bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucopolysaccharidosis Types IH, IS, II and IIIA: Glycosaminoglycans and Lipids of Isolated Brain Cells and Other Fractions from Autopsied Tissues
Authors: Constantopoulos George , Iqbal Khalid , Dekaban Anatole S.
Published: October 2006

Abstract: Brain cellular fractions were prepared in bulk from four non‐neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8‐month‐old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one‐half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four‐ to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides Gm2, Gm3, Gd3 and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of Gm3, Gm2 and Gd3 together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.
Subcellular Localization of Folate Turnover and Folate‐Dependent Enzyme Activity in Mouse Brain
Authors: Carl G. F. , Peterson J. D. , McClain L. D. , Bridgers W. F.
Published: October 2006

Abstract: Using 5‐[methyl‐14C]methyltetrahydrofolate and 5‐[2‐14C]methyl‐tetrahydrofolate, the rates of uptake and turnover of the methyl and folate moieties in mouse brain were compared. The methyl moiety was taken up and retained more readily than the folate moiety. Intracerebral injections of labeled folate were used to study the retention and subcellular turnover of folate. The injected folate exchanged slowly with the endogenous pool, stabilizing after 2 days. Analyses of the subcellular distribution of label with time showed that folate underwent intercompartmental exchange in all fractions except the synaptosomal particulate fractions, which appeared to contain a folate pool inert to exchange. The mitochondria] and synaptosomal soluble fractions exhibited a comparatively high content of labeled folate. The folate turnover rate was extremely slow. Subcellular localization of two folate‐dependent enzymes showed that /V5, Arlo‐methylenetetrahydrofolate reductase and N5‐methyltetrahydrofolate homocysteine methyltransferase activities were largely soluble, the latter exhibiting a very high specific activity in the synaptosomal lysate. The implications of these findings with respect to one‐carbon metabolism and the compartmentalization of folate function in mouse brain are discussed.
Competitive Inhibition of Catecholamine Uptake in Synaptosomes of Rat Brain by Rigid Bicyclo‐Octanes
Authors: Wong David T. , Bymaster Frank P. , Reid Leroy R.
Published: October 2006

Abstract: Cis‐3‐(3, 4‐dichlorophenyl)‐2‐N, N‐dirnethylaminomethyl‐bi‐cyclo‐[2, 2, 2]‐octane hydrochloride (LR5182) inhibited the uptake of dopamine by a synaptosomal fraction of corpus striatum of rat brain with an inhibitor constant (Ki value) of 6 nM. Kinetics analysis according to the methods of Lineweaver‐Burk and Dixon revealed a competitive inhibition of dopamine uptake of LR5182. When the uptake of dopamine by the noradrenergic terminals in the synaptosomal fraction of cerebral cortex was selectively abolished by desipramine, LR5182 competitively inhibited the uptake of dopamine by the dopaminergic terminals with a Ki value of 19 nM. LR5182 was also a competitive inhibitor of noradrenaline uptake by synaptosomes of hypothalamus and cerebral cortex with, however, one‐third to one‐tenth the effectiveness, as indicated by the larger Ki values, 52 and 58 nM, respectively. Structure‐activity studies with LR5182 and related compounds supported the idea that the uptake sites of dopamine favored a gauche conformer.
Cerebral Metabolic State During the Ethanol Withdrawal Reaction in the Rat
Authors: Hemmingsen R. , Chapman A. G.
Published: October 2006

Abstract: A severe ethanol withdrawal reaction was induced in rats by means of repeated intragastric intubation during a 4‐day period. At the peak of the withdrawal reaction cerebral cortical tissue was frozen in situ for analysis of glycogen, glucose, phosphocreatine, creatine, ATP, ADP, AMP, lactate, pyruvate, GAB A, β‐hydroxybutyrate, acetoacetate, cAMP and cGMP. Blood glucose concentration was also measured. The level of brain glycogen was decreased during ethanol withdrawal. Brain glucose concentration was increased, probably secondary to the increase in blood glucose concentration. The calculated NADH/NAD+ ratio was slightly increased during the withdrawal and brain ATP concentration and adenine nucleotide pool size were decreased. The adenylate energy charge remained unchanged. The overall changes in the metabolites were in agreement with the previously shown metabolic activation during ethanol withdrawal. The brain concentrations of ketone bodies (β‐hydroxybutyrate and acetoacetate) during withdrawal did not deviate from controls, indicating that no abnormal ketone metabolism had developed as a consequence of the long‐lasting ethanol intoxication. No changes were observed in the concentrations of GABA, cAMP, or cGMP in the rat cerebral cortex during ethanol withdrawal.
Calcium Binding Sites of Synaptosomes from Insect Nervous System as Probed by Trivalent Terbium Ions
Authors: Breer H. , Jeserich G.
Published: October 2006

Abstract: Terbium binding to synaptosomes from the central nervous system of the locust was studied by fluorescence spectroscopy and electron microscopy. The protein‐sensitized fluorescence of terbium was used to characterize the calcium binding sites of synaptosomes. As judged by electron microscopy and x‐ray analysis, terbium ions produced electron‐dense patches in regular arrays on the outer surface of synaptosomal membranes and induced marked aggregation of synaptic vesicles in isolated terminals.
Effects of Sodium and Bicarbonate Ions on γ‐Aminobutyric Acid Receptor Binding in Synaptic Membranes of Rat Brain
Authors: Kurioka Susumu , Kimura Yoshiaki , Matsuda Makoto
Published: October 2006

Abstract: Crude synaptic membranes treated with Triton X‐100 (TX) bound γ‐aminobutyric acid (GABA) to two classes of receptor site in Na+‐free 10 mM‐Tris‐sulfate buffer (pH 7.4), but to only a single class of receptor site in 10 MM Tris‐sulfate buffer (pH 7.4) containing 150 mM‐NaCl. The high‐affinity receptor site in TX membranes was specifically masked in the presence of Na+. However, TX membranes incubated in Krebs‐Ringer bicarbonate solution (pH 7.4) bound GABA to two classes of receptor site despite the presence of Na+. It was found that addition of bicarbonate ions to the Na+‐containing 10 mM‐Trissulfate buffer (pH 7.4) could restore the high‐affinity class of GABA receptors, rendering both classes detectable. This finding suggests that both Na+ and HCO‐3 may have a regulatory function on GABA binding to the receptor.
Glycolytic, Pentose‐Phosphate Shunt and Transaminase Enzymes in Gastrocnemius Muscle, Liver, Heart, and Brain of Two Mouse Mutants, 129 J‐dy and A2G‐adr, with Abnormal Muscle Function
Authors: Soothill Peter W. , Kouseibati Farid , Watts Rosemary L. , Watts David C.
Published: October 2006

Abstract: Aldolase and phosphoglycerate kinase activity were markedly reduced in muscle from two mouse mutants, 129 J‐dy and A2G‐adr, with abnormal muscle development. The pentose‐phosphate shunt enzymes, glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, were both greatly increased in the gastrocnemius of 129 J‐dy mice, but only the former was slightly increased in A2G‐adr muscle. Alanine and aspartate aminotransferase activities were normal or low in 129 J‐dy muscle but increased to approximately 200% in A2G‐adr muscle. Liver from 129 J‐dy mice showed increased activity of glucose‐6‐phosphate dehydrogenase. These findings are compatible with the well‐recognised lipid involvement in the 129 J‐dy mutant but indicate that an abnormality of amino acid metabolism in relation to energy supply is probably more important in the A2G‐adr mutant.
Determination of Picomole Amounts of Dopamine, Noradrenaline, 3,4‐Dihydroxyphenylalanine, 3,4‐Dihydroxyphenylacetic Acid, Homovanillic Acid, and 5‐Hydroxyindolacetic Acid in Nervous Tissue After One‐Step Purification on Sephadex G‐10, Using High‐Performance Liquid Chromatography with a Novel Type of Electrochemical Detection
Authors: Westerink B. H. C , Mulder T. B. A
Published: October 2006

Abstract: A determination of dopamine (DA), noradrenaline (NA), 3,4‐dihydroxyphenylalanine (DOPA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindolacetic acid (5‐HIAA) in nervous tissue is described. The method is based on a rapidly performed isolation of DA, NA, DOPA, DOPAC, HVA, and 5‐HIAA from one single nervous tissue sample on small columns of Sephadex G‐10, followed by reverse‐phase high‐performance liquid chromatography with electrochemical detection. A new type of electrochemical detector based on a rotating disk electrode (RDE) was used. The rotating disc electrode was found to be a reliable and sensitive amperometric detector with several advantages over the currently used thinlayer cells. The detector appeared very useful for routine analysis. Practical details are given for the routine use of the RDE. Brain samples containing no more than 75–150 pg (DA, DOPA, DOPAC, HVA, and 5‐HIAA) or 500 pg (NA) could be reproducibly assayed with high recovery (approx. 85%) and precision (approx. 5%), without the use of internal standards. Endogenous concentrations of DA, NA, DOPA, DOPAC, HVA, and 5‐HIAA were determined in eight brain structures.
Effect of Inhibitors of GABA Aminotransferase on the Metabolism of GABA in Brain Tissue and Synaptosomal Fractions
Authors: Löscher W
Published: October 2006

Abstract: Five inhibitors of the GABA degrading enzyme GABA‐aminotransferase (GABA‐T), viz., gabaculine, γ‐acetylenic GABA, γ‐vinyl GABA, ethanolamine O‐sulphate, and aminooxyacetic acid, as well as GABA itself and the antiepileptic sodium vdproate were administered to mice in doses equieffective to raise the electroconvulsive threshold by 30 V. The animals were killed at the time of maximal anticonvulsant effect of the respective drugs and GABA, GABA‐T and glutamate decarboxylase (GAD) were determined in whole brain and synaptosomes, respectively. The synaptosomal fraction was prepared from brain by conventional ultracentrifugation procedures. All drugs studied brought about significant increases in both whole brain and synaptosomal GABA concentrations, and, except GABA itself, inhibited the activity of GABA‐T. Furthermore, all drugs, except GABA and γ‐acetylenic GABA, activated GAD in the synaptosomal fraction. This was most pronounced with ethanolamine O‐sulphate, which induced a twofold activation of this enzyme but exerted only a weak inhibitory effect on GABA‐T. The results suggest that activation of GAD is an important factor in the mechanism by which several inhibitors of GABA‐T and also valproate increase GABA concentrations in nerve terminals, at least in the relatively non‐toxic doses as used in this study.
Solubilization of the Picrotoxinin Binding Receptor from Mammalian Brain
Authors: Davis William C , Ticku Maharaj K
Published: October 2006

Abstract: The binding sites for α‐dihydropicrotoxinin (DHP), which is a ligand for the picrotoxin‐sensitive component at the benzodiazepine‐γ‐aminobutyric acid‐receptor‐ionophore complex, has been solubilized from rat brain, using 1% Lubrol. A new assay, which involves precipitation of the [3H]DHP‐soluble protein complex by γ‐globulin and polyethylene glycol (PEG), followed by centrifugation, is described. The solubilized material bound DHP to two sites with apparent affinities of 0.038 and 1.85 μM. The binding of DHP to the solubilized receptors was inhibited by convulsant and depressant drugs with potencies similar to those required for membrane receptors. The ability of barbiturates to inhibit DHP binding to both solubilized and membrane receptors strongly suggests that barbiturates may interact with the picrotoxin binding component. These data suggest that ligand recognition properties of the picrotoxinin binding are not altered by solubilization. The binding was abolished by urea and partially destroyed by heating the soluble extract at 65°C for 30 min. This new method of measuring the binding of ligands to the solubilized receptors by PEG centrifugation might be used successfully in other solubilization studies.
Opioids, Noradrenaline and GTP Analogs Inhibit Cholera Toxin Activated Adenylate Cyclase in Neuroblastoma × Glioma Hybrid Cells
Authors: Propst Friedrich , Hamprecht Bernd
Published: October 2006

Abstract: D‐Ala2‐Met5‐enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma × glioma hybrid cells in a dose‐dependent manner even after the enzyme has been preactivated by cholera toxin. Half‐maximal inhibition and extent of inhibition are the same with native or cholera toxin‐activated enzyme. The inhibitions caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D‐Ala2‐Met5‐enkephalin on cholera toxin‐activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl‐5′‐yl‐imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 μM or above, inhibition even at 0.1 μM. The inhibitory effect of the non‐hydrolysable GTP analog is antagonized by GTP. Guanyl‐5′‐yl‐methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin‐stimulated or prostaglandin E1‐stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl‐5′‐yl‐phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide‐binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.
Developmental Changes in Polypeptide Composition of, and Precursor Incorporation into, Cellular and Subcellular Fractions of Rat Cerebral Cortex
Authors: Burgoyne R. D. , Rudge J. S. , Murphy S.
Published: October 2006

Abstract: Neuronal‐enriched and glial‐enriched fractions from rat cerebral cortex at 2. 5, 9, 14 and 23 days postnatally, and subcellular fractions from 2, 14 and 46 day old rat were prepared. The polypeptide composition of all fractions was analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electro‐phoresis and quantified by densitometry. Fifty‐nine polypeptides (mol. wts., 13,200–251,000) were resolved in the cell fractions of which the majority remained unchanged throughout postnatal development. Three polypeptides (mol. wts., 102,000, 56,000, 53,700) were found to increase in amount devel‐opmentally in both cellular fractions, the latter two showing a peak in relative amount on day 14 and a subsequent decline. Three polypeptides (mol. wts., 47,000, 28,200, 17,400) were found to be common to the glial‐enriched fraction as well as the myelin fraction, and all showed a developmental increase. The neuronal‐enriched fraction was found to be enriched in five polypeptides of which one (mol. wt., 51,900) showed a developmental increase after ten days postnatally, the others (mol. wts., 178,700, 142,000, 109,000, 24,000) showing a decrease. In vitro incorporation of [35S]‐methionine into the glial‐enriched fraction was carried out, and a developmental decline was observed in the labelling of a polypeptide of 42,000 mol. wt.
The Uptake of Carnitine by Slices of Rat Cerebral Cortex
Authors: Huth P. J. , Schmidt M. J. , Hall P. V. , Fariello R. G. , Shug A. L.
Published: October 2006

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue‐medium gradients of 38 ± 3 for L‐[14CH3]carnitine and 27 ± 3 for D‐[14CH3]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L‐ and D‐carnitine showed saturability. The estimated values of Km for L‐ and D‐carnitine were 2.85 mM and 10.0 mM, respectively; but values of Vmax (1 μmol/min/ml in‐tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L‐carnitine. Carnitine uptake was inhibited by structurally related compounds with a four‐carbon backbone containing a terminal carboxyl group. L‐Carnitine uptake was competitively inhibited by γ‐butyrobetaine (Ki= 3.22 mM), acetylcarnitine (Ki= 6.36 mM), and γ‐aminobutyric acid (Ki= 0.63 mM). The data suggest that carnitine and γ‐aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N2 atmosphere, 2,4‐dinitrophenol, carbonylcyanide‐N‐chlorophenylhydrazone, potassium cyanide, n‐ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na+‐dependent, but did not require K+ or Ca2+.
Effects of Conformationally Restrained Analogues of Serotonin on Its Uptake and Binding in Rat Brain
Authors: Friedman Eitan , Meller Emanuel , Hallock Marilyn
Published: October 2006

Abstract: Two series of serotonin analogues, in which the side chain amino group is constrained in the gauche or trans conformation, were utilized to study the preferred conformation of serotonin for interaction with two different neuronal sites. 6‐Hydroxytetrahydro‐β‐carboline and 6‐hydroxy‐3‐aminotetrahydrocarbazole were found to be potent inhibitors of serotonin uptake into hypothalamic synaptosomes, with IC50 values of 0.13 μM for each analogue. The type of inhibition, as determined by Dixon plots, was found to be competitive, with Ki's of 3.0 × 10−8 M and 4.6 × 10−8 M for the β‐carboline and carbazole derivatives, respectively. Methoxylation or lack of a hydroxy group at the 6 position of the carbazole derivative did not alter inhibitory potency, while methoxy or benzyloxy substitution decreased potency 22‐ to 326‐fold. The serotonin analogues were 20 to 30 times less potent in inhibiting the synaptosomal transport of the catecholamines. With regard to [3H]serotonin binding to membranes obtained from brain homogenates, both analogues exhibited poor affinity compared with the transmitter. However, the β‐carboline derivative was three times as potent as the carbazole analogue. These findings and earlier ones with regard to the effect of the serotonin analogues on brain monoamine oxidase activity support the idea that serotonin analogues interact differentially with the three different serotonergic sites examined.
Uptake, Release, and Metabolism of D‐ and L‐α‐Aminoadipate by Rat Cerebral Cortex
Authors: Charles A. K. , Chang Yung‐Feng
Published: October 2006

Abstract: Accumulation of L‐α‐aminoadipate by rat cerebral cortical slices is a stereospecific and Na+‐dependent process. The uptake of this compound is also temperature‐dependent, with a Km, of 1.6 × 10−4M for the high‐affinity system. D‐α‐Aminoadipate has characteristics similar to those displayed by the L‐isomer but to a lesser degree. L‐Glutamate and L‐aspartate inhibit the uptake of L‐α‐aminoadipate. D‐ and L‐α‐Aminoadipate are, respectively, weak uncompetitive and weak competitive inhibitors for the uptake of L‐glutamate and L‐aspartate. Both enantiomers inhibit GABA uptake but in quite different ways. The release of L‐α‐aminoadipate from the cerebral cortical slices is stimulated by a high concentration of K+ ions in the presence of Ca2+ in the perfusion buffer; the D‐isomer displays this property to a lesser degree. The omission of Ca2+ markedly reduces the release of these two compounds. Less than 10% of the preloaded D‐ and L‐α‐aminoadipate are metabolized by the cerebral cortex during 40 min of superfusion. The possibility of L‐α‐aminoadipate as a neurotransmitter candidate is discussed.
Lumiflavin and Lumichrome Transport in the Central Nervous System
Authors: Spector Reynold
Published: October 2006

Abstract: The transport of the lipid‐soluble sugarless flavins, [14C]lumiflavin and [14C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [14C] flavins by a saturable, energy‐requiring process that did not depend on binding or intracellular metabolism of the [14C] flavins. Both sugar‐containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [14C]lumiflavin and [14C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [14C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [14C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety‐five percent of the [14C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo, 2 h after the intraventricular injection of 6.5 nmol [14C]lumiflavin, almost all of the [14C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [14C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.
Preparation of Porcine Neurophysin Proteins by High Performance Liquid Chromatography
Authors: Richter W. , Schwandt P.
Published: October 2006

Abstract: The crude neurophysin containing extract from posterior lobes of porcine pituitaries was roughly purified by gel chromatography. 15 mg of the lyophilized neurophysin complex were completely separated by HPLC yielding in neurophysin I1 (3.6 mg), I2 (4.0 mg), II (4.6 mg) and III (1.9 mg). All of the neurophysins were homogenous by PAGE and SDS‐electrophoresis, isoelectrofocussing, amino‐acid composition and N‐ and C‐terminal amino acid analysis. In conclusion, HPLC is a reliable and quick method for the preparation of pure neurophysins.
Attachment of the Synapse‐Specific Phosphoprotein Protein I to the Synaptic Membrane: A Possible Role of the Collagenase‐Sensitive Region of Protein I
Authors: Ueda Tetsufumi
Published: October 2006

The purified synapse‐specific phosphoprotein Protein I was previously shown to be degraded by a bacterial collagenase, through a series of intermediates, to a collagenase‐resistant fragment of molecular weight about 48,000 containing a phosphorylated serine residue. In this study, a purified synaptic membrane fraction containing Protein I was treated with Cl. his‐tolyticum collagenase; membrane‐bound and membrane‐free proteins were then phosphorylated using [γ‐32P]ATP and analyzed by SDS‐polyacrylamide gel electrophoresis and autoradiography. It was observed that Protein I bound to the synaptic membrane was susceptible to the collagenase and degraded to fragments of molecular weights about 68,000, 62,000, and 48,000; the 68,000 fragment remained bound to the membrane whereas the 62,000 and 48,000 fragments were dissociated from the membrane. These observations suggest that the peptide moiety of mol. wt. 6000, present in the 68,000 fragment but absent from the 62,000 fragment, may play a crucial role in anchoring Protein I to the synaptic membrane.
Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis
Authors: Fando Juan L. , Domínguez Fernando , Herrera Emilio
Published: October 2006

The concentration of most amino acids was higher in the brains of 19‐ and 21‐day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19‐ and 21‐day pregnant rats but was higher in their fetuses, especially in the 21‐day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.
Inhibition of [3H]GABA Binding to Postsynaptic Receptors in Human Cerebellar Synaptic Membranes by Carboxyl and Amino Derivatives of GABA
Authors: Breckenridge Robin J. , Nicholson Sydney H. , Nicol Alan J. , Suckling Colin J. , Leigh Beatrice , Iversen Leslie
Published: October 2006

Abstract Fifty synthetic analogues of GABA were tested for their ability to interact with GABA receptors, using [3H]GABA binding to human cerebellar membranes as an in vitro model. The most active compounds were found to be aliphatic and heterocyclic aminosulphonic acids. Compounds with highly substituted nitrogen atoms were only weakly active unless a long alkyl chain, which can interact with the postsynaptic membrane, was present. It was concluded that a pyramidal nitrogen atom is favoured for binding of GABA analogues to human cerebellar membranes
Alterations of Phospholipid Metabolism in Rat Cerebral Cortex Mince Induced by Cationic Amphiphilic Drugs
Authors: Pappu Anuradha S. , Hauser George
Published: October 2006

Abstract Cationic amphiphilic drugs (CADs) of varied clinical use were screened to determine their capacity to alter the pattern of labeling with 32Pj of cerebral cortex mince phospholipids. The altered phospholipid labeling patterns were qualitatively similar, the prominent features being reduced incorporation into phosphatidylcholine and increased incorporation into phosphatidic acid. Relative potencies were: (±)‐propranolol > chlorpromazine = 4,4′‐bis(diethylaminoethoxy) α,β‐diethyldiphenylethane > desipramine > di‐bucaine > pimozide > oxymetazoline = fenfluramine = haloperidol = chloroquine > amphetamine = no drug added. Propranolol was used to study the action of CADs further. Its effect was time‐ and dose‐dependent, but in contrast with pineal gland, no label appeared in phosphatidyl‐CMP (CDP‐diacylglycerol), nor did dialysis of the mince to reduce diffusible substrates or exogenous addition of substrates cause appearance of liponucleotide. Thus lack of diffusible precursors is not responsible for CAD effects in vitro. Pulse‐chase experiments with 32P1 and [2‐3H]glycerol suggested that inhibition of phosphatidate phosphohydrolase may be partly responsible for the observed alterations in phospholipid labeling in the presence of CADs.
Interspecies Comparison of Brain Ganglioside Patterns Studied by Two‐Dimensional Thin‐Layer Chromatography
Authors: Hunter Gale D. , Wiegant Victor M. , Dunn Adrian J.
Published: October 2006

Abstract Brain gangliosides from four vertebrate classes (fish, amphibia, aves, and mammalia) were studied by patterns generated with the use of two‐dimensional thin‐layer chromatography. In an effort to retain possible alkali‐labile gangliosides, samples were not base‐hydrolyzed. Resultant chromatograms revealed complex patterns of the major known gangliosides and a number of minor and trace molecular species previously unresolved. More than 30 resorcinol‐positive components were detected in mammalian neural samples. Our data indicate both qualitative and quantitative differences in the chromatogram patterns between the vertebrate classes, but potentially greater qualitative similarity exists between the lower and higher classes than has previously been noted.
Identification with a Monoclonal Antibody of a Phylogenetically Conserved Brain‐Specific Determinant on a 130,000 Molecular Weight Glycoprotein of Human Brain
Authors: Lakin Kenneth H. , Fabre John W.
Published: October 2006

Abstract— Human brain glycoproteins depleted of Thy‐1 antigen were used to immunise Balb/c mice for monoclonal antibody production. The F3‐87‐8 antibody described in this paper interacts with a determinant present in large amounts on all human brain subregions studied (cerebral cortical grey matter, white matter, caudate, thalamus, dentate nucleus, putamen, cerebellar cortex) but absent from all other tissues examined (liver, heart, kidney, spleen, thymus, lymph node, erythrocyte, adrenal gland, and peripheral nerve). The determinant is conserved in mammalian evolution, as the brains of the rat and dog have amounts equal to that found in human brain. Balb/c mouse brain has approximately one‐third as much antigen activity as these other mammalian brains, whereas brains of the frog and chicken have no detectable antigenic activity. Developmental studies showed that 16‐week human foetal brain and neonatal dog brain had little or no antigen activity, indicating a dramatic increase in the amount of the determinant with brain maturation. Biochemical studies showed that the F3‐87‐8‐bearing molecule was a major sialoglyco‐protein of human brain with an apparent molecular weight of 130,000. It was shown by immunofluorescence to be particularly localised in what appeared to be fibre tracts in the thalamus and basal ganglia, and in the dentate nucleus, although all regions including grey matter were stained.
Cadaverine in the Rat Brain: Regional Distribution and Acylation of [14C]Cadaverine In Vivo and Uptake In Vitro
Authors: Salzman Steven K. , Stepita‐Klauco Matej
Published: October 2006

Abstract— The regional distribution and acylation of intraventricularly injected [14C]cadaverine was studied in the rat brain over a 48‐h period. The concentrations of labeled cadaverine and its acyl derivatives, N‐monoacetylcadaverine and N‐monopropionylcadaverine, were determined in the telencephalon, striatum, hypothalamus, midbrain, cerebellum, and medulla‐pons by TLC of their 5‐dimethylamino‐1‐naphthalenesulfonyl derivatives, followed by liquid scintillation spectrometry. The apparent passage of radioactivity from the ventricular space into brain tissue was slow, with the concentrations reaching a peak at 24 h after injection. The percentage of radioactivity in the acyl forms of cadaverine, however, was maximal 4 h after injection, with the propionyl form predominating. The telencephalon, striatum, and hypothalamus contained the highest concentrations of radioactivity, in all three forms, at all elapsed times. A high‐affinity uptake mechanism for cadaverine was demonstrated in slices of these tissues. This process was completely inhibited by equimolar concentrations of unlabeled putrescine.
ω‐Fluoromethyl Analogues of ω‐Amino Acids as Irreversible Inhibitors of 4‐Aminobutyrate: 2‐ Oxoglutarate Aminotransferase
Authors: Bey P. , Jung M. J. , Gerhart F. , Schirlin D. , van Dorsselaer V. , Casara P.
Published: October 2006

Abstract— ω‐Monofluoromethyl and ω‐difluoromethyl analogues of the known substrates of GABA‐T, β‐alanine, γ‐aminobutyric acid, and 5‐aminopentanoic acid, are time dependent inhibitors of purified 4‐aminobutyrate: 2‐oxoglutarate aminotransferase (GABA‐T). The inhibitory activity decreases with increasing chain length. In vitro, inhibitory activity decreases with increasing fluorine substitution of the methyl group. In vivo, β‐difluoromethyl‐β‐alanine and 2,4‐difluoro‐3‐aminobutyric acid are the most potent GABA‐T inhibitors ever reported. Trifluoromethyl derivatives are devoid of GABA‐T inhibitory activity in vitro or in vivo.
Biphasic Modulation by ACTH‐Like Peptides of Protein Synthesis in a Cell‐Free System from Rat Brain
Authors: Schotman Peter , Allaart Jeanne
Published: October 2006

Abstract— Brain protein synthesis in a cell‐free system was stimulated by 108m‐ACTH1‐24. This stimulatory effect was completely inhibited by aurintricar‐boxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N‐terminal peptide sequence 4‐ 10 exerted a biphasic modulation of cell‐free protein synthesis, i.e., a stimulation at low concentrations (10−8 and 10−10m) and an inhibition at a high concentration (10−4m). The d‐isomer, ACTH4‐10‐7‐d‐phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4‐10‐7‐l‐pheat 10−8m and 10−4m.
Effect of Ischemia on the Activity of DNA‐Dependent RNA Polymerase and DNA Polymerase
Authors: Gottlieb M. , Chavko M. , Maršala J.
Published: October 2006

Abstract: Ischemia, anoxia, and hypoxia of the brain have been shown to inhibit protein synthesis in the central nervous system. To obtain data on the changes in DNA‐dependent RNA and DNA polymerases as they pertain specifically to neurons and glia, nuclear enriched neuronal and glial fractions were prepared, by sucrose‐gradient centrifugation, from spinal cords of adult dogs that had been subjected to prolonged ischemia. The isolated fractions were assayed for enzyme activity by a radiochemical technique. RNA polymerase was affected more than DNA polymerase, activity being reduced considerably in both neurons and glia. Possible causes of the difference in sensitivity to ischemia are discussed.
Phosphate Ion Transport in Rabbit Brain Synaptosomes
Authors: Salamin A. , Deshusses J. , Straub R. W.
Published: October 2006

Abstract: Synaptosomes (vesicles of nerve endings) isolated from rabbit brain were studied as a model system for the uptake of inorganic phosphate. The phosphate uptake showed a sodium‐dependent, saturable component with a Kt of 0.29 mm, The sodium‐dependent component was larger at pH 6 than at pH 7.4 or 8. Application of potassium salts, ouabain, monensin, nigericin or FCCP decreased the uptake. The results indicate that the sodium‐sensitive phosphate influx is dependent on the Na+ gradient and on the membrane potential, which might act, preferentially, on the transport of the monovalent phosphate ion.
Glyoxylate Aminotransferase Activity in Vertebrate Retina
Authors: Dasgupta P. , Narayanaswami A.
Published: October 2006

Abstract: Glyoxylate aminotransferase activity with l‐glutamate, l‐glutamine and l‐alanine as substrates, has been estimated in rat, guinea pig and goat retinae. Enhanced enzyme activity was found in the presence of Triton X‐100. The presence of significant aminotransferase activity raises a possibility of glyoxylate as a precursor of glycine in vertebrate retina.
Characterization of 2′:3′‐Cyclic Nucleotide 3′‐Phosphodiesterase: Rapid Isolation, Native Enzyme Analysis, Identification of a Serum‐Soluble Activity, and Kinetics
Authors: Clapshaw Patric A. , Müller Hans Werner , Seifert Wilfried
Published: October 2006

Abstract: The enzyme 2′:3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.
Hypothalamic Neurons in Dissociated Cell Culture: The Mechanism of Increased Survival Times in the Presence of Non‐Neuronal Cells
Authors: Whatley S. A. , Hall C. , Lim L.
Published: October 2006

Abstract: Rat hypothalamic neurons were cultured in the presence of fluorodeoxyuridine to inhibit nonneuronal cell proliferation. Under these conditions, neuronal cell survival was dependent on contact with homologous nonneuronal cells. This phenomenon did not seem to be due to the release of diffusable trophic factors, since neither growth on polylysine in the close proximity of nonneuronal cells nor the use of preconditioned medium significantly increased neuronal survival. However, metabolically active cell layers were required, since growth on heat‐killed or fixed homologous nonneuronal cells did not increase neuronal survival. The increased survival of neurons seen here in the presence of homologous nonneuronal cells therefore appears to be due to metabolic co‐operation mediated by specific, direct cell‐cell contact.
Effect of Dietary Lipid on the Acyl Group Composition of Glycerophospholipids of Brain Endothelial Cells in the Developing Rat
Authors: Matheson Dugald F. , Oei Robert , Roots Betty I.
Published: October 2006

Abstract: The effects of dietary lipid on the acyl group composition of glycerophospholipids of isolated brain endothelial cell fractions have been determined in the developing rat. Rats were fed high‐protein diets containing differing dietary linoleic/linolenic acid ratios but having a similar caloric percentage, or were fed a fat‐free diet. With a diet supplemented with corn oil (linoleic/linolenic ratio, 47:1), the proportion of acyl groups of the linolenic acid series (n‐3) declines in all glycerophospholipid fractions compared with the controls (linoleic/linolenic ratio, 8.5:1), but the proportion of linoleic acid series (n‐6) tends to rise. Consequently, the n‐6/n‐3 ratio of these glycerophospholipids is markedly higher with corn oil than the control diet. N‐9 groups (oleic acid series) are consistently lower in proportion. With fat‐free diet, the proportion of n‐9 groups is higher in these glycerophospholipids than in the controls, but there is no change in the n‐6/n‐3 ratio. Comparing the changes produced in the adult and the developing cell fraction, the developing cell fraction is more responsive to dietary influence than that of the adult.
HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules
Authors: Schwandt P. , Richter W. O. , Reuschel‐Janetschek E.
Published: October 2006

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N‐ and C‐terminal amino acid residue determination, and amino acid composition, the neurophysins I1 I2, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I1 and I2 originated in the neurosecretory granules.
Local Protein Synthesizing Activity in Axonal Fields Regenerating In Vitro
Authors: Koenig E. , Adams P.
Published: October 2006

Abstract: The goldfish retinal explant system of Landreth and Agranoff was used to study endogenous protein synthesizing activity of retinal ganglion cell axons regenerating in culture. Light and electron microscopic examination of axonal fields showed that axons were free of nonneural cell investment. Decentralized axons were incubated with a mixture of tritiated amino acids, and direct quantitative microanalysis of protein and tritium radioactivity was carried out on individual axonal fields. Our findings showed that radioactive amino acids were incorporated into axonal protein in a manner that was inhibited significantly by cycloheximide, but not by chloramphenicol. Decentralized axons appeared to maintain their viability for at least 3–4 h. Axonal fields maintaining their central connections to the explant incorporated 3H‐amino acids at an apparent rate that was similar to decentralized axonal fields. Labeled material transported into axonal fields from ganglion cell bodies appeared in significant amounts after a delay of 2–3 h. Fluorographic patterns of axonal proteins after labeling with either 3H‐amino acids or [35S]methionine and separated by microelectrophoresis indicated that primarily tubulin and, to a lesser extent, actin were labeled. Our findings indicate that goldfish retinal ganglion cell axons regenerating in vitro exhibit measurable endogenous protein‐synthesizing activity.
5‐Hydroxytryptamine Stimulates [3H]Dopamine Release from the Fish Retina
Authors: Kato S. , Teranishi T. , Kuo C. H. , Negishi K.
Published: October 2006

Abstract: Serotonin (5‐hydroxytryptamine, 5‐HT; 0.5 μM and above) stimulated the release of [3H]dopamine ([3H]DA) from particulate fractions of the carp (Cyprinus carpio) retina. The 5‐HT effect was dose‐ and Ca2+‐dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6‐dihydroxytryptamine, 5,7‐dihydroxytryptamine, 5‐hydroxytrypto‐phan, or 5‐hydroxyindoleacetic acid) examined. An increase in [3H]DA release was elicited by addition of 5‐HT agonists (5‐methoxytryptamine, 5‐methoxy‐N,N‐ dimethyltryptamine, and tryptamine), but not antagonized by three 5‐HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 mM) produced a large increase in [3H]DA release from the particulate fractions, but this action was Ca2+‐independent. Further, no significant release of [3H]γ‐aminobutyric acid could be evoked by 5‐HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5‐HT stimulates [3H]DA release from the fish retina through a specific receptor‐mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.
Comparative Microdistribution of the Activity of Catecholamine‐Synthesizing Enzymes in Horizontal Sections of the Rat Lower Brainstem
Authors: Chamba Geneviève , Denoroy Luc , Renaud Bernard
Published: October 2006

Abstract: The activities of the three major catecholamine‐synthesizing enzymes were determined in brain tissue pellets dissected from 500‐μm thick horizontal sections of rat lower brainstem. The rostrocaudal distributions of the three enzymatic activities were generally not parallel, suggesting differences in the respective localization of the noradrenergic and adrenergic neurons. The difference was most important in the A2‐C2 region where the maximal activity of phenylethanolarnine‐N‐methyltransferase (EC 2.1.1.28) was located 1.5 mm more rostrally than the maximal activities of the tyrosine hydroxylase (EC 1.14.16.2) and dopamine β‐hydroxylase (EC 1.14.17.1). This result indicates that a more specific dissection of the adrenergic and noradrenergic neurons could be performed in the A2‐C2 area of the rat brainstem.
Rate and Duration of Stimulation Determine Presynaptic Effects of Haloperidol on Dopaminergic Neurons
Authors: Hoffmann Irene S. , Cubeddu Luigi X.
Published: October 2006

Abstract: Superfused rabbit neostriatal slices prelabeled with [3H]dopamine ([3H]DA) were depolarized with electrical pulses (12 V, 1 ms). Although transmitter release showed a proportional increase with a greater number of pulses (30‐360 pulses), flat frequency‐release curves were obtained. Haloperidol (0.03–0.3 μm) enhanced 3H overflow without affecting its metabolism or time course, and antagonized apomorphine‐induced inhibition of transmitter release. Maximal enhancement of release by haloperidol was obtained with 30–60 pulses delivered at a rate of 3 Hz, whereas much less facilitation of release was seen at 0.3 and 1 Hz (30–90 pulses) or with 360 pulses at either of the three frequencies. Therefore, the slope of the frequency‐release curve was markedly increased by haloperidol. These results indicate that activation of presynaptic DA receptors, and thus facilitation of release by haloperidol was highly dependent on the rate and duration of stimulation of striatal dopaminergic terminals. In these neurons the feedback loop seems to act physiologically to depress the slope of the frequency‐release curve.
Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices
Authors: Sterling Gerald H. , O'Neill Kevin J. , McCafferty Mary R. , O'Neill John J.
Published: October 2006

Abstract: The effect of chronic low‐level lead (Pb2+) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb2+‐exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs‐Ringer bicarbonate buffer with 10 mM glucose and tracer amounts of [6‐3H]glucose and either [6‐14C]glucose or [3‐14C]β‐hydroxybutyrate. Altering the concentration of Pb2+ in the drinking water produced a dose‐related increase in blood and brain lead levels. When tissue from Pb2+‐exposed rats was incubated with mixed‐labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the 3H/14C ratios. Similar effects of Pb2+ were found when 14C‐labeled β‐hydroxy‐butyrate was substituted for the [14C]glucose. It appears from these data that Pb2+ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb2+‐induced changes observed in cholinergic function.
Relationship Between Regional Brain Angiotensinogen a Local Blood Flow and Volume in the Adrenalectomize Rat: Application of Approach to Quantification of Brain Corticosterone Receptors
Authors: Wallis Cleatus J. , Gregory Timothy J. , Printz Morton P.
Published: October 2006

Abstract: Prior studies from this laboratory have established that angiotensinogen, the prohormone of angiotensin, is unevenly distributed in the rat brain and that adrenalectomy selectively perturbs levels of the prohormone in regions associated with cardiovascular neural pathways. However, plasma angiotensinogen levies are 102‐103 times higher in plasma than in brain, so that the observation of a unique distribution of brain angiotensinogen may reflect variable plasma contamination. Studies were therefore undertaken to establish whether adrenalectomy selectively alters regional blood flow, blood volume, or plasma contamination of brain tissue, thereby artifactitiously altering apparent angiotensinogen levels. Radioactive 2‐deoxyglucose, iodoantipyrine, and inulin were employed in these analyses. We conclude that variations in blood flow do not explain the selective effects of adrenalectomy, but that a variable extent of residual plasma contamination (remaining after transcardiac perfusion) is partially reflected in our earlier data. However, after correcting for plasma contamination, we still find significant changes in selected areas of the rat brain following adrenalectomy. Finally, our results demonstrate the necessity for direct quantitation of plasma contamination of brain tissue segments. This is shown to have relevance in other situations, such as corticosterone binding globulin contamination of brain corticosterone receptor binding.