No abstract

No abstract

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.

Intra-axonal recording and horseradish peroxidase labelling techniques were used to examine structure-function relationships for vibrissa-sensitive primary afferent fibers (N = 40) in rat trigeminal brainstem subnucleus interpolaris (SpVi). All responded at short (mean-0.42 ms) latencies to trigeminal ganglion shocks and to innocuous stimulation of an individual vibrissa in a slowly adapting type I, slowly adapting type IIa, slowly adapting type IIb, low-velocity-sensitive rapidly adapting, or high-velocity-sensitive rapidly adapting fashion. As in the medullary dorsal horn (Hayashi, '82; Jacquin et al., '86a), functionally distinct mystacial vibrissae-related fibers were morphologically indistinguishable. Each gave rise to up to ten collaterals that entered interpolaris perpendicular to the long axis of the nucleus and often overlapped to form a densely packed, highly circumscribed, and largely continuous column of terminal arbors. While some morphological variability was observed both within and between individual axons, variance within a given functional class was no greater than that between classes. Nonmystacial vibrissae afferent arbors also formed similar ovoid, dense circumscribed terminal plexuses. Presumably, these individual arbors collectively form a longitudinal tubelike vibrissae representation in this and other components of the trigeminal brainstem nuclear complex. The relative locations of each fiber's terminal field could be accurately predicted by the particular vibrissa innervated. Contrary to previous data obtained with similar methods (Hayashi, '82), but consistent with the findings of earlier transganglionic tracing (Arvidsson, '82) and histochemical (Belford and Killackey, '79) studies, the arbors of these fibers terminated throughout the mediolateral extent of SpVi. Axons innervating rostral vibrissae terminated medially, and those that supplied caudal vibrissae innervated the lateral SpVi. Dorsal vibrissae were represented in the ventral SpVi, while ventral vibrissae were represented more dorsally. This transverse topography extended largely throughout the rostrocaudal extent of interpolaris. In summary, the vibrissae primary afferent map in SpVi is inverted, faces medially, and is rostrocaudally consistent. In its most caudal aspect, as the displaced substantia gelatinosa swings laterally, the map is less orderly and incomplete. These generalizations also apply to the nonmystacial vibrissae afferents.