bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of 3‐[123I]iodo‐L‐α‐methyl tyrosine transport in astrocytes of neonatal rats
Authors: Kopka Klaus , Riemann Burkhard , Friedrich Matthias , Winters Silke , Halfter Hartmut , Weckesser Matthias , Stögbauer Florian , Ringelstein E. Bernd , Schober Otmar 
Published: March 2003

3‐[123I]Iodo‐L‐α‐methyl tyrosine (123I‐IMT) is used for diagnosis and monitoring of brain tumours by means of single‐photon emission tomography. As recently shown, 123I‐IMT is predominantly mediated into rat C6 glioma cells by sodium‐independent system L for large neutral amino acids. Until now, 123I‐IMT transport in non‐neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of 123I‐IMT uptake into astrocytes of neonatal rats. In particular sodium‐independent 123I‐IMT transport into neonatal astrocytes was compared with sodium‐independent 123I‐IMT uptake into neoplastic rat C6 glioma cells.Competitive inhibition experiments showed that 123I‐IMT is exclusively transported via sodium‐independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium‐independent 123I‐IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants KM = 13.9 ± 0.5 µm and KM = 33.9 ± 4.1 µm, respectively, which are in the same range of KM values as those recently determined for amino acid transport into neoplastic and non‐neoplastic glial cells. Indeed, the KM values in the micromolar range correspond to the expression of the LAT‐1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium‐independent maximum transport velocities (Vmax) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 ± 0.3 and 39.9 ± 3.3 nmol/mg protein/10 min, respectively).
A novel untranslated ‘exon H’ of the human choline acetyltransferase gene in placenta
Authors: Robert Isabelle , Quirin‐Stricker Christiane 
Published: March 2003

To investigate the existence of 5′‐region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5′‐untranslated regions (UTR) in human neuronal and non‐neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG‐3 and neuroblastoma CHP126 and MC‐IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT‐PCR). We used a sense primer located in the 5′‐flanking region, in the previously defined intronic sequence and an anti‐sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG‐3 cells whereas it was absent in spinal cord, CHP126 and MC‐IXC cells. It was designated ‘H‐type’ of ChAT mRNA. Whereas CHP126 produced the R‐ and N‐type of ChAT mRNAs, no transcript of the N‐and R‐type was detected in JEG‐3 and human placenta. In addition, CHP126 and JEG‐3 cells and placenta showed the expression of the M‐type of ChAT mRNA. The idendity of the amplified 567 bp product (H‐type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H‐type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M‐type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.
Divergent pathways account for two distinct effects of amyloid β peptides on exocytosis and Ca2+ currents: involvement of ROS and NF‐κB
Authors: Green Kim N. , Peers Chris 
Published: March 2003

AbstractAmyloid peptides (AβPs) are implicated in neuronal death associated with Alzheimer's disease. Their toxicity involves disruption cellular Ca2+ homeostasis, leading to activation of caspases and cell death. Antioxidants can prevent such cell death and show beneficial clinical effects in Alzheimer's disease patients. Using the model neurosecretory cell line, PC12, we have shown that AβPs cause enhancement of evoked exocytosis via formation of a Cd2+‐resistant Ca2+ influx pathway, and also cause selective, functional up‐regulation of current through L‐type Ca2+ channels. The involvement of reactive oxygen species (ROS) in these effects were investigated by examining the ability of various antioxidants to interfere with these responses. Both melatonin and ascorbic acid fully blocked the enhancement of catecholamine secretion caused by application of AβP(1−40), as monitored in real time amperometrically, but inhibition of the transcriptional regulator NF‐κB with SN‐50 did not affect secretion. Enhanced immunofluorescence, observed in AβP‐treated cells using a monoclonal antibody raised against the	N‐terminus of AβP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen also fully prevented augmentation of whole‐cell Ca2+ currents caused by application of AβP(1−40). By contrast, inhibitors of NF‐κB (sulfasalazine and SN‐50) were able to prevent AβP induced Ca2+ channel current enhancement, whilst inhibitors of mitogen‐activated protein kinase and protein kinase C could not. Our results indicate that augmentation or induction by AβPs of two important, distinct factors regulating Ca2+ homeostasis is mediated by increased ROS production, but only one of these (up‐regulation of native Ca2+ channels) requires activation of NF‐κB.
Further characterization of the molecular interaction between PSD‐95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating
Authors: Rutter A. Richard , Freeman Fiona M. , Stephenson F. Anne 
Published: March 2003

AbstractCoexpression of PSD‐95c‐Myc with NR1‐1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3H]MK801 binding similar to that previously described for l‐glutamate. The inhibition constants (KIs) for the binding of l‐glutamate and glycine to NR1‐1a/NR2A determined by [3H]CGP 39653 and [3H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1‐1a/NR2A receptors coexpressed ± PSD‐95c‐Myc. The increased EC50 for l‐glutamate enhancement of [3H]MK801 binding was also found for NR1‐2a/NR2A and NR1‐4b/NRA receptors thus the altered EC50 is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1‐4b but not the NR1‐1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1‐4b and NR1‐4b/NR2A expression was enhanced by PSD‐95c‐Myc but whole cell enzyme‐linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD‐95c‐Myc has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C‐terminal E(T/S)XV PSD‐95 binding motifs.
Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons
Authors: Holm Pontus C. , Åkerud Peter , Wagner Joseph , Arenas Ernest 
Published: March 2003

AbstractNoradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c‐ret, which binds ligands of the glial cell line‐derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)‐positive neurons in the LC express both GFRα1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down‐regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH‐positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6‐hydroxydopamine (6‐OHDA) lesion model, but induced the sprouting of TH‐positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.
Aspirin provides cyclin‐dependent kinase 5‐dependent protection against subsequent hypoxia/reoxygenation damage in culture
Authors: Vartiainen Nina , Keksa‐Goldsteine Velta , Goldsteins Gundars , Koistinaho Jari 
Published: March 2003

AbstractAspirin [acetylsalicylic acid (ASA)] is an anti‐inflammatory drug that protects against cellular injury by inhibiting cyclooxygenases (COX), inducible nitric oxide synthase (iNOS) and p44/42 mitogen‐activated protein kinase (p44/42 MAPK), or by preventing translocation of nuclear factor κB (NF‐κB). We studied the effect of ASA pre‐treatment on neuronal survival after hypoxia/reoxygenation damage in rat spinal cord (SC) cultures. In this injury model, COX, iNOS and NF‐κB played no role in the early neuronal death. A 20‐h treatment with 3 mm ASA prior to hypoxia/reoxygenation blocked the hypoxia/reoxygenation‐induced lactate dehydrogenase (LDH) release from neurons. This neuroprotection was associated with increased phosphorylation of neurofilaments, which are substrates of p44/42 MAPK and cyclin‐dependent kinase 5 (Cdk5). PD90859, a p44/42 MAPK inhibitor, had no effect on ASA‐induced tolerance, but olomoucine and roscovitine, Cdk5 inhibitors, reduced ASA neuroprotection. Hypoxia/reoxygenation alone reduced both the protein amount and activity of Cdk5, and this reduction was inhibited by pre‐treatment with ASA. Moreover, the protein amount of a neuronal Cdk5 activator, p35, recovered after reoxygenation only in ASA‐treated samples. The prevention of the loss in Cdk5 activity during reoxygenation was crucial for ASA‐induced protection, because co‐administration of Cdk5 inhibitors at the onset ofreoxygenation abolished the protection. In conclusion, pre‐treatment with ASA induces tolerance against hypoxia/reoxygenation damage in spinal cord cultures by restoring Cdk5 and p35 protein expression.
Tetrahydrobiopterin deficiency increases neuronal vulnerability to hypoxia
Authors: Delgado‐Esteban María , Almeida Angeles , Medina Jose M. 
Published: March 2003

AbstractTetrahydrobiopterin (BH4) is an essential co‐factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre‐incubating neurones with 5 mm 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia‐induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4‐deficient neurones. All these effects were counteracted whenneuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia‐induced damage in BH4‐deficient neurones was prevented when Nω‐nitro‐l‐arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability tohypoxia‐induced mitochondrial damage and necrosis.
Long‐term post‐synaptic consequences of methamphetamine on preprotachykinin mRNA expression
Authors: Johnson‐Davis Kamisha L. , Hanson Glen R. , Keefe Kristen A. 
Published: March 2003

AbstractExposure to repeated high doses of methamphetamine produces long‐term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by ∼20% in the middle striatum and ∼32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by ∼20% in the middle striatum and ∼14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either timepoint. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.
The human SH‐SY5Y neuroblastoma cell‐line expresses a functional P2X7 purinoceptor that modulates voltage‐dependent Ca2+ channel function
Authors: Per Larsson Kim , Jon Hansen Anker , Dissing Steen 
Published: March 2003

AbstractFura‐2 imaging of purinergic stimulation of non‐differentiated neuronal human SH‐SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 µm) was as follows: 2′,3′‐O‐(4‐benzoyl‐benzoyl)‐ATP (BzATP) > ATP4– > ATP; whereas 2‐(methylthio)‐ATP, ADP, UTP and α,β‐methylene‐ATP and β,γ‐methylene‐ATP were ineffective. The response to BzATP was inhibited by pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic‐acid (PPADS, 1 µm), 1‐(N,O‐bis[5‐isoquinolinesulfonyl]‐N‐methyl‐l‐tyrosyl)‐4‐phenylpiperazine (KN‐62, 100 nm) and 8‐(3‐benzamido‐4–4‐methylbenzamido)‐naphthalene‐1,3,5‐trisulfonic‐acid (suramin, 200 µm). The presence of a P2X7 receptor was confirmed by western blot studies using anti‐P2X7. EC50 for BzATP was 212 ± 6 µm. BzATP > 30 µm induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 µm only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 µm) and to a smaller degree by ω‐conotoxin GVIA (1 µm) despite an almost equal presence of L and N‐type Ca2+‐channels. In whole‐cell voltage‐clamp studies at − 90 mV, BzATP (300 µm) produced a fast activating inward current with a similar pharmacology as observed with Fura‐2 imaging. Current clamp studies showed a dose‐dependent depolarization to BzATP and ATP4–. BzATP also triggered transmitter release. Thus, the human neuronal SH‐SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+‐channels.
Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization
Authors: Le Crom Stéphane , Prou Delphine , Vernier Philippe 
Published: March 2003

AbstractAdenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS‐7 cells, long‐term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS‐15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long‐term desensitization time‐course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre‐treatment of cells with R(–)‐PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.
β‐Amyloid peptide induces formation of actin stress fibers through p38 mitogen‐activated protein kinase
Authors: Song Cheng , Perides George , Wang Dechun , Liu Ya Fang 
Published: March 2003

AbstractBased on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial β‐amyloid precursor protein APP‐V642I (IAPP) or mutant presenilin‐1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild‐type amyloid precursor protein or presenilin‐1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with β‐amyloid (Aβ) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a γ‐secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Aβ peptide induces actin stress fibers. Because activation of the p38 mitogen‐activated protein kinase (p38MAPK)–mitogen‐associated protein kinase‐associated protein kinase (MAPKAPK)‐2–heat‐shock protein 27 signaling pathway mediates actin polymerization, we explored whether Aβ peptide activates p38MAPK and MAPKAPK‐2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK‐2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Aβ peptide, IAPP or mPS1. Moreover, treatment with a γ‐secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK‐2. In summary, our data suggest that overproduction of Aβ peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.
Glutamate release by an Na+ load and oxidative stress in nerve terminals: relevance to ischemia/reperfusion
Authors: Tretter Laszlo , Adam‐Vizi Vera 
Published: March 2003

AbstractPreviously we have reported that oxidative stress induced by hydrogen peroxide exacerbates the effect of an Na+ load in isolated nerve terminals, with a consequence of an ATP depletion, [Ca2+]i and [Na+]i deregulation, and collapse of mitochondrial membrane potential. In the present study, the release of glutamate in response to a combined effect of an [Na+] load and oxidative stress was measured in isolated nerve terminals over an incubation for 15 min. Exposure to hydrogen peroxide (100 µm) had no effect on the release of glutamate, but significantly enhanced the Ca2+‐independent glutamate release induced by a small [Na+] load achieved with 10 µm veratridine. The effect of a larger Na+ load induced by 40 µm veratridine was not further increased by hydrogen peroxide; in contrast the external Ca2+‐dependent glutamate release was completely eliminated by the oxidant under this condition. The effects of oxidative stress superimposed on a Na+ load are consistent with at least two factors: (i) a relatively modest Na+ load induced by veratridine is augmented by H2O2 giving rise to an increased Ca2+‐independent release of glutamate (ii) oxidative stress in combination with a larger Na+ load causes severe ATP depletion limiting the Ca2+‐dependent vesicular glutamate release. Given the concurrent presence of an Na+ load and oxidative stress in ischemia/reperfusion these results indicate that the extent of the Na+ load developing during the ischemic period could determine the release of glutamate induced by an oxidative stress during reperfusion.
Synaptic concentration of type‐I adenylyl cyclase in cerebellar neurons
Authors: Wang Hongbing , Chan Guy C.‐K. , Athos Jaime , Storm Daniel R. 
Published: March 2003

AbstractSpecific subcellular targeting and spatial arrangement of signaling molecules are important for efficient signal transduction. The neuro‐specific type‐I adenylyl cyclase (AC1) is stimulated by Ca2+, and plays an essential role in neurodevelopment and neuroplasticity. We generated hemagglutinin (HA)‐tagged AC1 to study its subcellular localization in cultured neurons. The HA‐tagged AC1 has similar enzymatic activity and regulatory properties to that of non‐tagged protein. HA‐AC1 targeted to both apical and basolateral domains in the epithelial Madin–Darby canine kidney (MDCK) cells, and it was found in both axons and dendrites in cultured hippocampal neurons as well as in cerebellar granule neurons. Interestingly, AC1 showed a distinct punctate form of immunostaining in MDCK cells and transfected neurons, suggesting it targets to specific subcellular domains. By immunostaining with different synaptic markers, we found that AC1 puncta were located at the excitatory synapses in cerebellar granule neurons. Our data provide a possible cellular mechanism for the physiological role of AC1 in neuroplasticity.
Association of Caspr/paranodin with tumour suppressor schwannomin/merlin and β1 integrin in the central nervous system
Authors: Denisenko‐Nehrbass Natalia , Goutebroze Laurence , Galvez Thierry , Bonnon Carine , Stankoff Bruno , Ezan Pascal , Giovannini Marco , Faivre‐Sarrailh Catherine , Girault Jean‐Antoine 
Published: March 2003

AbstractCaspr/paranodin is an essential neuronal component of paranodal axoglial junctions, associated with contactin/F3. Its short intracellular domain contains a conserved motif (GNP motif) capable of binding protein 4.1 domains [FERM domains (four point one, ezrin, radixin, moesin)]. Schwannomin/merlin is a tumour suppressor expressed in many cell types, including in neurons, the function and partners of which are still poorly characterized. We show that the FERM domain of schwannomin binds to the paranodin GNP motif in glutathione S‐transferase (GST)‐pull down assays and in transfected COS‐7 cells. The two proteins co‐immunoprecipitated in brain extracts. In addition, paranodin and schwannomin were associated with integrin β1 in transfected cells and in brain homogenates. The presence of paranodin increased the association between integrin β1 and schwannomin or its N‐terminal domain, suggesting that the interactions between these proteins are interdependent. In jimpy mutant mice, which display a severe dysmyelination with deficient paranodal junctions, the interactions between paranodin, schwannomin and integrin β1 were profoundly altered. Our results show that schwannomin and integrin β1 can be associated with paranodin in the central nervous system. Since integrin β1 and schwannomin do not appear to be enriched in paranodes they may be quantitatively minor partners of paranodin in these regions and/or be associated with paranodin at other locations.