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Biomedical subjects

R M Pruss

Publications and source records attributed to R M Pruss.

At least 37 records · Page 2Linked to original sources
PubMed1985

Efficient detection of intermediate filament proteins using a panspecific monoclonal antibody: anti-IFA.

Anti-IFA, a panspecific monoclonal antibody which was raised against human glial fibrillary acidic protein (GFAP), recognizes a determinant common to GFAP and all other intermediate filament proteins. This antibody can be used to identify intermediate filament proteins from both vertebrate and invertebrate tissues. Its utility in immunoblot studies of intermediate filament proteins is enhanced by using cytoskeleton extracts and protease treatment to facilitate the transfer of high molecular weight (greater than 70 000) proteins from gels to nitrocellulose membranes.

Animals↗
PubMed1985

Immunochemical characterization of carboxypeptidase B-like peptide-hormone-processing enzyme.

Specific rabbit antisera against purified bovine pituitary carboxypeptidase processing enzyme (which has also been referred to as "enkephalin convertase") have been prepared and characterized. The antisera recognized both the purified soluble and the membrane-bound forms of the enzyme with equal affinity, suggesting that these two forms of the enzyme may possess many regions of structural homology. Since the antisera did not crossreact with carboxypeptidases B, N, A, Y, and P, the carboxypeptidase processing enzyme may be a structurally distinct form of carboxypeptidase. Carboxypeptidase immunostaining, as seen by light microscopy, was found throughout the rat brain and in bovine adrenal medulla, reflecting the widespread distribution of neuropeptides. Electron microscopic immunocytochemistry of rat paraventricular nucleus and other brain areas showed that the enzyme was present in some dendrites and nerve terminals, which contain storage vesicles. These findings support the hypothesis that this carboxypeptidase is involved in the processing of many peptide hormone precursors.

Adrenal Medulla↗
PubMed1985

Specific regulation of vasoactive intestinal polypeptide biosynthesis by phorbol ester in bovine chromaffin cells.

Two neuropeptides, enkephalin and vasoactive intestinal polypeptide (VIP), are simultaneously increased in cultures of bovine chromaffin cells after diverse treatments including elevation of cAMP, application of nicotine, or chronic depolarization. We now show that phorbol esters can specifically elevate VIP in cultured chromaffin cells without changing the amount of enkephalin. Peptide histidine isoleucine, a VIP-related peptide, is also expressed concomitantly with VIP after treatment with phorbol ester. Immunocytochemical examination of drug-treated cells defines a subpopulation of chromaffin cells which are responsive to phorbol ester stimulation. The unique ability of phorbol esters to selectively regulate VIP expression indicates the presence of independent mechanisms for controlling the expression of individual neuropeptides in chromaffin cells.

Animals↗
PubMed1982

Insulin is present in chicken eggs and early chick embryos.

We showed that insulin appears in the chick embryo before beta-cells are recognizable, as well as in the egg constituents even before fertilization. Acid-ethanol extracts of 2- to 8-day-old embryos were gel-filtered on Sephadex G-50. The peak of immunoreactive insulin chromatographed in a position corresponding to that of authentic insulin. The immunoreactive insulin extracted from embryos was approximately 2 ng/g wet wt during early embryogenesis (days 2, 3, and 4), with a 2- to 3- fold increase by days 5 and 6, in conjunction with pancreatic development. The heads of the embryos contributed 22-23% of the total insulin on days 3 and 4, but only 5% by day 5. Based on its reactivity in a pork insulin RIA, chicken insulin RIA, and rat adipocyte bioassay, we concluded that the material is very similar to avian (chicken or turkey) rather than mammalian type insulin. Similar immunological and biological insulin-like activity [but at much lower concentrations (0.2-0.8 ng/ml)] were recovered from the gel-filtered acid-ethanol extracts of yolk and white of unfertilized and fertilized eggs. This study, which shows that insulin is present at a very early stage in ontogeny, extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.

Adipose Tissue↗
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↗
PubMed1981

Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes and Schwann cells.

We have identified two growth factors for cultured rat astrocytes: fibroblast growth factor, a peptide derived from either whole bovine brain, or pituitaries, and a growth factor in extracts of bovine pituitary which was previously identified as a Schwann cell mitogen. Oligodendrocytes in primary cultures derived from neonatal rat central nervous system divide only rarely if at all. These growth factors did not stimulate primary oligodendrocytes to divide. Occasionally cells found in suspension in long-term cultures of the central nervous system were enriched for cells which were identified as oligodendrocytes by the presence of galactocerebroside on their surface and myelin basic protein in their cytoplasm. When provided with a monolayer of irradiated 3T3 cells, these oligodendrocytes were able to spread out and extend elaborate branched processes typical of oligodendrocytes in the primary cultures. Unlike their counterparts in the primary cultures, these suspension-derived oligodendrocytes are capable of cell division as demonstrated by the uptake of [3H]thymidine and autoradiography.

Animals↗
PubMed1980

Antisera to bovine oligodendroglia raised in guinea pigs bind to surface of rat oligodendroglia and Schwann cells.

Antisera against bulk isolated bovine oligodendrocytes was raised in 2 guinea pigs (GPaBO). The sera bound only to surface of rat oligodendrocytes in dissociated rat corpus callosum and cerebellar cultures and only to surface of Schwann cells in primary and secondary sciatic nerve and dorsal root ganglia cultures as determined by indirect immunofluorescence. No cells in muscle or retinal cultures bound the guinea pig antisera. The shared antigen does not seem to be either galactocerebroside (GalC) or a myelin basic protein. GPaBO can serve as a useful marker in double-label experiments to identify oligodendrocytes and Schwann cells in dissociated cultures.

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↗
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

Cholera toxin stimulates division of 3T3 cells.

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.

Animals↗
PubMed1978

Epidermal growth factor induced membrane changes in 3T3 cells.

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF.

Clone Cells↗
PubMed1978

Mitogens for murine embryo cell lines.

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2alpha, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T 1/2). Each cell has an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2alpha were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.

Animals↗
PubMed1977

Specific radiolabeling of a cell surface receptor for epidermal growth factor.

A photoreactive derivative of epidermal growth factor (EGF) has been used to identify and specifically label a membrane receptor for EGF on mouse 3T3 cells. Photoactivable EGF, labeled with 125I, was incubated with 3T3 cells and then photolyzed in situ to generate a nitrene capable of reacting with a wide variety of chemical bonds. Analysis of the system by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed, besides the band of EGF, only one other major radioactive band, at a position indicating an apparent molecular weight of 190,000. This band was absent when a nonresponsive and nonbinding variant of 3T3 was used. A direct proportionality between binding activity and crosslinked complex formation was demonstrated using a variety of binding conditions. "Down regulated" cells, in which EGF binding activity was greatly reduced by prolonged incubation with an appropriate concentration of EGF, also had a decrease in covalent complex formation proportional to the decrease in EGF binding activity.

Affinity Labels↗