bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of ELISA to G4 Antigen to Quantitate Neurite Outgrowth in the Chick Both In Vivo and In Vitro
Authors: Weikert Thomas , Rathjen Fritz G. , Layer Paul G.
Published: July 2010

Abstract: The G4 glycoprotein is found on the earliest developing neurite tracts of the chick embryo. An ELISA is introduced here to quantify the amount of G4‐expressing neurites in the picogram range. In this double‐sandwich assay, an anti‐G4 monoclonal antibody fixes the G4 antigen to the plastic surface, which then is detected by a polyclonal antiserum; nonspecific background is decreased by competitive displacement. The sensitivity of the assay allows us to follow quantitatively the very first neurite growth in embryonic heads, trunks, retinae, and brains. G4‐based neurite growth is shown to occur earlier in heads than in trunks; in brain it is nearly 10‐fold higher than in the retina by embryonic day 8. By determination of acetylcholinesterase (AChE) activities from the same homogenates, our earlier histochemical findings are verified now on a quantitative basis, again showing that AChE consistently precedes G4 antigen. Moreover, as an in vitro example, the G4 ELISA is applied to the nerve growth factor (NGF) standard bioassay on dorsal root ganglia; the half‐maximal response is reached at ∼10 ng/ml of NGF for G4‐based neurite growth and at ∼1 ng/ml of NGF for AChE expression, respectively.
Protein Kinase FA/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr97‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs
Authors: Yu Jau‐Song , Yang Shiaw‐Der
Published: July 2010

Abstract: In a previous study, protein kinase FA/glycogen synthase kinase‐3 (FA/GSK‐3) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase FA/GSk‐3 were further determined by two‐dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase FA/GSK‐3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C18 reverse‐phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr97‐Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase FA/GSK‐3, implicating a physiologically relevant role of FA/GSK‐3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT94(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [32P]MBP phosphorylated by kinase FA/GSK‐3, further indicating that kinase FA/GSK‐3 represents a Thr‐Pro motif‐directed MBP kinase involved in the phosphorylation of brain myelin.
Somataglycan‐S: A Neuronal Surface Proteoglycan Defines the Spinocerebellar System
Authors: Williams Celia , Hinton David R. , Miller Carol A.
Published: July 2010

Abstract: The formation and maintenance of functionally specific neuronal networks may depend on specific proteoglycans localized to the surface membranes of a subset of neurons. Monoclonal antibody (MAb) 6A2 labeled a distinct subset of CNS neurons: the somas and proximal dendrites of cells making up the spinocerebellar and reticular systems. These pathways contribute to proprioceptive and exteroceptive functions. Ultrastructurally, MAb 6A2 immunoreactivity was distributed focally along the cell surface membranes and the adjacent extracellular space. On western blots of immunoaffinity‐purified preparations from cerebellar homogenates, a major, broad band of ∼400 kDa is labeled by MAb 6A2. Increased electrophoretic mobility of the purified antigen after digestion with chondroitinase ABC and keratanase suggests that the antigen is a proteoglycan bearing chondroitin sulfate and keratan sulfate glycosaminoglycans. Unsulfated N‐acetyl‐ galactosamine residues linked to unsaturated uronic acid constituted the initial disaccharide in the chondroitin sulfate glycosaminoglycan chains. N‐ and O‐linked oligosac‐ charides on the core protein were detected by the biotinylated lectins wheat germ agglutinin and Jacalin, respectively, and by MAb anti‐HNK‐1. Lyase and glycosidase digests result in a 280‐kDa band. This proteoglycan, somataglycan‐S, may provide a key to the role of glycoconjugates in determining neuronal diversity and system specificity.
Rapid Communication Muscle Mitochondrial ATP Production in Progressive Supranuclear Palsy
Authors: Monte Donate A. Di , Harati Yadollah , Jankovic Joseph , Sandy Martha S. , Jewell Sarah A. , Langston J. William
Published: July 2010

Abstract: Six patients with progressive supranuclear palsy (PSP) and 12 age‐matched disease‐free subjects participated in this study designed to compare rates of ATP production by intact mitochondria from biopsied skeletal muscle. When pyruvate and malate were used as metabolic substrates, rates of ATP production were 0.184 ± 0.025 μmol/min/U of citrate synthase (CS) activity (a mitochondrial marker) in control subjects and 0.131 ± 0.051 μmol/min/U of CS in PSP patients. In the presence of succinate, rates of ATP formation were 0.137 + 0.02 μmol/min/U of CS in controls and 0.109 ± 0.04 /4mUmol/min/U of CS in patients. With N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD) and ascorbate, rates were 0.034 ± 0.008 μm Umol/min/U of CS in controls and 0.022 ± 0.01 μmol/min/U of CS in PSP subjects. Differences between the control and PSP populations reached statistical significance with pyruvate/malate and TMPD/ascorbate. No differences in either muscle histopathology or histochemistry were found between patient and control subjects. Results of this study suggest that oxidative phosphorylation defects occur in muscle mitochondria from patients with PSP.
Rapid Communication Chronic Ingestion of Ethanol Up‐Regulates NMDAR1 Receptor Subunit Immunoreactivity in Rat Hippocampus
Authors: Trevisan Louis , Fitzgerald Lawrence W. , Brose Nils , Gasic Gregory P. , Heinemann Stephen F. , Duman Ronald S. , Nestler Eric J.
Published: July 2010

We examined the effects of chronic ethanol exposure on the levels ofN‐methyl‐D‐aspartate receptor subunit 1 (NMDAR1) protein, an essential component ofN‐methyl‐D‐aspar‐ tate glutamate receptors, in rat brain. By immunoblotting procedures using a specific antibody for the NMDAR1 subunit, we found that ethanol dramatically up‐regulated (by 65%) NMDAR1 immunoreactivity in the hippocampus but not in the nucleus accumbens, cerebral cortex, or striatum. In contrast, ethanol did not alter the levels of glutamate receptor subunit (GLUR) 1 or GLUR2 protein, subunits that make up the α‐amino‐3‐hydroxy‐5‐methy4‐isoxazole propionic acid glutamate receptor, in the hippocampus. Because ethanol can potentially influence many different neurotransmitter systems, we examined whether chronic treatment with several psychotropic drugs with different pharmacological profiles (cocaine, haloperidol, SCH 23390, imipramine, and morphine) could mimic the effect of ethanol. None of these agents increased hippocampal NMDAR1 subunit immunoreactivity after chronic administration. Increased NMDAR1 subunit levels in the hippocampus after chronic ethanol exposure may represent an important neurochemical substrate for some of the features associated with ethanol dependence and withdrawal.
Rapid Communication Activation of Protein Kinase C Inhibits Kainate‐Induced Currents in Oocytes Expressing Glutamate Receptor Subunits
Authors: Dildy‐Mayfield J. E. , Harris R. A.
Published: July 2010

Abstract:The effect of protein kinase C (PKC) activation on maximal kainate (KA)‐induced currents was studied inXenopusoocytes expressing the glutamate receptor (GluR) subunits GluR3, GluR1+3, GluR2+3, and GluR6. The PKC activator phorbol 12‐ myristate 13‐acetate (PMA) inhibited peak KA responses in a time‐dependent manner. The magnitude of inhibition was greatest in GluR6‐expressing oocytes. Desensitizing KA currents characterized by a peak, transient current followed by a slower, desensitizing current were observed in oocytes expressing GluR3 and GluR 1+3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMA's inhibition of peak current amplitude. PMA‐mediated inhibition of both desensitization and peak current amplitude was prevented by intracellular injection of the protein kinase C (PKC) inhibitor peptide. These results suggest that the function of GluRs is regulated by PKC‐dependent phosphorylation
Choline Acetyltransferase: Celebrating Its Fiftieth Year
Authors: Wu Donghai , Hersh Louis B.
Published: July 2010

Abstract: It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/ translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein‐protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic‐specific elements and the characterization of the trans‐acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three‐dimensional structure of the enzyme.
Structural Studies on D2Dopamine Receptors: Mutation of a Histidine Residue Specifically Affects the Binding of a Subgroup of Substituted Benzamide Drugs
Authors: Woodward Robert , Daniell Sarah J. , Strange Philip G. , Naylor Louise H.
Published: July 2010

Abstract:A histidine residue (His394) that is likely to be located in the ligand‐binding region of the D2dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394residue with these compounds.
Insulin‐Like Growth Factor‐I‐Enhanced Secretion Is Abolished in Protein Kinase C‐Deficient Chromaffin Cells
Authors: Penberthy W. Todd , Dahmer Mary K.
Published: July 2010

Abstract: Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nM insulin‐like growth factor‐I (IGF‐I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF‐I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF‐I on secretion from these cells. PKC was down‐regulated in the cells by 16–18 h of treatment with β‐phorbol didecanoate (β‐PDD; 100 nM). Such treatment had no effect on high‐K+‐stimulated secretion from cells cultured without IGF‐I; however, secretion from cells cultured with IGF‐I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, α‐PDD (100 nM), had no effect on secretion from untreated or IGF‐I‐treated chromaffin cells. The effect of β‐PDD was time and concentration dependent, with 100 nM β‐PDD producing a maximal effect in 8–10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by∼40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both α‐and ε‐PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 μM) prevented the enhanced secretion normally seen in IGF‐l‐treated cells, whereas HA1004 had no effect. High‐K+‐stimulated 45Ca2+ uptake in IGF‐I‐treated cells was attenuated by long‐term treatment with β‐PDD (200 nM) or H7 (100 μM). Together these observations suggest that PKC is required for IGF‐I‐enhanced secretion from chromaffin cells.
Free Radicals Enhance Basal Release of D‐[3H]Aspartate from Cerebral Cortical Synaptosomes
Authors: Oilman Sara C. , Bonner Meta J. , Pellmar Terry C.
Published: July 2010

Abstract:Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide‐generated free radicals decrease calcium‐dependent high K+‐evokedl‐[3H]‐glutamate release from synaptosomes while increasing calcium‐independent basal release. The present study evaluates the nonyesicular release of excitatory amino acid neurotransmitters, usingd‐[3H]aspartate as an exogenous label of the cytoplasmic pool ofl‐glutamate andl‐aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium‐independent basal release ofd‐[3H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K+‐evoked release in the presence of Ca2+was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton‐generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.
Serotonin 5‐HT1AReceptor Activation Increases Cyclic AMP Formation in the Rat Hippocampus In Vivo
Authors: Cadogan A. K. , Kendall D. A. , Marsden C. A.
Published: July 2010

Abstract:In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5‐HT1Areceptor agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT). The basal level of hippocampal extracellular cAMP was 2.3 ± 0.2 pmol/ml (n = 6), after a 3‐h postsur‐ gery stabilisation period. Perfusion of forskolin (100 μM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8‐OH‐DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8‐OH‐DPAT‐induced increase in cAMP efflux was observed following a second administration of 8‐OH‐DPAT after a 4‐h interval. Administration of 8‐OH‐DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N‐tert‐butyl 3–4‐(2‐methoxyphenyl)piperazine‐1 ‐yl‐2‐phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5‐HT1Areceptor antagonist, prevented the 8‐OH‐DPAT‐induced increase in cAMP efflux. The data indicate that the 8‐OH‐DPAT‐induced increase in cAMP efflux in vivo is mediated by a 5‐HT1Areceptor.
Heterogeneity of Cortical and Hippocampal 5‐HT1AReceptors: A Reappraisal of Homogenate Binding with 8‐[3H]Hydroxydipropylaminotetralin
Authors: Nénonéné E. K. , Radja F. , Carli M. , Grondin L. , Reader T. A.
Published: July 2010

Abstract:The selective serotonin (5‐HT) agonist 8‐hydroxydipropylaminotetralin (8‐OH‐DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5‐HT1Areceptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [3H]8‐ OH‐DPAT (free ligand from 200 pMto 160 nM) revealed high‐affinity 5‐HT1Areceptors (KH= 0.7–0.8 nM) and lowaffinity (KL= 22–36 nM) binding sites. The kinetics of [3H]8‐OH‐DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high‐ and low‐affinity binding sites. When the high‐affinity sites were labeled with a 1 nMconcentration of [3H]8‐ OH‐DPAT, the competition curves of agonist and antagonist drugs were best fit to a two‐site model, indicating the presence of two different 5‐HT1Abinding sites or, alternatively, two affinity states, tentatively designated as 5‐HT1AHIGH and 5‐HT1ALOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5‐HT1Asites are modulated by 5′‐guanylylimidodiphosphate, inhibition experiments with 5‐HT were performed in the presence or in the absence of 100 μM5′‐guanylylimidodiphosphate. The binding of 1 nM[3H]8‐OH‐DPAT to the 5‐HT1AHIGHsite was dramatically (80%) reduced by 5′‐guanylylimidodiphosphate; in contrast, the low‐affinity site, or 5‐HT1ALOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high‐affinity 5‐HT1AHIGHsite corresponds to the classic 5‐HT1Areceptor, whereas the novel 5‐HT1ALOWbinding site, labeled by 1 nM[3H]8‐OH‐DPAT and having a micromolar affinity for 5‐HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5‐HT1Asites and the 5‐HT innervation, rats were treated withp‐chlorophenylalanine or with the neurotoxinp‐chloroamphetamine. The inhibition of 5‐HT synthesis byp‐chlorophenylalanine did not alter either of the two 5‐HT1Asites, but deafferentation byp‐chloroamphetamine caused a loss of the low‐affinity [3H]8‐OH‐ DPAT binding sites, indicating‐that these novel binding sites may be located presynaptically on 5‐HT fibers and/or nerve terminals.
Evidence that Prostaglandin E2Can Block Calcium‐Activated86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C‐Dependent Mechanism
Authors: Ren Jun , Benishin Christina G.
Published: July 2010

Abstract:The effects of prostaglandin E2(PGE2) on86Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K+) channel modulation. A selective dose‐dependent inhibition of the calcium‐activated charybdotoxin‐sensitive component of efflux was found upon application of PGE2. No significant effect was seen on basal and voltage‐dependent components over the concentration range of 10–8to 10–5M.The protein kinase C (PKC) inhibitors H‐7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40‐phorbol 12, 13‐dibutyrate, which has been reported to down‐regulate PKC, abolished the PGE2‐in‐ duced inhibition, whereas HA1004 (10 μM) andRp‐3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β‐Phorbol 12, 13‐dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca2+‐dependent component of86Rb efflux but also had no effect on the basal and voltage‐dependent components. These data suggest that PGE2can inhibit rat brain nerve ending calcium‐activated86Rb efflux, and this inhibition may involve PKC activation.
Ischemia‐Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat
Authors: Sun Dandan , Gilboe David D.
Published: July 2010

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli‐Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical‐mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.
Effect of the Platelet‐Activating Factor Antagonist BN 50739 and Its Diluents on Mitochondrial Respiration and Membrane Lipids During and Following Cerebral Ischemia
Authors: Sun Dandan , Gilboe David D.
Published: July 2010

Abstract:Recent evidence suggests that platelet‐activating factor plays a role in ischemia‐induced neural injury. The Pulsinelli‐Brierley four‐vessel occlusion model was used to study the effect of a synthetic platelet‐activating factor antagonist, BN 50739, and its solvents, either dimethyl sulfoxide or hydroxypropyl‐β‐cyclodextrin, on cerebral ischemia‐reperfusion. Rats were subjected to either 30 min of ischemia or 30 min of ischemia followed by 60 min of recirculation. Changes in the brain mitochondrial free fatty acid pool size, fatty acyl composition of phospholipids, and respiratory function were monitored. When the BN 50739 (2 mg of BN 50739/kg of body weight i.v.) was administered at the onset of recirculation, it significantly reversed the ischemia‐induced accumulation of mitochondrial free fatty acids and loss of polyunsaturated fatty acyl chains from phosphatidylcholine and phosphatidylethanolamine while simultaneously improving mitochondrial respiration. Dimethyl sulfoxide alone decreased the mitochondrial level of malonyldialdehyde and total free fatty acid pool size, but there was no improvement in mitochondrial respiration. Hydroxypropyl‐β‐cyclodextrin was reported to be pharmacologically inactive and capable of dissolving BN 50739. However, hydroxypropyl‐β‐cyclodextrin alone also caused a significant increase in content of cerebral mitochondrial membrane free fatty acids and hydrolysis of phosphatidylcholine in normoxic control animals. The overall effect of BN 50739 on mitochondrial structure and energy metabolism supports the hypothesis that platelet‐activating factor may play a key role in ischemia‐induced cerebral injury.
Cellular and Subcellular Localization of Hexokinase, Glutamate Dehydrogenase, and Alanine Aminotransferase in the Honeybee Drone Retina
Authors: Veuthey Anne‐Lise , Tsacopoulos Marcos , de Ruiz Lourdes Millan , Perrottet Philippe
Published: July 2010

Abstract: Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone‐insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2‐deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose‐6‐phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria‐poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.
Tyrosine and Serine Phosphorylation of Dystrophin and the 58‐kDa Protein in the Postsynaptic Membrane of Torpedo Electric Organ
Authors: Wagner Kathryn R. , Huganir Richard L.
Published: July 2010

Abstract: Dystrophin associates with a 58‐kDa and an 87‐kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87‐kDa protein is a major phosphotyrosine‐containing protein in these membranes. Immunoprecipitation of the 87‐kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphoproteins. These phosphoproteins are identified as dystrophin and the 58‐kDa protein. Monoclonal antibodies to dystrophin and the 58‐kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58‐kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58‐kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58‐kDa and 87‐kDa proteins, may be modulated by tyrosine and serine protein Phosphorylation.
Effects of C‐Type Natriuretic Peptide on Rat Astrocytes: Regional Differences and Characterization of Receptors
Authors: Deschepper Christian F. , Picard Sylvie
Published: July 2010

Abstract: We have compared the effects of atrial natriuretic peptide (ANP), brain natriuretic peptide (BMP), and C‐type natriuretic peptide (CNP) on the accumulation of cyclic GMP (cGMP) in secondary cultures of rat astro‐cytes. The order of potency of these peptides was CNP < ANP < BNP, which would be compatible with a predominance of guanylate cyclase B (GC‐B)‐versus guanylate cyclase A (GC‐A)‐type receptors in these cells. Accordingly, we found by northern blot analysis that the mRNA transcripts of GC‐B were much more abundant in astro‐cytes than the transcripts of GC‐A. In addition, astrocytes from diencephalon accumulated two times more cGMP in response to CNP than astrocytes from cortex. Binding experiments with 125l‐labeled ANP or [Tyr0]‐CNP established that these ligands recognized only clearance‐type receptors on astrocytes. However, the number of binding sites was ∼ 100 times higher in astrocytes from cortex than in astrocytes from diencephalon and thus was inversely correlated to the amplitude of the cGMP response in the same cells. We found no further evidence for differences in the levels of GC‐B receptors in astrocytes from the two regions because (a) the abundance of GC‐B mRNA was similar and (b) there was no difference in particulate guanylate cyclase activity in astrocytes from each region. In addition, occupancy of clearance receptors with C‐ANP4–23 did not affect the accumulation of cGMP in response to CNP; this makes it unlikely that the differences in cGMP responsiveness can be accounted for by binding and sequestration of CNP to the clearance receptors. Thus, the abundance of GC‐B receptors is not the only factor governing the amounts of cGMP they can generate on exposure to CNP. Other factors may regulate the ability of these receptors to generate cGMP in astrocytes.
Unusual Low Proton Permeability of Liposomes Prepared from the Endogenous Myelin Lipids
Authors: Díaz Ricardo S. , Monreal Jaime
Published: July 2010

Abstract: In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin‐induced potassium diffusion potentials as high as ‐177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [14C]‐methylamine, or the changes in the fluorescence of the probes 9‐aminoacridine, acridine orange, and pyranine. The presence of cations (Na+, K+, Ca2+) did not alter this behavior. Voltage clamping did not increase the trans‐membrane ApH‐driven proton permeability. However, II‐posome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H+ through the membrane.
Neuromelanin of the Human Substantia Nigra: A Mixed‐Type Melanin
Authors: Odh Gerd , Carstam Ragnar , Paulson Jan , Wittbjer Anna , Rosengren Evald , Rorsman Hans
Published: July 2010

Abstract: Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin‐derived compound, thiazole‐4,5‐dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole‐2,3,5‐tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed‐type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.
GABAAReceptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study
Authors: Thompson Christopher L. , Stephenson F. Anne
Published: July 2010

Abstract:The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABAAreceptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABAAreceptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABAAreceptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific Mr58,000 anti‐α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABAAreceptor α6 subunit in age‐matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [3H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam‐sensitive and diazepam‐in‐sensitive [3H]Ro 15–4513 binding changed. A time‐dependent enrichment of the diazepam‐insensitive binding site up to a maximum of 74% of total [3H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABAAreceptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABAAreceptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.
ADP‐Ribosylation of Brain Neuronal Proteins Is Altered by In Vitro and In Vivo Exposure to Inorganic Mercury
Authors: Palkiewicz Pawel , Zwiers Henk , Lorscheider Fritz L.
Published: July 2010

Abstract: ADP‐ribosylation is an essential process in the metabolism of brain neuronal proteins, including the regulation of assembly and disassembly of biological polymers. Here, we examine the effect of HgCl2 exposure on the ADP‐ribosylation of tubulin and actin, both cytoskeletal proteins also found in neurons, and B‐50/43‐kDa growth‐associated protein (B‐50/GAP‐43), a neuronal tissue‐specific phosphoprotein. In rats we demonstrate, with both in vitro and in vivo experiments, that HgCl2 markedly inhibits the ADP‐ribosylation of tubulin and actin. This is direct quantitative evidence that HgCl2, a toxic xenobiotic, alters specific neurochemical reactions involved in maintaining brain neuron structure.
Intracerebroventricular Administration of AT1Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II
Authors: Sakai R. R. , He P. F. , Yang X. D. , Ma L. Y. , Guo Y. F. , Reilly J. J. , Moga C. N. , Fluharty S. J.
Published: July 2010

Abstract:Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT1) receptors in cultured cells and brain. In both liver epithelial WB and neuro‐blastoma N1E‐115 cells AT1antisense oligomers substantially decreased AT1receptor density, whereas angiotensin type 2 (AT2) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT1antisense oligomers in rats decreased AT1receptor density in hypothalamic‐thalamic‐septal tissue, and AT2receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra‐cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.
Biochemical Characterization of Two Distinct Angiotensin AT2Receptor Populations in Murine Neuroblastoma N1E‐115 Cells
Authors: Siemens Ivo R. , Reagan Lawrence P. , Yee Daniel K. , Fluharty Steven J.
Published: July 2010

Abstract:The murine neuroblastoma N1E‐115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate. These solubilized binding sites exhibited high affinity for CGP‐42112A and not Losartan, indicating that they were of the AT2subtype. However, displacement of125I‐Angll with the AT2nonpeptide antagonist PD‐123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2receptor population in N1E‐115 cells. In support of this view, separation of two receptor populations was accomplished with heparin‐Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin‐Sepharose column, two of which bound125I‐Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ∼80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5MNaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2receptors insofar as they exhibited high affinity for CGP‐42112A and little or no affinity for the AT1‐selective antagonist Losartan. However, whereas the nonpeptidic AT2‐selective antagonist PD‐123319 completely displaced the binding of126I‐Angll from peak I in a monophasic fashion (IC50= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD‐123319 was much less effective in displacing125I‐Angll from peak III (IC50= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in125I‐Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high‐affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated125I‐Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin‐Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT2receptors.
Epidermal Growth Factor and Basic Fibroblast Growth Factor Have Independent Actions on Mesencephalic Dopamine Neurons in Culture
Authors: Casper Diana , Roboz Gail J. , Blum Mariann
Published: July 2010

Abstract:Epidermal growth factor (EGF) and basic fibro‐blast growth factor (bFGF) are both trophic for dopamine neurons s in cultures of dissociated embryonic rat mesen‐cephaion, but the significance of this apparent overlap in neurotrophic activity is not yet known. In this study, we investigated the mechanisms of action of these two growth factors and the potential relationship between them, Using a nuclease protection assay, we determined that bFGF mRNA was expressed in the cultures. Double‐label immunocytochemistry revealed that bFGF immunore‐active material could be detected in tyrosine hydroxylase immunoreactive neurons and glial fibrillary acidic protein immunoreactive astrocytes. EGF treatment increased bFGF mRNA content per culture dish. As we have previously demonstrated that EGF exerts its dopaminergic neurotrophic activity via an intermediate cell type, studies were designed to address whether the pathway by which EGF acts on dopaminergic neurons is mediated by the release of bFGF. However, the trophic action of EGF on dopamine neurons, represented by high‐affinity neuronal dopamine uptake, could not be blocked by immunoneutralization of bFGF, suggesting that the actions of EGF were not mediated by bFGF release. The time course of the effects of EGF and bFGF on dopamine uptake were similar, with significant increases detectable only after 5 days in culture. Both growth factors were active in the picomolar‐to‐nannomolar range with maximal trophic activity between 0.4 and 2.5 nM.EGF, however, was the more potent mitogen under these conditions. When cultures were simultaneously incubated with maximal concentrations of EGF and bFGF, the effect on dopamine uptake was significantly greater than with either growth factor alone and, in fact, approximated the sum of the individual effects. On the basis of these results we conclude that these growth factors have independent effects on dopamine neurons of the mesencephalon.
Catecholamine Release and Excretion in Rats with Immunologically Induced Preganglionic Sympathectomy
Authors: Brimijoin Stephen , Hammond Pamela , Khraibi Ali A. , Tyce Gertrude M.
Published: July 2010

Abstract:Plasma and urinary catecholamines were quantified to assess global sympathoadrenal function in rats with preganglionic lesions caused by antibodies to acetyl‐cholinesterase (AChE). Rats were given intravenous injections of normal mouse IgG or murine monoclonal anti‐acetylcholinesterase IgG (1.5 mg). Five or 16 days afterward, basal blood samples were taken through indwelling arterial cannulae. A few hours later, the rats were immobilized for 10 min in padded restrainers, and another blood sample was drawn. HPLC determinations showed low basal levels of norepinephrine and epinephrine (<0.2 ng/ml in all rat plasma samples). In control rats, immobilization stress increased levels of plasma catecholamines up to 35‐fold. In rats tested 5 days after injection of antibody, the norepinephrine response was much smaller (15% of control), and (he epinephrine response was nearly abolished (5% of control). There was some recovery at 16 days after antibody treatment, but stress‐induced catecholamine release was still markedly impaired. Reduced stress‐induced release: was not accompanied by major changes in tissue epinephrine or norepinephrine (heart, spleen, adrenal glands, and brain), although adrenal dopamine content dropped by 60%. Urinary excretion was studied in parallel experiments to gain insight into the effects of AChE anti‐bodies on basal sympathoadrenal activity. Epinephrine, norepinephrine, dopamine, and selected metabolites were quantified in 24‐h urine samples collected at frequent intervals for 30 days after antibody injection. No statistically gnificant changes were detected in the urinary output of dopamine, 3‐methoxytyramine, normetanephrine, or 3‐methoixy‐4‐hydroxyphenylglycol. On the other hand, epinephrine and norepinephrine output increased sharply at the time of antibody injection and then fell significantly below control levels. Norepinephrine output returned to normal after 2 weeks, but epinephrine output remained depressed. These results are consistent with previous evidence of widespread and persistent antibody‐mediated βmade to the preganglionic sympathetic system.
Aluminium Inhibits Muscarinic Agonist‐Induced Inositol 1,4,5‐Trisphosphate Production and Calcium Mobilization in Permeabilized SH‐SY5Y Human Neuroblastoma Cells
Authors: Wood P. C. , Wojcikiewicz R. J. H. , Burgess J. , Castleden C. M. , Nahorski S. R.
Published: July 2010

Abstract:The effects of aluminium (as Al3+) on carbachol‐induced inositol 1,4,5‐trisphosphate (lnsP3) production arid Ca2+mobilisation were assessed in electropermeabilised human SH‐SY5Y neuroblastoma cells. Al3+had no effect on lnsP3‐induced Ca2+release but appreciably reduced carbachol‐induced Ca2+release (lC50of ∼90 μM). Aβ3+also inhibited lnsP3production (lC60of ∼15 μM). Dimethyl hydroxypyridin‐4‐one, a potent Al3+chelator (K5= 31), at 100 μMwas able to abort and reverse the effects of Al3+on both Ca2+release and lnsP3production. These data suggest that, in permeabilised cells, the effect of Al3+on the phosphoinositide‐mediated signalling pathway is at the level of phosphatidylinositol 4,5‐bisphosphate hydrolysis. This may reflect interference with receptor‐G protein‐phospholipase C coupling or an interaction with phosphatidylinositol 4,5‐bisphosphate.
Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells
Authors: Katayama Shigenori , Shuntoh Hisato , Matsuyama Shogo , Tanaka Chikako
Published: July 2010

Abstract:The effect of heat shock on agonist‐stimulated intracellular Ca2+mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin‐induced [Ca2+], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin‐induced [Ca2+]irise was abolished and the [Ca2+]irise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca2+‐ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5‐trisphosphate (IP3) production was not altered by heat shock at 12 h after heat shock, whereas IP3receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP3receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs‐mediated intracellular Ca2+signal transduction system and that hsp72 is involved in the recovery of bradykinin‐induced [Ca2+]irise.
Regulation of NMDA Receptor mRNA During Visual Map Formation and After Receptor Blockade
Authors: Hofer Magdalena , Prusky Glen T. , Constantine‐Paton Martha
Published: July 2010

Abstract: The topographic refinement of the rat retinocollicular projection is dependent on normal NMDA receptor function. Here we examined the expression of NMDA and non‐NMDA glutamate receptor mRNA in the rat superior colliculus (SC) during this postnatal refinement period. The temporal expression pattern of mRNA coding for the NMDA receptor subunit NR1 in the superficial SC followed the time course of collicular synaptogenesis. A pronounced increase of NR1 mRNA levels occurred during the late stages of retinocollicular map refinement. In cortex, the time course of the expression of NR1 mRNA in cortex was found to be similar to that observed in SC, with low levels during the first postnatal week, a maximum at P19, and a decrease thereafter. In SC, but not in cortex, there was a change in the ratio of the two NR1 transcripts during the second postnatal week that parallels a previously demonstrated developmental change in the mean open time of NMDA channels in collicular neurons. In contrast, the mRNA expression pattern of the non‐NMDA receptor subunit GluR2 in the developing SC was not closely correlated with synaptic changes. Chronic treatment of the SC with the NMDA receptor antagonist 2‐amino‐5‐phosphonovalerate (APV) for 12 or 19 days, which disrupts retinocollicular map formation, appears to block the developmental rise in NR1 mRNA levels. These findings support a specific role for the NMDA receptor subtype of glutamate receptors in the control of synaptogenesis and developmental plasticity in the SC.