Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract

Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.

Neurite‐promoting activity from fetal skeletal muscle: Immunological comparison with laminin

“…laminin chain that is recognized by neurons is the same The bovine skeletal muscle PNPF is different from the in laminin and PNPF, but that nearby antigenic determineurite-promoting factor isolated from chicken gizzard nants recognized by blocking antibodies are conformasmooth muscle, in that the chicken factor has very weak tionally different between PNPF and laminin. Matthew immunological crossreactivity with mouse laminin [Miki et a1 [reported in Patterson, 19851 have mapped the bindet al, 1981;Hayashi et al, 1984; ing of a monoclonal antibody that blocks neurite promo-19851. However, these results indicate that the PNPF tion by PNPF to a determinant that consists of a heparan from bovine skeletal muscle is very similar to neurite-sulfate and laminin or laminin-like chain, but this deterpromoting factors isolated from culture media condi-minant is not present on laminin alone.…”

“…The purified PNPF from chicken gizzard smooth muscle tissue was different from PNPFs from other sources, in that it showed almost no crossreactivity with antilaminin antiserum, either under native conditions or in immunoblots of denatured preparations. From physical and immunoreactivity criteria this PNPF was designated as a unique molecule that is clearly distinguishable from murine laminin [Miki et al, 1981;Hayashi et al, 1984;Hayashi and Miki, 19851. Laminin has binding sites for heparin, and heparan sulfate proteoglycans are a component of at least several of these PNPF complexes.…”

Purification and characterization of an 82-kD membrane protein as a neurite outgrowth factor binding protein: possible involvement of NOF binding protein in axonal outgrowth in developing retina.

“…Indirect immunohistochemicai examination of thin sections onto gelatincoated coverslips cut in a cryostat was perforrned as described previously (7). Sections were incubated with antiserum against NOF binding protein at a dilution of 1:500 for 12 h at 4"C. After staining with fluoresceinconjugated goat anti-rabbit immunoglobulin at a dilution of 1:500 for 12 h at 40C, the samples were examined with an Olympus IMT-2 fluorescence microscope.…”

“…Bioassay was carried out as previously described (7). The preparation containing NOF was centrifuged for 30 rain at 10,000 g, and the supernatant was collected aseptically.…”