This is a revised proposal for five years of funding for the support of studies in the age-associated defect of neutrophil function in man. The Investigator proposes to continue with studies based on observations he and his coworkers have already made. The fundamental theme is the utilization of the neutrophil as a model of cellular aging, with the expectation that mechanistic studies of a defect in the function of """"""""aged"""""""" neutrophils, previously reported and studied by the Investigator, will lead to an understanding of the widespread and various defects in cellular function which accompany aging. The experiments are planned around four specific aims: 1) to define the age-related decline in signal transduction, 2) to explain why the age-related decrements in signal transduction are greater at early steps in the pathway than later events, 3) to define whether the age-related decline in function is heterogeneous among neutrophils, and 4) to identify the reversible change in the cell membrane that accounts for the age-related decrease in receptor-mediated responses. Further studies of the turnover of phosphoinositide precursors of DAG and IP3 were planned, and considerable progress has already been made with this, as described in the supplemental material. Also planned are studies of the number of high and low-affinity fMLP receptors of young and old cells. Studies of the effect of age on G protein regulation of receptor kinetics will concentrate on Gn, a CT binding protein thought to play a role in transducing the signal from the fMLP receptor to PLC. Experiments will be based on the concept that Gn is dissociated into three subunits on activation by flvILP, and that subsequently ribolsylated. The method for quantifying the GTP-ase activity will be measured by assessing the liberation of radioactive phosphorous from labeled GTP. ability of PT to catalyze ADP-ribosylation of the alpha subunit was described by Bokoch, who will act as a collaborator in the proposed studies. He has also developed immunoblot assays for the subunits1 and the Investigator includes a letter promising Dr. Bokoch's collaboration. Studies of the activity of PLC, PLD and PLA2 activity will also be carried out. Now in other studies, FACS will be used to assess membrane potential(di-1-C5(3)), formation of oxidative products (DCF), the mobilization of calcium (indo-1 AM) -and the binding of fluorescenated flVILF. An expert collaborator, Dr. Epstein at the University of Arkansas, will aid with this. Finally, membrane microviscosity will be estimated using the fluorescent probes, DPH, PRO-DPH, A-DPH, cis-parinarate and para-parinarate. Dr. Mrak will aid with this.
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