bluuebunny/crossref_metadata_2025 huggingface dataset:
PURIFICATION AND CHARACTERIZATION OF OX BRAIN NAD+ ‐DEPENDENT ISOCITRATE DEHYDROGENASE
Authors: Willson Vivian J. C. , Tipton K. F.
Published: October 2006

Abstract— The NAD+ ‐dependent isocitrate dehydrogenase from ox brain has been purified about 130‐fold by a method involving affinity chromatography on an NAD+ ‐derivative of agarose. The enzyme preparation is not homogeneous but it is free from contaminating enzyme activities that could interfere with kinetic studies. The kinetic properties of the enzyme did not appear to have been altered by the purification procedure involved. The initial velocity of the reaction showed a sigmoid dependence on the concentration of isocitrate, and ADP behaved as an allosteric activator. The kinetics with NAD+ as the substrate were hyperbolic. The molecular weight of the purified enzyme was found to be 285,000 ± 25,000.
AMMONIUM ION INHIBITION OF EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM HIPPOCAMPAL SLICES
Authors: Hamberger Anders , Hedquist Bo , Nyström Britta
Published: October 2006

Abstract— The effect of pathophysiological levels (2‐5 mm) of ammonium chloride on the efflux of endogenous and exogenous [14C]glutamate from hippocampal slices was studied. The evoked release of glutamate which occurs dring tissue depolarization with 56 mm‐KCl was greatly reduced when the tissue had been exposed to NH4Cl for 40–80 min. This effect was seen whether or not glutamine (0.5 mm) was present in the incubation medium. The effect was completely reversible. The spontaneous efflux and the evoked release of [14C]glutamate was, on the contrary, completely unaltered after exposure of the slice to ammonium ions. Nigher (20–36 mm) amounts of NH4Cl evoked a release of [14C]glutamate from the crude mitochondrial fraction, as did high concentrations of KCl. The results are discussed in relation to the compartmentation of glutamate metabolism and the pathogenesis of hepatic coma.
STUDIES ON AUTOIMMUNE ENCEPHALOMYELITIS IN THE GUINEA PIG—TIT. A COMPARATIVE STUDY OF TWO AUTOANTIGENS OF CENTRAL NERVOUS SYSTEM MYELIN1
Authors: Lebar René , Vincent Christine , Boubennec Elisabeth Fischer‐le
Published: October 2006

Abstract— A new CNS myelin autoantigen(s) (referred to as M2), different from the encephalitogenic basic protein (BP), can be detected with guinea‐pig demyelinating and complement fixing (CF) sera raised against guinea pig CNS tissue or myelin (Lebaret al., 1976). M2 and BP were present in mouse, rat, rabbit, bovine and human CNS tissues when tested with guinea‐pig homologous specific antisera; they were not present in non‐CNS tissues. Both autoantigens were also detected in newborn guinea‐pig myelin and myelin‐like fractions.

The CF activity of myelin with demyelinating (anti‐M2) sera was not altered by trypsin; however, absorption experiments showed that M2 was partly trypsin sensitive. Both antibodies against the trypsin sensitive and the trypsin resistant determinants of M2 were demyelinating. Both determinants of M2 were preselit in mouse, rat, rabbit, bovine‘and human CNS tissues and in guinea‐pig newborn myelin. CF BP activity of myelin was partially or even totally abolished by trypsin, but the persistent encephali‐togenicity of trypsin‐treated myelin could be attributed to non‐CF encephalitogenic peptides from BP.

In accordance with recent work our results tend to support an inner localization of BP in myelin; M2, on the other hand, would be a surface antigen(s).
THE EFFECT OF MONOAMINE OXIDASE INHIBITORS ON SOME ARYLALKYLAMINES IN RAT STRIATUM
Authors: Philips S. R. , Boulton A. A.
Published: October 2006

The trace amines phenylethylamine, tryptamine, p‐tyramine and m‐tyramine have been measured in the striatum of both control and MAO‐treated rats. Dose‐response and time‐response studies have been carried out with clorgyline and deprenyl, inhibitors which preferentially inhibit the A and B forms of MAO, respectively, and with tranylcypromine and phenylethylhydrazine, which are used clinically in the treatment of depression. Phenylethylamine was increased by 1 mg/kg of deprenyl, but was unaffected by clorgyline at doses up to 50 mg/kg, while the tyramines and tryptamine were increased by low doses of clorgyline, but were increased only by much greater doses of deprenyl than those required to affect phenylethylamine.

Phenylethylamine is oxidized by the B form of MAO, but tryptamine and the tyramines appear to be oxidized by both A and B MAO. The observed proportional increases in trace amine levels are much greater than those observed for the classical neurotransmitters, noradrenaline, dopamine and 5‐hydroxytryptamine. As these increases are differential, selective manipulation of trace amine concentrations is possible.
Human Brain Aldehyde Reductases: Relationship to Succinic Semialdehyde Reductase and Aldose Reductase
Authors: Hoffman Paula L. , Wermuth Bendicht , Wartburg Jean‐Pierre von
Published: October 2006

Human brain contains multiple forms of aldehyde‐reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH‐dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase („SSA reductase”) specifically reduces succinic semialdehyde (SSA) to produce γ‐hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions.
Rat, Mouse, and Guinea Pig Brain Development and Microtubule Assembly
Authors: Lennon A. M. , Francon J. , Fellous A. , Nunez J.
Published: October 2006

The development of in vitro microtubule assembly and of tubulin concentration have been studied during brain maturation in the mouse and the rat, two species which have postnatal brain development, and in one species which is mature at birth, the guinea pig. (a) The rate of tubulin assembly is very slow soon after birth in both the mouse and rat; it increases progressively with age until adulthood. In contrast, in the guinea pig this rate is maximal at birth and slower rates are seen only at foetal stages. (b) Postnatal changes in the lag period of assembly and in the minimal concentration of tubulin (Cc) required to obtain in vitro assembly are seen in the mouse and the rat; in contrast these parameters are constant at all postnatal stages in the guinea pig with longer lag periods and lower Cc values being seen only at foetal stages. (c) Maximal rates of assembly, minimal lag periods, and minimal Cc values are restored after addition of microtubule‐associated proteins to foetal guinea pig or young mouse and rat preparations, suggesting that the difference in the kinetic parameters of assembly between these species depends on differences in the concentration or activity of these proteins. (d) Maximal tubulin concentrations are observed before birth in the guinea pig and approximately at day 10 in the rat and mouse. Lennon A. M. et al. Rat, mouse, and guinea pig brain development and microtubule assembly. J. Neurochem.35, 804–813 (1980).
Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108‐15
Authors: McGee Richard
Published: October 2006

The neuroblastoma X glioma hybrid clone NG108‐15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108‐15 cells. The uptake system appears to bear little if any resemblance to the Na+‐dependent high‐affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high‐ and low‐ affinity choline uptake systems, neither system is dependent on Na+ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na+, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca2+ or Mg2+ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium‐3, but only at high concentrations of the drug (IC50= 30–80 μm). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30‐40%. Growth of the cells in N6,O2′ dibutyryladenosine‐3′,5′‐cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108‐15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high‐affinity choline uptake system normally associated with cholinergic neurons.
Pentobarbital Anesthesia Reduces Blood–Brain Glucose Transfer in the Rat
Authors: Gjedde Albert , Rasmussen Mette
Published: October 2006

Abstract: Pentobarbital anesthesia (40 mg kg–1) was accompanied by a 50% decrease of blood flow and a 40% decrease of unidirectional blood‐brain glucose transfer in the parietal cortex of the rat brain. The correlation was explained by a decrease of the number of perfused capillaries. The maximal transport capacity, Tmax, decreased from 409 to 235 μ mol 100 g–1 min–1 and the half‐saturation constant, Km, from 8.8 to 4.9 mm. At 8.3–8.7 mm‐glucose in arterial plasma, the transfer constant (clearance) for unidirectional blood‐brain transfer decreased from 0.195 ± 0.011 in awake rats to 0.132 ± 0.005 ml g–1 min–1 in anesthetized rats. Half of the decrease was due to less complete diffusion‐limitation of glucose uptake at the low plasma flow rate in brain, the other half to the decreased Tmax.
Studies on a Capillary‐Rich Fraction Isolated from Brain: Histaminic Components and Characterization of the Histamine Receptors Linked to Adenylate Cyclase
Authors: Karnushina I. L. , Palacios J. M. , Barbin G. , Dux E. , Joó F. , Schwart J. C.
Published: October 2006

Abstract: A fraction enriched in capillaries has been prepared from the guinea pig cerebral cortex. The purity of this fraction was checked by light‐ and electron‐microscopic examination and by its high enrichment in alkaline phosphatase and γ‐glutamyl transpeptidase. In the capillary‐rich fraction, the endogenous level of histamine was 1.9%’of that measured in the initial hornogenate. The histamine‐synthesizing enzyme, I‐histidine decarboxylase, and the metabolizing enzyme, histamine‐N‐methyltransferase, were barely detectable. In addition, histamine elicits a twofold stimulation in the accumulation of cyclic AMP in this capillary fraction with an EC50 of 5 γM. Agonists and antagonists of the two types of histamine receptors (H1 and H2) were used for the characterization of the receptors mediating this action: H2‐receptor agonists were able to activate the adenylate cyclase with “relative potencies” similar to that found on typical H2‐receptors, and cimetidine, a specific H2‐receptor antagonist, competitively inhibited the response to histamine with a K1 value reflecting its interaction with a single population of H2‐receptors. On the contrary, data obtained with H1‐receptor agonists and antagonists reflect their interaction with H2‐receptors rather than H1‐receptors. Thus H2‐receptors are involved in the activation of adenylate cyclase of the capillary fraction.
Nature of the Cytoplasmic Factor(s) Modulating Adenylate Cyclase in the Developing Rat Brain
Authors: Nijjar M. S.
Published: October 2006

Abstract: Adenylate cyclase activity in the particulate fraction from rat brain was markedly enhanced by the cytoplasmic fraction, which itself contained negligible enzyme activity, indicating the presence of some stimulatory factor(s) in the supernatant. Activation of adenylate cyclase was dependent on the supernatant concentration up to 1 mgiml, but higher concentration of the supernatant did not produce further activation of the enzyme. The supernatant retained its stimulatory activity after boiling for 5 min, extensive dialysis, and phospholipase A and DNAase treatments, but was completely inactivated by digestion with trypsin. Ability of the supernatant to activate adenylate cyclase was low during fetal life, increased severalfold neonatally, and declined somewhat thereafter to an adult level. Adenylate cyclase in the particulate fraction from 2‐day‐old rat brain was also activated by GTP, calcium‐dependent regulator (CDR) of cyclic AMP phosphodiesterase in the presence of 100 pM‐Ca1, and by NaF. The supernatant produced additive activation of the enzyme with NaF but not with GTP or CDR, suggesting a common site of action of the supernatant factor(s) and the latter two agents. DEAE‐cellulose chromatography of the boiled supernatant resolved the heat‐stable proteins into several peaks. Adenylate cyclase activator eluted in two distinct peaks, one of which also contained CDR activity. It is concluded that rat brain supernatant contains some factor in addition to CDR which activates particulate adenylate cyclase.
Separation of Two Dipeptidyl Aminopeptidases in the Human Brain
Authors: Kato Takeshi , Hama Tokiko , Nagatsu Toshiharu
Published: October 2006

Abstract: Soluble dipeptidyl aminopeptidases in the human cerebral cortex were purified by CM‐cellulose, Sephadex G‐200 and hydroxyapatite column chromatography. With hydroxyapatite chromatography two enzymes, dipeptidyl aminopeptidases A and B (DAP‐A and DAP‐B), were separated. DAP‐A and DAP‐B were different from each other in several properties: optimum pH, substrate specificity, Km values in 7‐(Gly‐Pro)‐4‐methylcoumarinamide and molecular weight. They were identified as dipeptidyl aminopeptidases based on the analysis of the products by thin‐layer chromatography. DAP‐A was similar to dipeptidyl aminopeptidase II, but DAP‐B was different from any of the previously described dipeptidyl aminopeptidases (I‐IV) and may be a new dipeptidylaminopeptidase. DAP‐B liberated N‐terminal Arg‐Pro and subsequently Lys‐Pro, from substance P as substrate. Although the physiological roles of these two enzymes in the human brain are not clear yet, they may act on regulation and degradation of biologically active peptides.
Presence of γ‐Aminobutyric Acid in Rat Ovary
Authors: del Rio Rafael Martin , Caballero Amparo Latorre
Published: October 2006

Abstract: As γ‐aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non‐neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.
Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications
Authors: Saborío José L. , Palmer Elizabeth
Published: October 2006

Abstract: We have studied the posttranslational processing of actin molecules synthesized in a cell‐free system. The results of these experiments indicate that during the in vivo synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH2‐terminal amino acid residues of actin molecules.
Organization of Brain Synaptic Vesicle Proteins
Authors: Smith Andrew P. , Loh Horace H.
Published: October 2006

Abstract: The topographical arrangement of proteins and glycoproteins of mouse brain synaptic vesicles was studied with trypsin and galactose oxidase, reagents known to be impermeable with respect to other membranes. Incubation of vesicles with trypsin at a concentration of 1 μg/ml extensively degraded seven polypeptides of molecular weights (M.W.) (×10‐3) 125, 107, 95, 83, 70, 60, and 36; higher concentrations degraded two additional species of 75,000 and 46,000 M.W., while leaving unaffected polypeptides of M.W. 66,000, 55,000, 33,000, 26,000, 22,000, 19,000, and 16,000. All of the trypsin‐sensitive species of greater than 70,000 M.W. stained positively with the periodic acid‐Schiff reagent; several other glycoproteins, all of M.W. less than 70,000, were identified, and all of these were insensitive to trypsin. Galactose oxidase‐NaB3H4 treatment of synaptic vesicles heavily and exclusively labeled material of greater than 70,000 M.W. All of the polypeptides studied were sensitive to each reagent when the synaptic vesicles were first treated with detergents. Extraction of vesicles with 0.05 M‐NaOH partially or completely removed a wide variety of polypeptides, including most of those in the M.W. range 46,000–83,000; none of the glycoproteins was solubilized. Essentially the opposite results were obtained when the vesicles were extracted with 0.5% Triton X‐100. Most of the vesicle's species were insensitive to several bisimidate cross‐linking reagents. These results suggest that: (a) The polypeptides of M.W. 125K, 107K, 95K, 83K, 75K, 70K, 60K, 46K, and 36K are externally oriented in the vesicle, whereas those of 66K, 55K, 33K, 26K, 22K, 19K, and 16K are internally oriented; (b) the vesicles contain two classes of glycoproteins, one consisting of high‐molecular‐weight, externally oriented species that are rich in galactose, and the other consisting of low‐molecular‐weight, internally oriented species of relatively low galactose content; (c) the vesicles contain a large class of nonglycosylated species that are relatively loosely attached to the membrane; and (d) most of the vesicles' polypeptides are probably freely mobile in the membrane. The organization of synaptic vesicle proteins is compared with that of the proteins of synaptosomal plasma membrane, with which the vesicle is believed to fuse.
Characterization of Water‐Soluble Products of Palmitic Acid β‐Oxidation by a Rat Brain Preparation
Authors: Kawamura Nariko , Kishimoto Yasuo
Published: October 2006

Abstract: (1) [1‐14C]Palmitic acid was oxidized to CO2 and a water‐soluble material by a rat brain preparation. The radioactive CO2 and water‐soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO2 production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water‐soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60–65% in citrate. The water‐soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10–40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat liver preparations was also camed out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of 14CO2. With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water‐soluble material.
Indolic Substances in Plasma, Cerebrospinal Fluid, and Frontal Cortex of Human Subjects Infused with Saline or Tryptophan
Authors: Gillman P. K. , Bartlett J. R. , Bridges P. K. , Hunt A. , Patel A. J. , Kantamaneni B. D. , Curzon G.
Published: October 2006

Abstract: Psychiatric patients undergoing the psychosurgical operation of stereotactic subcaudate tractotomy were infused intravenously with either saline or L‐tryptophan (15 mg/kg/h). Plasma, lumbar cerebrospinal fluid (CSF), ventricular CSF and a specimen of frontal cortex were collected. The relationships of plasma concentrations of substances claimed to influence brain tryptophan concentration (total tryptophan, free tryptophan, large neutral amino acids) with the concentration of tryptophan in the cortex and CSF were investigated. Tryptophan infusion resulted in plasma tryptophan values comparable to those found after oral doses used in treating depression or insomnia, and about sixfold increases of tryptophan in the cerebral cortex. Increased brain 5‐hydroxytryptamine synthesis was indicated by significant rises of CSF 5‐hydroxyindoleacetic acid. The concentration of plasma free tryptophan was a better predictor than plasma total tryptophan of cortex tryptophan concentration. As all correlation coefficients of plasma versus brain or plasma versus ventricular CSF tryptophan concentrations were decreased when allowance was made for differences of concentration of large neutral amino acids, the results suggest that the role of these substances within their physiological range as inhibitors of tryptophan transport to the brain may previously have been overemphasised.
Identification of β‐Adrenergic Receptor Binding Sites in Rat Brain Micro vessels, Using [125I]Iodohydroxybenzylpindolol
Authors: Kobayashi Hideyuki , Memo Maurizio , Spano Pier Franco , Trabucchi Marco
Published: October 2006

Abstract: Brain microvessels were prepared from rat cerebral cortex. The purity was confirmed by phase‐contrast microscopy and by the measurement of an enzymatic marker, γ‐glutamyltranspeptidase. The microvessel preparation was subjected to radioreceptor assay using a 125I‐labelled β‐adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80 μg per tube. It was saturated at 200 PM IHYP concentration. The KD, value calculated by Scatchard analysis was 69.4 ± 9.9 PM. The maximum binding (Bmax) was 107 ± 4 fmol/mg protein. The binding reached equilibrium within 30 min and was dissociated by addition of (−)‐propranolol. The inhibitory effects of isomers of propranolol and iso‐proterenol on this binding showed that (−)‐isomers were two orders of magnitude more potent than the (+)‐isomers. Other neurotransmitters did not affect IHYP binding. The characteristics of the binding, saturability, high affinity, reversibility and stereospecificity, suggest that IHYP is bound to β‐adrenergic receptor sites located on brain microvessels.
α‐Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax
Authors: Jones Stephen W. , Sudershan Puppala , O'Brien Richard D.
Published: October 2006

Abstract: House fly heads contain a site that binds α‐bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α‐bungarotoxin (labeled with [3H]pyridoxamine phosphate) reversibly with a Kd of 6 nM. The effects of 48 drugs have been compared on the α‐bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α‐bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl‐β‐methylcholine, but lower affinities for nicotine, atropine, and dihydro‐β‐erythroidine.
Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites
Authors: Lemaire Simon , Livett Bruce , Tseng Richard , Mercier Paulette , Lemaire Irma
Published: October 2006

Abstract: In isolated bovine adrenal chromaffin cells, β‐endorphin, dynorphin, and levorphanol caused a dose‐dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 μM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD= 0.59 nM). β‐Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5‐I2Tyr1]‐β‐endorphin, [3,5‐I2Tyr1]‐dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 of 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with β‐endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh‐evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 μM, respectively). Inhibition of CA release was also obtained with naloxone (ID50= 14 μM) and naltrexone (ID50 > 10−4 M), two classical antagonists of opiate receptors, and this effect was additive to that of β‐endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.
Structure of Bovine P2 Basic Protein: Sequence of a Carboxyterminal Segment That Is a Neuritogen in Rabbits
Authors: Hsieh D. L. , Weise M. J. , Levit S. , Powers J. M. , Brostoff S. W.
Published: October 2006

Abstract: The sequence of the carboxyterminal 18 amino acids released by cyanogen bromide digestion of the bovine P2 protein was determined. It has several interesting structural and immunological properties. It contains the only two half‐cystines in the molecule that have the capacity to form an intrachain disulfide bond. Using the rabbit as a test animal, this carboxyterminal peptide was capable of producing experimental allergic neuritis. The sequence of this peptide is Val‐Val‐Glu‐Cys‐Lys‐Met‐Lys‐Asp‐Val‐Val‐Cys‐Thr‐Arg‐Ile‐Tyr‐Glu‐Lys‐Val.
Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study
Authors: Ghalidour M. S. , Derer P. , Labourdette G. , Delaunoy J. P. , Langley O. K.
Published: October 2006

The glial cell contents of S lOO protein, 2′3′‐cyclic AMP, 3′‐ phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the SlOO protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S 100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.
Control of Noradrenaline Release from Hippocampal Synaptosomes
Authors: West D. P. , Fillenz M.
Published: October 2006

Abstract Potassium‐evoked tritiated noradrenaline (NA) release from hippocampal synaptosomes was measured with a superfusion method. A single 2‐min high‐K+ pulse released 39% of the vesicular NA by a Ca2+‐dependent mechanism; the Ca2+‐independent release was negligible. After changing the vesicular NA store size by pretreating rats with either α‐methyl‐para‐tyrosine, 500 mg/kg, or tranylcypromine, 10 mg/kg, a single K+ pulse released a constant percentage of the vesicular NA. With two K+ pulses, however, there was a reduction in the percentage of vesicular N A released in response to the second pulse.
Inhibition of Axoplasmic Transport in the Optic System by Kainic Acid
Authors: Gomez‐Ramos P. , Donoso J. A. , Samson F. E.
Published: October 2006

Abstract— Axoplasmic transport along the optic axons was studied after intraocular injections of kainic acid (KA). Transport of labeled material did not initiate from the eye when KA was injected simultaneously with the protein precursor [3H]proline. When KA was injected after axoplasmic transport of labeled proteins had begun, no additional radioactive material moved out of the retinal ganglion cells. However, the labeled material already present in the optic nerve at the time of KA injection continued to move, and accumulated at the nerve endings. Although KA reduces the incorporation of precursor, this effect of KA on axoplasmic transport appears to be more than a consequence of inhibition on precursor uptake or protein synthesis. Recovery from this KA action began 6 h after exposure to KA and was about 50% recovered by 36 h. The extent of the recovery remained at this level for as long as a week, which suggested a partial recovery of the ganglion cells. A second exposure to KA after the inner plexiform layer had virtually disappeared was as effective as the first exposure in preventing the appearance of transported protein in the optic nerve, suggesting a direct action of KA on the ganglion cells. We interpreted the results to indicate that KA interferes with the initiation phase of axoplasmic transport in ganglion cells and this effect is partially reversible.
The Characterization of Tubulin in CNS Membrane Fractions
Authors: Strocchi Paola , Brown Beverly A. , Young Janis D. , Bonventre Joseph A. , Gilbert Jeffrey M.
Published: October 2006

Abstract— Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two‐dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the β‐tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the β‐tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic β‐tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the β‐tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the β‐tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one‐dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic β‐tubulin pattern; however, the peptide maps of the PM and SER β‐tubulin region were identical to the cytoplasmic β‐tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic α‐tubulin. SER and PM contained proteins in the α‐tubulin region with a similar, but not identical, peptide analysis to cytoplasmic α‐tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule‐associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.
Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin
Authors: Rapaport Raymond N. , Benjamins Joyce A.
Published: October 2006

Abstract: Sciatic nerves from 9‐day‐old rat pups were removed, sliced into 0.4‐mm sections, and incubated with [3H]fucose or [14C]glycine precursors. The nerve slice system gave nearly linear incorporation of [3H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [3H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10−8m to 1.5 × 10−6m. Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P0, with no other major constituents. This system was used in fucose‐chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P0 into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P0 myelin.
The Action of Adenosine Analogs on PC12 Cells
Authors: Guroff Gordon , Dickens Geneva , End David , Londos Constantine
Published: October 2006

Abstract: PC12 cells, a nerve growth factor–responsive clone of rat pheochromocytoma, contain a membrane–bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase–stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5′‐N‐ethyIcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine–evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non–histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.
Dermorphins, Opioid Peptides from Amphibian Skin, Act on Opioid Receptors of Mouse Neuroblastoma x Rat Glioma Hybrid Cells
Authors: Glaser Thomas , Hübner Karin , Castiglione Roberto de , Hamprecht Bernd
Published: October 2006

Abstract: Dermorphin and its Hyp6 analogue are opiate‐like heptapeptides originally discovered in frog skin and characterized by the presence of a d‐Ala2 residue in their sequence. They were assayed for their capacity to compete with [3H]Leu‐enkephalin for binding to opioid receptors in membranes of neuroblastoma x glioma hybrid cells. In the presence of 7 nm‐[3H]Leu‐enkephalin, the concentrations at which they caused 50% inhibition of [3H]enkephalin binding (IC50 values) are 0.1 μm and 0.3 μm, respectively. In contrast, the synthetic l‐Ala2‐dermorphin shows very low affinity for the opioid receptors. In addition, like other opioid peptides, dermorphin and Hyp6‐dermorphin inhibit the elevation by prostaglandin E, (PGE1) of the level of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) (IC50 values 0.2μm and 0.4 μm, respectively). The inhibition is prevented by the opiate antagonist naloxone, l‐Ala2‐dermorphin is at least three orders of magnitude less potent in inhibiting the PGE1‐evoked increase in the level of cyclic AMP. The results show that peptides with an amino acid sequence quite different from that of the enkephalins can bind to opioid receptors of the hybrid cells.
Regionally Selective Metabolic Effects of Hypoglycemia in Brain
Authors: Ratcheson Robert A. , Blank Ann C. , Ferrendelli James A.
Published: October 2006

Abstract: Regional CNS levels of glucose reserves, glycolytic intermediates, and high‐energy phosphate reserves were measured in insulin‐treated, hypoglycemic rats and correlated with EEG activity. Intravenous administration of insulin to paralyzed, ventilated animals causes concomitant reduction of blood glucose levels and progressive abnormality and eventual loss of EEG activity. In all regions of brain examined, glucose and glycogen levels decrease until they are essentially depleted, and glucose‐6‐phosphate and fructose‐1,6‐biphosphate fall approximately 80%. Pyruvate levels decrease 50% in cerebral cortex and brain stem and a lesser amount in striatum, hippocampus, thalamus, and cerebellum. Lactate levels fall 50–60% in all regions except cerebellum, where no change is observed. ATP and phosphocreatine levels remain normal until the EEG is isoelectric, and then decrease in all regions except cerebellum. These results demonstrate that hypoglycemia does not have a uniform effect on brain glucose and energy metabolism, and cerebellum seems to be relatively protected.
Effects of Cyclic AMP Analogues and Phosphodiesterase Inhibitors on K+‐Induced [3H]Noradrenaline Release from Rat Brain Slices and on Its Presynaptic α‐Adrenergic Modulation
Authors: Werner Johan , Schoffelmeer Anton N. M. , Mulder Arie H.
Published: October 2006

Abstract: The possible role of cyclic AMP in the presynaptic α‐adrenoceptor‐mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyrylcyclic AMP (db‐cAMP) and 8‐bromo‐cyclic AMP (8‐Br‐cAMP) dose‐dependently (10−4‐10−2M) enhanced K+‐induced [3H]NA release, maximally to about 160% of control. In contrast, db‐cAMP had no effect on calcium‐induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3‐isobutyl‐1‐methylxanthine (IBMX), 7‐benzyl‐IBMX, 4‐(3‐cyclopentyloxy‐4‐methoxyphenyl)‐2‐pyrrolidone (ZK 62771), and 4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro 20‐1724) appeared to inhibit K+‐induced [3H]NA release in a dose‐dependent (10−5‐10−3M) manner. At a concentration of 10−4M, ZK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10−6M phentolamine nor by 10−3M db‐cAMP or 10−4M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitory effect of the a‐adrenoceptor agonist oxymetazoline (1 μM) and the enhancing effect of the antagonist phentolamine (1 μM) on [3H]NA release were significantly decreased in the presence of 10−3M db‐cAMP or 8‐Br‐cAMP, whereas 10−4M ZK 62771 had no effect. In the presence of 10−2M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 μM) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic α‐receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of the PDE inhibitors are discussed.
Comparison of Axonal Transport of Cytoplasmic‐ and Particulate‐Associated Tubulin in Rat Optic System
Authors: Goodrum Jeffry F. , Morell Pierre
Published: October 2006

Abstract: Adult rats were injected intraocularly with [35S]methionine and killed from 1 to 10 weeks later. Optic nerves, optic tracts, and superior colliculi were dissected and then homogenized and separated into soluble and particulate fractions by centrifugation. Radioactivity coelectrophoresing with tubulin in buffers containing sodium dodecyl sulfate was determined (in cytoplasmic fractions, preliminary enrichment was achieved by vinblastine precipitation). Accumulation of radioactive tubulin along the optic pathway occurred in parallel (and in approximately equal amounts) in cytoplasmic and particulate fractions. Transported tubulin peaked at approximately 2 and 4 weeks in the optic nerve and tract, respectively, corresponding to a transport rate of ∼ 0.4 mm/ day. There was little diminution in the amount of transported tubulin between optic nerve and tract, suggesting tubulin was not degraded in the axon. Accumulation in the superior colliculus reached a plateau by 4 weeks at less than 20% of the peak in the optic nerve, indicating turnover of tubulin at the nerve endings. The α/β subunit labeling ratio (radioactivity distribution between the tubulin subunits) was 0.57 for both cytoplasmic‐ and particulate‐transported tubulin. In contrast, this ratio was 0.69 for whole brain tubulin prepared by vinblastine precipitation of soluble material. Isoelectric focusing and two‐dimensional gel electrophoresis showed that the subunit compositions (microheterogeneity of the α and β bands) of transported tubulins in the cytoplasmic and particulate fractions were very similar. However, some differences relative to whole brain tubulin were noted; a tubulin subunit not identifiable in whole brain tubulin preparations but present in both soluble‐ and particulate‐transported tubulin was observed. Because of the compositional and metabolic similarities of transported tubulin in the soluble and particulate fractions, we conclude that they form a common metabolic pool. This suggests either that, at least for some membranes, the well‐characterized tight association between particulate tubulin and membranes may be artifactual or else that an equilibrium exists between soluble and particulate tubulin.
Effects of the Convulsant Methionine Sulfoximine on Striatal Dopamine Metabolism
Authors: Rothstein Jeffrey D. , Tabakoff Boris
Published: October 2006

Abstract: Experiments were conducted to investigate the effects of the convulsant l‐methionine‐dl‐sulfoximine (MSO) on striatal dopamine (DA) metabolism. Intraventricular injections of MSO produced a transient increase in striatal DA release followed by inhibition of DA release for up to 3 days, which paralleled the inhibition by MSO of the enzyme glutamine synthetase (GS). DA synthesis was decreased for up to 24 h after injection of MSO, but returned to normal within 3 days after MSO administration. Intrastriatal injections of MSO produced a pronounced decrease in striatal DA release and inhibition of striatal GS activity 24 h postinjection but, unlike intraventricular MSO, did not produce behavioral convulsions. Glutamate‐DA interactions may be responsible for the observed effects.
Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion
Authors: Kwok Y. N. , Collier B.
Published: October 2006

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl‐CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10−5M) and [3H], [1‐4C], or [2‐14C]acetate (103M); perfusion for 60 min or with [3H]acetate (10−2M) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca2+‐dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 mM K+, the labeling of acetylcholine from [3H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2‐14C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [3H]acetate was shown during a 15‐min resting period following a 60‐min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl‐CoA to choline acetyltransferase from a source other than acetate.
Binding of [3H] γ‐Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline
Authors: Jordan C. C. , Matus A. I. , Piotrowski W. , Wilkinson Diane
Published: October 2006

Abstract: The binding of [3H] γ‐aminobutyric acid ([3H]GABA) and [3H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ‐aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent KD and Bmax values for [3H]GABA at 4°C were KD1, 20 nM; KD2,165 nM; Bmax1, 0.48 pmol;Bmax2, 6.0 pmol. mg−1; for [3H]muscimol at 4°C they were: KD1, 1.75 nM; KD2, 17.5 nM; Bmaxl, 0.84 pmol. mg−1; Bmax2, 4.8 pmol.mg−1; and for [3H]muscimol at 37°C they were: KD1, 7.0 nM; Km, 60 nM; Bmax], 0.5 pmol‐mg−1; Bmax2, 7.2 pmol‐mg1. Under the experimental conditions used, the similar Bmilx values for [3H]GABA and [3H]muscimol binding to the SPM preparations suggests that the high‐ and low‐affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [3H]muscimol at 4CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high‐affinity site but noncompetitive at the low‐affinity site. At 37°C, antagonism was again competitive at the high‐affinity site but was of a mixed competitive/noncompetitive nature at the low‐affinity site. Assuming that binding to the high‐affinity site is associated with the pharmacological actions of bicuculline, the apparent KD values obtained suggest a pA2 value of 5.3 against [3H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [3H]GABA and [3H]muscimol suggest that [3H]GABA might be a more satisfactory ligand than [3H]muscimol in GABA radioreceptor assays.