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Axolemma-enriched fractions isolated from rat CNS stimulated cultured Schwann cells to divide without changing their morphology. Fluorescent activated cell sorter analysis of the axolemma-stimulated cells demonstrated an approximate 3-fold increase in the number of Schwann cells in the S-phase of the cell cycle. This increase correlated well with increases in the number of [3H]thymidine-labeled nuclei observed by light level radioautography. The membrane-bound mitogen was relatively heat-stable, but trypsin-sensitive, and was inactivated both by lipid extraction and sonication. Liver plasma membranes did not increase the mitotic index over that of untreated cells, indicating the axolemma-induced mitosis was not a general response to exogenous membranes. Increasing serum concentrations in the presence of a constant level of axolemma did not change the mitotic index, suggesting that the axolemma did not cause mitosis by removal of an inhibitory factor in serum. Potential mitogens such as gangliosides, myelin basic protein, heparin, an axolemmal lipid extract, and cGMP had no effect on the cultured Schwann cells. The characteristics of the axolemma-related mitogenic factor are discussed relative to other known mitogens for cultured Schwann cells.

Water-soluble proteins were extracted from individual retinas, optic nerves, combined optic tracts and lateral geniculate bodies, and superior colliculi of rabbits at 1, 3, and 18 days after injection of [3H]leucine into the right eye. The Ca2+-dependent protein modulator of 3′:5′-cyclic-AMP phos-phodiesterase (calmodulin) was isolated from these samples by a two-step polyacrylamide gel electrophoresis procedure. An analysis of the radioactivity incorporated into the total soluble proteins and the calmodulin revealed that most of the calmodulin was axonally transported at a slow rate (2–4 mm/day) and represented about 0.45% of the total transported soluble protein.

Polypeptides (233 micrograms) from brain synaptosomes resolved in SDS 7.5%--15% polyacrylamide gradient gels were electrophoretically transferred quantitatively to nitrocellulose sheets at 3 V/cm for 21 h in the following buffer: 25 mM Tris-192 mM glycine, pH 8.3/20% methanol/0.1% SDS. After immunoautoradiographic detection of antigens on this nitrocellulose replica with either rabbit anti-rat cerebrum immunoglobulins or a mouse monoclonal antibody to a rat synaptosomal protein, the antibody and [125I]protein A were removed from the replica by treatment with 8 M urea, 0.1 M 2-mercaptoethanol, and 5 mg/ml BSA at 60 degrees C for 1 h. The replicas were successfully re-used by re-exposing them to the antibody and [125I]protein A.

The ultrastructure of cuticle formation and the effect of actinomycin-D on cuticle formation were examined in the second molt of Nippostrongylus brasiliensis . At the end of the first molt the rough endoplasmic reticulum of the hypodermis (epidermis) had narrow cisternae. As cuticle formation began the RER cisternae dilated and accumulated a fibrous material. These large fibrous material-containing vesicles then decreased and by the time cuticle deposition was completed they had disappeared. Actinomycin-D prevented the proliferation of RER and the formation of fibrous material-containing vesicles. Actinomycin-D induced several ultrastructural alterations in the hypodermis, the most prominent of which were large crystalline-like inclusions. Other alterations involved the configuration of RER and structure of nucleoli. These morphological aspects of cuticle formation were correlated to the molecular events that control molting.

Light microscope radioautographic procedures were employed to investigate the binding patterns of lectins derived from Lotus tetragonolobus (Lotus A) and Ulex europeus (UEA I) to the surfaces of rat cerebellar cells maintained in dispersed cell culture. Lotus A bound extensively to the surfaces of neuronal cell somata and neuronal processes at all time points investigated; the lectin also bound to nonneuronal cell surfaces, but to an extent much less than that for neurons. Lectin binding was completely inhibited by the presence of 0.1 M alpha-L-fucose in the reaction medium. Conversely, UEA I did not appear to bind to any significant extent to the surfaces of cerebellar cells in vitro at any stage of maintenance. The incorporation of [3H]fucose into fucosyl-conjugates by cerebellar neurons in culture was investigated using light microscope radioautography and polyacrylamide gel electrophoretic methods. Radioautographs indicated that the fucose came to be distributed throughout neuronal cell somata and processes and that, with increasing time, the grain density appeared to be highest over areas of neuropil and fascicles of cellular processes. The electrophoresis data demonstrated that, among others, the label was incorporated into prominent classes of polypeptides having nominal molecular weights of 400,000, 125,000, 91,000 and 46,000 daltons.

The complement of cell surface proteins of differentiating rat cerebellar neurons maintained in dispersed cell cultures was evaluated by the combined methods of lactoperoxidase-catalyzed radioiodination and miniature sodium dodecyl sulfate polyacrylamide gel electrophoresis. Pure neuronal cell samples were obtained by direct dissection of such cells from the surfaces of the culture vessels. The electrophoretic profiles of cell surface proteins (CSP) from neurons maintained for 1, 7, 14, 21 and 28 days in vitro (DIV) were compared. It could be seen that while the pattern was relatively constant between 7 and 28 DIV, several differences existed between profiles from 1 and 7 DIV. The earliest stage was characterized by a prominent polypeptide triplet having molecular weight of approximately 48,000, 56,000 and 71,000 daltons. Later stages of development (7-28 DIV) were characterized by a greater number of high molecular weight surface polypeptides relative to the earliest stage. Comparison of electrophoretic profiles of CSPs of neurons and nonneuronal cells indicated that a number of polypeptides were not shared by these classes of cells.

The distribution of the large, external, transformation-sensitive (LETS; fibronectin) protein was investigated in rat cerebellum, both in vitro and in vivo, by biochemical and immunocytochemical methods. Biochemical analyses indicated that LETS protein is not demonstrable on the surfaces of cerebellar neurons from postnatal rats maintained in cell culture for varying periods of time, but is present on the surfaces of at least some fraction of the total nonneuronal cell population in vitro. Indirect immunofluorescence microscopy with an anti-LETS antiserum substantiated these observations and further indicated that LETS-bearing cells of cerebellum maintained in vitro are probably of endothelial and fibroblastic origin. The LETS protein is arranged in a reticular network of filaments spanning the surfaces of the cells, and the filaments are often extensively interdigitated with each other. At all stages of development investigated (two days postnatal to adult) LETS antigen was observed in vivo to be primarily localized in the meninges covering the surface of the cerebellum and between folia, and in the walls of blood vessels within the tissue. Neuroblasts and neurons of the external and internal granule layers of the cerebellum, respectively, were negative for the presence of LETS antigen.