bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse Brain DNA‐Dependent RNA Polymerases After Chronic Morphine Treatment
Authors: Stokes K. Bradley , Lee Nancy M. , Loh Horace H.
Published: October 2006

Abstract: Chronic morphine pellet implantation was found to decrease the specific activity of two forms of mouse brain RNA polymerase I and to alter the requirements of Mg2+ and Mn2+ for the activities of RNA polymerases II and III. DNA‐dependent RNA polymerases were partially purified from small dense nuclei isolated from brains of naive and morphine tolerant‐dependent mice, and three RNA polymerases were separated on a DEAE‐Sephadex A‐25 column. The three fractions, referred to as peak I, peak II, and peak III, were studied, characterized, and identified as being RNA polymerases I, II, and III, respectively. Chronic‐morphine pellet implantation resulted in a lower specific activity of RNA polymerase I, but the specific activities of RNA polymerases II and III were not affected. This effect was prevented by preimplantation of a naloxone pellet and thus was narcotic‐specific. Chronic morphine treatment lowered the concentration of Mg2+ required for optimal activity of RNA polymerase II and elevated the Mn2+‐Mg2+ activity ratios of RNA polymerases II and III. A second DEAE‐Sephadex A‐25 column chromatography of the peak I RNA polymerase was carried out, revealing five component activity peaks. Two of these contained lower specific activities as a result of chronic morphine pelletimplantation. These specific changes in RNA polymerase function in morphine tolerance‐dependence may be associated with the elevated chromatin template activities, altered chromatin phosphorylation, and elevated rates of cell‐free translation that have been reported by others.
The Origin of Indoleacetic Acid and Indolepropionic Acid in Rat and Human Cerebrospinal Fluid1
Authors: Young Simon N. , Anderson George M. , Gauthier Serge , Purdy William C.
Published: October 2006

Abstract: Using a new high performance liquid chromatographic method we have measured tryptophan, 5‐hydroxyindoleacetic acid (5HIAA), indoleacetic acid (IAA), and indolepropionic acid (IPA) in rat and human CSF. Experiments on rats indicate that IPA in CSF is not derived from the CNS but from bacterial metabolism in the intestine. However, IAA in CSF is derived from CNS tryptamine metabolism. Some tryptamine that is formed peripherally diffuses across the blood‐brain barrier and augments the tryptamine formed within the CNS. We have concluded from our data that (i) measurements on CSF are a useful way of studying trace amine metabolism in human CNS, but it is essential to establish the anatomical and metabolic origin of any metabolite found in the CSF; and (ii) tryptamine metabolism is more important in man than in the rat.
Biochemical Characterization of Myelin Isolated from the Central Nervous System of Xenopus Tadpoles
Authors: Trapp Bruce D. , McIntyre Laurence J. , Quarles Richard H. , Nonaka Genïchro , Moser Ann , Maser Hugo W. , Webster Henry de F.
Published: October 2006

Abstract: Myelin isolated from the central nervous system of Xenopus tadpoles was characterized biochemically and compared with Xenopus frog and mammalian myelins. Xenopus tadpole myelin contains the characteristic protein and lipid components of mammalian myelin, although quantitative differences exist. The biochemical composition of Xenopus tadpole myelin suggests that it is an immature form of XePnopus frog myelin. Basic protein and proteolipid protein are prominent components of Xenopus myelin, but isolated tadpole myelin contains a greater proportion of higher molecular weight proteins than Xenopus frog or mature mammalian myelin. The basic protein has a higher apparent molecular weight than mammalian myelin basic protein. The levels of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase are significantly higher in whole tadpole brain homogenate and purified myelin than in similar mammalian preparations. Tadpole myelin lipids contain a higher proportion of phospholipids and less galactolipid than mammalian myelin. Tadpole myelin galactolipids include a high (16%) percentage of monogalactosyl diglyceride, a component found in only trace quantities (0.9%) in bovine myelin.
Partial Purification of Nuclear Protein Kinase from Small Dense Nuclei of Mouse Brain and the Effect of Chronic Morphine Treatment
Authors: Hook Vivian Y. H. , Lee Nancy M. , Loh Horace H.
Published: October 2006

Abstract: To deduce whether or not the nuclear protein kinase activity may be responsible for the change in phosphorylation of chromatin proteins during morphine tolerance‐dependence, the nuclear protein kinases from small dense nuclei of mouse brain have been partially purified by ammonium sulfate fractionation and phosphocellulose column chromatography. Two peaks of cyclic AMP‐independent nuclear protein kinase activity are eluted from the phosphocellulose column by a linear NaCl gradient. During morphine tolerance‐dependence, the specific activity of peak I, but not of peak 11, is increased significantly relative to placebo controls. This increase in nuclear protein kinase activity may partially account for the elevated chromatin protein phosphorylation in small dense nuclei of mouse brain seen during morphine tolerance‐dependence.
The Action of Black Widow Spider Venom on Cholinergic Mechanisms in Synaptosomes
Authors: Baba Akemichi , Cooper Jack R.
Published: October 2006

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high‐affinity choline uptake into the preparation. Both activities occurred in the absence of [Ca2+]0. When Na+ in Krebs‐Ringer was replaced isotonically by sucrose, BWSV did not cause any release of [3H]ACh. On the other hand, BWSV was still effective if Na+ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10−5 M) failed to prevent the ACh‐releasing action of the venom. The uptake of [3H]norepinephrine and [3H]tyrosine into the P2 fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of [3H]deoxyglucose was slight. In addition, the venom‐induced release of [3H]norepinephrine was much higher than that of [3H]deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage‐sensitive fluorescence probe, 3, 3′‐dipentyl‐2, 2′‐oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca2+ ‐free or K+ ‐free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P2 fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein‐cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P2 preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.
Actin and Myosin Synthesis During Differentiation of Neuroblastoma Cells
Authors: Rein Diane , Gruenstein Eric , Lessard James
Published: October 2006

Abstract: The levels and rates of synthesis of actin and myosin in murine neuroblastoma S20 cells have been examined during neurite formation in the absence of serum. Two forms of actin (β, ‐γ) have been identified in these cells; they have isoelectric points of about 5.43 and 5.45, respectively, which differ from that of murine skeletal muscle actin (5.41). Although the neuroblastoma actins have the same molecular weight as skeletal muscle actin, they can be clearly distinguished from this protein by peptide maps. Actin comprises about 7% of the total protein in these cells and neither the level nor the proportion of the two isoelectric forms change during neurite extension (β: γ:60: 40). Myosin ATPase activity is also similar in morphologically differentiated and undifferentiated cells. The rates of synthesis of actin and myosin relative to total protein synthesis undergo small changes during differentiation. Actin synthesis increases about 30% in the 1st day of induction and falls off to the undifferentiated level by day 4. Myosin synthesis increases by about 30% within 2 days of induction and remains at that level for at least 2 days. In contrast, no change in the relative synthesis of tubulin occurs over 4 days of neurite induction. Thus, neurite extension by these cells is not accompanied by large changes in either the level or rates of synthesis of actin and/or myosin.
Acetylation of Homocholine by Rat Brain: Subcellular Distribution of Acetylhomocholine and Studies on the Ability of Homocholine to Serve as Substrate for Choline Acetyltransferase In Situ and In Vitro
Authors: Boksa P. , Collier B.
Published: October 2006

Abstract: In situ acetylation of homocholine by slices of rat cerebral cortex was about 34% of the in situ acetylation of choline. Acetylhomocholine synthesized by the cerebral cortical slices was distributed in the same subcellular fractions as was acetylcholine (ACh), although the relative distribution of acetylhomocholine and ACh between nerve‐ending‐free and nerve‐ending‐bound stores was different. Cerebellar slices acetylated homocholine <10% as well as did cerebral cortical slices. In vitro, choline acetyltransferase (ChAT; EC 2.3.1.1.6) either partially purified from whole rat brain, solubilized from lysed synaptosomes, or in a synaptosomal membrane‐associated form, did not acetylate homocholine at an appreciable rate. Under conditions of alkaline pH, an appreciable in vitro rate of homocholine acetylation by preparations of lysed synaptosomes was detected. However, analysis of this acetylation showed it not to be the result of ChAT catalysis and unlikely to occur by the same mechanism as that responsible for acetylation of homocholine in situ: the acetylation was not inhibited by ChAT inhibitors and occurred equally in the presence of preparations of lysed cerebral cortical or cerebellar synaptosomes. It is concluded that in situ acetylation of homocholine is probably catalyzed by ChAT and that acetylhomocholine is subsequently stored in the same subcellular sites as is ACh; the inability to detect ChAT‐catalyzed acetylation of homocholine in vitro might arise as an artefact of the procedures employed in isolation of the enzyme.
Sidedness of (Calcium, Magnesium) Adenosine Triphosphatase of Purified Torpedo Synaptic Vesicles
Authors: Michaelson Daniel M. , Ophir Idith
Published: October 2006

Abstract: Purified Torpedo synaptic vesicles contain ouabain‐insensitive Mg2+τ and Ca2+‐stimulated ATPase activity. The sidedness of the ATPase on the vesicular membranes was investigated. Addition of ATP and Mg2+ or Ca2+ to intact vesicles results in activation of the ATPase. Exposure of the vesicles to low concentrations of Lubrol‐PX and Triton X‐100, which do not solubilize the activity, results in the concurrent release of the vesicular contents and in an increase of the Mg2+‐ATPase activity, whereas the Ca2+‐dependent activity is drastically decreased. p‐Chloromercuribenzene sulphonate (PCMBS) almost completely inhibits the activity of detergent‐treated vesicles whereas that of the native material is only slightly affected. Tryptic digestion of intact vesicles and of vesicular ghosts results in partial reduction of the ATPase activity. These results suggest that the vesicles contain an outward oriented Ca2+/Mg2+ ATPase activity which can be modulated by detergents.
Uptake of l‐Glutamate and Taurine in Neuroblastoma Cells with Altered Fatty Acid Composition of Membrane Phospholipids
Authors: Balcar V. J. , Borg J. , Robert J. , Mandel P.
Published: October 2006

Abstract: Effects of altered composition of membrane lipids on high‐affinity uptake of l‐glutamate and taurine were studied in an established neuroblastoma cell line M1. Increase in participation of certain fatty acids (20: 5ω3 and 22: 5ω3) as constituents of membrane lipids resulted in a fourfold increase in the maximum initial rate of l‐glutamate uptake (Vmax) while increase in Vmax of taurine uptake was much smaller. Neither structural requirements of l‐glutamate uptake nor passive permeability of the membrane to l‐glutamate or taurine seemed to be influenced by the alterations in the lipid composition. Since increased proportions of 20: 5ω3 and 22: 5ω3 in the membrane phosphatides caused no dramatic changes in kinetic parameters of taurine uptake and incorporation of either linoleic or linolenic acid alone into the phosphatides had only a relatively small effect on some of the measured parameters, the possibility of a specific relationship between 22: 5ω3 and/or 20: 5ω3 and l‐glutamate uptake is discussed. Unlike l‐glutamate uptake systems in other preparations, the high‐affinity uptake system of l‐glutamate in neuroblastoma cells did not readily accept l‐aspartate as a substrate.
The Induction of Ornithine Decarboxylase by Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells
Authors: Guroff Gordon , Dickens Geneva , End David
Published: October 2006

Abstract: Both nerve growth factor and epidermal growth factor cause an induction of ornithine decarboxylase in the rat pheochromocytoma clone PC12. The induction by nerve growth factor is transcription‐dependent and occurs within 4 to 6 h. Antibody studies indicate that nerve growth factor must be present for 2–3 h to obtain full induction. Nerve growth factor is synergistic with either N6,O2‐dibutyryl cyclic 3′,5′‐adenosine monophosphate (dBcAMP) or 3‐isobutyl‐1‐methylxanthine (IBMX) in the induction. The magnitude of ornithine decarboxylase induction is influenced by the density of the culture. Synchronized cell populations show the greatest sensitivity to nerve growth factor just before, or immediately upon, entering S phase. The induction of ornithine decarboxylase by epidermal growth factor appears to be quite similar to that exhibited by nerve growth factor. Epidermal growth factor is active in the range of ng/ml. The time course of the induction is the same, as is the need for the peptide to remain in contact with the cells for several hours. Putrescine inhibits the induction and dBcAMP and IMBX accentuate it. Cells appear to be sensitive to epidermal growth factor also near the G1/S border. In spite of the marked similarities in these inductions, a maximal level of nerve growth factor plus a maximal level of epidermal growth factor yields greater induction than either alone, indicating the inductions occur by somewhat different mechanisms.
Characterisation of Inhibitory 5‐Hydroxytryptamine Receptors That Modulate Dopamine Release in the Striatum
Authors: Ennis Christine , Kemp Janet D , Cox Barry
Published: October 2006

Abstract: The effect of a series of indoleamines on the potassium‐evoked tritium release of previously accumulated [3H]dopamine from rat striatal slices has been investigated. The indoleamines 5‐hydroxytryptamine, 5‐methoxy‐tryptamine, 5‐methoxy‐N, N′‐dimethyltryptamine and tryptamine (10−7 to 10−3 M) all reduced potassium‐evoked release of tritium, to a maximum of 50%. The uptake of [3H]dopamine was unaffected by these compounds. A series of 5‐hydroxytryptamine antagonists were examined for their ability to reduce the inhibition of potassium‐evoked tritium release induced by 5‐methoxytryptamine. The relative order of antagonist potency obtained was methysergide > metergoline > methiothepin > cinanserin > cyproheptadine > mianserin, and was consistent with an action on 5‐hydroxytryptamine receptors. It is concluded that there are inhibitory 5‐hydroxytryptamine receptors located on the terminals of dopaminergic neurones in the striatum.
Dopamine and Noradrenaline Levels in Peripheral Tissues of Several Mammalian Species
Authors: Bell C. , Gillespie J. S.
Published: October 2006

Abstract: Tissue levels of dopamine (DA) and noradrenaline (NA) have been compared in atrium, spleen, mesenteric artery, vas deferens and renal cortex of sexually mature rats, guinea‐pigs, ferrets, rabbits and cats. The results suggest the presence of dopaminergic autonomic axons in the cat renal cortex, as has been previously reported for the dog. Such nerves are probably sparse or absent in the kidneys of the other species examined.
Presence of Conjugated Catecholamines in Rat Brain: A New Method of Analysis of Catecholamine Sulfates
Authors: Buu N. T. , Duhaime J. , Savard C. , Truong L. , Kuchel O.
Published: October 2006

Abstract: A new method of measuring catecholamine (CA) sulfate permitted us to detect its presence in rat brain for the first time. The procedure consisted of separating the CA sulfate from the free CA by alumina adsorption followed by passage through Dowex, and measuring the CA sulfate by a radioenzymatic assay in the presence of a sulfatase. This method permitted demonstration of the presence of dopamine sulfate, and occasionally, of norepinephrine and epinephrine sulfate in the hypothalamus, striatum, and hippocampus of rat brain.
Effects of a Single Therapeutic Dose of Glycerol on Cerebral Metabolism in the Brains of Young Mice: Possible Increase in Brain Glucose Transport and Glucose Utilization
Authors: Thurston Jean Holowach , Hauhart Richard E. , Dirgo John A.
Published: October 2006

Abstract: This is a study of the effects of a single “therapeutic” dose of glycerol [2 g(22 mmol)/kg i.p.] on brain carbohydrate and energy metabolism in normal nursing weanling mice. Findings were correlated with brain water and electrolyte content and with metabolite changes in plasma, red blood cells, and liver.

Plasma glycerol levels peaked at 21 mM 7.5 min after injection and returned to the control value, 0.16 mM, by 2 h. Plasma Na+ concentration decreased and plasma protein increased for as long as 2 h after injection. Although red blood cells were freely permeable to glycerol, there was no evidence for glycerol metabolism in these cells. Glycerol levels in liver paralleled those in plasma. Glycerol injection increased liver glucose concentration 23% and doubled hepatic glycerol‐1‐phosphate levels. Liver ATP levels were reduced 24% after glycerol injection.

Brain water concentration was significantly reduced from 7.5 min to 30 min after glycerol injection; brain Na+ and K+ levels were unchanged. There was no evidence for glycerol entry into brain (the amount detected in brain tissue could be explained by the glycerol content in the blood of the brain). While plasma glucose increased 33%, brain glucose increased 87%. Concomitantly there were statistically significant increases in fructose‐1,6‐diphosphate, lactate, α‐ketoglutarate, and malate levels.

The disproportionately high brain glucose value suggests increased transport of glucose from the blood to the brain. Increases in fructose‐1,6‐diphosphate, lactate, α‐ketoglutarate, and malate are compatible with an increased metabolic flux in the glycolytic pathway and Krebs citric acid cycle. As has been previously shown for urea and/or mannitol, these changes may result from the effects of the hyperosmolar glycerol solution on the blood‐brain barrier and on cerebral glucose utilization.

The sustained lowering of plasma Na+ concentration after a single “therapeutic” glycerol injection suggests a need for monitoring plasma Na+ levels in the clinical situation. Possible lowering of hepatic ATP levels by the use of glycerol in humans is another concern.
Ion Channels and Membrane Potential in Stimulus‐Secretion Coupling in Adrenal Medulla Cells
Authors: Kilpatrick Daniel L. , Slepetis Ruta , Kirshner Norman
Published: October 2006

Abstract: The role of Na+ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)‐sensitive Na+ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 μM). In Ca2+‐sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na+ with Li+ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na+ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM‐100 mM‐K+, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM‐K+. Neither dibutyryl‐cyclic AMP nor dibutyryl‐cyclic GMP evokes secretion. α‐Bungarotoxin (1 μM) did not inhibit nicotine‐induced secretion. These studies indicate that Na+ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca2+. The membrane potential appears to affect nicotine‐ and veratridine‐evoked secretion.
Culture and Characterization of Epithelial Cells from Bovine Choroid Plexus
Authors: Crook Richard B. , Kasagami Hiroshi , Prusiner Stanley B.
Published: October 2006

Abstract Epithelial cells were isolated from choroid plexus, which plays a major role in cerebrospinal fluid production and regulation. Incubation of bovine choroid plexuses with pronase released cells which attached to plastic dishes with a plating efficiency of 5%. The cells were predominantly polygonal as judged by phase‐contrast microscopy. These polygonal cells undergo limited cell division and survive for 1 – 2 weeks in culture before being overgrown by fibroblasts. The fibroblastic cells could be selectively removed from the cultures by the addition of 100 μg/ml cis‐hydroxyproline to the medium for several days. The specific activities of three membrane‐bound enzymes, γ‐glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were compared in selective cultures of polygonal cells and fibroblasts. Polygonal cells were found to have 4–5 times the γ‐glutamyl transpeptidase of fibroblasts, whereas fibroblasts have 2–3 times the alkaline phosphatase of polygonal cells. Leucine aminopeptidase levels in the two cultures were roughly equivalent. The polygonal cells rapidly lost γ‐glutamyl transpeptidase activity over a 4‐day period in culture but acquired increased levels of leucine aminopeptidase. Alkaline phosphatase remained roughly constant. Under similar conditions fibroblasts showed a 3‐ to 4‐fold increase in the specific activities of all three enzymes; these changes coincided with a substantial increase in cell density. Based on morphology, resistance to cis‐hydroxyproline, absence of antihemophilic factor antigen, and enzymatic characteristics, we believe the polygonal cells to be of epithelial origin.
Elevation of Brain GABA Content by Chronic Low‐Dosage Administration of Hydrazine, a Metabolite of Isoniazid
Authors: Perry Thomas L. , Kish Stephen J. , Hansen Shirley , Wright James M. , Wall Richard A. , Dunn William L. , Bellward Gail D.
Published: October 2006

Abstract: When γ‐aminobutyric acid aminotransferase (GABA‐T) activity was measured in vitro in rat brain, neither isoniazid (INH) nor four of its known metabolites (isonicotinic acid, acetylisoniazid, acetylhydrazine, diacetylhydrazine) inhibited the enzyme in concentrations (5 mM) far higher than those likely to be achieved when INH is administered to man. In contrast, hydrazine (5 μM) caused a 50% inhibition of GABA‐T without inhibiting glutamic acid decarboxylase (GAD). Rats were injected daily for 109 days with hydrazine (0.08 or 0.16 mmol/kg/day), after which amino acid contents and enzyme activities were measured in their brains. Both hydrazine doses caused significant elevations of whole brain GABA content and reductions of GABA‐T activity, but did not affect GAD activity. Chronic administration of hydrazine at thee doses did not reduce weight gain or alter rat behavior, nor did it produce any irreversible pathologic changes in liver or alterations in hepatic aryl hydrocarbon hydroxylase activity. However, hydrazine treatment caused changes in the contents of many brain amino acids besides GABA, and markedly increased concentrations of ornithine, tyrosine, and α‐aminoadipic acid in rat plasma. Inhibition of GABA‐T activity and the other biochemical alterations observed in patients given high doses of INH probably result from hydrazine formed in the metabolic degradation of INH. Thus administration of hydrazine might be a more direct means of elevating brain GABA content in patients where this seems indicated, and might not entail a greater risk of adverse effects.
Effect of Depolarizing Agents on the Incorporation of Amino Acids into Soluble Cytoplasmic and Membrane‐Bound Proteins of Synaptosome Fractions
Authors: Boyar M. M. , Coutinho‐Netto J. , Abdul‐Ghani A. S. , Bradford H. F.
Published: October 2006

Abstract: The incorporation of [U‐14C] protein hydrolysate and [U‐14C]leucine into the trichloroacetic acid (TCA)‐insoluble membrane and the soluble synaptoplasm proteins of synaptosomes was studied. Following treatment with the depolarizing agents veratrine, Tityus toxin, or potassium, the specific radioactivity of both precursor pool and proteins was measured to examine the link between protein labeling and the fall in the free amino acid pool due to depolarization‐induced release of glutamate and aspartate. By reducing the size of the fall in precursor pool due to depolarization by using a nontransmitter amino acid such as leucine (as compared with the usual use of protein hydrolysate), it was shown that the amount by which the pool is reduced is proportional to the change in the protein labeling observed. These results confirm that membrane depolarization causes a large increase in the labeling of membrane‐bound proteins as compared with the soluble synaptosomal proteins.
Synaptic Plasma Membrane Tubulin May Be an Integral Constituent
Authors: Babitch J. A.
Published: October 2006

Abstract: Mild detergent extraction of chick brain synaptic plasma membranes followed by gel electrophoresis suggests that synaptic plasma membrane tubulin is an integral component. Although some of the synaptic plasma membrane tubulin might be aggregates, that possibility is not supported by the observation that tubulin aggregates that are added to synaptosomes before synaptic subfractionation do not partition with synaptic plasma membranes during membrane isolation.
Postmortem Changes of Normetanephrine Determined by a Mass‐Fragmentographic Technique in Different Rat Brain Areas
Authors: Mocchetti I. , Angelis L. De , Racagni G.
Published: October 2006

Abstract: A specific and sensitive gas chromatographic–mass spectrometric assay for measurement of normetanephrine (NMN) in different rat brain regions is presented. The results show that hypothalamus contains the highest concentrations of the norepinephrine metabolite. An increase of NMN concentrations has been detected in animals killed by decapitation in comparison with microwave–irradiated rats. This finding has to be taken into consideration when NMN is measured in the brain in order to assess the functional activity of noradrenergic neurons.
Partial Purification and Characterization of Messenger RNA Coding 14‐3‐2 Protein from Rat Brain
Authors: Sakimura K. , Araki K. , Kushiya E. , Takahashi Y.
Published: October 2006

Abstract: 14‐3‐2 Protein (neuron‐specific enolase) is a neuron‐specific protein. Using a reticulocyte lysate cell‐free system for translation of 14‐3‐2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))‐Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14‐3‐2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8‐fold. The size of 14‐3‐2 protein mRNA appears to be about 19–20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.
Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine
Authors: Lacoste Lucille , Chaudhary Kapil Dev , Lapointe Jacques
Published: October 2006

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro‐2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10‐fold increase in glutamine synthetase specific activity (15‐fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 mM glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 mM). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C‐6 glioma cells and glial cells in primary cultures.
[3H]Nitrobenzylthioinosine Binding as a Probe for the Study of Adenosine Uptake Sites in Brain
Authors: Marangos P. J. , Patel J. , Clark‐Rosenberg R. , Martino A. M.
Published: October 2006

Abstract: The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ([3H]NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high‐affinity binding was demonstrated in both rat and human brain. The Kd in both was 0.15 nM with Bmax values of 140–200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X‐100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on [3H]NBI binding. The inhibitory potencies of copper and zinc were IC50= 160 μM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the [3H]NBI binding sites on synaptosomes, indicating that these sites were present in the synaptic region. A study of the tissue distribution of the [3H]NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of [3H]NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2‐chloroadenosine, were three to four orders of magnitude less potent. [3H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.
Metabolism of Phospholipids in Peripheral Nerve from Rats with Chronic Streptozotocin‐induced Diabetes Increased Turnover of Phosphatidylinositol‐4,5‐Bisphosphate
Authors: Bell Margaret E. , Peterson Richard G. , Eichberg Joseph
Published: October 2006

Abstract: The effect of chronic streptozotocin‐induced diabetes on phospholipid metabolism in rat sciatic nerve in vitro was investigated. In normal nerve incubated for 2 h in Krebs‐Ringer‐bicarbonate buffer containing [32P]orthophosphate, radioactivity was primarily incorporated into phosphati‐dylinositol‐4,5‐bisphosphate and phosphatidylcholine. Smaller amounts were present in phosphatidylinositol‐4‐phosphate, phosphatidylinositol, and phosphatide acid. As compared to controls, phosphatidylinositol‐4,5‐bisphosphate in nerves from animals made diabetic 2, 10, and 20 weeks earlier accounted for 30–46% more of the isotope, expressed as a percentage, incorporated into all phospholipids. In contrast, the proportion of radioactivity in phosphatidylcholine decreased by 10–25%. When the results were expressed as the quantity of phosphorus incorporated into phospholipid, only phosphatidylinositol‐4,5‐bisphosphate displayed a change. The amount of isotope which entered this lipid increased 60% and 67% for 2‐ and 10‐week diabetic animals, respectively. Increased phosphatidylinositol‐4,5‐bisphosphate labeling was observed when epineurial‐free preparations were used or when the composition of the incubation medium was varied. Sciatic and caudal nerve conduction velocities were decreased after 10 and 20 weeks but were unchanged after 2 weeks. We conclude that an increase in the turnover of phosphatidylinositol‐4,5‐bisphosphate in sciatic nerve from streptozotocin‐diabetic rats appears relatively early and persists throughout the course of the disease. This metabolic alteration may be related to a primary, defect responsible for the accompanying deficient peripheral nerve function.
Local Cerebral Glucose Utilization in Hypothermic and Hyperthermic Rats
Authors: McCulloch James , Savaki Helen E. , Jehle Jane , Sokoloff Louis
Published: October 2006

Abstract: Local rates of glucose utilization in 38 regions of the CNS were measured in conscious, lightly restrained rats during normothermia (rectal temperature, 37.4 ± 0.1°C), hypothermia (31.8 ± 0.1°C), and hyperthermia (40.2 ± 0.3°C). In 34 of the 38 regions examined (the four exceptions being primary auditory nuclei in the lower brainstem), a significant relationship could be demonstrated between the rate of glucose utilization and body temperature. The magnitude of temperature‐related alterations in glucose use displayed considerable regional heterogeneity. In hypothermic rats the reductions in glucose use were proportionately most marked (reduced 35–50% from normothermic) in thalamic nuclei, extrapyramidal and motor areas, septohippocampal formation, and some areas of neocortex and white matter; they were least pronounced in anterior hypothalamus (reduced by 13%), habenula (by 16%), and amygdala (by 22%). In hyperthermic rats, significantly increased glucose utilization was observed in only 16 of the 38 areas examined (e.g., hypothalamus, hippocampus, extrapyramidal system, and raphe nucleus), whereas in a number of major areas (such as the neocortex and thalamus) glucose use was minimally altered with hyperthermia. The regional heterogeneity in the alterations in glucose utilization suggests that caution must be exercised in the interpretation of autoradiographic 2‐deoxyglucose investigations in which body temperature disturbances occur.
Retinal Gangliosides in RCS Mutant Rats
Authors: Seyfried Thomas N. , Yu Robert K. , Miyazawa Nobuko , Lai Yin‐Lok
Published: October 2006

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, GD3, is discussed.
Ontogenesis of Monoamine‐Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration
Authors: Kato T. , Yamaguchi T. , Togari A. , Nagatsu T. , Yajima T. , Maeda N. , Kumegawa M.
Published: October 2006

Abstract: Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co‐factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands: the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12‐day‐old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co‐factor, biopterin, in rat midbrain.
Complex Binding of l‐[3H]Glutamate to Hippocampal Synaptic Membranes in the Absence of Sodium
Authors: Werling Linda L. , Nadler J. Victor
Published: October 2006

Abstract: Specific binding of L‐[3H]glutamate was investigated with a thoroughly washed synaptic membrane preparation from rat hippocampal formation, a region of brain densely innervated by putatively glutamatergic fibers. L‐[3H]Glutamate bound rapidly, saturably, and reversibly to these membranes in the absence of Na+. Specific binding was greatest around 38°C and at a slightly acidic pH. Saturation isotherms fit a model of two independent binding sites with dissociation constants of 11 and 570 nM and corresponding densities of 2.5 and 47 pmol/mg protein. All potent amino acid excitants, except N‐methyl‐D‐aspartate and kainate, and several excitatory amino acid antagonists inhibited specific radioligand binding with IC50 values between 10−1 M and 10−4 M. In contrast, weak amino acid excitants and an inhibitor of glutamate uptake were nearly inactive. Displacement curves were analyzed with a computer program that assumed the simultaneous contributions of two independent sites at which each compound competitively inhibited the binding of L‐[3H]glutamate. According to this analysis, ibotenate and the L‐ and D‐isomers of glutamate and aspartate bind preferentially to the high‐affinity site, whereas quisqualate, L‐α‐aminoadipate, and the L‐ and D‐isomers of homocysteate bind preferentially to the low‐affinity site. With the notable exception of γ‐D‐glutamylglycine, all of the more potent antagonists appear to bind preferentially to the low‐affinity site. Both sites exhibit marked stereoselectivity for L‐glutamate. D‐ and L‐Homocysteate and most excitatory amino acid antagonists increased specific binding at concentrations below those required to demonstrate inhibition. Some properties of the low‐affinity binding site resemble those of junctional glutamate receptors on insect muscle, but neither site appears to correspond to the “N‐methyl‐D‐aspartate receptor” or the “quisqualate receptor.”
Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord
Authors: Stine Susan M. , Yang Hsiu‐Ying , Costa Erminio
Published: October 2006

Abstract: Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro‐caudally, somatostatin content was about 50% higher in lumbar‐sacral cord than in cervical or thoracic levels. The dorso‐ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met‐enkephalin and substance P were also measured after the above surgical manipulations. Met‐enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the orgin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.
Carnosine Release from Olfactory Bulb Synaptosomes Is Calcium‐Dependent and Depolarization‐Stimulated
Authors: Rochel S. , Margolis F. L.
Published: October 2006

Abstract: The dipeptide carnosine (β‐alanyl‐L‐histidine) has been proposed as a neurotransmitter in the mammalian olfactory pathway. Therefore, the efflux of in vivo‐synthesized [14C]carnosine from mouse olfactory bulb synaptosomes was investigated. Carnosine was found to be released from the olfactory bulb synaptosomes by two mechanisms. The first is a slow spontaneous process that is independent of depolarization. The rate of this release was doubled in the presence of 1 mM external carnosine. Release by the second mechanism was markedly stimulated in the presence of calcium by depolarization with either 60 mM K+ or 300 μM veratridine. Omission of calcium abolished the stimulatory effect of both of these agents. Further, blockage of the veratridine‐induced depolarization by tetrodotoxin also inhibited carnosine release. These results are consistent with the hypothesis that carnosine acts as a neurotransmitter in the mouse olfactory pathway.
Ontogeny and Subcellular Distribution of Rat Brain Tele‐Methylhistamine
Authors: Hough Lindsay B. , Khandelwal J. K. , Green Jack Peter
Published: October 2006

Abstract: The whole brain content and subcellular distribution of histamine and its metabolite, tele‐methylhistamine, were studied during postnatal development of the rat. Brain methylhistamine levels were similar to or greater than histamine levels, indicating that histamine methylation is a major metabolic pathway in neonatal brain, as it is in adults. When calculated per brain, histamine, methylhistamine, and histamine methyltransferase were all maximal 10 days after birth. In neonates, brain histamine was found almost entirely in nuclear fractions, whereas methylhistamine was found almost exclusively in supernatant fractions. By day 20, however, a greater proportion of both amines was localized in subcellular fractions containing synaptosomes, a finding consistent with histamine's suggested transmitter role. The ontogenic pattern of brain methylhistamine questions the mast cell origin of neonatal histamine, but may be consistent with a role for histamine in brain development.
Identification of a Cholinergic‐Specific Antigen Chol‐1 as a Ganglioside
Authors: Richardson Peter J. , Walker John H. , Jones R. Theresa , Whittaker Victor P.
Published: October 2006

Abstract: An antiserum specific for cholinergic terminals was used to identify an antigen conserved between Elasmobranchs and mammals. Immunohistochemistry and a cytotoxicity test were used to assay the binding of antibody to mammalian terminals. Torpedo electric organ gangliosides totally abolished antibody binding. The highest inhibitory activity was associated with a single polysialoganglioside band on TLC plates. Neuraminidase altered the migration of the inhibitory activity on TLC plates. Antibody binding was inhibited by ganglioside fractions derived from chicken and mammalian brains. A summary of those tissues in which the antigen has been detected is presented. The possible function of the antigen is discussed.
The Contribution of Citrate to the Synthesis of Acetyl Units in Synaptosomes of Developing Rat Brain
Authors: Szutowicz A. , Kabata J. , Bielarczyk H.
Published: October 2006

Abstract: The activities of pyruvate dehydrogenase, citrate synthase, and choline acetyltransferase in rat brain synaptosomes increased during on‐togenesis by 3 and 14 times, respectively. Activity of ATP‐citrate lyase decreased by 26% during the same period. Pyruvate consumption by synapto‐somes from 1‐day‐old animals was 40% lower than that found in older rats; however, citrate efflux from intrasynaptosomal mitochondria in immature synaptosomes was over twice as high as that in mature ones. The rates of production of synaptoplasmic acetyl‐CoA, ATP‐citrate lyase were 1.03, 1.40, and 0.49 nmol/min/mg protein in 1‐, 10‐day‐old, and adult rats, respectively. 3‐Bromopyruvate (0.5 mM) inhibited pyruvate consumption by 70% and caused a complete block of citrate utilization by citrate lyase in every age group. Parameters of citrate metabolism in cerebellar synaptosomes were the same as those in cerebral ones. These data indicate that production of acetyl‐CoA. from citrate in synaptoplasm may be regulated either by adaptative, age‐dependent changes in permeability and carrier capacity of the mitochondrial membrane or by the inhibition of synthesis of intramitochondrial acetyl‐CoA. ATP‐citrate lyase activity is not a rate‐limiting factor in this process. Metabolic fluxes of pyruvate to cytoplasmic citrate and acetyl‐CoA. are presumably the same in both cholinergic and noncholinergic nerve endings. The significance of citrate release from intrasynaptosomal mitochondria as a regulatory step in acetylcholine synthesis is discussed.
Catabolism of Endogenous Dopamine in Peripheral Tissues: Is There an Independent Role for Dopamine in Peripheral Neurotransmission?
Authors: Lacković Zdravko , Relja Maja , Neff Norton H.
Published: October 2006

Abstract: Dopamine (DA) and its metabolites, homovanillic acid (HVA) and 3,4‐dihydroxyphenylacetic acid (DOPAC), have been measured in peripheral tissues of the rat and human by gas chromatography‐mass spectrometry. The content of HVA and DOPAC in peripheral tissue is higher than in blood and is usually higher than the content of DA. In the rat, chemical denervation with 6‐hydroxydopamine decreased the tissue content of DOPAC. inhibition of monoamine oxidase increased tissue DA. Apparently, in vivo, a large quantity of peripheral DA is catabolized rather than converted to norepinephrine (NE). These observations suggest that either NE synthesis is inefficient, with a large quantity of DA wasted and not converted to NE, or that DA is physiologically utilized as a neurotransmitter and/or cotransmitter in many peripheral nerves. A survey of the reported actions of DA on peripheral tissues suggests that the latter proposal is more likely.