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S D Skaper

Publications and source records attributed to S D Skaper.

At least 37 records · Page 2Linked to original sources
PubMed1998

Melatonin prevents the delayed death of hippocampal neurons induced by enhanced excitatory neurotransmission and the nitridergic pathway.

The mechanisms by which neurons die after stroke and status epilepticus and related neuropathological conditions are unclear, but may involve voltage-dependent Na+ channels, glutamate receptors, and nitric oxide (NO.). These questions were investigated using an in vitro primary cell culture model in which hippocampal pyramidal neurons undergo a gradual and delayed neurodegeneration induced by enhanced excitatory neurotransmission. When cells were treated with Mg2+-free, glycine-supplemented medium for a brief period (15 min) and examined 24 h later, approximately 30-40% of the neurons had died. Cell death could be inhibited by blockers of voltage-sensitive Na+ channels and by N-methyl-D-aspartate receptor antagonists. Application of either the endogenous antioxidant melatonin (EC50: 19.2+/-2.8 microM) or the NO. synthase inhibitor Nomega-nitro-L-arginine after, but not during, Mg2+-free exposure protected against delayed neuronal death; significant neuroprotection was observed when the addition was delayed for up to 4 h. This operational time window suggests that an enduring production of NO. and reactive oxygen species from neuronal sources is responsible for delayed cell death. A role for reactive oxygen species in this injury process was strengthened by the finding that, whereas neurons cocultured with astroglia were more resistant to killing, agents capable of lowering intracellular glutathione negated this protection. Because secretion levels of melatonin are decreased with aging, reductions in this pineal hormone may place neurons at a heightened risk for damage by excitatory synaptic transmission.

Animals↗
PubMed1997

The complete mature bovine prion protein highly expressed in Escherichia coli: biochemical and structural studies.

According to the 'protein only' hypothesis, modification of the 3-dimensional fold of the constituent cellular protein, PrP(C), into the disease-associated isoform, PrP(Sc), is the cause of neurodegenerative diseases in animals and humans. Here we describe the high-level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine-tagged full-length mature PrP (25-249) of bovine brain, termed His-PrP. Based on biochemical and spectroscopic data, His-PrP displays characteristics expected for the PrP(C) isoform. The reported expression system should allow the production of quantities of bovine PrP(C) sufficient to permit 3-dimensional structure determinations.

Amino Acid Sequence↗
PubMed1997

Quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: cooperative effects of ascorbic acid.

Oxidation reactions are essential biological reactions necessary for the formation of high-energy compounds used to fuel metabolic processes, but can be injurious to cells when produced in excess. Cutaneous tissue is especially susceptible to damage mediated by reactive oxygen species and low-density lipoprotein oxidation, triggered by dysmetabolic diseases, inflammation, environmental factors, or aging. Here we have examined the ability of the flavonoid quercetin to protect cutaneous tissue-associated cell types from injury induced by oxidative stress, and possible cooperative effects of ascorbic acid. Human skin fibroblasts, keratinocytes, and endothelial cells were cultured in the presence of buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione (GSH) synthesis. Depletion of intracellular levels of GSH leads to an accumulation of cellular peroxides and eventual cell death. Quercetin concentration-dependently (EC50: 30-40 microM) reduced oxidative injury of BSO to all cell types, and was also effective when first added after BSO washout. BSO caused marked decreases in the intracellular level of GSH, which remained depressed in quercetin-protected cells. Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO-induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue-type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage.

3T3 Cells↗
PubMed1997

Synthesis and cytotoxic profile of a diphtheria toxin-neurotrophin-4 chimera.

A diphtheria toxin-neurotrophin-4/5 (NT-4/5) chimera (DAB389-NT4), in which the native receptor binding domain of diphtheria toxin was replaced with a synthetic gene encoding rat NT-4/5, was expressed, refolded, and purified. This fusion toxin has a deduced molecular mass of 60,163 and is formed by joining the first 389 amino acids of diptheria toxin to amino acids 1-130 of mature rat NT-4/5, using an NH2-terminal bridge of 33 additional amino acids including six consecutive histidines. Neural cell types expressing only p75LNGFR or p75LNGFR and full-length or truncated TrkB were used to evaluate the cytotoxic efficacy of DAB389-NT4. The fusion toxin produced a concentration-dependent killing of all cell populations, with LC50 values that largely reflected the known NT-4/5 binding affinities for these receptor proteins. Mean LC50 values ranged from 2,960 pM in p75LNGFR-expressing neuro-2a neuroblastoma cells to 1,075 and 70 pM, respectively, in hippocampal astrocytes (p75LNGFR+/truncated TrkB+) and cerebellar granule cells (p75LNGFR+/TrkB+). The LC50 for DAB389-NT4 in receptor-negative 3T3 fibroblasts was 20 nM. NT-4/5 and brain-derived neurotrophic factor but not ciliary neurotrophic factor added in excess neutralized DAB389-NT4 cytotoxicity. NT-4/5, however, did not reduce the cytotoxicity of intact diphtheria toxin.

3T3 Cells↗
PubMed1997

Mast cells contain large quantities of secretagogue-sensitive N-acetylaspartate.

Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N-acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.

Animals↗
PubMed1997

Genetic construction, properties and application of a green fluorescent protein-tagged ciliary neurotrophic factor.

The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance. CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha). Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha. This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF. The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli. Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography. Under non-reducing conditions GFP-CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species. Despite these discrepancies, the molecular mass of GFP-CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536. Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain. Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield. Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other. GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons. GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF. GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat. This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner. GFP-CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.

Amino Acid Sequence↗
PubMed1997

Melatonin maintains glutathione homeostasis in kainic acid-exposed rat brain tissues.

Reduced glutathione (GSH) is a key component of the cellular defense cascade against injury caused by reactive oxygen species. Because kainic acid (KA) neurotoxicity is probably mediated at least in part by oxidative stress, we examined the influence of KA treatment on GSH content and GSH-related enzyme activities in adult rats. A single injection of KA (10 mg/kg i.p.) time-dependently decreased forebrain GSH (maximal reduction at 48 h). KA also markedly lowered GSH levels in amygdala and hippocampus, but not in the corpus striatum, which is resistant to KA injury. The pineal secretory product melatonin has been shown to exert neuroprotective effects against KA-induced excitotoxicity in rats. Melatonin (2.5 mg/kg i.p., administered four times) partially prevented all decreases in GSH of KA-treated rats. These neuroprotective effects of melatonin may result from a sparing of glutathione reductase, which decreased in KA-treated but not in KA/melatonin-treated animals. Moreover, KA caused a rapid decrease in the GSH content of cultured cerebellar granule neurons but not astrocytes. These cell types both express functional KA receptors, but only the former are sensitive to reactive oxygen species-dependent KA injury. Melatonin counteracted the changes in GSH induced by KA in cultured cerebellar granule neurons. Our results suggest that melatonin prevents the neurotoxic effects of reactive oxygen species linked to KA receptor activation by maintaining cellular GSH homeostasis.

Animals↗
PubMed1996

Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein.

Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.

Amino Acid Sequence↗
PubMed1996

Biotin deficiency facilitates kindling hyperexcitability in rats.

Biotin-deficient conditions are frequently associated with epileptic disorders. Biotin deficiency may be caused by long-term treatment with anticonvulsants or excessive ingestion of avidin. Absence of biotinidase activity can also lead to biotin deficiency, and is characterized by developmental delay as well as neurological and dermatological abnormalities. Because seizures are one of the most frequent signs of the latter, biotin-deficient conditions could conceivably facilitate convulsive disorders. To test this hypothesis, we investigated the occurrence of a latent kindling hyperexcitability in biotin-deprived rats. In these animals, duration of after-discharge on the first stimulation was longer at threshold amplitude, kindling development through its early stages was accelerated and duration of the forelimb clonus of fully kindled seizures was increased. Biotin deprivation in mixed cerebellar granule cell-astrocyte cultures also produced a tetrodotoxin-sensitive delayed loss of the glutamatergic neuronal population. The data thus support a facilitatory role for biotin-deficient conditions in convulsive disorders.

Animals↗
PubMed1996

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Amides↗
PubMed1996

Nerve growth factor: from neurotrophin to neurokine.

Nerve growth factor (NGF) is largely known as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurones and basal forebrain cholinergic nuclei during development and maturation. However, NGF also exerts a modulatory role on sensory, nociceptive nerve physiology during adulthood that appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other NGF-responsive cells are now recognized as belonging to the haemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with an increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general 'alert' molecule capable of recruiting and priming tissue defence processes following insult as well as systemic defensive mechanisms. Moreover, mast cells themselves produce NGF, suggesting that alterations in normal mast cell behaviours can provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states. This review discusses recent discoveries involving novel and diverse biological activities of this fascinating molecule.

Animals↗
PubMed1996

Mast cell activation causes delayed neurodegeneration in mixed hippocampal cultures via the nitric oxide pathway.

Mast cells are pleiotropic bone marrow-derived cells found in mucosal and connective tissues and in close apposition to neurons, where they play important roles in tissue inflammation and in neuroimmune interactions. Connective tissue mast cells, with which intracranial mast cells share many characteristics, contain cytokines that can cause inflammation. Here, we report that myelin basic protein, a major suspected immunogen in multiple sclerosis, as well as an antigenic stimulus, provokes mast cells to trigger a delayed cytotoxicity for neurons in mixed neuron-gila cultures from hippocampus. Neurotoxicity required a prolonged period (12 h) of mast cell incubation, and appeared to depend largely on elaboration of the free radical nitric oxide by astrocytes. Activation of astrocytes was mediated, in part, by mast cell-secreted tumor necrosis factor-alpha. Myelin basic protein and 17 beta-estradiol had a synergistic action on the induction of mast cell-associated neuronal injury. The cognate mast cell line RBL-2H3, when subjected to an antigenic stimulus, released tumor necrosis factor-alpha which, together with exogenous interleukin-1 beta (or interferon-gamma), induced astroglia to produce neurotoxic quantities of nitric oxide. A small but significant proportion of mast cell-derived neurotoxicity under the above conditions occurred independently of glial nitric oxide synthase induction. Further, palmitoylethanolamide, which has been reported to reduce mast cell activation by a local autacoid mechanism, decreased neuron loss resulting from mast cell stimulation in the mixed cultures but not that caused by direct cytokine induction of astrocytic nitric oxide synthase. These results support the notion that brain mast cells could participate in the pathophysiology of chronic neurodegenerative and inflammatory diseases of the nervous system, and suggest that down-modulation of mast cell activation in such conditions could be of therapeutic benefit.

Amides↗
PubMed1995

Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

Amides↗
PubMed1995

Update of the NGF saga.

Nerve growth factor (NGF), initially characterized for its survival and differentiating actions on embryonic sensory and sympathetic neurons, is now known to display a greatly extended spectrum of biological functions. NGF exerts a profound modulatory role on sensory nociceptive nerve physiology during adulthood which appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other newly detected NGF-responsive cells belong to the hematopoietic-immune and neuroendocrine systems. In particular, mast cells and NGF both appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general "alert" molecule capable of recruiting and priming both local tissue and systemic defense processes following stressful events. NGF can thus be viewed as a multifactorial mediator modulating neuroimmune-endocrine functions of vital importance to the regulation of homeostatic interactions, with potential involvement in pathological processes deriving from dysregulation of either local or systemic homeostatic balances.

Animals↗
PubMed1995

Inflammatory mediator stimulation of astrocytes and meningeal fibroblasts induces neuronal degeneration via the nitridergic pathway.

The role of inflammatory cytokines in the pathogenesis of neurological disorders is not entirely clear. The neurotoxic effects of cytokines, and perhaps indirectly bacterial endotoxins, could be mediated by the stimulation of immunocompetent cells in the brain to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. NO is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. Both constitutive and inducible NO synthase has been described in astrocytes in vitro. Here we demonstrate that newborn mouse cortical astrocytes, when coincubated with neonatal mouse cerebellar granule cells or hippocampal neurons, induced neurotoxicity upon stimulation with endotoxin (lipopolysaccharide) (ED50 30 ng/ml). Astrocytes were unresponsive to the cytokines tumor necrosis factor-alpha or interleukin-1 beta individually, but exhibited a marked synergistic stimulation in their combined presence. Moreover, meningeal fibroblasts treated with tumor necrosis factor-alpha, but not interleukin-1 beta or lipopolysaccharide, elaborated neurotoxicity for cocultured granule cells (ED50 30 U/ml). In cocultures of immunostimulated astrocytes or meningeal fibroblasts, neurotoxicity was blocked by the NO synthase inhibitors N omega-nitro-L-arginine and N omega-nitro-D-arginine methyl ester, and by oxyhemoglobin, which inactivates NO. Astroglial-induced neurotoxicity was not affected by N-methyl-D-aspartate receptor antagonists. Superoxide dismutase, which degrades superoxide anion, attenuated astrocyte- and fibroblast-mediated neurotoxicity, indicating that endogenous superoxide anion may react with NO to form toxic peroxynitrite and its breakdown products. These findings suggest a potentially important role for glial- and meningeal fibroblast-induced NO synthase in the pathophysiology of CNS disease states of immune or inflammatory origin.

Animals↗
PubMed1995

Production, characterization and cytotoxic properties of a diphtheria toxin-ciliary neurotrophic factor fusion protein.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. This study describes the genetic construction, expression, purification and properties of a diphtheria toxin-related CNTF fusion gene in which the native receptor binding domain of diphtheria toxin was genetically replaced with a synthetic gene encoding human CNTF. The fusion protein expressed from the chimeric tox gene was designated DAB389-CNTF. This fusion toxin has a deduced molecular weight of 67 440 and is formed by the fusion of the first 389 amino acids of diphtheria toxin to amino acids 15-200 of mature human CNTF (Cys17-->Ser), using a bridge of 34 additional amino acids including six consecutive histidine residues. This latter span allows for a single-step purification of the fusion protein by Ni(2+)-immobilized metal ion affinity chromatography, and provides a degree of flexibility which facilitates polypeptide refolding. DAB389-CNTF was selectively cytotoxic for clonal cells bearing CNTF receptors and for CNTF-responsive spinal sensory ganglion neurons in primary culture. The cytotoxic action of DAB389-CNTF, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The delivery of the catalytic domain to the target cell cytosol results in inhibition of protein synthesis and cell death. This latter point was confirmed by the observation that both CNTF and DAB389-CNTF increased c-fos mRNA expression, but only CNTF induced Fos protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Poly(ADP-ribose) polymerase: early involvement in glutamate-induced neurotoxicity in cultured cerebellar granule cells.

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.

Animals↗