Biomedical subjects

D E Pleasure

Publications and source records attributed to D E Pleasure.

At least 19 recordsLinked to original sources

PubMed2005

Schwann cell-autonomous role of neuropilin-2.

Neuropilins and group A plexins are components of receptor complexes for class 3 semaphorins, gradients of which help to guide migration of neural progenitor cells and axonal growth cones during development. We demonstrated previously that neuropilins and class 3 semaphorins are induced in sciatic nerve by crush or transection. We now report that in cultured rat Schwann cells, expression of mRNA encoding neuropilin-2 (NRP2) and plexin-A3 (PlexA3), proteins involved in semaphorin-3F (Sema3F) signal transduction, is diminished markedly by forskolin, an adenylate cyclase activator that, like axonal contact, induces Schwann cell synthesis of myelin lipids and proteins. Interestingly, Schwann cell expression of mRNA encoding NRP1, which participates in Sema3A signaling, is not downregulated by forskolin. Antibodies that recognize ectodomains of NRP2 but not control antibodies prevented cultured Schwann cells from aligning in parallel and forming columns. These results are consistent with the view that in nerves undergoing Wallerian degeneration, Schwann cell NRP2 facilitates assembly of Schwann cells into the tubular aggregates (bands of Büngner) that guide regenerating axons.

Animals↗

PubMed1998

A variant transcript encoding an isoform of the human protein tyrosine kinase EPHB2 is generated by alternative splicing and alternative use of polyadenylation signals.

We previously isolated and characterized cDNA clones of DRT (EPHB2), encoding a receptor protein-tyrosine kinase of the EPH family. Northern blot analysis showed that EPHB2 transcripts are expressed in three sizes of approximately 4, 5, and 11 kb, suggesting that these transcripts are generated by alternative splicing and/or alternative use of polyadenylation sites. To explore this possibility, we isolated additional EPHB2 cDNA clones, including clone 5K-1, by re-screening the human fetal brain cDNA library. Nucleotide sequence analysis of clone 5K-1 revealed that it represents a variant transcript of EPHB2 (EPHB2v). Relative to the EPHB2 cDNA sequence previously reported, clone 5K-1 has two coding region deletions of 3 and 93 nucleotides. Nucleotide sequence analyses of EPHB2 genomic DNA fragments corresponding to these deletions suggest that the EPHB2v transcript is generated by alternative splicing. The 3' end of clone 5K-1 contains a polyadenosine stretch preceded by a potential polyadenylation signal, which is not used to generate the EPHB2 transcript. Taken together, these data indicate that EPHB2v is generated by both alternative splicing and alternative use of polyadenylation sites. The EPHB2v protein lacks one arginine residue that resides immediately following the EPHB2 transmembrane domain. In contrast, as a result of the frame shift caused by the 93 nucleotide deletion, the C-terminus of the EPHB2v protein is longer by 70 amino acids than that of EPHB2. We also show that the human neuroblastoma cell line SY5Y and NTera-2N neurons express high levels of EPHB2 and lower levels of EPHB2v. These structural variations found between the EPHB2 and EPHB2v proteins may reflect functional heterogeneity of EPHB2.

Alternative Splicing↗

PubMed1998

Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors.

N-Methyl-D-aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1(011), conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.

Calcium↗

PubMed1997

cDNA cloning, chromosomal localization, and expression pattern of EPLG8, a new member of the EPLG gene family encoding ligands of EPH-related protein-tyrosine kinase receptors.

By screening a human fetal brain cDNA library under low stringency using cDNA encoding the mouse ligand of Cek5 as a probe, we have isolated a novel cDNA belonging to the EPLG gene family. This family encodes ligands of EPH-related tyrosine kinase receptors. Since the novel gene is the eighth member of the EPLG gene family, it is designated EPLG8. The deduced amino acid sequence of EPLG8 suggests that it encodes a transmembrane protein that is most related to those encoded by EPLG2 and EPLG5. We mapped the EPLG8 gene to human chromosome 17p11.2-p13.1 by PCR screening of human-rodent somatic cell hybrid panels. In the midterm fetus, EPLG8 mRNA is expressed at the highest level in brain, followed by heart, kidney, and lung. In the adult, EPLG8 mRNA expression is restricted to brain. These data suggest that LERK-8, the protein encoded by EPLG8, is important in brain development as well as in its maintenance. Moreover, since levels of EPLG8 expression were particularly high in several forebrain subregions compared to other brain subregions, LERK-8 may play a pivotal role in forebrain function.

Adult↗

PubMed1997

ECK, a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers.

Mouse eck, a member of the EPH gene family, has been mapped to mouse chromosome 4. The syntenic relationship between this chromosome and human chromosome 1 suggests that the human ECK gene maps to the distal short arm of human chromosome 1 (1p). Since this region is frequently deleted or altered in certain tumors of neuroectodermal origin, it is important to define the specific chromosomal localization of the human ECK gene. PCR screening of a rodent-human somatic cell hybrid panel by ECK-specific primers showed that ECK is indeed localized to human chromosome 1. Additional PCR screening of a regional screening panel for chromosome 1p indicated that ECK is localized to 1p36, distal to FUCA1. Furthermore, fluorescence in situ hybridization analysis with an ECK-specific P1 clone showed that ECK maps proximal to genetic marker D1S228. Taken together, the data suggest that ECK maps to 1p36.1, a region that is frequently deleted in neuroblastoma, melanoma, and other neuroectodermal tumors.

Animals↗

PubMed1997

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Calcium↗

PubMed1996

Effects of FGF-1 and FGF-2 on GD3 immunoreactive spinal neuroepithelial cells.

Embryonic central nervous system neuroepithelial cells are a transient population of cells that give rise to neuronal and glial progenitors. In the E12-E16 embryonic rat spinal neural tube we have identified neuroepithelial cells as radially oriented cells expressing the GD3 ganglioside as recognized by the monoclonal anti-GD3 ganglioside antibodies, R24 and LB1. In vitro, neuroepithelial cells, which migrate from the ventral aspect of E12 rat lumbosacral neural tube explants, also express GD3 ganglioside immunoreactivity, thus permitting their distinction from neural crest cells (NCC) which migrate from the dorsal aspect of such explants. Fibroblast growth factor-1 (FGF-1, acidic FGF) and FGF-2 (basic FGF) increase the migration of neuroepithelial cells and the extent to which they incorporate the thymidine analogue bromodeoxyuridine (BrdU). They do not, however, alter the rate at which these migrating neuroepithelial cells undergo cell death. Previous observations established the actions of FGF-1 and FGF-2 on neuronal and glial cells. The present study indicates that these growth factors also influence the motility and proliferation of progenitor cells at a developmental stage which precedes their divergence into neuronal and glial lineages.

Animals↗

PubMed1995

cDNA cloning, molecular characterization, and chromosomal localization of NET(EPHT2), a human EPH-related receptor protein-tyrosine kinase gene preferentially expressed in brain.

By screening a human fetal brain cDNA expression library using a monoclonal anti-phosphotyrosine antibody, we have isolated a cDNA clone encoding a receptor type protein-tyrosine kinase belonging to the EPH family, NET (neuronally expressed EPH-related tyrosine kinase). NET shows 87% homology in nucleotide sequence and 99% homology in the deduced amino acid sequence to rat elk, suggesting that NET is the human homologue of elk. The NET gene is mapped to human chromosome 3q21-q23 by PCR screening of a human-rodent somatic cell hybrid panel and by fluorescence in situ hybridization. Examination of NET mRNA expression in several human tissues has shown that the NET gene is expressed preferentially in brain as a 5-kb transcript. Steady-state levels of NET mRNA in human brain are greater in the midterm fetus than in the adult. Lower levels of NET mRNA are found in fetal kidney and adult skeletal muscle. The expression pattern of NET mRNA thus differs from that of elk, suggesting that these two gene products may perform distinct roles in human and rat. NET transcripts are detected in human NTera-2 teratocarcinoma cells after retinoic acid-induced neuronal differentiation. Several human tumor cell lines derived from neuroectoderm including primitive neuroectodermal tumor, small cell lung carcinoma, and neuroblastoma also express NET transcripts. Since the NET mRNA expression in human brain is developmentally regulated and is induced during neuronal differentiation, NET potentially plays important roles in human neurogenesis.

Adult↗

PubMed1995

Molecular characterization and chromosomal localization of DRT (EPHT3): a developmentally regulated human protein-tyrosine kinase gene of the EPH family.

By screening a human fetal brain cDNA expression library using a monoclonal antiphosphotyrosine antibody and by 5' RACE procedures, we have isolated overlapping cDNAs encoding a receptor-type tyrosine kinase belonging to the EPH family, DRT (Developmentally Regulated EPH-related Tyrosine kinase gene). The DRT gene is expressed in three different size transcripts (i.e. 4, 5 and 11 kb). DRT transcripts are expressed in human brain and several other tissues, including heart, lung, kidney, placenta, pancreas, liver and skeletal muscle, but the 11 kb DRT transcript is preferentially expressed in fetal brain. Steady-state levels of DRT mRNA in several tissues, including brain, heart, lung and kidney, are greater in the midterm fetus than those in the adult. DRT transcripts are detectable at low levels in a human teratocarcinoma cell line (NTera-2), but its expression is greatly increased after the NTera-2 cells are induced to become postmitotic neurons (NTera-2N) by retinoic acid treatment. These data suggest that DRT plays a part in human neurogenesis. A large number of tumor cell lines derived from neuroectoderm express DRT transcripts, including 12 neuroblastomas, two medulloblastomas, one primitive neuroectodermal tumor and six small cell lung carcinomas (SCLC). Interestingly, several neuroblastoma cell lines with 1p deletion and one SCLC cell line express DRT transcripts of aberrant size (i.e. 3, 6 and 8 kb) in addition to those found in normal tissues. We mapped the DRT gene to human chromosome 1p35-1p36.1 by PCR screening of human-rodent somatic cell hybrid panels and by fluorescence in situ hybridization. As the distal end of chromosome 1p is often deleted in neuroblastomas and altered in some cases in SCLCs, these chromosomal abnormalities may have resulted in the generation of aberrant size transcripts. Thus, the DRT gene may play a part in neuroblastoma and SCLC tumorigenesis.

Amino Acid Sequence↗

PubMed1990

Induction of severe experimental autoimmune neuritis with a synthetic peptide corresponding to the 53-78 amino acid sequence of the myelin P2 protein.

We generated a synthetic peptide (SP-26), corresponding to the amino acid residues 53-78 of bovine P2 protein, which induced severe clinical and pathological characteristics of experimental autoimmune neuritis (EAN) in Lewis rats. Lymph node cell populations from SP-26-immunized rats elicited a proliferative response to the peptide and to the P2 protein. After 16 cycles of antigen stimulation with the peptide, the SP-26 T cell line shows a decreased response to P2, but not to SP-26. Fluorescence-activated cell sorter (FACS) analysis of a SP-26 T cell line indicated the majority of cells to be of CD4+ CD8-. This report demonstrates that the synthetic peptide SP-26 can induce severe EAN in Lewis rats in a dose-dependent manner. Furthermore, specific T cell lines reactive to SP-26 can be generated from the lymph nodes of SP-26-immunized rats.

Animals↗

PubMed1990

Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.

Animals↗

PubMed1989

Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture.

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.

Animals↗

PubMed1988

Growth factors for fetal and adult human astrocytes in culture.

Using a recently developed method for the determination of cell proliferation in culture, we assessed the effects of several growth factors on their capability to promote proliferation of astrocytes derived from human fetuses or adults. Factors tested included fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, glial growth factor from bovine pituitary, nerve growth factor, interleukin-2, dibutyryl cyclic adenosine monophosphate, and 4 beta-phorbol 12,13-dibutyrate. The results show that astrocytes from human adults do not undergo proliferation in vitro even in the presence of these agents and that in the case of fetal astrocytes, only glial growth factor from bovine pituitary, platelet-derived growth factor and the phorbol ester were mitogenic. In addition, the capability of fetal astrocytes to proliferate appears to decrease with the increasing length of time they have been maintained in culture.

Adult↗

PubMed1988

Establishment of Schwann cell cultures from adult rat peripheral nerves.

Schwann cell cultures are difficult to obtain from adult rat because of the abundant amount of connective tissue and myelin. We have developed a method for isolation and culture of these cells by mechanical and chemical dissociation which represents a modification of a previously described procedure for human neurofibromas. Schwann cells were identified in indirect immunofluorescence by the capacity to bind antibodies to S-100, galactocerebroside, and laminin. The baseline cell proliferation index was assessed by immunofluorescence of bromodeoxyuridine. This method provides Schwann cells from adult rat nerves for at least 7 days and in sufficient numbers for (a) morphological and immunological characterization and (b) analysis of the effects of mitogenic factors.

Animals↗

PubMed1987

A monoclonal antibody to Schwann cell surface membrane recognizes a cAMP inducible epitope.

A monoclonal antibody to the surface membrane of rat Schwann cells was generated. This antibody bound to Schwann cell surface but not to fibroblasts in mixed neonatal sciatic nerve cultures. It did not bind to cells in newborn rat brain or corpus callosum cultures that contained oligodendrocytes, astrocytes, fibroblasts and neurons. The target antigen for this monoclonal antibody disappeared from Schwann cell surface after 5 days in culture. At this time, addition of 10(-3) M 8-bromo cyclic AMP or 10(-3) M dibutyryl cyclic AMP resulted in the reappearance within 2 days, of the target antigen on Schwann cell surface membrane. The reappearance of this epitope parallels that of surface galactocerebroside on Schwann cells when incubated with cAMP analogues. Re-expression of the target antigen for this monoclonal antibody in the presence of cAMP is evidence that cAMP may be a signal for synthesis of a variety of myelin molecules and as such could be a signal for myelination of axons by Schwann cells.

Animals↗

PubMed1986

Peripheral nerve grafts: the relationship of axonal growth cone and biomechanical properties.

Cat sciatic nerves containing 2 and 4 cm nerve grafts were subjected to tensile testing with the finding that a significant decrease in force at failure occurs at the distal suture line at a time after surgery that is graft length dependent. Tritiated labeling of regenerating axons and qualitative electromyography demonstrated that this effect was unrelated to the leading edge of the axonal growth cone, but temporally related to the functional reinnervation of denervated muscle.

Animals↗

PubMed1986

Astrocytes, oligodendrocytes, and Schwann cells share a common antigenic determinant that cross-reacts with myelin basic protein: identification with monoclonal antibody.

We have produced a monoclonal antibody against myelin basic protein that reacts with astrocytes, oligodendrocytes, and Schwann cells. This antibody was generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from adult rat corpus callosum. The antibody was characterized via solid-phase radioimmunoassay, immunoblot of SDS-PAGE, and by indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes, astrocytes, and Schwann cells. Myelin basic protein (MBP) was shown previously to be present only in myelin producing cells in CNS and PNS (oligodendroglia and Schwann cells) and not in astrocytes. The binding of this monoclonal antibody to all 3 cell types suggests that these cells share a common epitope. This epitope may be related to a common progenitor cell.

Animals↗

PubMed1985

Products of activated lymphocytes stimulate Schwann cell mitosis in vitro.

Culture of Schwann cells and endoneurial fibroblasts from newborn rat sciatic nerves in the presence of supernatants obtained from concanavalin A (Con-A)-stimulated rat mononuclear cells resulted in proliferation of both cell types. Con-A did not induce Schwann cell or fibroblast proliferation. Supernatant from a Gibbon T-cell lymphoma and chromatographically purified rat interleukin-2 (IL-2) induced fibroblast but not Schwann cell proliferation, and cloned human IL-2 did not induce proliferation of either cell type. Proliferation of Schwann cells and endoneurial fibroblasts induced by activated mononuclear inflammatory cells may be important in inflammatory demyelinative neuropathies.

Animals↗