bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain γ‐Aminobutyric Acid Turnover Rates After Spontaneous Chronic Ethanol Intake and Withdrawal in Discrete Brain Areas of C57 Mice
Authors: Simler S. , Clement J. , Ciesielski L. , Mandel P.
Published: December 2006

Abstract: The effect of 4 weeks of spontaneous chronic ethanol intake in drinking water and then ethanol withdrawal on the γ‐aminobutyric acid (GABA) steady‐state levels and turnover rates was investigated in 15 brain areas of C57 B1/6J alcohol‐preferring mice. These mice did not display typical ethanol withdrawal convulsions. There was no statistically significant difference in the brain GABA steady‐state levels among the control group, chronic ethanol‐treated mice, and mice after ethanol withdrawal. In contrast, chronic ethanol treatment induced significant variations in GABA turnover rate, as measured by gabaculine‐induced accumulation of GABA, in eight of 15 areas examined versus a decrease in seven brain areas [cerebellum (–29%), amygdala (–28%), olfactory tubercles (–24%), septum (–24%), striatum (–53%), frontal cortex (–21%), and hippocampus (–24%)]; an increase in turnover rate in the posterior colliculus (100%) was observed. At 26 h after ethanol withdrawal, in the seven areas in which GABA turnover rate decreased after spontaneous chronic ethanol intake, a return to the initial control value was observed; in the posterior colliculus, the turnover rate did not change, remaining higher than the control value. This persisting alteration of GABA turnover rate may be related to the absence of the ethanol withdrawal syndrome in the C57 mouse strain.
Characterization of l‐[3H]Nicotine Binding in Human Cerebral Cortex: Comparison Between Alzheimer's Disease and the Normal
Authors: Flynn Donna D. , Mash Deborah C.
Published: December 2006

Abstract: Putative nicotine receptors in the human cerebral cortex were characterized with l‐[3H]nicotine. l‐[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25°C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimer's disease were comparable to age‐matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimer's disease had a 50% reduction in the number of l‐[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimer's disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimer's disease.
Presynaptic Serotonergic Dysfunction in Patients with Alzheimer's Disease
Authors: Palmer A. M. , Francis P. T. , Benton J. S. , Sims N. R. , Mann D. M. A. , Neary D. , Snowden J. S. , Bowen D. M.
Published: December 2006

Abstract: Indices of presynaptic serotonergic nerve endings were assayed in neocortical biopsy samples from patients with histologically verified Alzheimer's disease. The concentrations of 5‐hydroxytryptamine (serotonin) and 5‐hydroxyindoleacetic acid, serotonin uptake, and K+‐stimulated release of endogenous serotonin were all found to be reduced below control values. Changes occurred in samples from both the frontal and temporal lobes, but they were most severe (at least a 55% reduction) in the temporal lobe. This is indicative of substantial serotonergic denervation. Values for serotonergic markers in Alzheimer's disease samples did not show correlations with rating of the severity of dementia, indices of cholinergic innervation, or senile plaque and cortical pyramidal neurone loss. However, neuronbrillary tangle count and an index of glucose oxidation (both probably reflecting pyramidal cells) correlated with the concentration of 5‐hydroxyindoleacetic acid.
Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study
Authors: Sieghart W. , Eichinger A. , Richards J. G. , Möhler H.
Published: December 2006

Abstract: Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15‐4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15‐4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51, 000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51, 000 (P51) and 55, 000 (P55) in hippocampus. Photolabeling was inhibited by 10 μM diazepam but not by 10 μM Ro 5‐4864. The BZ1 receptor‐selective ligands CL 218872 and ß‐carboline‐3‐carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15‐4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15–4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same pro‐teins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15‐4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51, 000 and/or 55, 000. In cerebellum, an additional protein (MW 57, 000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15‐4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15–4513 in the granular than the molecular layer.
Rapid Anterograde Spread of Premitotic Activity Along Degenerating Cat Sciatic Nerve
Authors: Oaklander Anne Louise , Miller Matthew S. , Spencer Peter S.
Published: December 2006

Abstract: Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio‐temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S‐phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo‐distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 ± 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.
Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine
Authors: Deacon C. F. , Agoston D. V. , Nau R. , Conlon J. M.
Published: December 2006

Abstract: The highest concentration of neurokinin A‐like immunoreactivity and substance P‐like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus‐containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 ± 0.003 g/ml was prepared from the myenteric plexus‐containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 ± 12‐fold in the concentration of neurokinin A‐like immunoreactivity and 43 ± 13‐fold in the concentration of substance P‐like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide‐containing vesicles (density, 1.154 ± 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that β‐pre‐protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.
Binding of [3H]Ro 16‐6491, a Reversible Inhibitor of Monoamine Oxidase Type B, to Human Brain Mitochondria and Platelet Membranes
Authors: Cesura A. M. , Galva M. D. , Imhof R. , Da Prada M.
Published: December 2006

Abstract: The reversible inhibitor of monoamine oxidase type B (MAO‐B) [3H]Ro 16‐6491 binds specifically and with high affinity to a single population of binding sites in human frontal cortex crude mitochondria and platelet membranes. In both tissues binding equilibrium was reached after l h incubation at 20°C. Dissociation of bound radioactivity was relatively fast at 20°C (t1/2= 90–120 min) whereas at 0°C [3H]Ro 16–6491 showed the characteristics of a slowly dissociating ligand. Inhibitors and substrates of MAO‐B inhibited binding of [3H]Ro 16‐6491, whereas MAO‐A blockers were much less potent. Ro 16‐6491 was also a substrate for MAO‐B and a stable unidentified intermediate of the oxidation of Ro 16‐6491 possessing high affinity for the enzyme may account for the marked MAO‐B inhibitory effect of the drug. According to this hypothesis Ro 16‐6491 would behave as a mechanism‐based reversible inhibitor. In conclusion, [3H]Ro 16‐6491 binds selectively to MAO‐B and represents an excellent new radioligand probe for studying the regional tissue distribution of this enzyme in normal and pathological conditions.
Dephosphorylation of Synaptosomal Proteins P96 and P139 Is Regulated by Both Depolarization and Calcium, but Not by a Rise in Cytosolic Calcium Alone
Authors: Robinson Phillip J. , Hauptschein Robert , Lovenberg Walter , Dunkley Peter R.
Published: December 2006

Abstract: Depolarization of intact synaptosomes activates calcium channels, leads to an influx of calcium, and increases the phosphorylation of several neuronal proteins. In contrast, there are two synaptosomal phosphoproteins labeled in intact synaptosomes with 32P1, termed P96 and P 139, which appear to be dephosphorylated following depolarization. Within intact synaptosomes P96 was found in the cytosol whereas P 139 was present largely in membrane fractions. Depolarization‐stimulated dephosphorylation was fully reversible and continued for up to five cycles of depolarization/repolarization, suggesting a physiological role for the phenomenon. The basal phosphorylation of these proteins was at least partly regulated by cyclic AMP, since dibutyryl cyclic AMP produced small but significant increases in P96 and P 139 labeling, even in the presence of fluphenazine at concentrations that inhibited calcium‐stimulated protein kinases. Depolarization‐dependent dephosphorylation was independent of a rise in intracellular calcium, since agents such as guanidine and low concentrations of A23187, which increase intracellular calcium without activating the calcium channel, did not initiate P96 or P 139 dephosphorylation. These agàents did sustain increases in the phosphorylation of a number of other proteins including synapsin I and protein III. The results suggest that the phosphorylation of these two synaptosomal proteins is intimately linked to the membrane potential and that their dephosphorylation is dependent on both the mechanism of calcium entry and calcium itself, rather than simply on a rise in intracellular free calcium.
γ‐Hydroxybutyric Acid in Subcellular Fractions of Rat Brain
Authors: Carter Snead O.
Published: December 2006

Abstract: The presence of γ‐hydroxybutyric acid (GHB) in synaptosome‐enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various γ‐aminobutyric acid (GABA) transaminase (GABA‐T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA‐T inhibitors, GABA, or ethosuximide. GHB was present in synaptosme‐enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA‐T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.
Enhancement of Stimulation‐Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin
Authors: Morita Katsuya , Dohi Toshihiro , Kitayama Shigeo , Koyama Yutaka , Tsujimoto Akira
Published: December 2006

Abstract: Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 μM) enhanced the ACh‐evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose‐dependently up to 1 μM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh‐evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mMK+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+‐free medium. The potentiation by forskolin of the ACh‐evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.
Isolation and Characterization of Chromogranins A, B, and C from Bovine Chromaffin Granules and a Rat Pheochromocytoma
Authors: Fischer‐Colbrie Reiner , Schober Maria
Published: December 2006

Abstract: Bovine chromaffin granules from adrenal medulla contain three acidic secretory proteins: chromogranins A, B, and C. For isolation of these proteins, methods based mainly on high performance liquid chromatography were developed. After removal of contaminating glycoproteins by lectin affinity chromatography, chromogranins were separated by high performance anion‐exchange, gel‐filtration, and reverse phase liquid chromatography. As a final purification step sodium dodecyl sulfate‐gel electrophoresis was performed. Amino acid analysis of isolated bovine chromogranins revealed a similar composition of all three proteins, with glutamic acid being the most prominent amino acid. The methods developed for bovine proteins also proved suitable for isolating rat chromogranins A and B from a transplantable pheochromocytoma. Chromogranin C was not present in sufficient amounts to be isolated from this tissue. The chromogranins purified by these methods were used to raise specific antibodies in rabbits. The use of purified chromogranins together with specific antisera may be valuable in understanding the still undiscovered function of these proteins.
In Vivo Phosphorylation of Axonal Proteins in Goldfish Optic Nerve During Regeneration
Authors: Larrivee Denis C. , Grafstein Bernice
Published: December 2006

Abstract: In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two‐dimensional gel electrophoresis. By 6–24 h after intraocular injection of H332PO4, ˜ 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45‐kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.
Glial Fibrillary Acidic Protein in the Cytoskeletal and Soluble Protein Fractions of the Developing Rat Brain
Authors: Malloch Gavin D. A. , Clark John B. , Burnet Frank R.
Published: December 2006

Abstract: The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme‐linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied. These findings, and the failure to detect a significant increase in the amount of soluble GFAP during development, strongly suggest that the very small pool of soluble GFAP is unlikely to be derived from the much larger pool of cytoskeletal GFAP. The possible role of soluble GFAP as a precursor to glial filaments is discussed.
Cloning of cDNA Derived from mRNA of the Electric Lobe of Torpedo marmorata and Selection of Putative Cholinergic‐Specific Sequences
Authors: Schmid Dieter W. , Girou Catherine
Published: December 2006

Abstract: With the aim of selecting cDNA sequences expressed in neurons utilizing exclusively cholinergic synaptic transmission, cDNA derived from Torpedo electric lobe was cloned and screened by hybridization with probes originating from another brain region known for its low content of cholinergic neurons (cerebellum) and a nonneuronal tissue (muscle). This method led to the isolation of 18 clones among 3, 200 showing no hybridization with probes other than those derived from electric lobe. These clones can therefore be considered to represent sequences involved in the expression of cholinergic function in neurons.
The American Society for Neurochemistry (ASN): Antecedents, Founding, and Early Years
Authors: Tower Donald B.
Published: December 2006

Abstract: Events leading to and the influences on the founding of the American Society for Neurochemistry are recounted, with emphasis on early activities of neurochemists in the United States, as well as the international activities, that led to the founding of both the International and American societies (in 1965 and 1969, respectively). The founding of the American Society for Neurochemistry in the period 1968–1969 and its first annual meeting in 1970 are described, together with significant developments during the early years of the Society.
Incorporation of Glycerol and Ethanolamine into Glycerophospholipid in Rat Brain Areas During Bicuculline‐Induced Convulsive Seizures
Authors: Marku Ndok , Corazzi Lanfranco , Piccinin Gian Luigi , Arienti Giuseppe
Published: December 2006

Abstract: The effect of bicuculline‐induced convulsive seizures on lipid metabolism has been studied in four brain areas (cerebellum, cerebral cortex, hippocampus, and brainstem) using [2‐3H]glycerol and [1,2‐14C]ethanolamine as radioactive lipid precursors administered simultaneously with bicuculline. Twelve minutes after the administration, the uptake of radioactivity depended both on brain area and treatment, being generally higher in convulsing rats. The uptake of glycerol was influenced to a larger extent than that of ethanolamine and increased during convulsions, but its incorporation into lipids did not. In contrast, the amount of ethanolamine incorporated into lipids increased during bicuculline‐induced seizures. The difference in behavior of glycerol and of ethanolamine is also indicated by the decrease of the 3H/14C ratio of phosphatidylethanolamine in various brain areas during convulsions. It is, therefore, evident that the metabolism of the two precursors is affected differently by seizures.
Characterization of a Dipeptidyl Aminopeptidase from Bovine Adrenal Medulla
Authors: Kecorius Elaine , Small David H. , Livett Bruce G.
Published: December 2006

Abstract: A dipeptidyl aminopeptidase was partially purified from a supernatant fraction of bovine adrenal medulla by gel filtration and anion‐exchange chromatography. From gel filtration, the apparent molecular weight of the enzyme was 68,100 and its pH optimum was 9.5. Its Km for hydrolysis of the synthetic substrate arginylarginine‐β‐naphthylamide was 5.5 × 10−6M. The enzyme was inhibited by metal ion chelating agents and thiol blocking agents. suggesting the requirement for both a metal ion and an active cysteine residue for its activity. Several peptides were cleaved by the dipeptidyl aminopeptidase involving the sequential removal of dipeptides from the N‐terminus. Biologically active peptides, such as leucine‐enkephalin, methionine‐enkephalin, and angiotensin II, were hydrolyzed by the dipeptidyl aminopeptidase although opioid peptides with a length greater than five amino acid residues were not susceptible to hydrolysis. Other peptides with a blocked N‐terminus (neurotensin, bombesin) or a proline residue adjacent to a potential cleavage site (substance P) were not hydrolyzed. The ability of this dipeptidyl aminopeptidase to degrade certain neuropeptides suggests that it could be involved in neuropeptide degradation.
Calmodulin Kinase II in Pure Cultured Astrocytes
Authors: Bronstein Jeff , Nishimura Robert , Lasher Robert , Cole Ruth , de Vellis Jean , Farber Debora , Wasterlain Claude
Published: December 2006

Abstract: Calcium‐ and calmodulin‐dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50‐ and 60‐kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II sub‐units (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50‐kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.
Mutual Antagonism of κ‐Opiate and α2‐Adrenoceptor Agonist Effects on Intrasynaptosomal Free [Ca2+]i
Authors: Adamson P. , McWilliam J. R. , Brammer M. J. , Campbell I. C.
Published: December 2006

Abstract: Synaptosomes prepared from rat cerebral cortices on Percoll discontinuous density gradients were loaded with the fluorescent EGTA analogue Quin 2 to allow measurement of intracellular free [Ca2+]i. When either κ‐opiate or α2‐adrenoceptor agonists were incubated with the synaptosomes, there was a highly significant (p < 0.004, p < 2.7 × 10−6, respectively) reduction in intrasynaptosomal free [Ca2+]i relative to controls. As these synaptosomes are not depolarised, the data suggest that both α2‐adrenoceptor agonists and κ‐opiate agonists inhibit neurotransmitter release, decreasing the availability of intraneuronal [Ca2+]i rather than altering Ca2+ entry. However, when these two agonists were coincubated, there was a complete abolition of the effects of either agonist; in fact, there was an apparent increase in the intrasynaptosomal free [Ca2+]i. Neither morphine nor [D‐Ala2‐D‐Leu5]enkephalin, μ and δ opiate agonists respectively, had any significant effect on intrasynaptosomal free [Ca2+]i. These results show that the individual effects of clonidine and dynorphin A1–13 are in keeping with the role of these substances at autoreceptors controlling neurotransmitter release. The mutual antagonism of their effects on [Ca2+]i is more difficult to explain but it may be a mechanism that prevents the occurrence of excessive inhibition of neuronal systems.
Myelin Composition and Activities of CNPase and Na+, K+‐ATPase in Hypomyelinated “pt” Mutant Rabbit
Authors: Domańska‐Janik K. , Gajkowska B. , de Nkhaud B. , Bourre J. M.
Published: December 2006

Abstract: A disorder of CNS myelination was found in paralytic tremor (“pt”) rabbits. The condition is inherited in a sex‐linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20–30% of control. Myelin isolated from 4‐week‐old “pt” rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two‐dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+, K+‐ATPase specific activity. 2′,3′‐Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in “pt” as in control myelin but differed by having greater sensitivity to detergent activation.
Cloning of Quail Tyrosine Hydroxylase: Amino Acid Homology with Other Hydroxylases Discloses Functional Domains
Authors: Fauquet Mireille , Grima Brigitte , Lamouroux Annie , Mallet Jacques
Published: December 2006

Abstract: A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C‐terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.
Effect of Chronic Lithium Treatment on 5‐Hydroxytryptamine Autoreceptors and Release of 5‐[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices
Authors: Friedman Eitan , Wang Hoau‐Yan
Published: December 2006

Abstract: The effect of acute and chronic lithium treatments on 5‐hydroxytryptamine (5‐HT, serotonin) release and on its regulation by presynaptic 5‐HT autoreceptors was studied in [3H]5‐HT preloaded superfused rat brain slices. The [3H]5‐HT overflow evoked by a 30‐s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium‐containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5‐HT release. The K+‐induced release observed in both control and chronically lithium‐treated animals was Ca2+‐dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5‐HT overflow in the cortex and hypothalamus and with an increase in spontaneous hippocampal 5‐HT overflow. The Ca2+‐independent overflow induced by fenfluramine was also decreased in cortical slices from lithium‐treated animals. The sensitivity of the inhibitory 5‐HT autoreceptors was assessed by the response to the 5‐HT agonist 5‐methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5‐HT release induced by 5‐methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5‐HT sites may be responsible for the increase in K+‐stimulated 5‐HT overflow in brain slices of animals treated chronically with lithium.
Effect of Crush Lesion on Radiolabelling of Ganglioside in Rat Peripheral Nerve
Authors: Guzman‐Harty Melinda , Warner Jean K. , Mancini Mary E. , Pearl Dennis K. , Yates Allan J.
Published: December 2006

Abstract: Left sciatic nerves of adult male Sprague‐Dawley rats were crushed and allowed to recover for 0, 1, 2, 4, 7, or 14 days. At each of these times both L‐5 dorsal root ganglia were injected with 100 μCi of [3H]glucosamine. Two days later, dorsal root ganglia, lumbosacral trunks, and sciatic nerves were removed bilaterally. The amounts of radiolabelled ganglioside in crushed lumbosacral trunks were consistently higher than in the controls, with the largest difference occurring within 2 days from simultaneous crush and injection to killing (specimens labelled day 0). The largest difference in the amount of radiolabelled ganglioside between crushed and control sciatic nerve (4–9 days from crush to killing) occurred later than that of lumbosacral trunk, but no significant difference occurred within the first 3 days following crush. There was only a slightly higher radioactivity in gangliosides totalled from all three anatomical specimens of crushed than in control nerves. The neutral nonganglioside lipid and acid‐precipitable fraction followed patterns of synthesis and accumulation similar to those of the gangliosides. These findings indicate that after nerve crush gangliosides, glucosamine‐labelled neutral nonganglioside lipids, and glycoproteins accumulate close to the proximal end of the regenerating axon. This accumulation could serve as a reservoir to increase the ganglioside concentration in the growth cone membrane.
[3H]MK‐801 Labels a Site on the N‐Methyl‐D‐Aspartate Receptor Channel Complex in Rat Brain Membranes
Authors: Wong Erik H. F. , Knight Antony R. , Woodruff Geoffrey N.
Published: December 2006

Abstract: The potent noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist [3H]MK‐801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]‐MK‐801 was considerably higher in 5 mM Tris‐HCl (pH 7.4) than in previous studies using Krebs‐Henseleit buffer. [3H]MK‐801 labels a homogenous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus > cortex > olfactory bulb = striatum > medulla‐pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]HIMK‐801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N‐allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high‐affinity sites labelled by the σ receptor ligand (+)‐[3H]SKF 10,047. [3H]MK‐801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK‐801 labels a high‐affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK‐801 and other noncompetitive NMDA receptor antagonists.
Modulation of Neuronal Signal Transduction Systems by Extracellular ATP
Authors: Ehrlich Y. H. , Snider R. M. , Kornecki E. , Garfield M. G. , Lenox R. H.
Published: December 2006

Abstract: The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 μM. The present study examined the effects of extracellular ATP in the concentration range of 0.1–1.0 mM on second‐messenger‐generating systems in cultured neural cells of the clones NG108‐15 and NIE‐115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin‐2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCI. Extracelluar ATP induced a concentration‐dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin‐2 signals. At the same concentration range (0.1–1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1‐stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15‐fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG 108–15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half‐maximal effects on Ca2+ uptake. Extracellular ATP at concentrations that can be produced in the synaptic cleft by repetitive stimulation but not during routine transmission can thus initiate a unique chain of events, which may play a role in the induction of long‐term adaptive changes in neuronal function.
Brain Coated Vesicle Destabilization and Phosphorylation of Coat Proteins
Authors: Georgieva‐Hanson Veneta , Schook William J. , Puszkin Saul
Published: December 2006

Abstract: Two basic polypeptides, bee venom melittin and poly‐L‐lysine, induced concentration‐dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly‐L‐lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: M, 33,000 and 100,000. Melittin‐stimulated phosphorylation was Ca2+ sensitive and inhibited by EGTA. The initiation of vesicle membrane segregation by melittin, followed by fusion and formation of elongated membrane cisternae, paralleled an increase of endogenous phospholipase A2 activity. The data suggest that a correlation exists between the state of assembly of the coat proteins on coated vesicles and protein phosphorylation.
Mutation in GM2‐Gangliosidosis B1 Variant
Authors: Ohno Kousaku , Suzuki Kunihiko
Published: December 2006

Abstract: Fibroblasts from a patient with GM2‐gangliosidosis B1 variant contained mRNA of normal size but in reduced quantity for the β‐hexosaminidase α subunit. The nucleotide sequence of a cDNA clone that included the entire protein coding sequence was completely normal except for a sinde base substitution from G to A at no. 533, resulting in a change from arginine to histidine at amino acid no. 178. The same mutation was found in two other cDNA clones. The position of the mutation is ∼90 amino acids from the N‐terminus of the mature, processed enzyme. Computer analysis predicated substantial alterations in the secondary structure of the enzyme protein. These results provide new insight into functional domains of this enzyme.
Agonist‐Selective Protection of the Opioid Receptor‐Coupled G Proteins from Inactivation by 5′‐p‐fluorosulphonylbenzoyl Guanosine
Authors: Wong Y. H. , Demoliou‐Mason C. D. , Hanley M. R. , Barnard E. A.
Published: December 2006

Abstract: The guanine nucleotide analogue, 5′‐p‐fluorosul‐phonylbenzoyl guanosine (FSBG), can react covalently with GTP‐binding proteins (G proteins). In rat brain membranes, FSBG causes a time‐dependent loss of β,γ‐imido[8‐3H]guanosine 5′‐triphosphate binding sites. Using 1 mM FSBG, the guanyl nucleotide modulation of opioid agonist binding is abolished, whereas the guanyl nucleotide sensitivity of neurotensin binding is retained. The action of FSBG can be prevented by the presence of opioid agonists, but not the antagonist naloxone. Iodoacetamide treatment of membranes in the presence of agonist, but not antagonist, can attenuate the action of FSBG in blocking guanyl nucleotide modulation of opioid agonist binding. These results suggest that FSBG covalently modifies essential thiol groups, whose exposure to the reagent is modified by agonist occupancy of the receptor, on a species of G protein linked to opioid receptors, but not on a species of G protein linked to neurotensin receptors. Thus, FSBG may have selectivity for the forms of Gi or Go, proteins associated with opioid receptors.
Characterization of Annexins in Mammalian Brain
Authors: Woolgar Julie A. , Boustead Catherine M. , Walker John H.
Published: December 2006

Abstract: Three annexins‐p68, endonexin, and p32‐have been isolated from porcine brain using their calcium‐dependent affinity for membranes. Large amounts (20‐50 mg/kg of tissue) of p68 and p32 can be isolated from cerebrum and cerebellum. The p68 is present as up to 0.3% of total porcine brain protein. The p68 and p32 from porcine brain bind to phosphatidic acid (half‐maximal binding at 6 and 34 μM free calcium, respectively) and to phosphatidylserine (8 and 34 μM, respectively). They do not bind to phosphatidylcholine at calcium concentrations up to 1 mM. Two other major proteins (Mr 180,000 and Mr 76,000) were isolated with the annexins in a calcium‐dependent manner but do not bind to phospholipids. The 180‐kilodalton protein is the heavy chain of clathrin. From immunohistochemical studies, p68 is strongly associated with the plasma membranes of Purkinje cell bodies and dendrites in porcine cerebellum. It is also an intracellular component of Purkinje cells localized to perinuclear structures. Staining of axons in the white matter and granule cell layer was also seen. In contrast, p32 is completely absent from Purkinje cells and their dendrites; it is predominantly located in the molecular layer and in white matter of the cerebellar folds. The distribution of p32 may be consistent with a predominantly glial localization.
Neurotransmitter Changes in Guinea‐Pig Brain Regions Following Soman Intoxication
Authors: Fosbraey Paul , Wetherell Janet R. , French Mary C.
Published: December 2006

Abstract: The effects of the organophosphate acetylcholinesterase (AChE) inhibitor soman (31.2 μg/kg s.c.) on guinea‐pig brain AChE, transmitter, and metabolite levels were investigated. Concentrations of acetylcholine (ACh) and choline (Ch), noradrenaline (NA), dopamine (DA), 5‐hydroxytryp‐tamine (5‐HT), and their metabolites, and six putative amino acid transmitters were determined concurrently in six brain regions. The brain AChE activity was maximally inhibited by 90%. The ACh content was elevated in most brain areas by 15 min, remaining at this level throughout the study. This increase reached statistical significance in the cortex, hippocampus, and striatum. The Ch level was significantly elevated in most areas by 60‐120 min. In all regions, levels of NA were reduced, and levels of DA were maintained, but those of its metabolites increased. 5‐HT levels were unchanged, but those of its metabolites showed a small increase. Changes in levels of amino acids were restricted to those areas where ACh levels were significantly raised: Aspartate levels fell, whereas γ‐Aminobutyric acid levels rose. These findings are consistent with an initial increase in ACh content, resulting in secondary changes in DA and 5‐HT turnover and release of NA and excitatory and inhibitory amino acid transmitters. This study can be used as a basis to investigate the effect of toxic agents and their treatments on the different transmitter systems.
Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells
Authors: Tanaka Teruo , Yokohama Hiromitsu , Negishi Manabu , Hayashi Hideya , Ito Seiji , Hayaishi Osamu
Published: December 2006

Abstract: We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+, K+‐ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1= E2 > F2α > D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+‐depleted medium and not observed in Ca2+‐free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose‐dependent manner with half‐maximal stimulation at 1 μM, and omission of K+ from the medium, a condition which suppresses the Na+, K+‐ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose‐dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+‐antiport, but not by the Na+‐channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 μM inhibited PGE2‐enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+‐antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+‐free medium. These results taken together indicate that PGE2 activates the Na+,H+‐antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+, K+‐ATPase and activation of Na+,H+‐antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.
Effects of the Amnesic Agent 2‐Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins
Authors: Bullock Sarah , Potter Jenny , Rose Steven P. R.
Published: December 2006

Abstract: The interaction of the amnesic agent 2‐deoxygalactose with fucose incorporation into glycoproteins in day‐old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2‐Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate‐polyacrylamide gradient gel analysis revealed that intracerebrally injected 2‐[3H]deoxygalatose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy‐sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylan‐thranilate‐coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150–180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training‐induced changes are discussed.