bluuebunny/crossref_metadata_2025_split huggingface dataset:
Toxic Effects of Iron for Cultured Mesencephalic Dopaminergic Neurons Derived from Rat Embryonic Brains
Authors: Michel Patrick P. , Vyas Sheela , Agid Yves 
Published: October 2006

AbstractIron, a transition metal possibly involved in the pathogenesis of Parkinson's disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum‐free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochem‐istry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 μM. High‐affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50= 67 μM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and γ‐aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferriox‐amine and apotransferrin, two ferric iron‐chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably vianonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glyco‐protein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxida‐tion into its ferric form, and (b) that the presence of intra‐and extracellular DA is not required for the observed toxic effects.
Glucocorticoids Exacerbate Hypoxic and Hypoglycemic Hippocampal Injury In Vitro: Biochemical Correlates and a Role for Astrocytes
Authors: Tombaugh Geoffrey C. , Yang Susan H. , Swanson Raymond A. , Sapolsky Robert M. 
Published: October 2006

AbstractThe acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure‐induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4–6 h of hypoxic treatment could damage age‐matched astrocytes if glucose was reduced or omitted. Ischemic‐like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT‐mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high‐glucose pretreatment. Identical 24‐h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.
Manipulation of Intracellular Calcium in NCB‐20 Cells
Authors: Garritsen Anja , Cooper Dermot M. F. 
Published: October 2006

AbstractA number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB‐20 cells. However, to date, the regulation of [Ca2+]i homeosta‐sis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2‐receptor, elevated [Ca2+]i by a pertussis‐toxin‐insensitive mechanism. The BK‐stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pre‐treatment with thapsigargin and ionomycin—compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK‐sensitive stores; the BK‐sensitive Ca2+ pool was a subset of the thapsigargin‐sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK‐induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.
Muscarinic Receptor Stimulated GTPase Activity in Synaptic Membranes from Bovine Retina
Authors: McIntosh Helen , Blazynski Christine 
Published: October 2006

AbstractGTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 μM oxotremorine and 100μM carbachol. (4‐Hydroxy‐2‐butynyl)‐1‐trimethylammonium m‐chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4‐Diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low‐affinity site (IC50±SEM, 0.63 ±0.18 μM) which accounted for 63% of the inhibition and a high‐affinity site (IC50 < 1 nM) which accounted for the remaining 37%. When carba‐chol‐stimulated GTPase activity was assayed, a single 4‐DAMP inhibitory site was apparent (IC50±SEM, 2.0 ±0.9 μM). Pirenzepine inhibited GTPase activity at a single site (IC50 values ±SEM, 46.9 ±11 and 25.4 ±6.5 μM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 ±1.8 and 6.2 ±1.5 μM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4‐DAMP = methoctramine > pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.
Evidence for Metabotropic Excitatory Amino Acid Receptor Heterogeneity: Developmental and Brain Regional Studies
Authors: Vecil Giacomo G. , Li Peter P. , Warsh Jerry J. 
Published: October 2006

AbstractTo examine whether multiple subtypes of the excitatory amino acid (EAA) receptor coupled to phosphoino‐sitide (PPI) hydrolysis exist, we have pharmacologically characterized the PPI response in neonatal and adult rat brain. Activation of PPI hydrolysis was determined by the accumulation of [3H]inositol monophosphate in brain slices prelabeled with [3H]inositol. In neonatal hippocampus, D,L‐2‐amino‐3‐phosphonopropionic acid (AP3; 1 mM) inhibited the m‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (IUPAC nomenclature; ACPD; 100 μM)‐ and quisqualate (Quis; 100 μM)‐stimulated PPI hydrolysis by 73 and 66%, respectively, but had no effect in neonatal cerebellum. In adult hippocampus, AP3 stimulated PPI hydrolysis with potency and efficacy comparable to those of Quis and ACPD and completely masked the Quis concentration‐response curve. In adult cerebellum, only Quis behaved as a full agonist on the PPI response. The Quis concentration‐response curve was shifted rightward with a fourfold decrease in potency in the presence of ACPD (5 mM), whereas it was nearly additive with the PPI response induced by AP3 (5 mM). Thus, our data reveal significant developmental and brain regional differences in metabotropic EAA receptor responses and support the notion that this receptor is heterogeneous, in both a regionally specific and a develop‐mentally dependent manner.
Evidence that Axonal tRNAs Are Resistant to RNase and ATPase and Can Be Aminoacylated in the Absence of Exogenous ATP
Authors: Chakraborty G. , Nicola A. , Ingoglia N. A. 
Published: October 2006

AbstractA high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein‐tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNAArg was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNALys did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNAArg and tRNALys was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme‐resistant, ATP‐tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNAArg and tRNALys were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase‐luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overho‐mogenizing the tissue. These procedures failed to render brain tRNALys susceptible to RNase. But phenol‐extracted, ethanol‐precipitated brain tRNALys was sensitive to RNase, suggesting that the protection of tRNALys may be by a protease‐ and heat‐resistant polypeptide or by a nonprotein‐aceous mechanism.
Nerve Growth Factor‐Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells
Authors: Katoh‐Semba Ritsuko , Oohira Atsuhiko , Kashiwamata Shigeo 
Published: October 2006

AbstractStructural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC 12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC 12 cells after 3 days of culture was in the range of 90–190 kDa for the intact form (Kav= 0.38 on Sepharose CL‐6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90–190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell‐associated CSPG had apparent molecular mass ranges of 120–150 kDa and 120–190 kDa (Kav= 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell‐associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell‐associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE‐cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav= 0.22 on Sephacryl S‐300 [40–84 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE)]. The CS and core protein of this CSPG had apparent molecular masses of 26 kDa and 15–17 kDa, respectively. The peak I fraction was separated further into three fractions of different molecular sizes on Sephadex G‐200 (I‐1,I‐2,I‐3). In the presence of NGF, peak I‐1 prepared after 3 days of culture consisted of mostly CSPG (130–170 kDa by SDS‐PAGE; Kav= 0.13 on Sephacryl S‐300) with CS of 32 kDa and a core protein of 105 kDa. Peaks 1–2 and 1–3 contained, respectively, CSPGs of 38–74 kDa and 31–56 kDa for their intact forms, with CSs of 25 kDa and 15 kDa. The apparent molecular masses of the core proteins of their CSPGs were 15–17 kDa. These CSPGs had similar molecular sizes, irrespective of the presence of NGF or the duration of culture. (4) These results indicate that structural changes in PGs during the neuritogenesis in PC12 cells induced by NGF occurred mostly in cell‐associated PGs and preceded elongation of neurites.
Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol‐O‐Methyltransferase Inhibitor Ro 40–7592
Authors: Acquas E. , Carboni E. , de Ree R. H. A. , Da Prada M. , Di Chiara G. 
Published: October 2006

AbstractThe effect of the systemic administration of a novel, orally active, catechol‐O‐methyltransferase (COMT) inhibitor, Ro 40–7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxy‐phenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40–7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long‐lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L‐β‐3,4‐dihydroxyphenylalanine (L‐DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L‐DOPA + benserazide with Ro 40–7592 (30 mg/ kg p.o.) resulted in a significant increase in DA output. Ro 40–7592 prevented the L‐DOPA‐induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extra cellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L‐DOPA, partially reduced it in the presence of L‐DOPA + Ro 40–7592 and even more so after L‐DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L‐DOPA but not of endogenous DA. Therefore, inhibition of COMT results in a potentiation of L‐DOPA effects not only by inhibition of its peripheral metabolism (conversion to 3‐methoxy‐DOPA), but also by inhibition of the metabolism of its active metabolite, DA, in the brain.
Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation
Authors: Winder Danny G. , Conn P. Jeffrey 
Published: October 2006

AbstractThe selective metabotropic glutamate receptor agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (trans‐ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans‐ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis‐linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans‐ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R‐ and 1S,3S‐ACPD (but not 1R,3S‐ACPD) induce a concentration‐dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L‐2‐amino‐3‐phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1R,3S‐ACPD‐stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.
Inositol 1,4,5‐Trisphosphate Receptor Immunoreactivity in SH‐SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation
Authors: Wojcikiewicz Richard J. H. , Nakade Shinji , Mikoshiba Katsuhiko , Nahorski Stefan R. 
Published: October 2006

AbstractInositol 1,4,5‐trisphosphate (InsP3) receptor immunoreactivity in SH‐SY5 Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, ∼275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half‐maximal and maximal at ∼2 and ∼6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12‐myristate 13‐acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol‐induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH‐SY5 Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.
In Memoriam: Henry McIlwain 1912–1992
Authors: Bachelard Herman 
Published: October 2006

It is with great sadness that the Editors, the Editorìal Board, the Publications Committee, and the Officers and Council of the ISN report the death of Professor Henry McIlwain. Henry was an eminent neurochemist and one of the fathers of the ISN and this Journal.The Editors
Myelin P0‐Glycoprotein: Predicted Structure and Interactions of Extracellular Domain
Authors: Wells Christopher A. , Saavedra Raul A. , Inouye Hideyo , Kirschner Daniel A. 
Published: October 2006

AbstractProtein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0‐ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N‐linked glycosylation. The x‐ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x‐ray diffraction data on the orientation of P0‐ED, and we propose how this immunoglobulin‐like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β‐strand predictions for P0‐ED are virtually superimposable with those of the VH domain of the phosphocholine‐binding immunoglobulin M603 of mouse, which has a similar number of residues as P0‐ED and a structure that has been solved crystallographically. The dimensions of P0‐ED from the space‐filling model, developed using PC‐ based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P0‐ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0‐ED from apposed membranes if the molecules were oriented so that the β‐ strands were approximately perpendicular to the membrane surface. The apposed P0‐EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P0‐ED regions analogous to the complementarity‐determining regions of immunoglobulins can account for the recognition of P0‐ED from apposed membranes in the incompletely swollen state. Two of the faces of P0‐ED that show charge complementarity could account for the homophilic interactions of P0‐ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N‐ linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P0‐ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P0 at the membrane surface. Our model shows how the single immunoglobulin‐like domain of P0 can account for distinct structural states of myelin membrane packing by homophilic interactions.
[125I]RTI‐55 Binding to Cocaine‐Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain
Authors: Little Karley Y. , Kirkman Jacob A. , Carroll F. Ivy , Breese George R. , Duncan Gary E. 
Published: October 2006

Abstract[125I]RTI‐55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI‐55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI‐55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI‐55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two‐phase processes. A curve‐fitting analysis of the data derived in saturation experiments found a high‐affinity site with KD= 66 ± 35 pM and Smax= 13.2 ± 10.1 pmol/g of tissue and a low‐affinity site with KD= 1.52 ± 0.55 nM and Bmax of 47.5 ± 11‐2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI‐55 > GBR‐12909 > mazindol > WIN 35428 > = methylphenidate > (−)‐cocaine > buproprion > (±)‐amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD= 370 ± 84 pM. It appears that [125I]RTI‐55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.