Biomedical subjects

G H DeVries

Publications and source records attributed to G H DeVries.

At least 19 recordsLinked to original sources

PubMed2001

Identification and functional characterization of thromboxane A2 receptors in Schwann cells.

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Isolation and characterization of unmyelinated axolemma from bovine splenic nerve.

Bovine splenic nerve was used as a source of axolemma-enriched fractions derived from mammalian unmyelinated axons. By electron microscopy, splenic nerve consisted entirely of fascicles of unmyelinated axons and associated Schwann cells. The epineurium and blood vessels were stripped from the dissected nerve, which was then homogenized followed by preparation of a microsomal fraction by differential centrifugation. The microsomes were fractionated on a 10% to 40% continuous sucrose gradient. The individual fractions were combined into six fractions based on sucrose concentration and each fraction was analyzed for membrane markers. The 20% to 23% region of the sucrose gradient was enriched approximately sevenfold in acetylcholinesterase activity and twofold enrichment in saxitoxin binding activity was noted in the same fraction. Relative to other microsomal fractions, this same fraction was less enriched in a microsomal marker (cytochrome c reductase) and only moderately enriched in the activity of a myelin membrane marker (2',3' cyclic nucleotide 3' phosphohydrolase, CNPase). Polyacrylamide electrophoresis of the axolemma-enriched fraction revealed five prominent peptides ranging in molecular weight from 40 kDa to 130 kDa. Lipids, comprising 59.4% of the dry weight, were enriched in cholesterol and sphingomyelin, consistent with the origin from a peripheral nervous system (PNS) plasma membrane. On a molar basis, the major gangliosides were G(T1b), G(D1a), and G(M1). As a whole, these molecular characteristics are consistent with the origin of the axolemma-enriched fraction in the unmyelinated splenic nerve axons. This membrane preparation should prove useful in future studies of the myelinogenic potential of mammalian unmyelinated axolemma.

Acetylcholinesterase↗

PubMed1999

Neuregulins and ErbB receptors in cultured neonatal astrocytes.

Neuregulins (NRGs) are a family of growth factors involved in signaling between neurons and glial cells of the peripheral and central nervous system. NRGs are synthesized and secreted by a number of cell types including Schwann cells, neurons, and oligodendrocytes. NRG transduction signals are mediated by the erbB family of receptor tyrosine kinases. These NRGs may be important for paracrine or autocrine signaling during development, injury, and the normal functioning of the central nervous system. In this study, we characterize the NRGs and erbB receptors expressed by cultured neonatal rat astrocytes. Using immunoblotting protocols with pan-specific antibodies, we identified eleven NRG molecular weight isoforms from approximately 16 kDa to 105 kDa in cultured neonatal rat astrocytes. Immunocyotchemistry with isoform-specific antibodies revealed the expression of both major isoform families (NRGalpha, NRGbeta). Additionally, astrocyte-conditioned media contained two molecular weight isoforms of NRGs. We detected mRNA expression of NRGalpha and NRGbeta in astrocytes by amplifying mRNA transcripts with reverse transcription polymerase chain reaction. Furthermore, we confirm that cultured astrocytes express all four erbB receptors as detected by immunocytochemical and immunoblotting techniques. These data indicate that astrocytes contain and secrete NRGs.

Animals↗

PubMed1999

Phosphorylation of CREB in axon-induced Schwann cell proliferation.

Axonal contact regulates Schwann cell (SC) proliferation during development. However, the intracellular signal transduction pathways involved in the axon-induced proliferation of SC have not been described. We have previously shown that SC proliferation induced by axolemma-enriched fractions (AEF) is accompanied by increased expression of cyclic AMP-responsive element binding protein, CREB. We now report the AEF and dorsal root ganglion neuritic-induced signal transduction pathway(s) which regulate the phosphorylation of CREB that correlate with the SC proliferative response. The phosphorylated form of CREB was significantly increased after 16 hr of axonal stimulation, continued to increase for 48 hr, and subsequently decreased as monitored by immunocytochemistry and Western blot analysis. Treatment with protein kinase A (PKA) inhibitor, H89, completely abolished both the CREB activation and SC proliferation. In contrast, treatment with protein kinase C (PKC) inhibitor (bisindolylmaleimide) inhibited AEF-induced SC proliferation, but did not immediately affect CREB phosphorylation. These data are consistent with the view that PKA and PKC pathways are essential for AEF-induced SC proliferation. Since PKC can influence SC proliferation without initially affecting CREB phosphorylation, PKC may regulate SC proliferation at pathways distal to the immediate CREB activation.

Animals↗

PubMed1999

Myelin basic protein and myelin basic protein peptides induce the proliferation of Schwann cells via ganglioside GM1 and the FGF receptor.

Myelin basic protein (MBP) and two peptides derived from MBP (MBP(1-44) and MBP(152-167)) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of 125I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of 125I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP(1-44)) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP(1-44) and MBP(152-167) associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.

Animals↗

PubMed1998

Quantitation of changes in P0 mRNA by polymerase chain reaction in primary cultured Schwann cells stimulated by axolemma-enriched fraction.

A requirement for large numbers of primary culture cells has frequently restricted investigations of gene expression in glial cells. We have developed a non-radioactive method based on reverse transcription-polymerase chain reaction (RT-PCR) to accurately assess small changes in the expression of the myelin specific gene P0 in Schwann cells. Using axolemma-enriched fraction (AEF) as an inducing agent, we demonstrate that RT-PCR can be used to detect 4-8-fold increases in P0 mRNA levels occurring in a time and dose dependent manner, utilizing only 250000 cells per assay. Initial experiments used an in vitro transcribed RNA for P0 constructed with a 300 bp deletion for quantitation by competitive RT-PCR. Relative quantitation by co-amplification of the housekeeping gene glyceraldehyde-phosphate dehydrogenase was established and provided similar results. Product evaluation was enhanced 50-100-fold by the incorporation of primers labelled with biotin at the 5' end, allowing for the sensitive detection of PCR product by enhanced chemiluminescence and autoradiography. This technique provides sensitivity to detect and evaluate picogram amounts of DNA. Our results validate the assay for P0 gene expression and indicate that the technique should facilitate the study of multiple genes of interest in glial cell systems.

Animals↗

PubMed1998

Neuregulins in glial cells.

The role of growth factors in controlling the development of glial cells in both the peripheral and central nervous systems has been investigated for a number of years. The recent discovery of a new family of growth factors termed the neuregulins (NRGs) has led to an explosion of information concerning the putative role of these growth factors in the development of Schwann cells (SC), oligodendrocytes (OLG), and astrocytes. Many of these previous studies have focused on the effects of exogenous NRGs on glial cell development and differentiation. We now review the evidence that these glial cells themselves produce NRGs and discuss the major implications of these findings with respect to glial cell development and diseases which affect glial cell function. We also discuss the potential role of endogenous NRGs following neural injury.

Animals↗

PubMed1998

Regulation of glucose transport in cultured Schwann cells.

Glucose is the major source of metabolic energy in the peripheral nerve. Energy derived from glucose is mostly utilized for axonal repolarization. One route by which glucose may reach the axon is by crossing the Schwann cells that initially surround the axons. Considering the ability of neurons to control many glial cell functions, we postulated that Schwann cell glucose transporters might be transiently regulated by axonal contact. Glucose transport was studied in a cultured, differentiated rat Schwann cell line stably expressing SV40 T antigen regulated by a synthetic mouse metallothionein promoter. 3[H]-2-deoxy-D-glucose uptake was measured in cultured cells in basal and in various experimental conditions. Glucose transporter gene expression was determined after RNA isolation from cultured cells through Northern and RNAse protection assay. In vitro, Schwann cells were found to express high-affinity, insulin-insensitive, facilitative glucose transporters and predominantly GLUT1 mRNA. Schwann cell 2-deoxyglucose uptake was increased by axolemmal membranes or forskolin but unchanged by elevated glucose levels. Regulation of Schwann cell glucose transporters by axolemma and their resistance to glucose-induced down-regulation suggest extrinsic rather than intrinsic regulation that might enhance Schwann cell vulnerability to glucotoxicity.

Animals↗

PubMed1997

Effect of neu differentiation factor isoforms on neonatal oligodendrocyte function.

Previous studies have suggested that neu differentiation factor (NDF), a member of the neuregulin (NRG) family of growth factors, may regulate the development of PNS and CNS glial cells. There is limited information concerning the potential role of NDF on the development of neonatal (immature) oligodendrocytes (OLG) into adult OLG. We now report the effect of the two major isoform families of NDF (NDF alpha and NDF beta) on the development of cultured rat neonatal OLG. Immunocytochemical and western blot analyses of neonatal OLG using anti-erb-B antibodies revealed that these immature OLG express all four members of NRG (erb-B) receptors. Treatment of neonatal OLG with varying concentrations of either NDF alpha or NDF beta did not have a mitogenic effect on cultured neonatal OLG. Pretreatment of immature OLG with either of the NDF isoforms also did not influence the subsequent mitogenicity of other known OLG mitogens. However, treatment of neonatal OLG with either isoform of NDF influenced the survival of these cells by protecting the cells from apoptosis. Additionally, treatment of neonatal OLG with either NDF alpha or NDF beta resulted in more extensive process formation compared to control, non-treated OLG.

Animals↗

PubMed1997

Lipid composition and phospholipid asymmetry of membranes from a Schwann cell line.

We report the total lipid composition and phospholipid asymmetry of a plasma membrane preparation isolated from a Schwann cell line (NF1T) derived from a human neurofibroma. The specific activities of three plasma membrane markers (5'-nucleotidase, Na-K-ATPase, and CNPase) were 8-fold, 12-fold, and 16-fold higher, respectively, in the plasma membrane fraction compared to the specific activities found in the total homogenate. The specific activities of the marker enzymes of intracellular membranes in the isolated plasma membrane fraction indicated little contamination with intracellular organelles. The enrichment of cholesterol (3-fold), sphingomyelin (3-fold), and glycolipids (cerebrosides 8-fold, sulfatides 5-fold) also indicated a high degree of purity of the plasma membrane fraction. The high content of phosphatidylinositol and phosphatidylcholine (10% and 44% of total phospholipid) and the low phosphatidylserine and phosphatidylethanolamine content (3% and 14% of the total phospholipid) were also characteristic of the plasma membrane fraction derived from this cell line. The transbilayer phospholipid distribution of the plasma membrane in intact cells and in the isolated plasma membrane fraction was investigated by using phospholipase A2 (bee venom) and sphingomyelinase (S. aureus). The phospholipid asymmetry of NF1T plasma membrane followed the general features of phospholipid asymmetry in eukaryotic cells: sphingomyelin and phosphatidylcholine were preferentially located in the outer leaflet (90% and 89%, respectively) while the aminophospholipids phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were in the inner half of the membrane (85%, 96%, and 69%, respectively). A high percentage of the total plasma membrane phosphatidylinositol (31%) was found in the outer side of the membrane indicating a decreased asymmetric distribution for this negatively charged phospholipid. The phospholipid asymmetry found in the plasma membrane vesicle fraction corroborated the phospholipid asymmetry of the intact cells, thus confirming that the plasma membrane vesicles maintained the original orientation and lipid asymmetry after homogenization and/or sonication.

Biomarkers↗

PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors.

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.

Animals↗

PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: I. Effect of treatment with growth factors.

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma-enriched fraction, cAMP, or NGF showed concentration-dependent inhibition of both mu calpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated mu calpain (37%) and mcalpain (58%) of tSC while PDGF-aa and PDGF-bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF-aa, and PDGF-bb compared to control while this activity was 40% with FGF-treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain-calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Animals↗

PubMed1997

Neonatal oligodendrocytes contain and secrete neuregulins in vitro.

The factors that influence the development of oligodendrocyte (OLG) progenitors into mature OLGs remain elusive. Recent evidence has suggested that neu differentiation factor (NDF), which is a member of the neuregulin family of growth factors, influences the development of glial cells, including Schwann cells, astrocytes, and OLGs. Neurons are postulated to be the source of neuregulins, because neurons closely interact with these glial cells during development. In this report, we have identified the mRNA for both isoform families of NDF in cultured neonatal (immature) OLGs. We have also demonstrated that cultured neonatal OLGs contain and secrete NDF protein. These data raise the possibility that NDF could be used in an autocrine/paracrine loop by neonatal OLGs during development for survival, proliferation, and/or differentiation.

Animals↗

PubMed1997

Antibodies to the axolemma-enriched fraction in the cerebrospinal fluid and serum of patients with multiple sclerosis and other neurological diseases.

Antibodies to an axolemma-enriched fraction (AEF) antigen have been detected in the cerebrospinal fluid (CSF) and serum of patients with Multiple Sclerosis (MS) using an enzyme-linked immunosorbent assay (ELISA). A marginal elevation (P < 0.08) of anti-AEF IgG was found in MS CSF when compared with OND samples. When CSF was diluted to a standardized IgG concentration, the anti-AEF IgG level in MS CSF was significantly elevated (P=0.007) when compared to OND CSF. MS serum was also found to contain a significantly higher level (P < 0.001) of anti-AEF IgG when compared to OND serum using the ELISA technique.

Adult↗

PubMed1996

Cultured neonatal Schwann cells contain and secrete neuregulins.

Neuregulins have become the focus of intense research due to their putative roles in the etiology of certain cancers and to their evolving roles in PNS and CNS development. Evidence has been presented that neuregulins are present in neuronal cells where they act as mediators of neuron-glial signaling. Consistent with this view, we now report that there is a dramatic differential response of Schwann cells to the two major isoforms of neu differentiation factor (NDF alpha and NDF beta). NDF beta is a potent mitogen, whereas NDF alpha is nonmitogenic for Schwann cells. In addition, we report that Schwann cells contain endogenous NDF as well as the mRNA for both isoforms of NDF. Evidence is also presented that several isoforms of NDF are released from cultured Schwann cells. Our data suggest that in addition to functioning as a neuron-glial mediator, endogenous neuregulins may function in an autocrine/paracrine loop or in a juxtacrine- (cell-to-cell) mediated signal between individual Schwann cells or between Schwann cells and neurons.

Animals↗

PubMed1996

Treatment of oligodendrocytes with antisense deoxyoligonucleotide directed against CREB mRNA: effect on the cyclic AMP-dependent induction of myelin basic protein expression.

We have shown previously that in oligodendrocytes, the transcription factor cyclic AMP response element binding protein (CREB) is maximally expressed immediately prior to the most rapid period of myelination in rat brain. We have begun to investigate the role of this protein during myelination by downregulating CREB synthesis in cultured oligodendrocytes using an antisense deoxyoligonucleotide directed against CREB mRNA. Neonatal oligodendrocytes were grown for 4 days in a chemically defined medium (CDM) after which intracellular delivery of CREB antisense oligonucleotide was facilitated by using a liposome preparation. Control cultures were treated in a similar manner but in the presence of CREB sense oligomer. Immediately after transfection, cells were cultured for 3 days in CDM in the presence or absence of the cyclic AMP (cAMP) analogue N6, O21-dibutyryl cAMP (db-cAMP). In these cultures, myelin basic protein (MBP) expression was investigated by immunocytochemistry and Western blot analysis. Treatment of control cultures with db-cAMP resulted in a significant increase in the number of MBP positive cells which was abolished when the cells were treated with CREB antisense oligonucleotide. MBP positive cells in control cultures treated with db-cAMP have extended and highly branched MBP positive processes. In contrast, MBP positive cells in either control cultures grown in the absence of db-cAMP or cultures grown in the presence of db-cAMP but treated with CREB antisense oligonucleotide showed shorter and less complex processes and the MBP immunoreactivity appeared to be concentrated in the cell body. These observations suggest that CREB is at least one of the mediators in the induction of oligodendrocyte differentiation by cAMP.

Activating Transcription Factor 2↗

PubMed1996

Neurofilament phosphorylation is modulated by myelination.

Axons undergo substantial changes in radial growth during the course of development. Recent evidence suggests that axonal diameter may be controlled by the state of neurofilament (NF) phosphorylation. Using dorsal root ganglion (DRG)-Schwann cell co-cultures, we provide direct evidence that phosphorylation of NF is regulated by myelination. NF phosphorylation increased upon myelination of DRG neurons by Schwann cells. The increase in NF phosphorylation was reflected both as an increase in immunoreactivity with the antibody SMI31, specific for phosphorylation-dependent NF epitopes, and a concomitant decrease in immunoreactivity with SMI32, specific for nonphosphorylated NF epitopes. The increase in NF phosphorylation induced by myelination in the neuron-glia co-cultures was similar to NF phosphorylation seen in sciatic nerve extracts of mice with normal myelination compared to Trembler J mouse littermates in which myelination of peripheral nerves is compromised. Using an in situ gel kinase assay, we have detected changes in individual NF kinase activities during myelination. In particular, a 35-kDa kinase activity was induced by myelination, whereas a 42-kDa kinase decreased in activity. We discuss the possibility that these and other kinases may be involved in signaling processes between neurons and glia during myelination.

Animals↗

PubMed1995

Two mitogenic regions of myelin basic protein interact with different receptors to induce Schwann cell proliferation in a cAMP dependent process.

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1-44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152-167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of 125I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-beta nor PDGF-BB displaced cell surface bound 125I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP interacted with ganglioside GM1, whereas MBP did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP, while the FGF receptor mediates the mitogenic effect of MBP. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens.

Animals↗