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S D Skaper

Publications and source records attributed to S D Skaper.

At least 55 records · Page 3Linked to original sources
PubMed1994

Structure-function studies of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a polypeptide that promotes the survival and/or differentiation of a number of neural cell types. Here we present a structural and functional analysis of the human CNTF molecule. Variant proteins were synthesized by Escherichia coli transformed with mutant cDNA constructs, and purified by SDS-polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography. Most variant CNTF proteins lacked neurotrophic activity, but two N- and C-terminal deletions (delta 2-14 and delta 173-200, respectively) actually displayed a several-fold increase in specific activity. Loss of biological activity was accompanied by changes in the alpha-helical nature of CNTF as measured by circular dichroism. These data strengthen the proposed similarity between CNTF and the family of hematopoietic cytokines.

Animals↗
PubMed1994

Characterization and growth-dependent regulation of the nerve growth factor receptor gp140trk in rat C6 glioma cells.

The glioma cell line C6 was used to study the expression and growth-dependent regulation of the nerve growth factor (NGF) tyrosine kinase receptor gp140trk, which is the mature protein product of the trk proto-oncogene. Chemical cross-linking of 125I-NGF to C6 cells, followed by immunoprecipitation with polyclonal anti-NGF antibodies and separation by polyacrylamide gel electrophoresis, revealed the presence of 90-95 and 150 kDa species. Immunocytochemical staining of C6 cells with antibodies directed against either the low-affinity NGF receptor gp75NGFR or trk proto-oncogene products demonstrated a heterogeneous cellular distribution of both antigens. Brief treatment of C6 cells with NGF led to the tyrosine phosphorylation of 80, 110 and 140 kDa protein species, as detected on anti-phosphotyrosine Western blots. Similar molecular weight species were found with anti-Trk antibodies in the NGF-treated cells. Intracellular localization of Trk-like immunoreactivity in C6 cells released from a growth-arrested state indicated an initial immunostaining of the nuclear periphery, progressing to cytoplasmic vesicles and finally to the plasma membrane. These observations at the light microscopic level were confirmed using immunoelectron microscopy with the same anti-Trk antibodies, and showed clearly the trafficking of Trk-like immunostained particles from the endoplasmic reticulum to the plasmalemma. The cellular localization of trk gene products also appeared to depend on their glycosylation state. Such growth-dependent expression of NGF receptors on glial cells may be important in controlling autocrine regulatory processes of glia to NGF, which these cells produce.

Animals↗
PubMed1994

Establishment and characterization of a CHO cell line producing a secreted form of human ciliary neurotrophic factor: neuroprotective effects of the recombinant protein.

Human ciliary neurotrophic factor (CNTF) was inserted into a mammalian expression vector linked to the prepro sequence of human nerve growth factor. A Chinese hamster ovary cell line was established by resistance to neomycin and the plasmid integrated DNA was amplified using the metallothionein gene. This cell line contained several hundred copies of the human CNTF gene and produced an NH2 terminal truncated form of human CNTF (22 kDa) which was secreted into the medium. Although the copy number of the human CNTF gene was high and its mRNA was actively transcribed, the recombinant protein secreted into the medium constituted only 35-40% of the total amount of human CNTF synthesized by these cells. Both wild-type human CNTF produced in bacterial cells and the human CNTF obtained by forced secretion were effective in protecting hippocampal pyramidal neurons from injury induced by glucose deprivation, a form of excitotoxic neurodegeneration.

Amino Acid Sequence↗
PubMed1994

Production and characterization of recombinant rat brain-derived neurotrophic factor and neurotrophin-3 from insect cells.

Rat brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were engineered for expression in a baculovirus-infected Spodoptera frugiperda insect cell system. The BDNF and NT-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. The purification procedure yielded approximately 2 mg of pure rat BDNF or NT-3 per liter of culture supernatant. A single N-terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell-produced rat BDNF and NT-3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT-3, the BDNF and NT-3 recovered after purification were > 95% fully processed, mature protein. Mature recombinant rat BDNF and NT-3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT-3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT-3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.

Amino Acid Sequence↗
PubMed1994

N-acetylaspartylglutamate selectively inhibits neuronal responses to N-methyl-D-aspartic acid in vitro.

Canavan's disease is an autosomal recessive disorder characterized by a deficiency of aspartoacylase and accumulation of N-acetylaspartic acid (NAA), leading to a severe leukodystrophy and spongy degeneration of the brain. N-Acetylaspartylglutamate (NAAG), the presumed product of NAA, also accumulates in this disease. The endogenous dipeptide NAAG has been suggested to have low potency at NMDA receptors. Here we have tested the actions of NAAG and NAA on NMDA-evoked responses in cultured cerebellar granule cells. In differentiating granule cells grown in low-K+ medium, NAAG negated the survival-promoting effects of NMDA but not K+ depolarization. Neither NAAG nor NAA alone promoted cell survival in low-K+ medium. The modest trophic action of 50 microM kainic acid in low-K+ medium was reinforced by the NMDA receptor antagonist dizocilpine maleate and by NAAG. In K(+)-differentiated granule cells, NAAG raised the threshold of NMDA neurotoxicity but not that of kainate. The observed activities of NAAG were overcome by excess NMDA and were not mimicked by NAA. These data raise the possibility that disruption of NMDA receptor processes by NAAG may be of pathophysiological relevance.

Animals↗
PubMed1994

Nerve growth factor and autoimmune diseases.

The initiation of a humoral immune response to a foreign antigen is a complex biologic process involving the interaction of many cell types and their secreted products. Autoimmune diseases, which are characterized by an abnormal activation of the immune system, probably result from the failure of normal self-tolerance mechanisms. The etiology of such illnesses, however, is far from being understood. While there have been extensive studies on the participation of the immune and endocrine systems in autoimmune diseases, few have dealt with nervous system-mediated immunoregulation in such situations. Evidence continues to grow suggesting that nerve growth factor (NGF), first identified for its activity in promoting the growth and differentiation of sensory and sympathetic neurons, may exert a modulatory role on neuroimmunoendocrine functions of vital importance in the regulation of homeostatic processes. Newly detected NGF-responsive cells belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. NGF levels are elevated in a number of autoimmune states, along with increased accumulation of mast cells. NGF and mast cells both appear to be involved in neuroimmune interactions and tissue inflammation. Moreover, mast cells themselves synthesize, store, and release NGF, proposing that alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature. This review focuses on these cellular events and presents a working model which attempts to explain the close interrelationships of the neuroendocrinoimmune triade via a modulatory action of NGF.

Autoimmune Diseases↗
PubMed1993

A semisynthetic glycosphingolipid (LIGA20) reduces 2,4, 5-trihydroxyphenylalanine neurotoxicity in primary neuronal cultures.

The semisynthetic glycosphingolipid derivative II3Neu5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-chloro-acetamid e-4-trans- octadacene (LIGA20) attenuated injury induced by the excitotoxic L-dopa metabolite 2,4,5-trihydroxyphenylalanine (TOPA) in cultures of rat cerebellar granule cells when presented simultaneously with TOPA (EC50; 9 microM LIGA20). The natural glycosphingolipid ganglioside GM1 up to 200 microM was not neuroprotective as cotreatment, although pretreatment of cells for 2 h was efficacious. This greater potency and speed of LIGA20 action extended to limiting TOPA-induced death of cultured mesencephalic dopaminergic neurons. These data suggest that LIGA20 may have therapeutic potential for the treatment of disorders associated with excitotoxic processes.

Animals↗
PubMed1993

Gangliosides and neurotrophic factors in neurodegenerative diseases: from experimental findings to clinical perspectives.

A large body of experimental data suggests that neurotrophic molecules and/or substances that facilitate their action could be pharmaceutical agents for neurodegenerative pathologies. In particular, it has been demonstrated that nerve growth factor (NGF) exerts a physiological role for forebrain cholinergic neurons, while brain-derived neurotrophic factor (BDNF) seems to play a relevant role in rescuing dopaminergic neurons following damage. In addition, gangliosides are reported to potentiate neurotrophic factor effects in vitro as well as in vivo. In this study we examined the effects of the monosialoganglioside GM1 in different experimental models. The responsiveness of forebrain cholinergic neurons following NGF +/- GM1 was evaluated by assessing choline acetyltransferase (ChAT) activity in hippocampus, septal area and striatum of behaviorally impaired 24-month-old rats. NGF was intracerebroventricularly (i.c.v.) infused for 2 weeks while GM1 was given systemically for 3 weeks, starting from the beginning of NGF infusion. Moreover, the possible protective effects of GM1 were assessed following exposure of cultured cerebellar granule cells and dopaminergic mesencephalic neurons to different doses of 6-OH-DOPA, a metabolite of the dopamine pathway which has excitotoxic properties and has been hypothesized to participate in the pathology of Parkinson's disease. GM1 treatment to aged rats was seen to potentiate the NGF-induced increase of ChAT activity in the striatum ipsilateral to the NGF infusion. Moreover, in the striatum contralateral to the NGF infusion, GM1 increased ChAT activity above the control values, whereas NGF treatment alone did not affect enzymatic activity. GM1 treatment of cerebellar granule cells and mesencephalic neurons counteracted the dose- and time-dependent neurotoxicity of 6-OH-DOPA. These data support the notion that GM1 might prove useful in treating those pathological conditions where trophic factor deficits and/or excitotoxin-related toxicity play an important role.

Aging↗
PubMed1993

Ganglioside GM1 cooperates with brain-derived neurotrophic factor to protect dopaminergic neurons from 6-hydroxydopamine-induced degeneration.

Brain-derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopaminergic neurons in cultures derived from the embryonic rat mesencephalon. In the present study BDNF was found to protect cultured dopaminergic neurons from injury induced by acute exposure to the dopaminergic-selective neurotoxin 6-hydroxydopamine. The BDNF effect was concentration (ED50 approximately 10 ng/ml) and time-dependent, as determined by tyrosine hydroxylase immunocytochemistry. More importantly, subthreshold amounts of BDNF were rendered efficacious in the presence of ganglioside GM1: loss of tyrosine hydroxylase positive cells was reduced from 80% to only 20%. Thus GM1 may provide a fruitful treatment strategy for disorders of dopamine function such as Parkinson's disease.

Animals↗
PubMed1993

Brain-derived neurotrophic factor selectively rescues mesencephalic dopaminergic neurons from 2,4,5-trihydroxyphenylalanine-induced injury.

Brain-derived neurotrophic factor (BDNF) supports the survival of sensory neurons as well as retinal ganglion cells, basal forebrain cholinergic neurons, and mesencephalic dopaminergic neurons in vitro. Here we examined the ability of BDNF to confer protection on cultured dopaminergic neurons against the neurotoxic effects of 6-hydroxyDOPA (TOPA or 2,4,5-trihydroxyphenylalanine), a metabolite of the dopamine pathway suggested to participate in the pathology of Parkinson's disease. Cells prepared from embryonic day 14-15 rat mesencephalon were maintained with 10-50 ng/ml BDNF for 7 days prior to addition of TOPA (10-30 microM) for 24 hr. In BDNF-treated cultures, the extensive loss (> 90%) of tyrosine hydroxylase immunopositive cells was virtually (< 10%) eliminated, while the equally drastic loss (> 90%) of the overall cell population was limited to only a 25-30% recovery. Furthermore, the monosialoganglioside GM1 (1-10 microM), although inactive alone, acted synergistically with subthreshold amounts of BDNF to rescue tyrosine hydroxylase-positive cells against TOPA neurotoxicity. These results add impetus to exploring the therapeutic potential of gangliosides and BDNF in Parkinson's disease.

Animals↗
PubMed1993

Basic fibroblast growth factor modulates sensitivity of cultured hippocampal pyramidal neurons to glutamate cytotoxicity: interaction with ganglioside GM1.

Basic fibroblast growth factor (bFGF), a polypeptide originally identified as a mitogen for a variety of cells including astroglial cells, also exhibits neurotrophic (survival) effects on a number of neuronal populations, among the latter being hippocampal pyramidal cells. The present study investigated the effects of bFGF on the sensitivity of pyramidal neurons to the excitatory neurotransmitter, glutamate, and possible modulation by monosialoganglioside GM1. Cultures were generated from embryonic day 18 rat hippocampus, and first treated with bFGF at 4-5 days in vitro. Twenty-four hours later, cells were exposed to glutamate (100 microM-1 mM) for a further 24 h in the continued presence of bFGF. The cytotoxic action caused by 200-500 microM glutamate, which normally is present at this culture stage, was reduced by bFGF in a concentration- and time-dependent manner. GM1 (100 microM), given alone 2 h prior to glutamate, also limited this neuronal loss by 50-80%. At lower concentrations, neither bFGF (0.3 ng/ml) nor GM1 (1-10 microM) alone for 24 h was effective in altering neuronal sensitivity to glutamate. However, given together for 24 h these levels of bFGF and GM1 were almost as efficacious as bFGF alone at 3-10 ng/ml. Similar results were obtained with more mature (12 day) cultures. The ability of GM1 to modulate trophic factor actions towards excitatory amino acids makes gangliosides useful tools in the study of central nervous system plasticity and repair processes.

Animals↗
PubMed1993

Nerve growth factor antibodies recognize neurotrophin-3.

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the beta subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.

Animals↗
PubMed1993

Neurotrophin-3 upregulates NGF receptors in a central nervous system glial cell line.

The low-affinity nerve growth factor (NGF) receptor (LNGFR) binds the neurotrophins NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) with similar affinities. Here we report on the ability of NT-3 to regulate the expression of the LNGFR in C6 glioma cells. LNGFR-like immunoreactivity (LNGFR-IR) was examined in C6 cells treated for 16 h with NT-3 and exposed to the antibody 192-IgG followed by immunoglobulins conjugated with colloidal gold by means of ultrastructural morphometric analysis. Untreated C6 cells exhibited some positive LNGFR-IR, while C6 cells treated with NT-3 displayed significantly increased (2.3 fold) LNGFR-IR. The increase in LNGFR protein was accompanied by a greater quantity of LNGFR mRNA in NT-3-treated cells. Thus, LNGFR can be upregulated by the structurally related neurotrophin NT-3.

Animals↗
PubMed1993

Glucocorticoids depress activity-dependent expression of BDNF mRNA in hippocampal neurones.

Glucocorticoid hormones are important regulators of brain development and ageing, and can impair the capacity of hippocampal neurones to survive various neurological insults. Here we show that dexamethasone, a synthetic glucocorticoid, prevents activity-dependent increases of brain-derived neurotrophic factor (BDNF) mRNA in cultures of rat hippocampal neurones. In situ hybridization was used to evaluate the levels of BDNF mRNA. Up-regulation of BDNF mRNA triggered by depolarization with high potassium, or exposure to the glutamate receptor agonist kainic acid, resulted both from higher levels of expression in neurones and from new recruitment of cells. These data suggest that the known ability of glucocorticoids to exacerbate neuronal injury following ischaemia and other metabolic insults could be due to antagonism of regulatory mechanisms governing neurotrophin levels in the brain.

Animals↗
PubMed1993

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.

Animals↗
PubMed1992

Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli.

The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.

Animals↗
PubMed1992

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

Animals↗