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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. MEMORANDUM TO: Dr S.C. Sommers and Staff From: D.H. Ford and D. Stone Re: Site visit with Dr. V. Richmond, March 20th, 1984 at the Pacific Northwest Research Foundation, Seattle, WA. Grant No. 1451R1 entitled "Microfibrillar protein structure." Site visitors: Drs. J. Feldman, D.H. Ford and D. Stone Goal: To elucidate the structure of a putative microfibrillar protein. Rich- mond suggests that this is a glycoprotein associated with elastin and is believed to be laid down just before or concurrently with elastin. Procedure: This involves an extraction process wherein the success of the extraction of the microfibrillar protein from elastin is determined by elect- ronmicroscopy of the elastin residue after extraction. If there is no electron dense material associated with the residue, the microfibrillar protein is assumed to be extracted, though it is not clear what other materials might also be extracted. The solvent phase is then assumed to contain the microfibrillar protein. The presence of a protein in this solute fraction is then determined by HPLC techniques, which Dr. Richmond is still learning. A large and several smaller peaks were obtained (see Fig.2 of progress report# 2). Dr. Richmond believes the large peak is the microfibrillar protein and that it is uncontaminated by elastin•due to the absence of desmosine. It was suggested by the site visitors that before she spend innumerable hours determining the structure of this protein, it might be well to develop monoclonal antibodies to the protein and other fractions and determine by EM immunocytochemical techniques whether or not the antibodies associate with what is microfibrillar pro- tein in intact elastin. In other words, is her fraction the true microfibrillar protein or some other fraction? The question is relevant because what she extracts and believes to represent the fibrillar protein may be two materials. One from within the elastin and one which appears to condense around the outside of the elastin and may represent some form of condensation of proteins in the extracellular fluid which become precipitated during fixation. This may be distinctly different from th, intraelastin fibrillar structures. Her HPLC separation of her extract shows one large and two intermediate sized peaks + several smaller peaks. Which is the micro_ fibrillar protein? Which should she use to determine the structure? Here is where we recommended the monoclonal approach. Other issues: The slaughter house from which she obtained her pig aorta has closed and while she feels she will be able to find another, she was not too clear about this. Her knowledge of electronmicroscopy is minimal at best, the work being done by others. Whether or not she will be able to develop a series of monoclonal systems to her 3 plus protein fractions and then have EM immunocytochemistry performed is debatable. While the earlier problem with numerous crystals appearing in the EM micrographs has been resolved, she is not clear as to how or why. In general, she appears to have difficulty in defining her problem and its goals. Then once she has

arrived at some sort of an idea as to the nature of the problem, she appears un- certain as to how to establish relevant techniques to answer the questions raised. She appears to be a biochemist embarked on a problem, which requires morphological and immunological skills as well initially. If there truely is a microfibrillar protein associated with elastin, the mechansims for the control of its synthesis, turnover and physiological role will also have to be determined. Richmond apparently would be satisfied with determining its structurP, but first she has to know what it is of which she is determining the structure. Comment: Since this is a low budget program, we would recommend that she be funded for her final year in the hope that she may make some progress toward develop- ing a monoclonal system from her proteins and determine whether or not these proteins fractions which she is extracting do relate to what investigators are referring to as the microfibrillar protein of elastin. D. Ford and D. Stone :mla