The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress–generated NO and activation of p38, and the significance of the PSD95–nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95–nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95–nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.
Targeting viral entry is one of the major goals in the development of vectors for gene therapy. Ideally, the coupling of each new targeting motif would not require changes in vector structure. To achieve this, we developed novel metabolically biotinylated baculoviral vectors by displaying a small biotin acceptor peptide (BAP) fused either to different sites in the baculovirus glycoprotein gp64 or to the transmembrane anchor of vesicular stomatitis virus G protein. Baculoviral particles were biotinylated during vector production by coexpression of Escherichia coli biotin ligase (BirA). The insertion of BAP at amino acid position 283 of gp64 resulted in the most efficient biotin display. Unlike vectors with lower biotin display, these vectors also showed improved transduction when retargeted to transferrin, epidermal growth factor, and CD46 receptors overexpressed on rat glioma and human ovarian carcinoma cells. Biotinylated baculoviral vectors could also be concentrated by one-step magnetic particle-based capture to reach titers up to 10(10) plaque-forming units/ml. These results demonstrate the utility of metabolically biotinylated baculovirus for vector targeting and viral purification applications.
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