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The activation of cultured Raw 264.7 murine macrophages with interferon gamma and lipopolysaccharide results in the expression of inducible nitric oxide synthase (i_NOS) and the subsequent production of nitric oxide. In the present study, the i-NOS expressed in these activated cells was characterized for possible post-translational protein modification by endogenous tyrosine protein kinases. Western-blot analysis using phosphotyrosine antibodies revealed that i-NOS was phosphorylated on tyrosine residues and that this was an early event coinciding with the appearance of newly synthesized i-NOS. A brief exposure of activated cells to vanadate, a tyrosine phosphatase inhibitor, significantly increased the level of i-NOS tyrosine phosphorylation, suggesting that tyrosine phosphatases are dynamically involved in the regulation of this process. Vanadate treatment of activated cells also resulted in a rapid increase in enzyme activity, occurring within 5 min of exposure. Taken together, these results demonstrate that tyrosine kinases and phosphatases are involved in the post-translational modification of i-NOS and may potentially play a role in modulating the functional activity of the enzyme in macrophages.

Alzheimer's disease (AD) pathology is characterized by senile plaques containing amyloid-b (Ab) peptide, a protein with neurotoxic and glial immune activating potential. In addition to the highly amyloidogenic peptides Ab(1±40/42), plaques contain amino-terminal truncated Ab peptides including the alpha secretase-generated p3 fragments Ab(17±40/42). In the present study, Ab(17±40/42), Ab(1±40/42), Ab(1±16), and Ab(25±35) aged in different solvents exhibited varying capacity to activate the murine microglia cell line MG-7 depending on solvent, peptidèaging', and peptide sequence that did not strictly correlate with b-sheet formation. Ab(17±40/42) or Ab(1±42) stimulated production of the pro-in¯ammatory cytokines interleukin (IL)-1a, IL-1b, IL-6 and tumor necrosis factor-a (TNF-a), and the chemokine MCP-1 from differentiated human monocytes (THP-1) while little or no stimulation was observed with the other Ab fragments. MG7 cells also produced these ®ve pro-in¯ammatory proteins in response to Ab(1±42) whereas Ab(17±40/42) elicited mainly TNF-a and MCP-1. Murine and human astrocyte cell lines (D30 and U373, respectively) were generally less responsive to Ab fragments producing mainly IL-6 and MCP-1 in response to Ab(1±42) or Ab(17±40/42) fragments. In mice, an intracerebroventricular infusion of Ab(1±42) signi®cantly increased IL-1a, IL-1b, IL-6 and MCP-1 while Ab(17±40/42) increased MCP-1 and Ab(17±40) increased IL-1b. These results demonstrate that p3 and p4 Ab fragments are pro-in¯ammatory glial modulators and thus may play a role in development of the immunopathology observed in AD.