The goal of this proposal is to determine the exact roles of lipid mediators (i.e. diacylglycerols [DAG's] and Presqualene Diphosphate [PSDP] in periodontal inflammation, and to develop novel molecular strategies to pharmacologically interrupt the inflammatory cascade. We will focus on Localized Juvenile Periodontitis (LJP), which presents neutrophils with a classically """"""""primed"""""""" phenotype, i.e. hyper-responsiveness in superoxide production during cell stimulation. Previous work by this group (see Project 1) has revealed that a new class of compounds, the so called aspirin triggered lipoxins that are potent natural inhibitors of inflammation in various models, and block superoxide production in neutrophils. These compounds are thought to exert their effects, at least in part, by increasing the cellular levels of PSDP. To accomplish our goals, we will characterize the biochemical basis for the primed state in LJP neutrophils and elucidate the molecular mechanism(s) responsible for PSDP inhibition of superoxide production by determining: 1. the chemical nature and the source of the elevated diacylglycerol (DAG) in LJP neutrophils; 2. the biochemical basis for the reduce activity of DAG kinase in neutrophils from LJP, including preparation of specific anti- peptide antibodies to the various isoforms and subtypes of DAG kinase to identify the species present in neutrophils, determining if the pronounced diminution of DAG kinase in neutrophils from LJP is due to the absence of one or more isoforms of this kinase, characterizing the isoform of DAG kinase that associates with Rac and Cdc42 in normal neutrophils, and determining if this situation is altered in LJP; 3. the mechanism(s) by which PSDP inhibits superoxide production in PMN stimulated permeabilized neutrophils, including inhibition of the phosphorylation of p47-phox, p67-phox or p40-phox, blocking assembly of the NADPH oxidase complex by inhibiting translocation of p47-phox, p67-phox and/or Rac to the plasmalemma, blocking activation of Rac (and other small GTPases) by inhibiting nucleotide exchange, finally comparing these experiments in normal neutrophils to those from LJP; and 4. characterizing the phosphatase that catalyses the conversion of PSDP to PSMP, including the subcellular location and substrate specificity of the PSDP-phosphatase (i.e. will this enzyme also catalyze dephosphorylation of phospatidate and sphingosine-1-P?), the kinetic (Km, Vmax) and physical properties (molecular weight, isoelectric point) of the enzyme, and the PSDP-phosphatase activity during cell stimulation.
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