Oligodendroglia express PDGF beta-receptor protein and are stimulated to proliferate by PDGF.
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Animals↗
Biomedical subjects
D Pleasure
Publications and source records attributed to D Pleasure.
The neuroendocrine and neural profiles of neuroblastomas, ganglioneuroblastomas, and ganglioneuromas.
To establish the neuroendocrine and neural features of peripheral neuroblastic tumors, a prospectively collected group of 12 neuroblastomas (NB), 2 ganglioneuroblastomas (GNB), and 4 ganglioneuromas (GN) was probed with a panel of monoclonal antibodies (MAbs) to neuroendocrine and neural antigens. All tumors expressed the pan-neuroendocrine markers synaptophysin and chromogranin A. They also showed extensive expression of neuronal antigens, ie, of each of the neurofilament (NF) triplet proteins and of the microtubule-associated proteins (MAPs) MAP2 and tau-protein. However, only in the GNBs and GNs was the pattern of NF phosphoisoforms relatively mature. In the latter tumors glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) could be demonstrated as well, suggesting the presence of nonmyelinating and myelinating Schwann cells, respectively. The glial markers did not colocalize with the neural markers. On the basis of these data, it was concluded that all peripheral neuroblastic tumors manifest molecular characteristics of neuroendocrine cells and of neurons. The latter were most developed in GNBs and GNs, in which they were accompanied by Schwann cell differentiation in a separate population of cells. The above-outlined neuronal profile of peripheral neuroblastic tumors, including NBs, distinguishes this group of tumors from the much-less neuronally differentiated primitive neuroectodermal tumors of the central nervous system.
Abdominal Neoplasms↗
Cerebral white matter contains PDGF-responsive precursors to O2A cells.
Cells dissociated from the cerebral white matter of immature rats were maintained in monolayer culture. Treatment with platelet-derived growth factor (PDGF) caused a large increase in the numbers of "O2A" oligodendroglial precursor cells (which bind the monoclonal antibody A2B5) and subsequently in the numbers of galactocerebroside (galC)-positive oligodendroglia. A2B5-negative "pre-O2A cells" in cerebral white matter cultures in which O2A cells and oligodendroglia had been killed by antibody-dependent complement-mediated cytolysis were induced by PDGF to proliferate and to differentiate into O2A cells and subsequently into oligodendroglia and type 2 astroglia. The most mature pre-O2A phenotype in these cultures was a small, round, process-bearing cell which expressed vimentin but not glial fibrillary acidic protein or galC. Cells of this phenotype were not observed upon PDGF treatment of immature rat optic nerve monolayer cultures from which O2A cells and oligodendrocytes had been depleted, and PDGF also failed to elicit the accumulation of O2A cells and oligodendroglia in such cultures.
Animals↗
Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines.
A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.
Antibodies, Monoclonal↗
Neuronal modulation of Schwann cell glial fibrillary acidic protein (GFAP).
Adult rat sciatic nerves contain cytoskeletal peptides that resemble CNS glial fibrillary acidic protein (GFAP) in immunoreactivity and molecular weight. Immunohistological examination of teased nerve fascicles indicated that these peptides are expressed selectively by Schwann cells related to small axons. Radiolabelled mouse and rat CNS GFAP cDNA probes hybridized with a single, 2.7 kb RNA band in Northern blots prepared from total RNA from both rat sciatic nerve and rat brain. Sciatic nerve GFAP mRNA was detectable by this means in adult, 2 month, or 21 day postnatal rats, but not in 3,6, or 10 day postnatal rats. Sciatic nerve transection caused a marked reduction in the level of GFAP mRNA in the axotomized distal stump. We conclude that Schwann cell synthesis of GFAP is developmentally regulated and that Schwann cells, unlike astroglia, require continued trophic input from small axons in order to express GFAP.
Aging↗
Rapid quantitative immunohistochemical assessment of human peripheral neuropathies using a monoclonal antibody against nerve growth factor receptor.
An analysis of nerve growth factor (NGF) receptor expression and density in human sural nerve biopsies was performed by immunocytochemistry with a murine monoclonal antibody against the human NGF receptor. Quantitative assessment of immunostaining density was made by histospectrophotometry on frozen sections. Although there was enhanced expression of NGF receptor within endoneurium in all patients with clinical neuropathies, expression was highest in nerves with axonal disease, consistent with the proposal that disruption of axon-Schwann cell interactions triggers the re-expression of the NGF receptor. These results with human nerves, together with previous studies with animal models, suggest that NGF and NGF receptor play important roles in the general response to neuronal injury.
Adult↗
Effects of palmitate on astrocyte amino acid contents.
The effects of palmitate on intracellular and extracellular amino acid concentrations of cultured astrocytes was studied. Exposure of astrocytes to either 0.72 mM or 0.36 mM palmitate was associated with a significant reduction in the intracellular pool of glutamine and taurine. In contrast, the intracellular concentration of histidine, glycine, citrulline, isoleucine and leucine were increased in the presence of 0.72 mM palmitate. Comparable changes in the extracellular amino acid pool were not observed. The data suggest that palmitic acid, which accumulates in the brain during periods of anoxia, alters the metabolism of several amino acids in cultured astrocytes. These changes may be of significance in terms of the pathophysiology of a stress such as anoxia.
Amino Acids↗
Nerve growth factor receptor immunoreactivity in the neuronal perikarya of human sensory and sympathetic nerve ganglia.
We examined immunohistochemically the dorsal root ganglia, sympathetic ganglia, spinal cord, ventral and dorsal roots, and sciatic nerves obtained at autopsy from adult humans, using a monoclonal antibody against the human nerve growth factor receptor. We observed labelling in a granular pattern in the neuronal perikarya of dorsal root and sympathetic nerve ganglia. Ventral horn cells and axons were not labelled.
Antibodies, Monoclonal↗
Expression of nerve growth factor receptor in human peripheral neuropathies.
Nerve growth factor (NGF) receptors in human sural nerve biopsies were detected immunohistochemically using a monoclonal anti-human NGF receptor antibody. NGF receptors were not visualized within the endoneurium of normal adult nerves but were readily demonstrable within the endoneurium of nerves undergoing active axonal degeneration. Immunostaining of adjacent sections for S-100 beta protein, a specific Schwann cell marker, suggested that Schwann cells expressed the NGF receptors induced by axonal degeneration. Residual Schwann cells in nerves in which axons were completely depleted also expressed NGF receptors. NGF receptors were not detected in relation to thinly myelinated regenerating axons, nor were they detected in the endoneurium of nerves that had undergone segmental demyelination and remyelination. The increased expression of NGF receptors in human axonal neuropathies may contribute to regeneration by NGF-responsive neurons in these disorders.
Adult↗
Regulation of myelin P0 glycoprotein synthesis in cultured rat Schwann cells and continuous rat PNS cell lines.
We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.
Animals↗
Astrocyte metabolism of [15N]glutamine: implications for the glutamine-glutamate cycle.
The metabolism of glutamine was studied in cultured astrocytes by incubating these cells with [2-15N]-glutamine and using gas chromatography-mass spectrometry to quantitate the transfer of 15N to other amino acids. We found that astrocytes simultaneously synthesize and consume [2-15N]glutamine, with the respective synthetic and utilization rates being approximately equal (ca. 13.0 nmol min-1 mg protein-1). Considerable 15N was transferred to alanine and a significant amount to the essential amino acids leucine, tyrosine, and phenylalanine, the latter process denoting active reamination of cognate ketoacids. A net export of alanine into the medium was noted. Astrocyte glutamine utilization appeared to be mediated via both the phosphate-activated glutaminase (PAG) pathway and the glutamine aminotransferase pathway, the activity of which was about half that of PAG. The glutamine concentration in the incubation medium determined whether net synthesis or utilization of this amino acid occurred. When glutamine was omitted from the medium, net synthesis occurred. When it was present at a high (5 mM) level, net consumption was observed. At a physiologic (0.5 mM) concentration, neither net synthesis nor consumption was noted, although the 15N data indicated that glutamine was actively metabolized. An implication of this work is that astrocytes clearly are capable of both synthesizing and utilizing glutamine, and current concepts of a glutamate-glutamine cycle functioning stoichiometrically between astrocytes and neurons may be an oversimplification.
Animals↗
A cyclic AMP analogue induces synthesis of a myelin-specific glycoprotein by cultured Schwann cells.
Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170K. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.
8-Bromo Cyclic Adenosine Monophosphate↗
Cultured rat Schwann cells express low affinity receptors for nerve growth factor.
Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.
Animals↗
[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.
The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.
Adenine Nucleotides↗
Rat brain microvessel extracellular matrix modulates the phenotype of cultured rat type 1 astroglia.
Astroglia from immature rat cerebral white matter which were plated on the insoluble extracellular matrix (ECM) secreted by rat cerebral microvessel endothelial cells (RCMEC) and maintained in a defined medium were induced to become stellate and to express glutamine synthetase. These effects were not elicited by RCMEC-conditioned medium. ECM secreted by rat pleural mesothelium elicited a lesser proportion of stellate astroglia and did not induce glutamine synthetase.
Animals↗
Schwann cell galactocerebroside of unmyelinated fibers is inducible by derivatives of adenosine 3',5'-monophosphate.
By using indirect immunofluorescence, galactocerebroside (galC) was detected on the surface of Schwann cells cultured from unmyelinated fibers of 10- to 12-day-old rat cervical sympathetic nerve trunk. By day 4 in vitro, galC-positive cells disappeared from the culture. When the 4-day cultures were treated with 1 mM 8-bromo cyclic AMP or dibutyryl cyclic AMP, galC reappeared in 72 h. The proportion of Schwann cells expressing galC was dependent on the concentration of cyclic AMP derivatives used.
8-Bromo Cyclic Adenosine Monophosphate↗
Characterization of rat schwannoma-Schwann cell hybrids.
Sciatic nerve Schwann cells from strain LEC rats, homozygous for the c form of 6-phosphogluconate dehydrogenase (6-PGD), and RN22 rat Schwannoma cells, a subclone of RN2 deficient in hypoxanthine phosphoribosyltransferase and expressing the s form of 6-PGD, were fused to produce 'RNS' hybrid clones which proliferate rapidly in a medium containing hypoxanthine, aminopterin and thymidine (HAT) and express c, s and c/s heterodimeric forms of 6-PGD. RNS cells, like both parents, maintain a high baseline activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase and, as in RN22, activity of this enzyme is further inducible by 1 mM N6, O2'-dibutyryl 3', 5'-cyclic AMP. The RNS clones resemble normal Schwann cells in the capacity to bind radioiodinated axolemmal fragments to their plasma membranes.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Utilization of [15N]glutamate by cultured astrocytes.
The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.
Adenosine Triphosphate↗