eotide. While he still has to prove it, Dr. Jones now believes that the whole system is turned off by the release of adenosine nucleotides derived from digested bacteria. Thus, what appeared to be fairly straightforward problem in biochemistry has has become a fairly complicated one. Thus, the PM will not go on digesting things (including the host cells or tissues) forever, but may be "self regulating" by the products of their own appetites. Dr. Jones has also made some progress in investigating the pro- tease regulator protein. He has developed an assay for the enzyme activity and noted that the enzyme is active at a neutral pH and that it is located in the PNIlV cytosol. Eventually Dr. Jones hopes to introduce cell-free systems in to the investigation of this problem. Two manuscripts have been submitted for publication since the inception of the grant, both of which ACK CTR. They are: "Allopurinol and superoxide production by neutrophils, "H. Jones, et, al.; and Calmodulin-dependent NAD kinase of humazz neutrophils," M.B. Williams and H.P. Jones. The material in both manuscripts has been presented at the FASEB meeting this past April and the abstracts ACK CTR. CocrKttent: A bright and enthusiatic young investigator who is showing excellent progress in an area of research of interest to CTR. His future plans appear to be logically derived from his concluded studies. Study merits continued CTR support. D.H. Ford and D. Stone DHF/DS/mla