bluuebunny/crossref_metadata_2025 huggingface dataset:
Generalized N‐Acetylneuraminic Acid Storage Disease: Quantitation and Identification of the Monosaccharide Accumulating in Brain and Other Tissues
Authors: Hancock Larry W. , Thaler M. M. , Horwitz Allen L. , Dawson Glyn
Published: October 2006

Abstract: Brain and other tissues from a patient with extensive neonatal as‐cites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3–21 μmol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas‐liquid chromatography, thin‐layer chromatography, and GLC‐mass spectrometry positively identified the saccharide as free N‐acetylneuraminic acid. Based on the accumulation of only free N‐acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.
Rapid and Sensitive Single‐Step Radiochemical Assay for Catechol‐O‐Methyltransferase
Authors: Zürcher G. , Da Prada M.
Published: October 2006

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol‐O‐methyltransferase activity is described. The method is based on the conversion of catechol to [3H]guaiacol by catechol‐O‐methyltransferase in the presence of Mg2+, adenosine deaminase and S‐adenosyl l‐[methyl‐3H]methionine. Incubation and direct extraction of [3H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol‐O‐methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.
Determination of Dopamine and Its Acidic Metabolites in Brain Tissue by HPLC with Electrochemical Detection in a Single Run After Minimal Sample Pretreatment
Authors: Van Valkenburg Cees , Tjaden Ubbo , Van der Krogt Jacques , Van der Leden Bram
Published: October 2006

Abstract: A method, based on reverse‐phase liquid‐liquid chromatography, has been developed for the determination, in a single run, of dopamine (DA) and its acidic metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), combined with electrochemical detection (ECD). If applied to brain tissue, sample pretreatment can be reduced to centrifugation, filtration and adjustment of pH and perchlorate concentration prior to introduction into the liquid chromatograph. The relation between the perchlorate (counterion) concentration of the mobile phase and the retention (k′) of the amines is linear, as is the relation between the H− concentration of the mobile phase and the retention of the acidic metabolites. This flexible phase system, combined with a simple and therefore reproducible sample pretreatment, warrants a high throughput of samples. The procedure offers good possibilities for routine analysis of catecholamines and their acidic metabolites in the picogram range. Some typical examples of the behaviour of this phase system and the electrochemical detector are presented and discussed.
Enkephalin Dipeptidyl Carboxypeptidase (Enkephalinase) Activity: Selective Radioassay, Properties, and Regional Distribution in Human Brain
Authors: Llorens C. , Malfroy B. , Schwartz J. C. , Gacel G. , Roques B. P. , Roy J. , Morgat J. L. , Javoy‐Agid F. , Agid Y.
Published: October 2006

Abstract: The compound [3H‐Tyr1,D‐Ala2,Lcu‐OH5]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase (enkephalinase) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [3H]Tyr‐D‐Ala‐Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly3‐Phe4 amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5‐enkephalin levels.
Biochemical and Morphological Comparison of Postsynaptic Densities Prepared from Rat, Hamster, and Monkey Brains by Phase Partitioning
Authors: Gurd James W. , Gordon‐Weeks Phillip , Evans W. Howard
Published: October 2006

Abstract: A new procedure for the preparation of postsynaptic densities (PSDs) is described. A synaptic membrane fraction was homogenized in an aqueous two‐phase polymer system containing Poly(ethylene glycol) 6000 (5% wt/wt) and Dextran T500 (6% wt/wt) containing 1% I‐o‐n‐octyl‐β‐D‐glucoside. Following a brief centrifugation to separate the phases, highly purified PSDs banded at the interface of the two phases. Using this procedure PSDs have been isolated from rat and hamster cerebral cortex and from the frontal cortex, cerebral cortex, hippocampus, and pooled caudateiputamen regions of Macaca mulatta Rhesus monkeys. The isolated PSDs appeared as curved bars when sectioned or as discs when viewed en face in the electron microscope. The hamster PSDs were associated with large numbers of small rod‐like structures 4.5 nm thick and 28 nm long. Similar structures were present, although in fewer numbers, in the rat and monkey preparations. Polyacrylamide gel electrophoresis showed that the PSDs contained a complex population of proteins with major components having molecular weights of 180,000, 130,000, 110,000, 94,000, 65,000, 60,000, and 51,000. Reaction of polyacrylamide gels with 125I‐ concanavalin A (Con A) identified two major (apparent Mr 180,000 and 130,000) and three minor (apparent Mr 230,000, 145,000, and 110,000) Con A‐binding glycoproteins in the PSD fractions. Although some quantitative variation between species and brain regions was apparent, the overall protein and glycoprotein composition was similar for all fractions studied.
On the Blood‐Brain Barrier to Peptides: [3H]βCasomorphin‐5 Uptake by Eighteen Brain Regions In Vivo
Authors: Ermisch A. , Ruhle H.‐J. , Neubert K. , Hartrodt B. , Landgraf R.
Published: October 2006

Abstract: After intracarotid injection of [3H]β‐casomorphin‐5 (βCM5) in rats, the accumulation of radioactivity was determined in 18 brain regions and the anterior pituitary. The relative accumulation in all regions significantly exceeded that of [3H]inulin by a factor of 2.5, indicating a low but measurable brain uptake of the peptide. In blood‐brain barrier‐free areas, the accumulation of radioactivity was 15‐fold higher than in blood‐brain barrierprotected areas. The relative accumulation was not dependent on the total βCM5 concentration in the range of 0.3–1.1μM, and was not depressed by 400μM L‐tyrosine. We conclude that βCM5, like other peptides, is accumulated in the blood‐brain barrier‐free areas to a relatively high but differing degree, whereas in the areas with a tight endothelium the accumulation is relatively low and nearly uniform. A binding to endothelial cells may contribute to the low accumulation of βCM5, especially in blood‐brain barrier‐protected areas.
[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake
Authors: Whitaker Patricia M. , Vint Carley K. , Morirn Rosemarie
Published: October 2006

Abstract: Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to erotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax= 202 fmol/mg protein) and with high affinity (KD= 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 μ.M and a Vmax of 1080 fmol/106 cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.
Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons
Authors: Yu A. C. H. , Drejer J. , Hertz L. , Schousboe A.
Published: October 2006

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol min−1 mg−1 protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol min−1 mg−1). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.
A Possible Role for Copper‐Mediated Oxidation of Thiols in the Regulation of the Release of Luteinizing Hormone Releasing Hormone from Isolated Hypothalamic Granules
Authors: Rice Gregory E. , Barnea Ayalla
Published: October 2006

Abstract: A role for copper in the release of luteinizing hormone releasing hormone (LHRH) from hypothalamic neurons has been previously proposed. To elucidate further the mechanism of action of copper, we addressed two questions: (a) what is the active form of copper that interacts with the LHRH granule (ionic or chelated)? and (b) is copper‐stimulated LHRH release a result of an interaction of copper with thiol groups and, if so, does it require oxygen? Granules were isolated from hypothalami of adult male rats and were then incubated at 37°C for 3–5 min in a buffered medium. When granules were incubated with various copper complexes, CuATP stimulated LHRH release by 45 ± 4% (mean ± SE), copper tartrate by 44 ± 4%, CuBSA by 27 ± 7%, and copper histidine by 16 ± 6%. Neither CuEDTA nor CuCl2 stimulated LHRH release. CuATP‐stimulated LHRH release from granules incubated under N2 was 50% of that incubated under air. Furthermore, the CuATP‐stimulated release of LHRH was completely inhibited by dithiothreitol or glutathione (10−3M each), partially (40–50%) by iodoacetate or 5,5‐dithiobis‐(2‐nitrobenzoic acid), and not at all by oxidized dithiothreitol. Thus, chelated copper, rather than ionic copper, is the active form of the metal, and the action of copper involves an oxidation reaction and granule thiol groups. The precise mechanism of action of copper, however, has yet to be elucidated. We propose that copper may affect LHRH release as follows: copper, bound to an intracellular chelator (protein, peptide, or amino acid), oxidizes thiols of the LHRH granule, leading to a change in granule‐membrane permeability and hence to LHRH release.
In Vivo Measurement of Dopamine and Its Metabolites by Intracerebral Dialysis: Changes After d‐Amphetamine
Authors: Zetterström T. , Sharp T. , Marsden C. A. , Ungerstedt U.
Published: October 2006

Abstract: By using a new technique, intracerebral dialysis, in combination with high performance liquid chromatography and electrochemical detection, it was possible to recover and measure endogenous extracellular dopamine, together with its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) from the striatum and nucleus accumbens of anaesthetized or freely moving rats. In addition, measurements of extracellular 5‐hydroxyindoleacetic acid, ascorbic acid, and uric acid were made. Basal extracellular concentrations of dopamine and DOPAC in the striatum were estimated to be 5 × 10−8M and 5 × 10−6M, respectively. d‐Amphetamine (2 mg/kg s.c.) increased dopamine levels in the striatum perfusates by 14‐fold, whereas levels of DOPAC and HVA decreased by 77% and 66%, respectively.
Specific Binding of [3H]Rauwolscine to α2‐Adrenoceptors in Rat Cerebral Cortex: Comparison Between Crude and Synaptosomal Plasma Membranes
Authors: Diop Laurent , Dausse Jean‐Pierre , Meyer Philippe
Published: October 2006

Abstract: [3H]Rauwolscine, a specific, potent, radiolabelled α2‐antagonist, binds to distinct high‐ and low‐affinity α2‐adrenoceptors in crude membrane preparations of the rat cerebral cortex. The concentration of high‐affinity α2‐adrenoceptors was increased by addition of sodium ions or guanylnucleotides. In synaptosomal plasma membrane preparations, only the low‐affinity component was found. Neither sodium or guanylnucleotides caused any increase in the concentration of these low‐affinity receptors for [3H]rauwolscine.
Adenosine A1 Receptors Are Associated with Cerebellar Granule Cells
Authors: Wojcik W. J. , Neff N. H.
Published: October 2006

Abstract: The cerebellum of mouse appears to have only the adenosine A1 receptor, which decreases adenylate cyclase activity, and not the A2 receptor, which increases adenylate cyclase activity. The adenosine analog N6‐(l‐phenylisopropyl)adenosine (PIA), stimulates the Al receptor in a membrane preparation and decreases basal adenylate cyclase activity by 40%. The EC50 for PIA is approximately 50 nM. To associate the A1 receptor with a cerebellar cell type, three different neurological mutant mouse strains were studied: staggerer (Purkinje and granule cell defect), nervous (Purkinje cell defect), and weaver (granule cell defect). PIA was unable to effect a maximal decrease in adenylate cyclase activity of membranes prepared from cerebella of the staggerer and weaver mice in comparison with the respective littermate control mice. In contrast, membranes from nervous mice and their littermates showed similar PIA dose‐response curves. Moreover, the diminished PIA response observed in the weaver cerebellum, when compared with the control littermate, was not detected in the striatum. This suggests no overall brain defect in the adenosine A1 receptors coupled to adenylate cyclase of the weaver mouse. We conclude that a loss of granule cells coincides with an attenuated response to PIA, implying that the A1 receptors are associated with the granule cells of the cerebellum.
A Stimulatory Subunit in the Polypeptide Elongation Factor‐1 of the Chick Brain
Authors: Murakami K. , Miyamoto K.
Published: October 2006

Abstract: The higher‐molecular‐weight elongation factor‐1 (EF‐1H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF‐1H consisted of aggregates of low‐molecular‐weight elongation factor‐ 1 (EF‐1L). Crude EF‐1H, obtained from 20‐day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF‐1βγ, was obtained using a DEAE‐Sephadex column equilibrated in 0.025 mM GTP. Both EF‐1β, and EF‐1γ, were isolated by means of a DE‐52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 104, respectively. EF‐1β and EF‐1γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF‐1α was 5 × 104. It was found that EF‐1β stimulated the two EF‐1α‐dependent reactions, i.e., phenylalanyl‐tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF‐ 1α‐guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF‐1β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF‐1α alone, respectively. The amino acid compositions of EF‐1α ‐1β, and ‐1γ and EF‐2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF‐1β, and EF‐1, had not been reported in the brain. A possible physiological significance of EF‐1β during brain development is also discussed.
Adenosine Transport by Primary Cultures of Neurons from Chick Embryo Brain
Authors: Thampy K. George , Barnes Eugene M.
Published: October 2006

Abstract: The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [3H]adenosine. During the 5‐25‐s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis‐Menten relationship and yielded a Km of 13 μM and Vmax of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10‐100‐fold higher than [3H]adenosine. Adenosine derivatives (N6‐cyclohexyl‐, N6‐benzyl‐, N6‐methyl‐, and 2‐chloroadenosine) were more effective inhibitors; p‐nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high‐affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.
Decarboxylation of p‐Tyrosine: A Potential Source of p‐Tyramine in Mammalian Tissues
Authors: Bowsher Ronald R. , Henry David P.
Published: October 2006

The question of the existence of a p‐tyrosine decarboxylase pathway for the formation of p‐tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p‐tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl‐Sepharose chromatography, DEAE‐Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L‐DOPA, 5‐hydroxytryptophan, 3,4‐dihydroxyphenylserine, o‐tyrosine, m‐tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L‐amino acid decarboxylase. This conclusion was supported by a constant ratio of 5‐hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p‐tyrosine is decarboxylated by aromatic L‐amino acid decarboxylase and that this metabolic transformation may be an important source of p‐tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p‐tyramine and other biogenic amines.
Evoked Release of Proteins from Central Neurons In Vivo
Authors: Greenfield Susan A. , Chéramy André , Glowinski Jacques
Published: October 2006

Push‐pull cannulae were implanted in both substantiae nigrae and caudate nuclei of the halothane‐anesthetized cat. The release of total protein, acetylcho‐linesterase, and nonspecific cholinesterases was examined. Following direct application of potassium to one substantia nigra, changes occurred in the local release of total protein and acetylcholinesterase, but not nonspecific cholinesterases; changes also were observed in both caudate nuclei and the contralatera/ substantia nigra. The local evoked release of acetylcholinesterase and of total protein differed in the extent to which they were calcium‐dependent. Control studies suggest that release of these compounds, both spontaneous and evoked, is related, at least in part, to neuronal activity. The significance of the neuronal release of proteins is discussed.
Changes in the Concentration of Enolase Isozymes and S‐100 Protein in Degenerating and Regenerating Rat Sciatic Nerve
Authors: And Kanefusa Kato , Satoh Toyohiko
Published: October 2006

Levels of enolase isozymes (ãã, ãγ, and γγ forms) and S‐100 protein in rat sciatic nerves were determined during their degeneration and regeneration processes. The sciatic nerves were unilaterally crushed or severed. The rats were killed 1,2,6, and 8‐9 weeks later, and both the proximal and distal portions of the damaged nerves were dissected. Control samples were obtained from the untreated contralateral hindlimbs. Enolase isozymes and S‐100 protein in the nerve segments were determined with the enzyme immunoassay method. The control nerves contained about 40, 90, and 30 pmol/mg protein of ãã, ãγ, and γγ enolases, respectively, and 0.85 μg/mg protein of S‐100 protein. These levels were not affected by repetitive electrical stimulation of the nerve fibers in vivo. The levels of the nervous system‐specific forms of enolase (ãγ and γγ) and S‐100 protein decreased markedly within a week in the distal portion of the crushed nerve (ãγ, 27 pmol/mg; γγ, 5.5 pmol/mg; S‐100 protein, 0.36 μg/mg) with apparently no change in the concentration of aa enolase. These levels in the proximal portion of the crushed nerve remained unaltered. The sensory and motor functions impaired by the sciatic nerve crush showed a recovery more or less after 4‐9 weeks. This recovery was accompanied by a gradual regaining of the specific proteins in the distal portion of injured nerves (ãγ, 64 pmol/mg; γγ, 13 pmol/mg; S‐100 protein, 0.63 μg/mg at the 8‐9th week).
The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone
Authors: Fowler Christopher J. , Benedetti Margherita Strolin
Published: October 2006

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO‐A toward dopamine (120 μM) is lower than the Km, of MAO‐B toward this substrate (340 μM). The activity of MAO‐A was lower in old rats than in young rats, and the same degree of decrease was found for 5‐hydroxy‐tryptamine as for dopamine as substrates for this enzyme form. The activity of MAO‐B was higher in the old rats, the degree of increase being the same for dopamine as for β‐phenethylamine as substrates for this enzyme form. The K1 values of the inhibition of MAO‐A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the K1 values for the inhibition of MAO‐B by these compounds.
Alterations in the Content of Amino Acid Neurotransmitters Before the Onset and During the Course of Methoxypyridoxine‐Induced Seizures in Individual Rabbit Brain Regions
Authors: Nitsch Cordula , Schmude Bernd , Haug Pat
Published: October 2006

In rabbits, generalized seizures were induced by methoxypyridoxine, and changes in amino acid concentrations of 15 brain regions were investigated before seizure onset and during the course of sustained epileptiform activity. As previously reported, γ‐aminobutyric acid (GABA) concentration decreased preictally in most regions. At the same time, taurine level was elevated in the hypothalamus, thalamus, hippocampus, caudatum, and frontal cortex. After 90 min of seizures, it was significantly decreased in the hypothalamus, periaqueductal grey, substantia nigra, frontal cortex, and cerebellum. Glycine content was reduced preictally only in the substantia nigra; after seizure onset its concentration rose in all brain areas. Glutamate content in the frontal cortex decreased before seizure onset; after 1.5 h of seizures, its concentration in cerebellum, caudatum, and hippocampus was reduced. Aspartate level was decreased in most areas after sustained seizures; in putamen, however, it was elevated. In contrast, glutamine content increased preictally in the superior colliculus and in all brain areas by ˜200% after 90 min of seizures. Alanine and valine content also rose markedly in most brain areas after prolonged seizures, and threonine showed the same tendency. The single brain regions were observed to respond to methoxypyridoxine in highly individualistic ways. For example, the glycine content of the substantia nigra, which is believed to utilize this amino acid as a neurotransmitter, decreased preictally. The potential importance of the superior colliculus in seizure induction is considered in view of the early rise in glutamine level. The antagonistic preictal behavior of taurine and GABA is discussed with respect to synthesis, uptake from the blood, and antiepileptic properties.
Microanalysis of Endogenous Acetylcholine Released from the Hemidiaphragm of the Rat
Authors: Häggblad J. , Eriksson H. , Heilbronn E.
Published: October 2006

A chemiluminescence method for the determination of endogenous acetylcholine was adapted to transmitter released from the hemidiaphragm of the rat. Released choline and acetylcholine were isolated and concentrated using KI3 precipitation. Between 1 and 20 pmol of acetylcholine may be measured without sample dilution. The detection limit for acetylcholine luminescence is 1.0 pmol; thus, with an observed precipitation recovery of ˜30%, the lowest detectable released amount is 3.5 pmol. The limitations of the method are discussed.
Protein Synthesis and Rapid Axonal Transport During Regrowth of Dorsal Root Axons
Authors: Perry G. W. , Krayanek Susan R. , Wilson David L.
Published: October 2006

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two‐dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes inproteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.
Identification of Intrinsic Sialidase and Sialoglycoprotein Substrates in Rat Brain Synaptic Junctions
Authors: Cruz Tony F. , Gurd James W.
Published: October 2006

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5–10% of the total sialidase activity recovered from SM following extraction with Triton X‐100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12–15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A‐agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.
An Aminopeptidase from Mouse Brain Cytosol That Cleaves N‐Terminal Acidic Amino Acid Residues
Authors: Kelly J. A. , Neidle E. L. , Neidle A.
Published: October 2006

An aminopeptidase with specificity directed toward peptides with acidic N‐terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion‐exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl‐phenylala‐nine methyl ester at a rate of 13.2 μu,mol/min/mg protein at pH 7.5, an increase in specific activity of 1000‐fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is ˜450,000. Dipeptides with N‐terminal aspartyl residues are cleaved preferentially to glutamic‐containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For pep‐tides of the form aspartyl‐X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripep‐tides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N‐terminal aspartyl residue of angiotensin II, aspartyl‐phenylalanine methyl ester or aspartyl‐alanine, and the N‐terminal glu‐tamyl residue of adrenocorticotropin(5‐10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o‐phenanthroline and EDTA. The latter effect could be reversed completely by Zn2+ and partially by Mn2+ or Mg2+; Co2+ and Fe2+ had no effect; Ca2+ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.
Loss of Ascorbic Acid from Injured Feline Spinal Cord
Authors: Pietronigro Dennis D. , Hovsepian Movses , Demopoulos Harry B. , Flamm Eugene S.
Published: October 2006

Abstract: Feline spinal cord contains 0.97 mM ascorbic acid, as measured by the dinitrophenylhydrazine method. Greater than 90% is maintained in the reduced form. When functioning normally, the CNS conserves its ascorbic acid with a turnover rate of 2% per h. Following contusion injury severe enough to produce paraplegia, ascorbic acid is rapidly lost from injured spinal tissue. Thus, ascorbic acid is decreased 30% by 1 h and 50% by 3 h following injury. Oxidized ascorbic acid is increased at 1, but not 3, h following impact. As a consequence of its many functions in CNS, loss of ascorbic acid may contribute to derangements in spinal cord function following injury.
Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride
Authors: Betz A. Lorris
Published: October 2006

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of 22Na + was compared with that of the relatively impermeable molecule, [3H]l‐glucose. At a Na + concentration of 1.4 mM, Na + uptake was 1.74 ± 0.07 times greater than l‐glucose uptake. This decreased to 1.34 ± 0.04 at 140 mM Na +, indicating saturable Na + uptake. Relative Na + extraction was not affected by pH but was inhibited by amiloride (Ki= 3 ± 10−7M) and by 1 mM furosemide. The effects of these two inhibitors were additive. Brain uptake of 86Rb +, a K + analogue, was measured to study interaction of K + with Na + transport systems. Relative 86Rb + extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na + to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na + from blood to brain.
Early and Dose‐Dependent Decrease of Retrograde Axonal Transport in Acrylamide‐Intoxicated Rats
Authors: Jakobsen J. , Sidenius P.
Published: October 2006

Abstract: The effect on retrograde axonal transport of doses of acrylamide ranging from 50 to 500 mg/kg was studied in sensory nerve of rats. Accumulation of trichloroacetic acid‐phosphotungstic acid‐insoluble label was measured in a collection segment distal to a double ligature placed on the sciatic nerve at intervals 9–15 h and 9–24 h following injection into the dorsal root ganglion of the fifth lumbar root of [35S]methionine and [3H]fucose. After a dose of 100 mg/kg of acrylamide no neurological signs of neuropathy had yet appeared, but retrograde buildup of protein label was significantly reduced for the long interval (2.20 ± 0.49 arbitrary units (AU) (mean ± SD) versus 2.81 ± 0.57 AU in controls, 2p = 0.034). No abnormality of the short interval appeared before a dose of 500 mg/kg was reached. The retrograde transport abnormality was dose‐related (r =−0.85, n = 28 and 2p = 1.2 × 10‐8), as was the degree of neuropathy evaluated by “blind” neurological scoring (r = 0.88, n = 14 and 2p = 2.8 × 10‐5). After a dose of 500 mg/kg, when the rats were severely disabled with almost total incoordination of the hindlegs, the retrograde accumulation of the long interval was profoundly depressed (1.08 ± 0.28 AU versus 2.81 ± 0.57 AU in controls, 2p = 1.2 × 10‐7). Similar changes were seen in accumulation of glycopro‐tein label. After the rats had recovered for 4–10 weeks neurological signs of neuropathy had disappeared and the transport abnormality had improved. To test the specificity of acrylamide on the retrograde transport defect N‐hydroxymethylacrylamide and methylene‐bisacrylamide, which do not induce neuropathy, were studied. None of these related compounds influenced the transport. These observations imply that in acrylamide intoxication a defect in the amount of material carried by retrograde axonal transport rather than in “turnaround” time or in transport velocity is present, that the transport abnormality precedes the development of neuropathy and that it is related to the degree of the neurological disability. We suggest that the retention of protein in the distal axons is the functional counterpart of the well‐known accumulation of vesicular organelles in the pre‐terminals.
Noradrenalin‐Inducible Cyclic‐AMP Accumulation in Rat Cerebral Cortex: Changes During Complete Global Ischemia
Authors: Lin Marina R. , Henteleff Harvey B. , Nemoto Edwin M.
Published: October 2006

Abstract: Neurologic dysfunction after cerebral ischemic insults may be due not only to neuronal death, but also to a possibly reversible failure in synaptic transmission. Because noradrenaline (NA)‐inducible cyclic‐AMP (cAMP) accumulation in brain may reflect the integrity of synaptic transmission mechanisms and brain viability, we studied its changes in cerebral cortex after various durations of decapitation ischemia. Unanesthetized rats were decapitated and the brains were kept at 37°C for times ranging from 0 to 60 min. Cerebral cortical slices were incubated in vitro and NA (11.2 μM)‐induced cAMP accumulation was evaluated over 10 min. At 0 min of ischemia, NA‐induced cAMP accumulation was 56 pmol/mg protein/10 min. Between 0 and 20 min of ischemia, a linear eightfold increase, to 435 × 49 pmol/mg protein/10 min, occurred in NA‐induced cAMP accumulation, with no further increase after longer durations of ischemia. The mechanisms modulating the increase in cortical NA‐inducible cAMP accumulation with a maximum response after 20 min of ischemia remain to be defined.
Guinea Pig Brain Histamine N‐Methyltransferase: Purification and Partial Characterization
Authors: Matuszewska Bozena , Borchardt Ronald T.
Published: October 2006

Abstract: Histamine N‐methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE‐cel‐lulose chromatography, and affinity chromatography on an S‐adenosylhomocysteine‐agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29,000 ± 1000 as determined by both gel filtration through Sephadex G‐100 and by electrophoresis in sodium dodecyl sulfate‐polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the Kms for histamine and S‐adenosyl‐l‐methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the Ki for S‐adenosyl‐l‐homocysteine was 24.5 ± 1.45 μM.
Monitoring the Stimulated Release of Dopamine with In Vivo Voltammetry. I: Characterization of the Response Observed in the Caudate Nucleus of the Rat
Authors: Kuhr Werner G. , Ewing Andrew G. , Caudill W. Lowry , Wightman R. Mark
Published: October 2006

Abstract: Microvoltammetric electrodes were employed in the brain of an anesthetized rat to monitor chemical substances in extracellular fluid following electrical stimulation of the medial forebrain bundle. An increase in concentration of an easily oxidized substance is observed in the caudate nucleus and in the nucleus accumbens. A large amount of evidence suggests that the substance that is observed following stimulation is dopamine. (1) The location of the stimulating electrode must be in known dopaminergic tracts to induce release. (2) Release is most easily observed in brain regions that contain significant numbers of dopamine‐containing neurons. (3) Two voltammetric electrodes with very different electrochemical responses provide voltammograms of the released species that are unique for catechols in one case and catecholamines in another case. (4) The amount of 3,4‐dihydroxyphenylacetic acid found in striatal tissue by postmortem analysis correlates with the calculated amount of dopamine released. (5) Inhibition of tyrosine hydroxylase, and thus dopamine synthesis, decreases the observed release while inhibition of monoamine oxidase, and thus formation of dopamine metabolites, does not (6) The dependence of release on stimulation parameters agrees with results obtained with perfusion techniques. Thus, a new method has been developed to characterize endogenous dopamine release in the rat brain and can be used on a time scale of seconds.
Monitoring the Stimulated Release of Dopamine with In Vivo Voltammetry. II: Clearance of Released Dopamine from Extracellular Fluid
Authors: Ewing Andrew G. , Wightman R. Mark
Published: October 2006

Abstract: Microvoltammetric electrodes implanted in the caudate nucleus of the anesthetized rat have been used to monitor dopamine released following electrical stimulation of the medial forebrain bundle. These electrodes are fabricated from unmodified carbon fibers and have been used with normal pulse voltammetry. Dopamine appears in the vicinity of the electrode when the stimulation is initiated, and disappears almost immediately when the stimulation is terminated. The data suggest that the effective diffusion distance is < 100 μm. Postmortem analysis using liquid chromatography with electrochemical detection shows that dopamine released in this manner is metabolized to 3,4‐dihydroxyphenylacetic acid (DOPAC); however, neither substance is observed electrochemically in the extracellular fluid within seconds after the stimulation. In addition, inhibitors of neuronal uptake of dopamine, amphetamine (1.8 or 15 mg · kg−1) or benztropine (25 mg · kg−1), or of dopamine metabolism, pargyline (150 mg · kg−1) or tropolone (100 mg · kg−1), do not significantly affect the rate at which dopamine disappears from extracellular fluid, although they can affect the amount released. These results suggest that dopamine cannot freely diffuse in the extracellular fluid because an extraneuronal uptake mechanism exists that clears dopamine from extracellular fluid into an extraneuronal pool where metabolism to 3,4‐dihydroxyphenylacetic acid occurs. Dopamine can be observed during electrical stimulation of the ascending fibers because neuronal and extraneuronal uptake systems are unable to remove dopamine on these short time scales.
Thyrotropin‐Releasing Hormone Enhances Choline Acetyltransferase and Creatine Kinase in Cultured Spinal Ventral Horn Neurons
Authors: Schmidt‐Achert K. Martin , Askanas Valerie , Engel W. King
Published: October 2006

Abstract: The effect of 0.1 mM thyrotropin‐releasing hormone (TRH) on ventral horn neurons was investigated in eight experimental sets of tissue cultures established from ventral and dorsal portions of spinal cords of 13–15‐day rat embryos. Cultures were treated with TRH from day 1 for 2–5 weeks. TRH‐treated ventral spinal cord cultures (VSCC), compared with control VSCC, had more numerous and more healthy‐appearing neurons and thicker bundles of long cell processes. In TRH‐treated VSCC, choline acetyltransferase (ChAT) activity was >16 times (p < 0.005) and creatine kinase >3 times (p < 0.005) that of control VSCC. Morphologic and biochemical parameters of dorsal spinal cord cultures remained unchanged by TRH treatment. Since lower motor neurons are numerous in the ventral spinal cord (and not present in the dorsal cord) and since lower motor neurons are the major ChAT‐containing spinal cord cells, our data demonstrating a beneficial effect of TRH on VSCC suggest a tropic effect of TRH on lower motor neurons.
Structure of the Proteolipid Protein Extracted from Bovine Central Nervous System Myelin with Nondenaturing Detergents
Authors: Smith Ross , Cook Jo , Dickens Paul A.
Published: October 2006

Abstract: As a basis for attempts to define the structures of the proteins within myelin, methods have been developed for their extraction and isolation in solutions of non‐denaturing detergents. With use of solutions of deoxycholate or Triton X‐100, up to 90% of the protein has been extracted from bovine CNS myelin, along with most of the phospholipid. The proteolipid protein has been purified in deoxycholate solutions by chromatography on a blue dye‐ligand column, which retained all of the basic protein and 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase, and then on Sephacryl S300, which separated proteolipid protein from phospholipid and high‐molecular‐weight proteins. The proteolipid protein was isolated from Triton X‐100 extracts of myelin by adsorption onto phosphocellulose resin, with subsequent elution by 0.5 M sodium chloride. Gel permeation chromatography was used as the final purification step. Sedimentation equilibrium experiments gave a monomer molecular weight of 134,000 ± 8000 in deoxycholate and 145,000 ± 17,000 in Triton X‐100 solutions. On the basis of an apparent subunit molecular weight of 23,500 it was deduced that the native protein is probably hexameric. Above 0.2 gL−1 in Triton X‐100 solutions and 0.5 gL−1 in deoxycholate solutions the protein aggregated. In deoxycholate solutions the protein adopts the highly helical conformation expected for an intrinsic membrane protein.
Hormones and Neurotransmitters Control Cyclic AMP Metabolism in Choroid Plexus Epithelial Cells
Authors: Crook Richard B. , Farber Michael B. , Prusiner Stanley B.
Published: October 2006

Abstract: The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 μg/ml cholera toxin, a 50‐fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10−5M isoproterenol, 10−4M prostaglandin E1, 10−5M histamine, and 10−5M serotonin caused increases of intracellular cyclic concentrations of 100‐, 50‐, 20‐, and 4‐fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol‐mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin‐dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.
γ‐Aminobutyric Acid Stimulates the Release of Endogenous Ascorbic Acid from Rat Striatal Tissue
Authors: Bigelow James C. , Brown David S. , Wightman R. Mark
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) was found to induce the release of ascorbic acid from rat striatal homogenates and minces. This release was studied with the use of a rapid supervision system with an on‐line amperometric detector that monitors for the presence of easily oxidized substances (i.e., ascorbate, 3,4‐dihydroxyphenylethylamine). The release was found to be calcium‐independent and depolarization‐dependent. This releasable pool of ascorbate could be replenished through nonstereospecific uptake. The releasing action of GABA was mimicked by the GABA agonist, muscimol, and was completely inhibited by the GABA antagonist, picrotoxin. The structural analogues of GABA, β‐alanine and γ‐hydroxybutyric acid, had no effect. These data indicate that ascorbate release is GABA‐receptor mediated and syn‐aptically localized.
Biochemical Expression of Mosaicism in Female Mice Heterozygous for the Jimpy Gene
Authors: Benjamins Joyce A. , Skoff Robert P. , Beyer Kevin
Published: October 2006

Abstract: Expression of the jimpy gene in heterozygous females was analyzed by measuring galactolipid synthesis in brain during early myelination. Sulfatide labeling in brains of heterozygous females at 13–14 days is decreased to 40–80% that of female littermates who do not carry the jimpy gene. The activities of ceramide galactosyl transferase and cerebroside sulfotransferase and levels of myelin basic protein were similarly depressed. Since the jimpy gene was maintained with the Tabby gene in these studies, the effect of the Tabby gene on these parameters was examined and found to have no effect. These biochemical findings indicate that myelination is retarded in the brains of heterozygous jimpy females during the second week of development. However, at 18 days and thereafter, sulfatide labeling is less reduced, suggesting that the oligodendrocytes in brain attempt to compensate, in agreement with morphologic studies which show that myelin is decreased in brain during early development, but appears normal in adult animals.