We have generated a hybridoma cell line secreting a monoclonal antibody that specifically binds to the surfaces of oligodendrocytes and Schwann cells, the cells involved in myelin formation in the central and peripheral nervous systems, respectively. Binding studies using purified sphingolipids showed that this antibody reacts strongly with galactocerebroside (GalC), the major galactosphingolipid of myelin. The antibody was used in conjunction with rabbit antisera against sulfatide, the sulfated form of GalC, to examine the developmental appearance of these lipids on the surfaces of oligodendrocytes and Schwann cells. In addition, the loss of GalC and sulfatide from freshly dissociated Schwann cells was compared. These studies showed that GalC is expressed on the cell surface prior to sulfatide on both of these cell types in vivo and in vitro. Conversely, dissociated Schwann cells lose their cell surface sulfatide more rapidly than they lose their surface GalC under nonmyelinating conditions. Certain lipids and proteins are thought to be predominantly found in myelin (1). Among these are the galactosphingolipids, galactocerebroside (GalC) and sulfatide. Despite their myelin association, the relationship of these lipids to the development ofoligodendrocytes and Schwann cells (the myelin-forming cells ofthe central and peripheral nervous systems, respectively) has not been studied on a cellular level in detail. Previous work, using either chemical analysis of extracted lipid or radiotracer studies, has described the developmental appearance ofmyelinspecific components in the nervous system (2). However, the techniques used were unable to establish the sequence in which components appear in individual cells or to resolve when such lipids are first inserted into the plasma membrane. Given that these lipids may play a role in the neuronal-glial interaction that results in myelination (3), it is important to ascertain their exact sequence and onset of appearance.One method that allows developmental studies on a cellular level is immunohistochemical labeling of cells with specific, characterized antisera. It has been established that in culture, polyclonal antisera against GalC and sulfatide specifically stain oligodendrocytes and Schwann cells (4). However, such work has suffered from the difficulty of adequately characterizing such antisera, which are often of low titer and questionable specificity.In the present study, we describe a hybridoma cell line that produces a monoclonal antibody binding specifically to the surfaces of oligodendrocytes and Schwann cells. A modification to existing enzyme-linked immunosorbent assay (ELISA) techniques was developed that allows the characterization of antibodies to lipid determinants. With this assay, the monoclonal antibody was shown to have a high specificity for GalC and, consequently, was designated mGalC. Various polyclonal rabbit antisera were similarly characterized and shown to bind to either GaiC or sulfatide. We used mGalC and rabbit antisera in double-labeli...
The dependence of 3[H]-L-glutamate uptake on the presence of sodium, chloride, or calcium ions or on a combination of the three was investigated in astrocyte primary cultures. A stimulating effect on glutamate uptake by each of the ions tested was found. In addition to the comparably small effect by calcium alone, calcium exhibits a synergistic effect on the sodium- and chloride-dependent uptake. The sodium-dependent transport accumulates the glutamate analogue D-aspartate as well as L-glutamate. L-aspartate is taken up by about 50% of the values observed for L-glutamate transport. Sodium-dependent glutamate uptake is strongly inhibited by aspartate-beta-hydroxamate (A beta H) and threo-beta-hydroxyaspartate (T beta H). Quisqualate is less potent in inhibiting this uptake. In contrast, the chloride- and the calcium-dependent uptake systems do not handle D- and L-aspartate as substrates. A beta H and T beta H are only poor inhibitors of these transporters while quisqualate reduces glutamate uptake almost completely. Kinetic data of all uptake systems were estimated. High and low affinity components of each individual system are demonstrated by Eadie-Hofstee analysis. Hill plots indicate that high and low affinity uptake may be due either to two respective uptake sites for Na(+)-, Cl(-)-, and Ca(++)-dependent glutamate transport, or to two glutamate binding sites for each single transport system with negative cooperativity.
A nearly pure neuronal culture from embryonic rat brain (hippocampus) has been established in order to observe individual neurons during their in vitro development. This neuronal culture has been used as a test system for a neurotrophic factor (NTF) which is released into a serum-free defined medium, presumably by astrocytes, the dominant cell type in a primary glial culture from rat brain. NTF is essential for the development of the hippocampal neurons in cell culture. The number of neurons responding to NTF by neurite extension is highly dependent on the concentration of the factor in the culture medium. The amount of factor in serum-free glial conditioned medium can be estimated in a rapid (20-hour) bioassay.
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