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Biomedical subjects

M C Raff

Publications and source records attributed to M C Raff.

At least 109 records · Page 6Linked to original sources
PubMed1983

Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics.

Two types of glial fibrillary acidic protein-positive (GFAP+) astrocytes were found in cultures of developing rat optic nerve. Type 1 astrocytes had a fibroblast-like morphology, did not bind tetanus toxin or the monoclonal antibody A2B5 (both of which bind to specific polysialogangliosides), and were stimulated to divide by an extract of bovine pituitary and by epidermal growth factor (EGF). Type 2 astrocytes had a neuron-like morphology, bound tetanus toxin and A2B5 antibody, and were not stimulated to divide by bovine pituitary extract or by EGF. Although both types of astrocytes were present in cultures of white matter, only type 1 astrocytes were found in cultures of gray matter. Astrocytes did not convert from one type to the other in culture: while many type 1 astrocytes adopted a neuron-like morphology when exposed to dibutyryl cyclic adenosine 3':5'-monophosphate, or pituitary or brain extracts, especially in serum-free medium, such morphologically altered cells did not bind tetanus toxin or A2B5 antibody. Although small numbers of tetanus toxin-binding, A2B5+, GFAP+ cells were present in suspensions of freshly dissected, neonatal optic nerves, most of the type 2 astrocytes in cultures of such optic nerves developed from tetanus toxin-binding, A2B5+, GFAP- cells, which were induced to express GFAP by the culture conditions. Since type 2 astrocytes have a neuron-like morphology and bind tetanus toxin and A2B5 antibody, these ligands cannot be used on their own as neuron-specific markers in central nervous system cultures.

Animals↗
PubMed1981

Rat neural antigen-2 (RAN-2): a cell surface antigen on astrocytes, ependymal cells, Müller cells and lepto-meninges defined by a monoclonal antibody.

We have immunized mice with enriched populations of cultured rat astrocytes and fused their spleen cells with NS-1 myeloma cells to generate antibody-secreting hybridomas. We have isolated two stable hybridoma clones which secrete monoclonal IgG2 antibodies that react with the surface of the great majority of rat astrocytes in culture. We have studied one of these antibodies in indirect immunofluorescence assays and show that it binds to the surface of rat ependymal cells, retinal Müller cells and leptomeningeal cells as well as to astrocytes, but not to cultured neurones, oligodendrocytes, Schwann cells, microglia or various non-neural cells. The antigen defined by this monoclonal antibody is protease-sensitive and rat-specific and we have called it rat neural antigen-2 (Ran-2). We also show that isolated rat ependymal cells and cultured rat Müller cells do not express other neural cell-type-specific markers, such as tetanus toxin receptors, rat neural antigen-1 (Ran-1), galactocerebroside or glial fibrillary acidic protein (GFAP). Nor do these cells express cell surface Fc receptors for IgG, phagocytose latex beads or make detectable amounts of the Thy-1 or fibronectin glycoproteins.

Animals↗
PubMed1981

All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody.

We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.

Animals↗
PubMed1980

Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins.

Rabbit antisera to the rat myelin proteins P0 and P1 were used to assay for the presence of these components by both immunochemical and immunofluorescence methods. The antiserum to P0 did not react detectably with polyacrylamide gels containing central myelin, or with P1 and P2 in peripheral myelin; it did react with P0 in peripheral myelin, and in extracts of adult and neonatal sciatic nerve. When reacted with frozen tissue sections using indirect immunofluorescence, it did not stain central myelin but did stain myelin in adult sciatic nerve, the myelinated fibres in cervical sympathetic trunk and occasional areas in neonatal sciatic nerve where Schwann cells had presumably begun to form myelin. Antiserum to basic protein reacted with both of the basic protein bands in central and peripheral myelin, but not P0; P1 and P2 were detectable in adult and neonatal sciatic nerve. In indirect immunofluorescence assays, the antiserum stained both central and peripheral myelin, the few myelinated fibres of sympathetic trunk and myelinating regions of neonatal sciatic nerve. Cultured secondary rat Schwann cells showed no detectable reaction with either reagent, using either technique. We conclude that these three proteins are probably expressed as a consequence of the neuron-Schwann cell interaction that initiates myelination.

Animals↗
PubMed1980

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture.

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.

Aging↗
PubMed1980

Cell type-specific markers for human glial and neuronal cells in culture.

We have used cell type-specific markers to identify and study the major classes of neural cells in dissociated cell cultures of optic nerve, spinal cord, and dorsal root ganglion from 15- to 21-week-old human fetuses. Astrocytes were identified by the intracellular expression of glial fibrillary acidic protein, oligodendrocytes by the cell surface expression of galactocerebroside, dorsal root ganglion neurons by their ability to bind tetanus toxin at their surface, and macrophages (including microglia) by cell surface Fc receptors and their phagocytic properties. Oligodendrocytes continued to express galactocerebroside (and some of them, also myelin basic protein) for up to 1 week in culture in the absence of neurons. While some dorsal root ganglion Schwann cells initially expressed galactocerebroside on their surface, and myelin basic protein and the major peripheral myelin glycoprotein (PO) intracellularly, they no longer expressed detectable amounts of any of these myelin-specific molecules after several days in culture. The Thy-1-like glycoprotein was found on the surface of fibroblasts, dorsal root ganglion neurons, and some astrocytes but not oligodendrocytes or the majority of leptomeningeal cells. Fibronectin was only expressed by fibroblasts and leptomeningeal cells.

Antigens, Surface↗
PubMed1979

Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture.

We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.

Animals↗
PubMed1979

Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve.

We have previously reported that in dissociated cultures of neonatal rat sciatic nerve, all of the cells could be identified by indirect immunofluorescence with two antisera to cell surface antigens. The Schwann cells, but not the fibroblasts, expressed the Ran-1 antigen, while the fibroblasts, but not the Schwann cells, expressed the Thy-1 antigen. We have exploited this difference to derive pure populations of Schwann cells. A combination of [3H]thymidine autoradiography and immunofluorescence marking showed that in Modified Eagle's Medium with 10% foetal calf serum, the Schwann cells divided slowly while the fibroblasts divided rapidly. Accordingly, two day old cultures were exposed to cytosine arabinoside to select against the fibroblasts, followed by growth in medium containing an extract of bovine pituitary which stimulated division of the Schwann cells. After 7 days the confluent cultures, which contained 80-90% Schwann cells, were passaged after treatment in suspension with antiserum to Thy-1 and rabbit complement. After continued growth in medium with pituitary extract, the secondary cultures contained greater than 99.5% Schwann cells. These purified populations have been maintained in culture for as long as 150 days (6 passages) and retained the Ran-1 marker. The cultured Schwann cells expressed the S100 antigen, as shown by indirect immunofluorescence and complement fixation, and receptors for cholera toxin. They did not express the large external transformation sensitive protein, the glial fibrillary acidic protein, or receptors for tetanus toxin.

Animals↗
PubMed1979

Studies on cultured rat Schwann cells. II. Comparison with a rat Schwann cell line.

Cultured rat Schwann cells do not exhibit the ring-like changes in cell shape previously reported to be induced in the Schwann cell line RN22 by elevation of intracellular cyclic AMP. They do, however, undergo different shape changes on treatment with cholera toxin or low serum concentration. Furthermore, DNA synthesis in the cell line is inhibited by treatment with cholera toxin and unaffected by bovine pituitary extract, though both of these agents stimulate DNA synthesis in normal Schwann cells. Our results, therefore, do not support the hypothesis that elevation of intracellular cyclic AMP is a positive signal for myelination by the Schwann cell. Moreover, they illustrate the need for caution in drawing conclusions about normal cells of the nervous system from studies on neural cell lines.

Animals↗
PubMed1979

Some membrane events occurring during fusion and exocytosis in rat peritoneal mast cells.

We have used the mast cell as a model system for studying some of the membrane events which occur during exocytosis. Our observations indicate that the maximum cluster size of IgE molecules necessary for the 'on' signal to activate a mast cell is ten or less, and that the 'off' signal is not associated with the gross patching or pinocytosis of IgE and its Fc receptors. Furthermore, the use of Con A-Sepharose beads to stimulate mast cells has shown that such signalling is localized to the areas of stimulus, but this localization is not a function of desensitization over the rest of the cell since the subsequent addition of soluble Con A to locally released cells induced generalized degranulation. Ca2+ influx therefore acts in a localized manner to initiate degranulation. Following receptor cross-linking, most of the membrane proteins and the layer of intervening cytoplasm are laterally displaced away from the areas of membrane interaction. This displacement may act as the signal for fusion to occur. The resulting fused bilayers are predominantly lipid, a situation which may be common in all transient membrane fusion.

Animals↗
PubMed1978

Schwann cell growth factors.

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.

Animals↗