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D Pleasure

Publications and source records attributed to D Pleasure.

At least 55 records · Page 3Linked to original sources
PubMed1995

Partial purification of a novel mitogen for oligodendroglia.

A protein with a MWapp of 50-70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS-polyacrylamide gel, this protein ("oligodendroglial trophic factor," OTF) elicited half-maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet-derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage.

Animals↗
PubMed1995

Myelin gene expression in glia treated with oligodendroglial trophic factor.

Oligodendroglia synthesize myelin in the CNS. In vitro, oligodendroglia may be identified by the binding of monoclonal antibodies against galactocerebroside, a myelin-specific galactolipid. Oligodendroglial trophic factor is a protein mitogen for cells of the oligodendroglial lineage. When oligodendroglia in cerebral white matter cultures are treated with oligodendroglial trophic factor, galactocerebroside-positive cells undergo mitosis but fail to express the myelin structural proteins, myelin basic protein and proteolipid protein. Oligodendroglia treated with oligodendroglial trophic factor, however, do express 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein in a manner similar to oligodendroglia treated with platelet-derived growth factor. Oligodendroglial trophic factor, therefore, generates a population of somewhat 'immature' oligodendroglia, which are galactocerebroside, myelin-associated glycoprotein and 2', 3'-cyclic nucleotide 3' phosphodiesterase positive but myelin basic protein and proteolipid protein negative.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1995

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage.

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.

Animals↗
PubMed1994

Expression of P0 protein mRNA along rat sciatic nerve during development.

The temporal and spatial expression of P0 protein mRNA in the spinal nerve roots and in distal (popliteal trifurcation) segments of the developing rat sciatic nerve was examined using in situ hybridization. P0 mRNA was undetectable in the PNS portions at day 16 post-fertilization (E16) but present in the roots as well as in the sciatic nerve by E18. While Schwann cells were labelled by in situ hybridization at E18, it was not until birth (P1) that cells revealed the typical clusters of silver grains in a perinuclear distribution. Hybridization signal rose between P5 and P10, than decreased at P40, with no significant difference of P0 mRNA levels between the different PNS portions at each age studied. These findings demonstrate the lack of proximo-distal gradient in abundance of P0 mRNA during rat PNS development and add further insight in understanding the dynamics of axonal regulation of myelination in the PNS.

Animals↗
PubMed1994

Cloning of a novel putative protein kinase having a leucine zipper domain from human brain.

We report a novel putative serine/threonine protein kinase containing a leucine-zipper domain, isolated from an human neuronal cell line. The teratocarcinoma cell line NT2 was differentiated to postmitotic NT2-N neurons by treatment with retinoic acid, and degenerate oligonucleotide primers to the catalytic domains of protein kinases were employed to PCR amplify subtractive cDNAs. We identified a clone, represented at higher abundance in NT2-N neurons than in the parental cell line, which encodes a putative serine/threonine kinase of 859 [corrected] amino acids, the leucine-zipper protein kinase (zpk). Zpk protein contains a leucine-zipper domain, found in many DNA-binding proteins, but few protein kinases. Steady-state mRNA levels for zpk are high in human brain and kidney. Further studies are required to evaluate the role of zpk in neuronal differentiation.

Amino Acid Sequence↗
PubMed1994

The expression of a NMDA receptor gene in guinea-pig myenteric plexus.

Muscle tension studies of guinea-pig ileum longitudinal muscle-myenteric plexus preparations suggest that the N-methyl-D-aspartate (NMDA) receptor may be present in the enteric nervous system. Therefore, we investigated the expression of a gene for the NMDA receptor in guinea-pig taenia coli. The gene product was amplified using polymerase chain reaction (PCR) and its synthesis localized using in situ hybridization. A NMDA receptor PCR product from the myenteric plexus was demonstrated with nearly identical sequence characteristics to that in the brain. In situ hybridization studies identified myenteric neurons which express NMDA receptor messenger RNA. Demonstration of the genetic expression of the NMDA receptor supports a role for glutamate as a neurotransmitter in the enteric nervous system.

Amino Acid Sequence↗
PubMed1994

Protein growth factors as potential therapies for central nervous system demyelinative disorders.

Demyelinative diseases are frequently accompanied by loss of oligodendroglia; in such instances, oligodendroglial regeneration must precede remyelination. Recent studies indicate that extracellular proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) profoundly influence the oligodendroglial lineage. PDGF stimulates the formation of oligodendroglia from partially differentiated progenitor cells, whereas bFGF induces mature oligodendroglia to proliferate and dedifferentiate. Manipulations of the central nervous system concentrations of these and other protein growth factors may prove of therapeutic value in multiple sclerosis.

Adult↗
PubMed1994

Expression of the low-affinity NGF receptor during human muscle development, regeneration, and in tissue culture.

The expression of the low-affinity NGF receptor (LNGFR) during human muscle development, regeneration, and in tissue culture was analyzed using a murine monoclonal antibody to human LNGFR (MAb ME 20.4). Muscle cells from 12-22-week fetuses stained strongly for LNGFR. In adult normal muscle, only intramuscular nerve endings showed immunoreactivity with MAb ME 20.4, but no staining was detected in muscle fibers. In Duchenne muscular dystrophy, immunohistologically demonstrable LNGFR was present in regenerating muscle fibers. In these fibers, LNGFR gene expression was also demonstrated at the transcriptional level using in situ hybridization with riboprobes coding with human LNGFR. Cultures from human fetal and adult muscle were studied by double label indirect immunofluorescence microscopy with MAb ME 20.4 and antisera against human fetal myosin. Most myosin-positive cells, both at the myoblast and myotube stages, displayed surface LNGFR immunostaining. In cells from fetal muscle, LNGFR was detected during the first 2 weeks in vitro, whereas in cells from adult muscle the expression of LNGFR was observed for up to 7 weeks. These findings suggest a potential involvement of LNGFR in human muscle development and regeneration.

Culture Techniques↗
PubMed1994

Developmental expression of P0 mRNA and P0 protein in the sciatic nerve and the spinal nerve roots of the rat.

Expression of myelin P0 protein by myelinating Schwann cells in vivo is dependent on axonal influences. This report describes P0 gene expression during development of rat sciatic nerve and spinal nerve roots using Northern blotting, in situ hybridization and immunohistochemistry. We demonstrate that: (1) the appearance of P0 mRNA and P0 protein in Schwann cells during nerve development in the rat begins prenatally, at day 18 post-fertilization (E18); (2) P0 mRNA and P0 protein have essentially identical developmental profiles, and are expressed in Schwann cells that are many days prior to myelin formation; (3) initial P0 gene expression is greatest in Schwann cells at the periphery of nerve bundles and in Schwann cells in contact with motor axons; (4) the decline in P0 expression with nerve maturation is accompanied by a sharp decline in P0 message levels in most Schwann cells, but a small subpopulation of these cells continue to synthesize very high levels of P0 mRNA. This study provides data on myelin P0 protein gene expression and distribution during PNS development and adds further insights into the axonal influences controlling Schwann cell behaviour during myelination of the rat PNS.

Aging↗
PubMed1994

Fc receptor for IgG (FcR) on rat microglia.

Receptor for IgG (FcR) was demonstrated on rat microglia in vivo and in vitro by immunohistochemical staining with immune complexes of horseradish peroxidase (HRP) and rabbit IgG anti-HRP. Astrocytes, oligodendrocytes and neurons did not express FcR. Microglia in culture also showed FcR-mediated agglutination and phagocytosis of IgG-sensitized erythrocytes. A radiolabelled cDNA probe for rat FcRIII hybridized with a 1.4-kb RNA band in Northern blots prepared from total RNA from rat brain. FcRIII mRNA-positive cells in rat brain, presumably microglia, were demonstrated by in situ hybridization. FcR participates in the initiation of cytotoxic responses and of phagocytosis by microglia and is therefore likely to be important in mediating immune reactions in the brain.

Animals↗
PubMed1994

Interrelationships of leucine and glutamate metabolism in cultured astrocytes.

The aim was to study the extent to which leucine furnishes alpha-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with alpha-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the "reverse" direction and intraastrocytic [glutamate] was reduced by approximately 50% in only 5 min. Extracellular concentrations of alpha-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was approximately 17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary "buffering" of intracellular glutamate.

Amination↗
PubMed1994

Expression of non-NMDA glutamate receptor channel genes by clonal human neurons.

Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor channels. We now show by reverse transcriptase-polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non-NMDA glutamate receptor genes (GluR1-7, Ka-1, and Ka-2) and that four of these genes (GluR2, GluR6, GluR7, and Ka-1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non-NMDA glutamate receptor channels are readily isolated from NT2-derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2-derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. We expect that the NT2-derived human neuronal culture system will facilitate studies of human neuronal non-NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Inhibition of astrocyte glutamine production by alpha-ketoisocaproic acid.

We have evaluated the effect of alpha-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5-15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic alpha-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1993

Trophic effects of basic fibroblast growth factor (bFGF) on differentiated oligodendroglia: a mechanism for regeneration of the oligodendroglial lineage.

We have investigated the effect of basic fibroblast growth factor (bFGF) on the proliferation and phenotype of differentiated oligodendroglia. Using primary cell cultures enriched in oligodendrocytes but containing few O2A-oligodendrocyte progenitor cells, we demonstrate that bFGF treatment greatly increases the proportion of O2A cells while decreasing the proportion of galactocerebroside +(GalC+), myelin basic protein +(MBP+) oligodendrocytes, and the steady state levels of MPB mRNA. Complement mediated cell lysis experiments using the A2B5 antibody to deplete existing O2A cells or the R-Mab antibody to deplete existing oligodendroglia show that bFGF elicits a rapid increase in the number of O2A cells in cultures previously depleted of O2A cells, but does not cause an early increase in O2A cells in cultures from which oligodendroglia had been removed, indicating that the oligodendrocytes are the source of the newly recruited O2A cells. This bFGF-mediated transition from oligodendrocyte to O2A cells occurs with a time course similar to the bFGF-induced increase of the proliferation rate of the GalC+ oligodendrocytes. Studies with purified, passaged cells of the oligodendroglial lineage show that bFGF augments oligodendroglial dedifferentiation and proliferation in chronologically adult oligodendrocytes and in the virtual absence of other cell types. We have thus demonstrated that mature oligodendrocytes are induced by bFGF to dedifferentiate and proliferate, suggesting a mechanism for regeneration of the oligodendroglial lineage following demyelinating disease.

Animals↗
PubMed1993

Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.

Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5' flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5' of the initiation of transcription was sufficient to direct oligodendrocyte-specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis-acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis-acting segment were identified. The distal segment behaved more negatively in Cos-7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis-acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos-7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos-7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte-specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin-specific promoters.

Animals↗
PubMed1993

Protein growth factor requirements of rat neural crest cells.

Nascent neural crest cells derived from explanted E12 embryonic rat caudal neural tubes were used as an assay system to investigate the effects of fibroblast growth factors on neural crest cell (NCC) survival, proliferation, migration, and differentiation. In vitro and in vivo all NCC express low affinity nerve growth factor receptors (p75-LNGFR), whereas a subpopulation of NCC expresses the carbohydrate epitope recognized by the monoclonal antibody HNK-1 (Bannerman and Pleasure, manuscript in preparation). Both acidic and basic fibroblast growth factor (FGF) promoted the survival of proportionally greater numbers of p75-LNGF+/HNK-1- than P75-LNGFR+/HNK-1+ NCC. An as yet uncharacterized factor present in neural tube-conditioned medium was also required for NCC survival. Mitosis was frequent in those NCC closest to the neural tube, less so as the cells migrated away. Neither basic nor acidic fibroblast growth factor (FGF) influenced rates of NCC mitosis in either of these locations, nor did these FGFs alter the rate at which nascent NCC migrated away from the neural tube. However, acidic and basic FGFs did delay the differentiation of neural crest derived neurons in the cultures. FGF is abundant in the embryonic rat neural crest outgrowth zone, and the present study strongly supports an essential role for FGF in early development of the mammalian neural crest.

Animals↗
PubMed1993

Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.

2-Amino-5-phosphonovalerate↗