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W D Matthew

Publications and source records attributed to W D Matthew.

34 records · Page 2Linked to original sources
PubMed1988

Immunoreactivity in pituitary gonadotropes and hypothalamic vasopressinergic neurons for the monoclonal antibody INO.

The monoclonal antibody INO (inhibitor of neurite outgrowth) has been shown to bind to a complex of laminin and a heparan sulfate proteoglycan and to block the action of this complex in promoting neurite outgrowth. We now report that the same antibody binds to cytoplasmic constituents in rat adenohypophyseal gonadotropes, as well as to vasopressinergic neurons in the hypothalamus and their terminals in the neurohypophysis. INO immunoreactivity in fixed sections of pituitary does not colocalize with the immunoreactive laminin in blood vessels and glandular basement membranes, although when unfixed tissue is washed in buffer prior to fixation, the INO immunoreactivity appears in these laminin-rich structures. These observations suggest similarities between the INO hypophyseal antigen and the neurite-promoting proteoglycan complex characterized in conditioned media. Presence of this complex in specific neurosecretory cell types suggests that it is involved with specific secretory products with function yet to be determined.

Animals↗
PubMed1988

Expression of X hapten immunoreactivity by human and rat adenohypophyseal cells.

The monoclonal antibody Hy2D4 was found to label a previously undescribed subset of rat and human anterior pituitary cells. The antibody binding site appears to be a carbohydrate moiety previously named "X hapten." Double-label immunofluorescence studies in both normal rat and postmortem human pituitaries showed that this antigen is distributed on a subset of adrenocorticotropic hormone (ACTH)-positive cells, but is not detectable in cells immunoreactive for growth hormone, prolactin (PRL), thyroid-stimulating hormone, or luteinizing hormone. Since X hapten labeling revealed a biological subdivision of corticotroph cells, it was expected that some ACTH-positive tumors would be immunoreactive, but that tumors of other hormonal types would be negative. Instead, in 21 surgical specimens examined, tumors of all hormonal types were found to show immunoreactivity. To determine whether experimental proliferative changes in the pituitary could explain the shift in the cell type expressing the antigen, PRL-cell hyperplasia was induced in rats through chronic (8-week) exposure to diethylstilbestrol. The fraction of X-positive cells increased in these hyperplastic glands almost ninefold and, as in human adenomas, many non-corticotroph cells expressed the X marker in this model. However, the non-corticotroph cells expressing X were predominantly growth hormone cells, not the proliferative PRL cells. Thus, expression of the antigen does not necessarily imply that a cell is in a proliferative mode. While it is not known what role an altered expression of this antigen might play, the antibody offers a probe into cellular biology of human and experimental pituitary tumors.

Adenoma↗
PubMed1987

Substrate-bound nerve growth factor promotes neurite growth in peripheral nerve.

Nerve growth factor (NGF), in addition to its well-known effects as a soluble neurite growth-promoting factor, also appears to promote the elongation of neurites when it is adsorbed to tissue culture substrates. Peripheral nerve Schwann cells appear to possess a receptor for NGF on their surfaces which is induced substantially after axotomy. We have found that the adsorption of NGF onto cryostat sections of the distal stump of previously severed sciatic nerve enhances neurite growth over this tissue. This finding, coupled with the two previous observations, suggests that Schwann cell surface NGF receptors serve to bind to NGF-like growth factors so as to provide favorable surfaces for regenerating peripheral nerve axons.

Animals↗
PubMed1987

An in vitro neurite-promoting antigen functions in axonal regeneration in vivo.

The function of the neurite growth-promoting antigen INO has been tested in an in vivo neurite regeneration system, the rat iris. The sympathetic innervation of the irides was removed by a single systemic injection of 6-hydroxydopamine. The subsequent regeneration of sympathetic axons into the iris of one eye bathed by the INO antibody, which inhibits neurite growth in vitro, was compared with the regrowth of sympathetic axons into the iris of the animal's other eye, which contained control antibody. Antibodies were released within the eye by implanted hybridoma cells. Neurite regeneration was measured by assaying [3H]norepinephrine uptake into freshly explained irides. The blockage of the function of the INO antigen by the antibody resulted in a decreased rate of axonal regeneration, thus suggesting the involvement of the INO antigen in the process of neurite regeneration in vivo.

Animals↗
PubMed1987

Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies.

After immunization with a complex mixture of antigens, a considerable bias toward obtaining monoclonal antibodies to immunodominant determinants exists. By selectively killing antigen-stimulated lymphocytes, the cytotoxic drug cyclophosphamide can be used to manipulate the bias of the normal immune response. Cyclophosphamide has been used to tolerize mice to one set of antigens followed by immunization with a similar but slightly different set of antigens. This approach yields an enhanced frequency of antibodies that distinguish the two sets of antigens. Cyclophosphamide treatment has also allowed us to produce monoclonal antibodies to weakly immunogenic glycosaminoglycans and to obtain a high frequency of apparently anti-idiotypic antibodies.

Animals↗
PubMed1987

Identification of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay.

The effective regeneration of severed neuronal axons in the peripheral nerves of adult mammals may be explained by the presence of molecules in situ that promote the effective elongation of neurites. The absence of such molecules in the central nervous system of these animals may underlie the relative inability of axons to regenerate in this tissue after injury. In an effort to identify neurite growth-promoting molecules in tissues that support effective axonal regeneration, we have developed an in vitro bioassay that is sensitive to substrate-bound factors of peripheral nerve that influence the growth of neurites. In this assay, neonatal rat superior cervical ganglion explants are placed on longitudinal cryostat sections of fresh-frozen sciatic nerve, and the regrowing axons are visualized by catecholamine histofluorescence. Axons are found to regenerate effectively over sciatic nerve tissue sections. When ganglia are similarly explanted onto cryostat sections of adult rat central nervous system tissue, however, axonal regeneration is virtually absent. We have begun to identify the molecules in peripheral nerve that promote effective axonal regeneration by examining the effect of antibodies that interfere with the activity of previously described neurite growth-promoting factors. Axonal elongation over sciatic nerve tissue was found to be sensitive to the inhibitory effects of INO (for inhibitor of neurite outgrowth), a monoclonal antibody that recognizes and inhibits a neurite growth-promoting activity from PC-12 cell-conditioned medium. The INO antigen appears to be a molecular complex of laminin and heparan sulfate proteoglycan. In contrast, a rabbit antiserum that recognizes laminin purified from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, stains the Schwann cell basal lamina of peripheral nerve, and inhibits neurite growth over purified laminin substrata has no detectable effect on the rate of axonal regeneration in our assay.

Animals↗
PubMed1986

Presynaptic elements formed on polylysine-coated beads contain synaptic vesicle antigens.

Cell cultures of the rat cerebellum were immunostained with antibodies to synaptic vesicle antigens, Synapsin I and SV48. Light microscopic immunocytochemistry showed that the initial appearance of demonstrable SV48 and Synapsin I immunoreactivity occurred at different times. Synapsin I immunostaining, unlike SV48 immunostaining, was first seen at 3 days in vitro as occasional punctate immunofluorescence in neurites, while SV48 immunostaining was first seen at 5 days in vitro. Both SV48 and Synapsin I punctate immunostaining became frequent at 7 days in vitro. Double labelling experiments showed coexistence of the above proteins in punctate swellings and growth cones. Using the electron microscope, either SV48 or Synapsin I immunostaining was demonstrated within presynaptic elements in the neuropil. When cultures were incubated with polylysine-coated beads, both types of immunostaining were found in the vesicle containing presynaptic elements formed on the bead surface. It is concluded that Synapsin I and SV48 are co-localized in the same populations of presynaptic elements, co-localized in some growth cones and found in presynaptic elements on beads.

Animals↗
PubMed1986

A monoclonal antibody that blocks the activity of a neurite regeneration-promoting factor: studies on the binding site and its localization in vivo.

Work from several laboratories has identified a proteoglycan complex secreted by a variety of non-neuronal cells that can promote neurite regeneration when applied to the surface of culture dishes. Using a novel immunization protocol, a monoclonal antibody (INO) was produced that blocks the activity of this outgrowth-promoting factor (Matthew, W. D., and P. H. Patterson, 1983, Cold Spring Harbor Symp. Quant. Biol. 48:625-631). We have used the antibody to analyze the components of the active site and to localize the complex in vivo. INO binding is lost when the complex is dissociated; if its components are selectively reassociated, INO binds only to a complex containing two different molecular weight species. These are likely to be laminin and heparan sulfate proteoglycan, respectively. On frozen sections of adult rat tissues, INO binding is present on the surfaces of glial cells of the peripheral, but not the central, nervous system. INO also binds to the basement membrane surrounding cardiac and skeletal muscle cells, and binding to the latter greatly increases after denervation. In the adrenal gland and kidney, INO selectively reacts with areas rich in basement membranes, staining a subset of structures that are immunoreactive for both laminin and heparan sulfate proteoglycan. In general, the outgrowth-blocking antibody binds to areas known to promote axonal regeneration and is absent from areas known to lack this ability. This suggests that this complex, which is active in culture, may be the physiological substrate supporting nerve regeneration in vivo.

Animals↗
PubMed1985

Atypical distribution of asymmetric acetylcholinesterase in mutant PC12 pheochromocytoma cells lacking a cell surface heparan sulfate proteoglycan.

We studied the distribution of the molecular forms of acetylcholinesterase (AChE) in a stable variant (F3) of the rat pheochromocytoma cell line, PC12, that lacks a heparan sulfate proteoglycan on the cell surface. After treatment with nerve growth factor F3 cells synthesize less 4S enzyme, and more 10S and 16S enzyme than normal PC12 cells. This distribution is similar to that seen in normal cells after incubation with beta-D-xylosides, molecules that interfere with proteoglycan assembly. Using collagenase treatment and membrane-permeable and -impermeable inhibitors of AChE, we determined the cellular location of the AChE forms. Although in normal cells greater than 90% of the 16S AChE is on the cell surface, approximately 60% is present in an internal pool in the variant. Following irreversible inhibition of all forms of AChE in the variant, the newly synthesized 16S AChE appears in the internal pool after a 1-h lag, but is not detected on the cell surface until after 2.5 h. Our results thus show that 16S AChE is assembled internally within neuronal cells and that alterations in the synthesis and distribution of proteoglycans affect the total amount and cellular localization of the 16S AChE form.

Acetylcholinesterase↗
PubMed1985

Immunopurification and characterization of a neuronal heparan sulfate proteoglycan.

We have identified a unique heparan sulfate (HeS) proteoglycan synthesized by the neuronal-like cell line PC12. The proteoglycan, purified with monoclonal antibodies from medium conditioned by PC12 cells, has an apparent molecular weight of 350,000, and it contains a Mr 80,000 core protein and HeS side chains of Mr 15,000 each. The purified molecule has the same apparent size and density as it has in conditioned medium. HeS proteoglycans that are indistinguishable antigenically but very difficult to solubilize are found on the external surface and in the interior of PC12 cells and neurons. Mild proteolysis converts the surface proteoglycan into a molecule closely resembling that found in the medium. The same surface antigens are also present on a subpopulation of T-cells and on a non-neuronal accessory cell found in dorsal root ganglion cultures. The PC12 cell line and the non-neuronal dorsal root ganglion cells secrete a factor into medium that, after adsorption to polylysine-coated surfaces, induces rapid neurite out-growth by primary sympathetic neurons. The monoclonal antibodies used to purify the neuronal HeS proteoglycan from PC12 cells are capable of depleting this conditioned medium of its neurite-promoting activity. These studies suggest that a HeS proteoglycan synthesized and secreted by neurons and certain accessory cells plays a role in regulating neurite outgrowth.

Animals↗
PubMed1982

Development and application of an efficient procedure for converting mouse IgM into small, active fragments.

A simple method is described for generating active molecules with molecular weights between 110 and 230 kilodaltons from mouse immunoglobulin M. The majority of these molecules have a 1 : 1 ratio of intact heavy and light chains. Approximately 70% of the specific IgM protein will still bind antigen after digestion with only a small decrease in binding affinity. Both anti-kappa and anti-mu chain specific antisera recognize these molecules. These low molecular weight molecules are much more efficient in immunocytochemistry and have localized antigens that could not be detected with undigested IgM.

Animals↗
PubMed1981

Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue.

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.

Animals↗