Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.

Abstract. We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of ot and/3 integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (CtO~OL). This complex is immunoprecipitated by a polyclonal anti-human/~ integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including c~1/~, ~3/~1, and cts/~l complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The/3 subunit of the oligodendrocyte integrin (/3OL) and rat fibroblast/~1 have different electrophoretic mobilities in SDS-PAGE. However, the two/3 subunits appear to be highly related based on immunological cross-reactivity and one-dimensional peptide mapping. After removal of N-linked carbohydrate chains, /3OL and/~ comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of/31 and/3OL accounts entirely for their size differences. The oligodendrocyte oL subunit, O~OL, was not immunoprecipitated by antibodies against well characterized o~ chains which are known to associate with 13~ (o~3, ou, and Ors). However, an antibody to o~s, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to CXOL and /3OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containlng synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation.

Abstract. Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr ,030,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.

We have investigated the expression of integrins in C6 glioma, a chemically-induced glial tumor cell line from rat brain. Immunochemical analysis revealed that C6 cells express sets of integrin receptor complexes which immunologically and electrophoretically are indistinguishable from those expressed by normal rat skin fibroblasts. These include the well-characterized fibronectin (alpha 5 beta 1) and the multi-specific laminin, collagen and fibronectin (alpha 3 beta 1) receptors. Assay of cell adhesion indicated that C6 cells adhere to fibronectin-coated surfaces or matrix deposited by the C6 glioma cells (CGM) in an RGD- and divalent cation-dependent fashion. However, anti-fibronectin antibodies, which are able to inhibit fibroblast adhesion to fibronectin, did not inhibit adhesion of the C6 cells to fibronectin or CGM. This may reflect differences in functional properties and/or distribution patterns of integrins in C6 cells and normal fibroblasts.

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