bluuebunny/crossref_metadata_2025 huggingface dataset:
Postmortem Accumulation of Tubulin in Postsynaptic Density Preparations
Authors: Carlin Richard K. , Grab Dennis J. , Siekevitz Philip
Published: October 2006

Abstract: Postsynaptic density (PSD) preparations isolated from canine cerebral cortex that had been left at 0–37°C for various times were found to become enriched in two bands in a time‐ but not temperature‐dependent manner. The two bands were identified as tubulin subunits by gel mobility and immunology. Of all the isolated synaptic structures the increase in tubulin occurred primarily in the PSD fraction. The increase of tubulin also occurred in PSD preparations isolated from canine cerebellum and rat forebrain. Results obtained when PSD fractions were isolated from canine brain obtained as rapidly as possible after the death of the animal indicate that the maximum amount of tubulin in the PSD preparations is 2.5% of total Coomassie blue‐stained protein as determined by scanning of gel electrophoretograms. These results imply that tubulin is probably not a major structural protein of the PSD as it exists in situ.
An Analysis of GABA Receptor Changes in the Discrete Regions of Mouse Brain After Acute and Chronic Treatments with Morphine
Authors: Sivam Subbiah P. , Nabeshima Toshitaka , Ho Ing K.
Published: October 2006

Abstract: The effects of morphine on the affinity and distribution of GABA receptors in the mouse regions (striatum, medulla, diencephalon, cortex, and cerebellum) were investigated in relation to: (a) acute administration, (b) chronic administration (tolerance), (c) precipitated withdrawal by naloxone, an opiate antagonist, and (d) abrupt withdrawal for 8 and 24 h. The alterations in the affinity as reflected by the dissociation constant (KD) and the number of receptors (Bmax) in the synaptic membranes obtained from controls and various treatments were determined by radioligand binding assay using [3H]muscimol as a ligand. Significant changes were observed in striatum, medulla, and diencephalon, whereas other regions including whole brain exhibited marginal changes. In general the number of GABA receptors increased after tolerance development, which upon abrupt withdrawal returned to control levels except in the case of naloxone‐induced precipitated withdrawal. The affinity changes in different regions were diverse in nature and were not evident in the whole brain membranes. These results indicate that: (a) the regional alterations in the affinity and distribution of GABA receptors may play a role in the induction, maintenance, and regression of morphine tolerance; (b) abrupt withdrawal and antagonist precipitated withdrawal affect the GABA system differently, (c) chronic morphine treatment appears to influence the GABA receptors in the cerebellum, a region generally known for its lack of opiate receptors.
Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)
Authors: Mikoshiba Katsuhiko , Akagawa Kimio , Takamatsu Ken , Tsukada Yasuzo
Published: October 2006

Abstract: The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K‐ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K‐ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.
Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization
Authors: Sugiyama Hiroyuki , Yamashita Yukiko , Murakami Fujio
Published: October 2006

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α‐bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.
Specific Muscarinic‐Cholinergic Desensitization in the Neuroblastoma‐Glioma Hybrid NG108‐15
Authors: Green David A. , Clark Richard B.
Published: October 2006

Abstract: The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E1 (PGE1)‐stimulated adenylate cyclase in homogenates from the neuroblastoma‐glioma hybrid NG108‐15. Pretreatment of the hybrid with 100 μM carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (tM1/2< 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 μM atropine. Pretreatment with carbachol (1–24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE1‐stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol‐desensitized or of control NGlOS‐15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)‐deficient cyclymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor‐associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.
The Differential Effects of GABA‐Transaminase Inactivation in the Chick Retina and Brain
Authors: Rando Robert R. , Coburn Jenifer , Parkinson David
Published: October 2006

Abstract: The inactivation of γ‐aminobutyrate (GABA)‐transaminase by the highly specific and potent neurotoxin gabaculine leads to different neurochemical consequences in the chick brain as opposed to the chick retina. In the brain, GABA levels continually climb, reaching approximately eightfold increases over control values after 24 h. The elevation in GABA levels leads to a time‐dependent and coincident fall in glutamate decarboxylase and cysteine‐ sulfinatc decarboxylase activities, to approximately 50% of control values. On the other hand, in the retina GABA levels only increase to a plateau level two‐ to threcfold that of control after inactivation of GABA‐transaminase. Further‐ more, although the glutamate decarboxylase activity decreases to about 50% of control values, cysteinesulfinate decarboxylase activity is not affected. These studies show that the processing of GABA in the retina differs from that in the brain, and that cysteinesulfinate and glutamate decarboxylase activity probably reside in different enzyme molecules in the retina, although they may reside in the same enzyme in the brain.
Multiple Molecular Forms of Dopamine β‐Hydroxylase in the C1300 Mouse Neuroblastoma Tumor and in the Serum of Tumor‐Bearing Mice
Authors: Fraeyman Norbert H. , Van de Velde Eric J. , De Smet Frits H. , De Schaepdryver Andre F.
Published: October 2006

Abstract: The distribution of the enzymatic activity of dopamine β‐hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C1300 mouse neuroblastoma tumor, for the serum of tumor‐bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high‐molecular‐weight form, denoted as DBHAA, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower‐sedimenting species (7.1 S), denoted as DBHBB. In tumor supernatants and in the serum of tumor‐bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHRB), while only 35% was recovered as the high‐molecular‐weight form (DBHAA). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBHC. The ratio DBHR/DBHA is significantly higher in tumor tissue and in serum of tumor‐bearing mice than in controls. The three enzymically active forms of DBH in the C1300 tumor are considered to represent the tetrameric (DBHAC), dimeric (DBHB), and monomeric (DBHC) forms of the enzyme.
Inhibition by l‐Phenylalanine of Tyrosine Transport by Synaptosomal Plasma Membrane Vesicles: Implications in the Pathogenesis of Phenylketonuria
Authors: Aragón M. C. , Giménez C. , Valdivieso F.
Published: October 2006

Abstract: The effect of l‐phenylalanine on the transport of tyrosine was studied using membrane vesicles from rat brain synaptosomes. Phenylalanine, which is accumulated in phenylketonuria, competitively inhibits tyrosine uptake at concentrations similar to those found in phenylketonuric patients, with a K1 of the same order of the Km for tyrosine. This inhibition could be responsible for the depletion of catecholamines observed in phenylketonuria.
Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ‐Aminobutyric Acid in Rat Hippocampal Slices
Authors: Corradetti R. , Moneti G. , Moroni F. , Pepeu G. , Wieraszko A.
Published: October 2006

Abstract: The release of endogenous aspartic, glutamic, and γ‐aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometnc method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+‐dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2 from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation‐evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.
Affinity‐Purified Anti‐B‐50 Protein Antibody: Interference with the Function of the Phosphoprotein B‐50 in Synaptic Plasma Membranes
Authors: Oestreicher Anna Beate , Van Dongen Carla J. , Zwiers Henk , Gispen Willem Hendrik
Published: October 2006

Abstract: Affinity‐purified anti‐B‐50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B‐50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B‐50 protein and its adrenocorticotropin‐sensitive protein kinase, obtained from rat brain. Anti‐B‐50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B‐50 protein isolated by an improved procedure. The purified antibodies reacted only with the B‐50 and B‐60 protein, a proteolysis derivative (of B‐50), as assessed by the sodium dodecyl sulfate‐gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B‐50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5‐diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4‐phosphate was not altered. Inhibition by ACTH 1–24 of the endogenous phosphorylation of B‐50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4‐phosphate to phosphatidylinositol 4,5‐diphosphate. These data support our hypothesis on the functional interaction of B‐50 protein and phosphatidylinositol 4‐phosphate kinase in rat brain membranes. The evidence shows that purified anti‐B‐50 protein antibodies can be used to probe specifically the function of B‐50 protein in membranes.
Quantification of the D2‐Glycoprotein in Amniotic Fluid and Serum from Pregnancies with Fetal Neural Tube Defects
Authors: Ibsen Susanne , Berezin Vladimir , Nørgaard‐Pedersen Bent , Bock Elisabeth
Published: October 2006

Abstract: D2 is a glycoprotein existing in both membrane‐bound and soluble forms. Employing a specific rabbit antibody against purified human brain D2, we developed an enzyme‐linked immunosorbent assay (ELISA) for the quantification of D2 and applied it to amniotic fluids from 87 normal and 36 pathological pregnancies. With a cut‐off point of 150 ng D2/ml, no false positive D2 values were obtained in any of the amniotic fluids from normal fetuses, although the alpha‐fetoprotein concentrations were slightly increased in 13 cases. No false negative D2 values were found in any of the 18 investigated amniotic fluids from fetuses with anencephaly. Of 8 amniotic fluids from fetuses with spina bifida, 2 false negative D2 values were found. No false negative alphafetoprotein values were found in any of the cases with neural tube defects in this study. In 10 amniotic fluids from fetuses with other malformations, 5 samples showed raised D2 concentrations. The D2 level in sera from 10 women carrying normal fetuses and 16 women carrying malformed fetuses was also determined, but no statistically significant difference in D2 level was found in the pathological sera when compared with normal sera. It was concluded that the determination of D2 concentrations in amniotic fluid by means of the D2‐ELISA may be used as an additional test in the screening of fetal malformations in early pregnancy.
Tetanus and Botulinum Toxins Inhibit, and Black Widow Spider Venom Stimulates the Release of Methionine‐Enkephalin‐Like Material In Vitro
Authors: Janicki Piotr K. , Habermann Ernst
Published: October 2006

Abstract: The actions of tetanus toxin, botulinum A toxin, and black widow spider venom on the release of methionine‐enkephalin‐like immunoreactivity have been studied; a particulate fraction prepared from rat striata was used. Depending on the duration of preincubation, tetanus toxin diminished the release evoked by veratri‐dine (50 μM final concentration), and abolished it at final concentrations between 0.1 and 1 μg/ml. Botulinum A toxin was about 10 to 20 times less potent. Heating or pretreatment with antitoxin inactivated the clostridial toxins. The particulate fraction pretreated with V. cholerae neuraminidase retained its toxin sensitivity. Tetanus toxin also depressed the release due to sea anemone toxin II and high K+. Spider venom stimulated the release in a concentration‐dependent manner and required the presence of Ca2+; its effects were depressed by tetanus toxin. These results support the view that both clostridial toxins and spider venom act as broad‐range presynaptic neurotoxins on peptidergic transmitter systems.
Evidence that Pituitary Cation‐Sensitive Neutral Endopeptidase Is a Multicatalytic Protease Complex
Authors: Wilk Sherwin , Orlowski Marian
Published: October 2006

Abstract: Pituitary cation‐sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin‐like activity), basic amino acids (trypsin‐like activity), and acidic amino acids (peptidyl‐glutamyl‐peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high‐molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2‐mercaptoethanol dissociates this complex into five low‐molecular weight components. Incubation of the complex at 37°C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl‐glycyl‐glycyl‐leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin‐like activity, exclusively inhibits that activity (Ki= 2.5 × 10−4M), while markedly activating the trypsin‐like activity. The trypsin‐like activity is inhibited by leupeptin (Ki= 1.2 μM) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl‐peptide hydrolyzing activity is activated almost 10‐fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation‐sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high‐molecular weight protein.
Differences Between d‐Fenfluramine and d‐Norfenfluramine in Serotonin Presynaptic Mechanisms
Authors: Borroni E. , Ceci A. , Garattini S. , Mennini T.
Published: October 2006

Abstract: The abilities of d‐fenfluramine (d‐F) and that of d‐norfenfluramine (d‐NF) to inhibit [3H]serotonin ([3H]5‐HT) accumulation in normal and reserpinized synaptosomes were compared to establish to what extent the serotonin‐releasing activity of the two drugs might contribute to reduced accumulation of [3H]5‐HT. The results indicate that the inhibitory action of (d‐NF) on [3H]5‐HT accumulation is due principally to its ability to release [3H]5‐HT. In contrast, the interference of release in accumulation studies does not seem to play an important role for d‐F, suggesting that release from the granular pool and true uptake inhibition are two different mechanisms by which d‐F affects serotonin neurons in vitro.
Identification of Ca2+‐Activated Neutral Protease in the Peripheral Nerve and Its Effects on Neurofilament Degeneration
Authors: Kamakura Keiko , Ishiura Shoichi , Sugita Hideo , Toyokura Yasuo
Published: October 2006

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca2+ and was inhibited by the addition of E‐64‐c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca2+‐activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 ± 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.
Striatal GABAergic Neuronal Activity Is Not Reduced in Parkinson's Disease
Authors: Perry Thomas L. , Javoy‐Agid France , Agid Yves , Fibiger Hans C.
Published: October 2006

The content of y‐aminobutyric acid (GABA) and the activities of glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) were measured in whole putamen obtained at autopsy from 13 patients dying with idiopathic Parkinson's disease and 13 appropriate control subjects. Mean GABA content was significantly elevated (by 28%) in the putamen of the Parkinson's disease patients. TH activity was markedly reduced, while there was no significant reduction of GAD activity in the putamen of these patients. GABA content was also measured in both sides of the striatum in rats which had received unilateral injections of 6‐hydroxydopamine (6‐OHDA) in the vicinity of the axons of the nigrostriatal projection. Mean GABA content was found significantly elevated (by 33%) in the ipsilateral striatum. Loss of dopaminergic nigrostriatal neurons, in both human Parkinson's disease and in the rat 6‐OHDA model, is accompanied by increased striatal GABA content. The assumption that GABAergic neurotransmission is reduced in the striatum in Parkinson's disease may not be correct.
Isolation of a Ca2+‐Dependent Actin‐Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones
Authors: Petrucci T. C. , Thomas C. , Bray D.
Published: October 2006

Extracts of ox spinal cord and chicken brain were fractionated by ion‐exchange chromatography and assayed for their ability to reduce the viscosity of muscle F‐actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase‐actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin‐fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F‐actin filaments at Ca2+ concentrations greater than 10‐7M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N‐(1‐pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G‐actin. On the basis of its actin‐fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase‐actin affinity column and by immune blotting and by immuno‐fluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin‐based movements of these cells.
In Vitro Protein Synthesis in the Goldfish Retinotectal Pathway During Regeneration: Evidence for Specific Axonal Proteins of Retinal Origin in the Optic Nerve
Authors: Quitschke Wolfgang , Schechter Nisson
Published: October 2006

Abstract: Four proteins with molecular weights of 58,000 can be separated as a linear array by two‐dimensional gel electrophoresis. They are highly concentrated in the goldfish optic nerve and are designated as ON1, ON2, ON3, and ON4. Proteins ON1 and ON2 are undetectable in the optic nerve after disconnection and their concentration is gradually restored during regeneration. In vitro incubations of retinas, optic nerves, or tecta in the presence of [35S]methionine indicate that proteins ON1 and ON2 are of retinal origin. The labeling rate of these proteins in the retina increases fourfold after optic nerve crush whereas the overall labeling rate in the retina remains largely constant. Their synthesis cannot be detected in tissues de void of retinal ganglion cells. This is consistent with the view that ON1 and ON2 are synthesized by retinal ganglion cells and are consequently of neuronal origin in the optic nerve. In contrast, similar experiments indicate that ON3 and ON4 are of nonneuronal origin. They are synthesized in the optic nerve in the absence of retinal ganglion cells.
Identification of 5‐Hydroxy Eicosatetraenoic Acid in Cerebrospinal Fluid After Subarachnoid Hemorrhage
Authors: Suzuki Noboru , Nakamura Tetsuya , Imabayashi Sukehiro , Ishikawa Yoko , Sasaki Tomio , Asano Takao
Published: October 2006

Abstract: To detect and identify lipid peroxides in the CFS following subarachnoid hemorrhage (SAH), CSF samples were obtained sequentially from 10 patients who developed typical vasospasm and were analyzed by HPLC and gas chromatography‐mass spectrometry. One of the peaks appearing on the 7th day after SAH was identified as 5‐hydroxy eicosatetraenoic acid. On HPLC, an identical peak was detected in samples from other SAH patients. The results gave unequivocal evidence that peroxides of arachidonic acid are present in the CSF following SAH, and a correlation between them and the occurrence of vasospasm seemed likely. The hypothesis that lipid peroxides are involved in the genesis of vasospasm deserves further investigation.
Cimoxatone Is a Reversible Tight‐Binding Inhibitor of the A Form of Rat Brain Monoamine Oxidase
Authors: Fowler Christopher J. , Benedetti Margherita Strolin
Published: October 2006

Abstract: Cimoxatone is a fully reversible inhibitor selective for the A form of monoamine oxidase. The inhibition is so potent against this enzyme form that it acts as a tight‐binding inhibitor. Use of this inhibitor indicates that in rat brain homogenates the concentration of monoamine oxidase A is approximately 8–11 pmol‐mg protein−1. Values similar to this were obtained by clor‐gyline titration and both methods gave values similar to those found with a [3H]harmaline binding assay.
Frequency‐Dependent Release of Acetylcholine and Dopamine From Rabbit Striatum: Its Modulation by Dopaminergic Receptors
Authors: Cubeddu Luigi X. , Hoffmann Irene S.
Published: October 2006

Abstract: The release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) was monitored from single slices of the rabbit striatum. In all cases, the evoked overflow of ACh showed a higher peak and was of shorter duration than that of 3H products. For ACh, the release per pulse showed a marked decline with increasing frequency of stimulation, whereas flat frequency‐release curves were obtained for DA. At 0.1 and 1 Hz the evoked overflows of ACh were 15 and 7 times greater, respectively, than those of DA. Haloperidol (0.03 μM) and sulpiride (1 μM) produced large increases in the evoked overflow of DA and ACh at 3 and 10 Hz; little effect was observed at lower frequencies. These results indicate that the frequency‐release curves for DA and ACh are different and that at high frequencies the slope of the curves is modified by activation of pre‐ and postsynaptic DA receptors. Apomorphine inhibited in a concentration‐dependent fashion the evoked overflow of DA and ACh; greater inhibition was obtained at lower frequencies of stimulation. At 0.3 Hz the‐ DA agonist was two times more potent in inhibiting DA than ACh overflow (IC50: 12.0 ± 2.2 versus 22.0 ± 2.8 nM; p < 0.01). The greater sensitivity of pre‐than postsynaptic sites to apomorphine was also seen at higher frequencies (3 Hz). Benztropine (1/μ) reduced the evoked overflow of ACh at 10 Hz, and enhanced that of 3H products at all rates of stimulation (0.3–10 Hz). These results suggest that the release of DA and ACh is regulated by dopaminergic receptors. They also indicate that the effects of DA agonists and antagonists and of uptake inhibitors on DA and ACh release are highly dependent on the frequency of stimulation used.
Biosynthesis of Long‐Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve
Authors: Natarajan V. , Schmid H. H. O. , Sastry P. S.
Published: October 2006

Abstract: Cell‐free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane‐bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro‐lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long‐chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10‐day‐old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.
Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two‐Dimensional Gel Analysis
Authors: Strocchi Paola , Gilbert Jeffrey M. , Benowitz Larry I. , Dahl D. , Lewis Ellen R.
Published: October 2006

Abstract: High resolution 2DGE (two‐dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with l‐[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of l‐[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the α‐and β‐tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high‐molecular‐weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free poly some population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.
Neurochemical Changes in Murine Trisomy 16: Delay in Cholinergic and Catecholaminergic Systems
Authors: Ozand P. T. , Hawkins R. L. , Collins R. M. , Reed W. D. , Baab P. J. , Oster‐Granite M. L.
Published: October 2006

Abstract: Two strains of Mus musculus musculus, C57BL/6J and CD‐1, and Mus musculus poschiavinus, the tobacco mouse, were used to study the effects of increased gene dosage of mouse chromosome 16 (MMU 16). A developmental delay has been found in the brains of murine trisomy 16 (Ts 16) fetuses. Both the brain weight (in all three strains) and DNA content (in CD‐1) were reduced, while protein content was unchanged in Ts16 compared to normal littermates. The daily increments of weight and protein (except in M. m. poschiavinus) were significantly greater in Ts16. The activities of choline acetyltransferase and acetylcholinesterase and nuscarinic receptor binding were reduced. Their daily increments were also reduced to less than 56% that of littermates in Ts16 brains. The rate limiting enzymes of Catecholaminergic neurons, tyrosine hydroxylase and do‐pamine β‐hydroxylase, and the concentration of catecholamines in the brains of Ts16 animals were lower. The activities of three other Catecholaminergic enzymes, DOPA decarboxylase, catechol O‐methyltransferase, and monoamine oxidase, were generally elevated in Ts16 brain, as were their daily increments. These observations indicate a significant developmental alteration in the maturation of the trisomic brain and suggest future avenues for defining the effect of increased gene dosage of MMU 16 in the CNS.
Regional Thymidine Transport and Incorporation in Experimental Brain and Subcutaneous Tumors
Authors: Molnar Peter , Groothuis Dennis , Blasberg Ronald , Zaharko Daniel , Owens Ernest , Fenstermacher Joseph
Published: October 2006

Abstract: The regional distribution and local incorporation of [14C]thymidine into a nonextractable tissue fraction, probably DNA, was measured in normal and neo‐plastic tissues. We studied brain tumors induced by avian sarcoma virus and ethylnitrosourea, and transplanted RG‐2 intracerebral and subcutaneous gliomas. An incorporation quotient, Q, was calculated for different tumor regions and brain from the methanol nonextractable radioactivity in the tissue and the plasma concentration‐time integral of thymidine. The incorporation quotient represents the rate of clearance of thymidine from blood and its incorporation into macromolecules (probably I NA). The values of Q were compared with a labeling index measured in the same tissue regions with conventional autoradiography. The following observations were made: (1) the mean plasma half‐life of thymidine was 6.5 min; (2) the regional incorporation quotient in tumors varied from values comparable to normal brain to more than 100 times higher; (3) RG‐2 tumors had significantly higher Qs than the other tumor models; (4) Q in subcutaneous tumors varied most widely (>500‐fold range); (5) the labeling index reflected the values of Q in some tumor regions but not in others; differences between the two were most frequently related to tumor cell density and the intensity of individual tumor cell labeling. A comparison of these data with previous studies of capillary permeability and blood flow in these tumor models indicates that the incorporation of [14C]thymidine into a nonextractable tissue fraction can be limited by transcapillary transport in brain tumors and by blood flow in systemic tumors, and that thymidine disposition in these tumors is not always indicative of the rate of DNA synthesis.
Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine
Authors: Horwitz Joel , Perlman Robert L.
Published: October 2006

Abstract: We have investigated the effect of veratridine on DOPA (3,4‐dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 μM) causes a 10‐fold increase in the rate of DOPA accumulation. Veratridine‐stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8‐bromo cyclic GMP does not increase DOPA accumulation, and 8‐bromo cyclic AMP causes only a 2‐fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell‐free extracts of veratridine‐treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.
The Activities of Some Energy‐Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions
Authors: Leong S. F. , Lai J. C. K. , Lim L. , Clark J. B.
Published: October 2006

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD‐linked isocitrate dehydrogenase (EC 1.1.1.41), NADP‐linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD‐linked malate dehydrogenase (EC 1.1.1.37), D‐3‐hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.
Posttranslational Protein Modification by Amino Acid Addition in Intact and Regenerating Axons of the Rat Sciatic Nerve
Authors: Zanakis M. F. , Chakraborty G. , Sturman J. A. , Ingoglia N. A.
Published: October 2006

Abstract: Experiments were performed to determine whether ppsttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 ·g, and analyzed for the ability to incorporate 3H‐amino acids into proteins. No incorporation of amino acids into proteins was found in the high‐speed supernatant, but when the supernatant was passed through a Sephacryl S‐200 chromatography column (removing molecules less than 20 kD), [3H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15–150 times that in intact nerves. The results indicate that the components of this reaction are transported axonally in rat sciatic nerves and that the reaction is increased dramatically in growing axons during nerve regeneration.
Increased Cytosolic Androgen Receptor Binding in Rat Striated Muscle Following Denervation and Disuse
Authors: Bernard Phyllis A. , Ranee Naomi E. , Fishman Paul S. , Max Stephen R.
Published: October 2006

Abstract: We investigated the effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle. Denervation of the extensor digitorum longus and tibialis anterior muscles caused a 40–50% increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120% of the control level 72 h after denervation. A 40% increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone‐sensitive levator ani muscle. The effect of denervation on androgen receptors was not blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.
Use of [3H]Triphenylmethylphosphomum Cation for Estimating Membrane Potential in Neuroblastoma Cells
Authors: Milligan Graeme , Strange Philip G.
Published: October 2006

The lipophilic permeant cation [3H]triphenylmethylphosphonium (TPMP) was used to estimate membrane potential in neuroblastoma N1E 115 cells under carefully controlled conditions. The cation distributes into the cells only in the presence of a lipophilic anion, and tetraphenylboron and picrate have been used for this purpose. The potassium salt of tetraphenylboron is poorly soluble, so that studies in high [K+] media are difficult with this anion whereas picrate, at the concentrations required, hyperpolarises the cells. The effect of muscarinic receptor activation was investigated by treating cells with carbachol but no effect was seen either on [3H]TPMP distribution or electrophysiological parameters. The use of [3H]TPMP for qualitative and quantitative evaluation of membrane potential in these cells is discussed.
Lesch‐Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain
Authors: Miñana M. D. , Portolés M. , Jordá A. , Grisolía S.
Published: October 2006

Rats ingesting high doses of caffeine reproduce the self‐destructive behaviour observed in the Lesch Nyhan syndrome. This syndrome includes a deficit in hypoxanthine‐guanine phosphoribosyltransferase. We have observed, however, that the activity of hypoxanthine‐guanine phosphoribosyltransferase increases in direct proportion to the concentration of caffeine found in rat brain. It appears, therefore, that the caffeine model is not a true model for the Lesch‐Nyhan syndrome, or alternatively, that the deficit in hypoxanthine‐guanine phosphoribosyltransferase is coincidental and not a main key to the multifarious aspects of the syndrome, particularly the self‐mutilation. The possibility that levels of dopamine are increased in the caffeine model are discussed as a basis for the destructive behaviour. We have found also that ingestion of large amounts of caffeine increases the activities in rat brain of adenosine deaminase, purine nucleoside phosphorylase, aspartate carbamoyltransferase, dihydroorotase, and dihydroorotate oxidase.
Studies of Rat Brain Metabolism Using Proton Nuclear Magnetic Resonance: Spectral Assignments and Monitoring of Prolidase, Acetylcholinesterase, and Glutaminase
Authors: Middlehurst Carl R. , King Glenn F. , Beilharz Georg R. , Hunt Glenn E. , Johnson Gordon F. S. , Kuchel Philip W.
Published: October 2006

The first application of inversion‐recovery spin‐echo proton nuclear magnetic resonance spectroscopy to the monitoring of reactions in rat brain preparations is presented. The initial report of the assignment of proton spin‐echo nuclear magnetic resonance spectra from rabbit brain homogenates (C. R. Middlehurst et al., J. Neurochem. 42, 878–879, 1984) was used to assist in the assignment of spectra acquired from rat brain homogenates that were obtained from animals killed by cervical fracture or focussed microwave irradiation. Microwave‐irradiated brains were divided into four major anatomical regions. Differences in metabolite levels were detected when spectra from fresh tissue and from various regions were compared. The in situ steady‐state kinetics of prolidase in whole brain homogenate was determined.

The procedure relies on the spectral differences between enzyme substrates and reaction products. The concentration dependence of the rate of hydrolysis of glycyl‐l‐proline was describable by the Michaelis‐Menten expression with a Michaelis constant of 1.90 mmol L‐1 and a maximal velocity of 9.30 μmol min‐1 mg‐1 protein. The reactions catalysed by glutaminase and acetylcholinesterase in the brain were also monitored.
Ionophore A23187, Verapamil, Protonophores, and Veratridine Influence the Release of γ‐Aminobutyric Acid from Synaptosomes by Modulation of the Plasma Membrane Potential Rather than the Cytosolic Calcium
Authors: Sihra Talvinder S. , Scott Ian G. , Nicholls David G.
Published: October 2006

The release of GABA induced by veratndine shows no correlation with the synaptosomal Ca content and is therefore not mediated by the release of mitochondrial Ca. Instead, with both Ca‐repleted and‐depleted synaptosomes, the extent of GABA efflux is correlated with the decrease in plasma membrane potential. The slow release of GABA induced by protonophores and the Ca‐dependent release induced by ionophore A23187 are also consequences of the depolarization of the plasma membrane, rather than of elevated cytosolic Ca. Finally, the ability of verapamil to inhibit the release of GABA induced by low veratridine concentrations is due to the ability of the Ca channel inhibitor to antagonize the action of veratridine, rather than to inhibit Ca entry into the synaptosome. It is concluded that it is essential to monitor plasma membrane potentials in experiments in which amino acid efflux from synaptosomes is induced.
Norepinephrine Stimulation of Phosphoinositide Hydrolysis in Rat Cerebral Cortex Is Associated with the Alpha1‐Adrenoceptor
Authors: Schoepp Darryle D. , Knepper Sheila M. , Rutledge Charles O.
Published: October 2006

Norepinephrine (NE) and the selective alpha‐adrenoceptor agonist phenylephrine (PE) both markedly stimulate the formation of [3H]inositol phosphates in a concentration‐dependent manner upon incubation with [3H]myo‐inositol. The selective alpha2 agonist, clonidine, did not significantly alter [3H]inositol phosphate formation, even at concentrations as high as 10‐3M. The alpha, antagonist prazosin (IC50, 0.036 μM) was 300 times more potent than the alpha2 antagonist yohimbine (IC50, 10.7 μM) as an inhibitor of NE (10‐4M)‐stimulated phospha‐tidylinositol (PI) hydrolysis. These results indicate that the alpha1‐, but not the alpha2‐adrenoceptor subtype in rat brain is coupled to phosphoinositide hydrolysis.
The Effects of Detergents on the Composition of Postsynaptic Densities
Authors: Somerville Robert A. , Merz Patricia A. , Carp Richard I.
Published: October 2006

Abstract: A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3–14 removed most of the β‐tubulin but not α‐tu‐ bulin. Sodium N ‐ lauroyI sarcosinate completely destroyed the structural integrity of the PSD when the invitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.