bluuebunny/crossref_metadata_2025 huggingface dataset:
Alteration in Neuronal‐Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid
Authors: Krespan Barbara , Berl Soil , Nicklas William J.
Published: October 2006

Abstract: The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with d‐[2‐14C]glucose, [U‐14C]‐γ‐aminobutyric acid, [3H]acetate, [U‐14C]glutamate, and [U‐14C]glutamine as precursors. Kainic acid (1 mm) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2‐14C]glucose and [U‐14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotox‐in. Kainic acid stimulated efflux of both glucose‐and acetate‐labeled glutamate. In contrast, veratridine released glucose‐labeled glutamate preferentially via a tetrodotoxin‐sensitive mechanism. Kainic acid did not release [U‐14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.
Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants
Authors: Seyfried Thomas N. , Yu Robert K. , Miyazawa Nobuko
Published: October 2006

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver (wv) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation (pcd) destroys the vast majority of Purkinje cells. The staggerer (sg) and lurcher (Lc) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of GD1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of GTIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of GD3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg, and Lc/+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that GDT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for GTla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.
Alteration of 5′‐Nucleotidase and Na+, K+‐ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler
Authors: Bourre J. M. , Chanez C. , Dumont O. , Flexor M. A.
Published: October 2006

Abstract: 5′Nucleotidase and Na+,K+‐ATPase are very probably myelin‐associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5′nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21‐day‐old and adult mutants, except Trembler. It was normal in 8‐day‐old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5′‐Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10‐day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin‐related enzymes and a myelin‐specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5′‐nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5′‐nucleotidase increased (42% and 190% of the control in 60‐day‐old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.
Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions
Authors: Murphy Mary G. , Spence Matthew W.
Published: October 2006

Abstract: We measured long‐chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl‐CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.
Activation of Brain Guanylate Cyclase by Phospholipase A2
Authors: Zwiller J. , Ciesielski‐Treska J. , Ulrich G. , Revel M. O. , Mandel P.
Published: October 2006

Abstract: Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane‐bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.
Effect of Dopamine Agonists and Antagonists on DOPA Formation in the Substantia Nigra
Authors: Argiolas A. , Melis M. R. , Fadda F. , Serra G. , Gessa G. L.
Published: October 2006

Abstract: The effect of different psychotropic drugs on the rate of DOPA accumulation after administration of a decarboxylase inhibitor (NSD 1015) was compared in the substantia nigra (SN) and caudate nucleus (CN) by a new radioenzymatic method. Inhibition of monoamine oxidase with pargyline or stimulation of dopamine (DA) receptors with apomorphine, N‐n‐propyl‐norapomorphine or d‐amphetamine reduced DOPA formation in the CN and SN to the same extent. Vice versa, both inhibition of DA receptors with haloperidol or (‐)sulpiride and depletion of DA concentration with reserpine enhanced DOPA formation to a greater extent in the CN than in the SN. Apomorphine antagonized not only the effect of haloperidol and (‐)sulpiride, but also, and even more effectively, that of reserpine. The results indicate that DA synthesis in the SN is controlled by both end‐product inhibition and DA receptor‐mediated mechanisms.
The Role of Chloride in Acetylcholine Metabolism
Authors: Ksiezak‐Reding Hanna J. , Goldberg Alan M.
Published: October 2006

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 mm‐potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 mm‐potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride‐containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium‐stimulated release of ACh was markedly reduced as compared with a chloride‐containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 mm‐potassium and was totally inhibited when the potassium concentration was increased to 35 mm. Choline uptake studies revealed that with propionate only a low‐affinity component of the choline transport system existed. Further, the Vmax was markedly reduced when the potassium concentration was increased to 35 mm. The results suggest that under certain conditions choline transported by a low‐affinity system might provide a substantial source of choline for ACh synthesis.
On the Nature of the Interaction Between Serotonin and Serotonin Binding Protein: Effect of Nucleotides, Ions, and Sulfhydryl Reagents
Authors: Tamir Hadassah , Liu Kuo‐Peing
Published: October 2006

Abstract: Rat brain serotonin binding protein (SBP) was found to have essential ‐S‐S and ‐SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of ‐SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe2+ and phosphate was found to bind to either an ‐SH group or to a site in very close proximity. Addition of serotonin protected ‐SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected ‐SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe2+. The ionic environment had a marked effect on the binding: intracellular ions such as K+ were found to enhance the binding, and extracellular ions such as Na+ and Ca2+ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP‐S‐Fe‐S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe2+ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.
Ca2+‐Guanine Nucleotide Interactions in Brain Membranes. I. Modulation of Central 5‐Hydroxytryptamine Receptors in the Rat
Authors: Mallat M. , Hamon M.
Published: October 2006

Abstract: The present study indicates that central 5‐hydroxytryptamine (5‐HT; serotonin) receptors can be modulated in opposite directions by Ca2+ and guanine nucleotides [guanosine triphosphate (GTP), β, γ‐imidoguanosine 5′‐triphosphate (GppNHp)]. Thus CaCl2 (≥0.5 mm) inhibited whereas GTP and GppNHp (10 μm) stimulated the 5‐HT‐sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (Kd−1) and the number (Bmax) of [3H]5‐HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca2+ (≥0.25 mm); GTP (≥0.1 mm) and GppNHp (≥0.3; μm) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [3H]5‐HT binding was counteracted by Ca2+: the addition of this cation (5mm‐CaCl2) to the assay mixture resulted in a 40‐fold increase in the IC50 for GTP; the IC50 for GppNHp increased five‐fold under the same condition. The examination of the respective effects of Ca2+ and of GTP on the specific binding of [3H]5‐HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca2+‐induced increase and of the GTP‐induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid‐treated rats because [3H]5‐HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca2+‐induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[3H]5‐HT binding sites to adenylate cyclase in the rat brain.
Ca2+‐Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ‐Imidoguanosine 5′‐Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum
Authors: Hamon M. , Mallat M. , El Mestikawy S. , Pasquier A.
Published: October 2006

Abstract: With [3H]guanosine triphosphate ([3H]GTP) and [3H]β, γ ‐imidoguanosine 5′‐triphosphate ([3H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (KD= 0.1‐0.5 μm) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [3H]guanine nucleotides. Calcium (0.1–5 mm) partially prevented the binding of [3H]GTP and [3H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [3H]GTP (into [3H]guanosine) and the likely formation of Ca‐guanine nucleotide2‐ complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [3H]GTP to hippocampal membranes in the presence of 0.1 mm‐ATP or 0.1 mm‐GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [3H]GTP and [3H]GppNHp catabolism (into [3H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [3H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [3H]5‐hydroxytryptamine ([3H]5‐HT) in the rat brain.
Purification and Characterization of Posterior Pituitary Calmodulin and Its Activation of Neurosecretosome Ca2++ Mg2+‐ATPase Activities
Authors: Sheaves R. M. , Bowman D. , Hope D. B.
Published: October 2006

Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin‐deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2++ Mg2+‐ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin.
Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells
Authors: Will Horst , Benenson Alexander , Devilliers Ginette , Mandel Paul
Published: October 2006

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a‐methyl‐D‐mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase‐catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem‐ brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a “lighter”)membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a “heavier” membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of 125I‐labeled cell surface proteins. Their specific (Na++ K+)‐ATPase activity was seven‐ to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo‐ plasmic reticulum, as judged from the activity 0: NADPH‐dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.
Striatal Dopamine Release In Vitro: A β‐Adrenoceptor‐Regulated Response Not Mediated Through Cyclic AMP
Authors: Reisine Terry , Chesselet Marie‐Francoise , Glowinski Jacques
Published: October 2006

Abstract: The effects of (‐)isoproterenol(106 M), dibutyryl cyclic AMP (10−2 M), and the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) (10−4 M) on in vitvo [3H]dopamine ([3H]DA) cllux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The β‐adrenoceptor agonist (‐)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (‐)isoproterenol. Additionally, the blockade of [3H]DA synthesis by α‐methyl‐p‐tyrosine (10−1 M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (‐)so proterenol was still able to increase [3H]DA efflux. The results suggest that (‐)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.
Competitive Inhibition of γ‐Aminobutyric Acid Synaptosomal Uptake by 4‐(4′‐Azidobenzoimidylamino)butanoic Acid
Authors: Tunnicliff Godfrey , Ngo That T.
Published: October 2006

Abstract: 4‐(4′‐Azidobenzoimidylamino)butanoic acid (ABBA) is a potent inhibitor of rat brain synaptosomal [3H]γ‐aminobutyric acid uptake. K1 values were calculated to be 8 μM and 16 μM with respect to the high‐affinity and the low‐affinity uptake processes. These values are of the same order as those reported for nipecotic acid and guvacine, which until now have been the most potent uptake inhibitors available. Since ABBA contains a phenyl group, it might be capable of penetrating the blood‐brain barrier, thus becoming a useful GABA mimetic.
Quantitative Analysis of Axonal Transport of Cytoskeletal Proteins in Chicken Oculomotor Nerve
Authors: Filliatreau G. , Di Giamberardino L.
Published: October 2006

Abstract: We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1–15 and 1–10 mn/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.
Purification and Properties of Bovine Brain Dopamine β‐Hydroxylase
Authors: Matsui Hiroaki , Yamamoto Chosaburo , Nagatsu Toshiharu
Published: October 2006

Abstract: Dopamine β‐hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X‐100, ammonium sulfate fractionation, and serial chromatographies with Concanavalin A (Con A)‐Sepharose, Biogel A‐1.5 m, DEAE‐Sephadex, and phenyl‐Sepharose. The overall purification was approximately 4200‐fold and the final specific activity was 147 nmol/min/mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. Furthermore, the enLyme bound to phenyl‐substituted agarose by hydrophobic interaction. An approximate molecular weight was estimated to be 400,000 by gel filtration; the protein eluted earlier than bovine adrenal DBH with a molecular weight estimated to be 290,000. The Km values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. Our data showed a close similarity between the bovine brain and adrenal enzymes.
Cerebellar Hypoplasia in Gunn Rats: Effects of Bilirubin on the Maturation of Glutamate Decarboxylase, Na,K‐ATPase, 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase, Acetylcholine and Aryl Esterase, Succinate and Lactate Dehydrogenase, and Arylsulfatase Activities
Authors: Aoki Eiko , Katoh‐Semba Ritsuko , Kashiwamata Shigeo
Published: October 2006

Abstract: We have investigated the postnatal development of cerebellar glutamate decarboxylase (GAD), acetylcholine esterase (AChE), 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase), arylsulfatase, succinate dehydrogenase (SDH), Na,K‐ATPase, arylesterase, and lactate dehydrogenase (LDH) activities in homozygous (jj) Gunn rats, comparing them with those in heterozygotes. Also examined were GAD activities in the anterior and posterior parts of the vermis cerebelli on day 13. The specific activities of GAD on the basis of wet weight (g) were lower in jj rats after day 10, and remained unchanged from days 10 to 15 in jj rats with severe hypoplasia. On day 13, GAD on wet weight and protein (mg) bases in the anterior part OfJJ rats was lower than that in the posterior parts of both rats. On the contrary, AChE activities based on wet weight after day 10 and on protein after day 5 in jj rats were significantly elevated. While correlations of GAD on a wet weight basis with the cerebellar wet weight were positive after day 8, those of AChE on wet weight and protein bases were inverse after days 10 and 5, respectively. CNPase and SDH activities based on protein in jj rats were higher after day 15 and showed inverse correlations with the cerebellar wet weight after days 15 and 10, respectively. Arylsulfatase activities on wet weight and protein bases in jj rats, which had a peak on day 20, were significantly high after days 10 and 8, respectively. Arylsulfatase activities by wet weight on days 10 to 20 and by protein after day 8 were inversely correlated with cerebellar wet weight in jj rats. These results suggest that the cerebellar inhibitory neurons, the axons of which make synaptic connections to the target cells from days 10 to 15, are selectively affected by bilirubin inji rats with severe hypoplasia. The enhanced arylsulfatase activity in jj rats on days 10 to 20 may be due to the increased number of lysosomes, suggesting that cell damage by bilirubin followed by cell destruction occurs. A high level of AChE activity in jj rats appears to show a relative increase in density of the mossy fibers in the hypoplastic cerebellum.
Limited Digestion of Guinea Pig Myelin Basic Protein and Its Carboxy‐Terminal Fragment (Residues 89–169) with Staphylococcus aureus V8 Protease
Authors: Deibler Gladys E. , Nomura Kohji , Kies Marian W.
Published: October 2006

Abstract: Staphylococcub aureus
V8 protease has been reported to have a strict specificity for cleavage of the Glu‐X bond in ammonium bicarbonate (pH 7.9). With myelin basic protein and one of its major peptic fragments (residues 89–169) as substrates, selective cleavage of Asp(32)‐Thr(33), Asp(37)‐Ser(38), and Glu(118)‐Gly(119) bonds was observed, as well as the unusual cleavage of the Gly(127)‐Gly(128) bond. The Asp‐Glu and Glu‐Asn bonds in the sequence of Gln‐Asp‐Glu‐Asn‐Pro(81–84) were resistant to V8 protease attack. The following peptides were identified as products of limited cleavage of basic protein by V8 protease: (1–32), (1–37), (33–169), (38–169), (33–118), (38–118), (33–127), (38–127), (119–169), and (128–169). Cleavage of the peptic peptide (89–169) yieldedfragments(89–118),(89–127),(119–169), and (128–169). All peptides were identified by amino acid analysis, as well as NH2‐and COOH‐terminal analyses. Time course studies with basic protein showed that V8 protease initially attacked the bonds between Asp(32) and Thr(33) and Asp(37) and Ser(38). With peptide (89–169) the initial cleavage was between Glu(118) and Gly(119). Peptides (89–118) and (89–127) were encephalitogenic in the Lewis rat. The activity of these peptides in the rat confirms the presence of a minor encephalitogenic site in guinea pig basic protein. Peptide (89–127) was encephalitogenic in the guinea pig, as expected, because it contains the intact encephalitogenic site. V8 protease digestion of basic protein yields some interesting new fragments, not previously available for biologic studies.
Concentrations of GABA and Other Amino Acids in CSF from Torsion Dystonia Patients
Authors: Perry Thomas L. , Hansen Shirley , Quinn Niall , Marsden C. David
Published: October 2006

Abstract: Free amino acid concentrations were measured by conventional amino acid analysis, and γ‐aminobutyric acid (GABA) concentrations were determined, by an ion‐exchange fluorometric technique, in CSF specimens from 16 patients with torsion dystonias and in CSF from a large number of control subjects. The mean CSF GABA concentration of the dystonia patients (97 ± 11 nmol/L) did not differ significantly from the means for CSF GABA in two groups of adult control subjects. Mean concentrations of all commonly determined amino compounds were normal in the CSF of torsion dystonia patients, except for ornithine, which was modestly but significantly reduced.
Evidence for a Selective Processing of Proenkephalin B into Different Opioid Peptide Forms in Particular Regions of Rat Brain and Pituitary
Authors: Seizinger Bernd R. , Grimm Cornelia , Höllt Volker , Herz Albert
Published: October 2006

Abstract: The distribution of five major products of proenkephalin B [dynorphin1–17, dynorphin B, dynorphin1–8, α‐neo‐endorphin and β‐neo‐endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of irdynorphin1–17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1–17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1–32 on HPLC. In contrast with all other tissues, anterior pituitary ir‐dynorphin B and ir‐dynorphin1–17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1–8 were several times higher than those of dynorphin1–17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1–17 was detected in the anterior pituitary. Concentrations of β‐neo‐endorphin were similar to those of α‐neo‐endorphin in the posterior pituitary. In contrast, in all brain tissues α‐neo‐endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of β‐neo‐endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids ‐Lys‐Arg‐ as the “typical” cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.
Binding Characteristics and Interactions of Depressant Drugs with [35S]t‐Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site
Authors: Ramanjaneyulu R. , Ticku M. K.
Published: October 2006

Abstract: [35S]r‐Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin‐like activity, binds to rat brain membranes to a single site with an apparent KD of 25.1 ± 5.6 nM and a Bmax of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [3H]a‐dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC50 values of (+)etomidate (9 μM) and pentobarbital (90 μM) are similar to the EC50 values at which they enhance both [3H]‐γ‐aminobutyric acid and [3H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC50= 10 μM). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine‐GABA receptor‐ionophore complex.
Identification of Neuron‐Specific Enolase and Nonneuronal Enolase in Human and Rat Brain on Two‐Dimensional Polyacrylamide Gels
Authors: Heydorn William E. , Creed G. Joseph , Marangos Paul J. , Jacobowitz David M.
Published: October 2006

The location of the enzymes neuron‐specific enolase and nonneuronal enolase on two‐dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: (1) comigration on polyacrylamide gels with the appropriate purified protein and (2) staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron‐specific enolase from rat and human brain is highly pure, as only one spot is obtained on two‐dimensional gels. Further, the antiserum to neuron‐specific enolase is highly specific, as it reacts only with neuron‐specific enolase on nitrocellulose blots derived from two‐dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two‐dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two‐dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.
Binding of Lanthanum Ions and Ruthenium Red to Synaptosomes and Its Effects on Neurotransmitter Release
Authors: Tapia Ricardo , Arias Clorinda , Morales Ezequiel
Published: October 2006

Abstract: A technique for studying the binding of La3+ to synaptosomes in a double‐beam spectrophotometer, using murexide as indicator, is described. The binding of La3+ was very rapid and Scatchard plots revealed two components, with KD values of 0.6 and 27 μM in a Na+‐free medium (sucrose medium) and 2.3 and 63 μM in an ionic medium containing 135 mM Na+. The binding of the cationic dye ruthenium red (RuR) showed only one site, with a KD of 3.7 μM. La3+ binding was partially inhibited by RuR and vice versa, and La3+ was also capable of partially displacing RuR previously bound to the synaptosomes, particularly in the sucrose medium. The release of labeled γ‐aminobutyric acid (GABA) stimulated by K+ depolarization was inhibited by La3+ concentrations at or above 1 μM. in the ionic medium, whereas in the sucrose medium 2.5 μM or higher La3+ concentrations notably stimulated the spontaneous release of both GABA and glutamic acid. It is concluded that La3+ and RuR share at least one type of binding site, which is probably the high‐affinity La3+ site. Since both La3+ and RuR at low concentrations have been shown to block the depolarization‐induced Ca2+ entry in synaptosomes, this site might be related to the voltage‐dependent Ca2+ entry involved in neurotransmitter release.
Manganese Stimulates the Incorporation of [3H]Inositol into a Pool of Phosphatidylinositol in Brain That Is Not Coupled to Agonist‐Induced Hydrolysis
Authors: Schoepp Darryle D.
Published: October 2006

Abstract: Mn2+ greatly increases the incorporation of myo‐[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI‐myoinositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+‐stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo‐[3H]inositol for 60 min in an N‐2‐hydroxyethyl piperazine‐N′‐2‐ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo‐inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist‐stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight‐ and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate ([3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+ ‐treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo‐[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist‐induced hydrolysis.
γ‐Aminobutyric Acid Affects the Developmental Expression of Neuron‐Associated Proteins in Cerebellar Granule Cell Cultures
Authors: Meier Eddi , Jørgensen Ole Steen
Published: October 2006

Abstract: The effect of γ‐aminobutyric acid (GABA) on the expression of the neuron‐associated D2 and neuronspecific enolase (NSE) was studied during development in culture of cerebellar granule cells. It was found that the presence of GABA during culture development increased the overall protein content. D2 content was also increased but not above the general increase in protein whereas NSE increased above the general level of protein.The presence of GABA in the growth medium also appeared to accelerate the changes in molecular forms of D2 and NSE seen during neuronal development. This suggests that GABA promotes or accelerates the general maturation of neurons, as these two neuron‐associated proteins otherwise differ from each other with respect to their subcellular localization and their physiological and biochemical properties.
Hyperglycemia Preserves Brain Mitochondrial Respiration During Anoxia
Authors: Wagner Kenneth R. , Myers Ronald E.
Published: October 2006

Abstract We examined brain mitochondrial function in normo‐ (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD‐Iinked substrates gluta‐mate or pyruvate (both plus malate) was inhibited 30–50%. The uncoupler carbonylcyanide P‐trifluoromethoxyphe‐nylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rote‐none) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo‐ and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high‐energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.
Distribution of the Glucose Transporter in the Mammalian Brain
Authors: Dick Arthur P. K. , Harik Sami I.
Published: October 2006

Abstract: We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D‐glucose‐displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000–64,000 daltons.
Altered Mitochondrial Respiration in Selectively Vulnerable Brain Subregions Following Transient Forebrain Ischemia in the Rat
Authors: Sims Neil R. , Pulsinelli William A.
Published: October 2006

Abstract: Mitochondrial respiratory function, assessed from the rate of oxygen uptake by homogenates of rat brain subregions, was examined after 30 min of forebrain ischemia and at recirculation periods of up to 48 h. Ischemia‐sensitive regions which develop extensive neuronal loss during the recirculation period (dorsal‐lateral striatum, CA1 hippocampus) were compared with ischemia‐resistant areas (paramedian neocortex, CA3 plus CA4 hippocampus). All areas showed reductions (to 53–69% of control) during ischemia for oxygen uptake rates determined in the presence of ADP or an uncoupling agent, which then recovered within 1 h of cerebral recirculation. In the ischemia‐resistant regions, oxygen uptake rates remained similar to control values for at least 48 h of recirculation. After 3 h of recirculation, a significant decrease in respiratory activity (measured in the presence of ADP or uncoupling agent) was observed in the dorsal‐lateral striatum which progressed to reductions of greater than 65% of the initial activity by 24 h. In the CA 1 hippocampus, oxygen uptake rates were unchanged for 24h, but were significantly reduced (by 30% in the presence of uncoupling agent) at 48 h. These alterations parallel the development of histological evidence of ischemic cell change determined previously and apparently precede the appearance of differential changes between sensitive and resistant regions in the content of high‐energy phosphate compounds. These results suggest that alterations of mitochondria activity are a relatively early change in the development of ischemic cell death and provide a sensitive biochemical marker for this process.
Relationship Between Dopamine Receptor Occupation by Spiperone and Acetylcholine Levels in the Rat Striatum After Long‐Term Haloperidol Treatment Depends on Dopamine Innervation
Authors: Korf Jakob , Sebens Jantiena B.
Published: October 2006

Abstract: The effect of chronic neuroleptic treatment on the relationship between the blockade of dopamine (DA) receptors by the neuroleptic drug spiperone and the decline in acetylcholine (ACh) levels was determined in the rat striatum in vivo. In rats, a unilateral lesion of the nigrostriatal pathway was produced with 6‐hydroxydopamine. The rats were treated for 6 weeks with haloperidol (twice a day at 1 mg kg−1). Partial and complete receptor occupation was determined with radioactive spiperone (a D2 antagonist), given in various doses of different specific activity 2 h before death. ACh, choline, and radioactivity contents were measured in the same striatum. Following long‐term haloperidol treatment, an increase in the maximal number of binding sites for spiperone was found. Virtually identical negative (linear) correlations between striatal ACh content and the number of receptors occupied by spiperone were found in saline‐ or subchronic haloperidol‐treated rats when DA innervation was intact. The slope of the line describing the decrease in ACh content per occupied receptor, however, was much lower in haloperidol‐treated rats than in saline‐treated animals. After lesioning of the dopaminergic pathway, there was no longer a correlation between the receptor occupation and ACh levels in the striatum. These results show that receptor occupation by a neuroleptic correlates highly with function only when dopaminergic innervation is intact. Also, it appears that there is no fixed number of striatal ACh molecules per DA receptor, and, finally, that in vivo receptor detection methods distinguish differences in receptor density (as do in vitro techniques).
Adenosine Inhibition of Histamine‐Stimulated Inositol Phospholipid Hydrolysis in Mouse Cerebral Cortex
Authors: Kendall D. A. , Hill S. J.
Published: October 2006

The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine‐stimulated accumulation of inositol phosphates in a concentration‐dependent manner. The responses to carbachol, noradrenaline, 5‐hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine‐stimulated hydrolysis of inositol phospholipids in guinea pig brain.
Lithium Stimulation of Membrane‐Bound Phospholipase C from PC 12 Cells Exposed to Nerve Growth Factor
Authors: Volonte Cinzia , Racker Efraim
Published: October 2006

Abstract: LiCl stimulated the formation of inositol mono‐phosphate in PC 12 cells that had been exposed to nerve growth factor (NGF) for 4–5 days. Half‐maximal accumulation was observed at ∼8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inos′itol trisphosphate was half‐maximal at ∼ 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol4,5‐bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half‐maximal at ∼0.7 mM and the second half‐maximal at −15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from ∼200 to ∼70 μM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.