bluuebunny/crossref_metadata_2025 huggingface dataset:
MODIFICATIONS OF MYELIN BASIC PROTEIN WHICH OCCUR DURING ITS ISOLATION1,2
Authors: Chou F. C‐H. , Chou C‐H. J. , Shapira R. , Kibler R. F.
Published: October 2006

Abstract— Chromatography of myelin basic protein (BP) on carboxymethylcellulose gives a pattern of multiple components, of which three are major. Component 1 is considered the unmodified species of BP while component 2 has been found to be modified primarily by deamidation and component 3 by phosphorylation (Chouet al., 1976).3 In contrast to BP prepared from tissue delipidated in the standard fashion in chloroform–methanol (CM powder), BP prepared from tissue delipidated first in acetone and then in chloroform–methanol (ACM powder) gave an elution pattern on carboxymethylcellulose characterized by a decrease in component 1 and an increase in the earlier eluting, less basic components. Studies with radiolabelled component 1 showed that this difference in elution patterns was due to the partial conversion of component 1 to less basic components during the extraction of ACM powder at neutral pH. The components derived from component 1 (D2, D3 and D4) were then isolated and subjected to tryptic peptide map analyses and determination of their carboxy‐terminal arginine content and content of phosphorus. None of the derived components contained phosphorus but tryptic peptide map analyses did show the presence of two minor peptides, T14M2 and T20M, previously found in component 2 from CM powder and considered to be the deamidation products of their parent peptides T14 and T20 (Chouet al., 1976). In addition components D3 and D4 were shown to have lost appreciable arginine from their carboxy‐termini. Since none of the efforts to reduce enzyme activity in vitro had any appreciable effect on components 2 and 3 it was concluded that phosphorylation probably occurs exclusively in vivo, that deamidation occurs both in vivo and in vitro and that loss of carboxy‐terminal arginine occurs exclusively in vitro.
PURIFICATION AND CHARACTERIZATION OF CARNOSINE SYNTHETASE FROM MOUSE OLFACTORY BULBS
Authors: Horinishi Hiroo , Grillo Mary , Margolis Frank L.
Published: October 2006

Carnosine synthetase was purified about 500‐fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800‐fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β‐alanine analogues tested, the purified mouse enzyme incorporated only γ‐aminobutyric acid and β‐amino‐n‐butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1‐methyl histidine and 3‐methyl histidine, with Ki values which were at least 40 times the Km value for histidine (16 μM). Ornithine and lysine were more efficient β‐alanine acceptors than 1‐methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher Ki values for 1–methyl histidine than the Km value for histidine. In contrast, carnosine‐anserine synthetase from chicken pectoral muscle gave Km values for histidine, 1‐methyl histidine and 3‐methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.
ON THE STRUCTURE OF A NEW, FUCOSE CONTAINING GANGLIOSIDE FROM PIG CEREBELLUM
Authors: Sonnino S. , Ghidoni R. , Gall G. , Tettamanti G.
Published: October 2006

A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer.

Fucose is α‐glycosidically linked to the 2‐position of external galactose and one N‐acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC‐IUB Commission on Biochemical Nomenclature is indicated as II3α(NeuAc)2 IV2αFuc‐GgOse4Cer.
THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES
Authors: Gundersen Cameron B. , Howard Bruce D.
Published: October 2006

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K+ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K+ media. When labeled choline was present during the release incubation, a‘newly‐synthesized’pool of acetylcholine was formed in the tissue. In toxin‐treated slices exposed to high K+, both the production and the release of this‘newly‐synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [3H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.
MEMBRANE POTENTIAL DEPENDENT BINDING OF SCORPION TOXIN TO THE ACTION POTENTIAL SODIUM IONOPHORE. STUDIES WITH A 3‐(4‐HYDROXY 3‐[125I] IODOPHENYL) PROPIONYL DERIVATIVE
Authors: Ray Radharaman , Catterall William A.
Published: October 2006

Abstract— A polypeptide toxin purified 80‐fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na+ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [125I] by reaction with N‐succinimidyl 3‐(4‐hydroxy 3‐[125I] iodophenyl) propionate. The [125I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [125I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na+ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na+ ionophore in variant neuroblastoma clones that specifically lack the action potential Na+ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na+ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm2 of cell surface membrane. A single action potential Na+ ionophore transports 1 × 108 ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K+ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na+ medium (130mm‐Na+, 5mm‐K+) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na+ ionophore. whose conformation is dependent on membrane potential.
CEREBRAL METABOLIC CHANGES IN PROFOUND, INSULIN‐INDUCED HYPOGLYCEMIA, AND IN THE RECOVERY PERIOD FOLLOWING GLUCOSE ADMINISTRATION
Authors: Agardh C.‐D. , Folbergrovaa J. , Siesjou B. K.
Published: October 2006

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°o N2O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence.

All animals with an isoelectric EEG showed extensive deterioration of the cerebral energy state. and gross perturbation of amino acid concentrations. The latter included a 4‐fold rise in aspartate concentration and reductions in glutamate and glutamine concentrations to 20 and 5oo of control levels respectively. There was an associated rise in ammonia concentration to about 3μmol‐g‐1.

Administration of glucose brought about extensive recovery of cerebral energy metabolism. For example, after an isoelectric period of 30 min tissue concentrations of phosphocreatine returned to or above normal, the accumulation of ADP and AMP was reversed, there was extensive resynthesis of glycogen and glutamine and full normalisation of tissue concentrations of pyruvate. α‐ketoglutarate. GABA and ammonia. However, even after 3 h of recovery there was a reduction in the ATP concentration and thereby in adenine nucleotide pool, moderate elevations of lactate content and the lactate pyruvate ratio, and less than complete restoration of the amino acid pool. It is concluded that some cells may have been irreversibly damaged by the hypoglycemia.
EFFECTS OF THE ANTICONVULSANT SODIUM VALPROATE ON γ‐AMINOBUTYRATE AND ALDEHYDE METABOLISM IN OX BRAIN
Authors: Whittle S. R. , Turner A. J.
Published: October 2006

Abstract—

Succinic semialdehyde dehydrogenase and NADPH‐dependent aldehyde reductase have been purified from ox brain by affinity chromatography on 5′ AMP–Sepharose and 2′5′ ADP–Sepharose respectively. Aldehyde reductase has also been purified using chromatography on Procion Red HE3B–Sepharose. The effects of the anticonvulsant drug sodium valproate were examined on these enzymes, and also on GABA‐aminotransferase partially purified from ox brain. Aldehyde reductase was inhibited by valproate in an uncompetitive manner with respect to aldehyde substrates (Ki= 38–85 μM). The inhibitions of succinic semialdehyde dehydrogenase and GABA aminotransferase were approx 2 orders of magnitude weaker, suggesting that aldehyde reductase may be an important site of action of anticonvulsant drugs.
DEVELOPMENT OF (Na+‐K+)‐ATPase IN RAT CEREBRUM: CORRELATION WITH Na + ‐DEPENDENT PHOSPHORYLATION AND K +‐para NITROPHENYLPHOSPHATASE1
Authors: Bertoni John M. , Siegel George J.
Published: October 2006

Abstract— The activities of (Na+ K+)‐ATPase and its proposed partial reactions, K +‐pNPPase and Na +‐dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI‐treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie‐stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na +‐dependent phosphorylation. The fraction of total phosphorylation that is Na +‐dependent increases steadily during development. suggesting a precursor role for some of the Na +‐independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na +‐K +)‐ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age‐related changes of Mg 2+ ‐ATPase activity. Mg 2+ ‐paranitrophenylphosphatase activity, and Na+‐independent phosphorylation levels. The most rapid changes in (Na+‐K+)‐ATPase take place during the period corresponding to glial proliferation and neuronal arborization.
THE EFFECT OF HIBERNATION ON THE LIPIDS OF BRAIN MYELIN AND MICROSOMES IN THE SYRIAN HAMSTER
Authors: Blaker William D. , Moscatelli Ezro A.
Published: October 2006

Abstract— Microsomal and myelin membrane fractions were prepared from the brains of warm‐adapted (room temperature) and hibernating Syrian hamsters (Mesocricetus auratus). Lipid extracts of these preparations were assayed for phospholipid and galactosphingolipid composition, and for cholesterol levels. In both myelin and microsomes, plasmenlethanolamine levels decreased while total ethanol‐amineglycerophospholipid levels remained constant with hibernation. Cerebroside levels changed slightly, increasing in microsomes while decreasing in myelin. No changes in cholesterol levels were detectable. Fatty acid analyses of microsomal ethanolamineglycerophospholipids and phosphatidylserine showed predominantly increases in 18:1 and 20:4 (n‐6), and decreases in 18:0 and 22:6 (n‐3), in both lipid classes with hibernation. Myelin ethanolamineglycerophospholipids exhibited a decrease in 20:1 and an increase in 20:4 (n‐6). Aldehyde analyses of plasmenylethanolamines revealed a decrease in 16:0 and an increase in 18:1 in microsomes, and an increase in 18:O in myelin. The hydroxylated fatty acids of myelin cerebrosides showed no discernible changes in composition with hibernation. It is proposed that these lipid changes aid in the maintenance of the structure and function of brain membranes at the reduced temperatures encountered during hibernation.
CEREBRAL BLOOD FLOW AND CEREBRAL METABOLIC RATES FOR OXYGEN, GLUCOSE, AND KETONE BODIES IN NEWBORN DOGS
Authors: Gregoire N. M. , Gjedde A. , Plum F. , Duffy T. E.
Published: October 2006

Abstract—

Cerebral blood flow (CBF) and the cerebral metabolic rates for oxygen, glucose, acetoacetate, β‐hydroxybutyrate and lactate were measured in 1‐ to 5‐day old Beagle dogs under nitrous oxide anesthesia. CBF was determined by 133Xe washout with mechanically integrated blood samples withdrawn simultaneously from a femoral artery and from the posterior one‐third of the superior sagittal sinus. CBF and CMRO2 in normocapnia (PaCO2 40 × 1 mm Hg) were 48 × 5 ml/100 g/min and 2.15 ml/100 g/min, respectively. There was a positive, linear relationship between CBF and PaCO2, calculated for PaCO2 values ranging from 26 to 70 mm Hg. Induced hypocapnia (PaCO2 31 × 1 mm Hg) or hypercapnia (PaCO2 58 × 2 mm Hg) did not alter the CMRO2. Glucose and acetoacetate were taken up by the brain at all PaCO2 levels examined; however, the cerebral uptake of glucose always exceeded the combined uptake of ketone bodies by more than a factor of ten. The cerebral metabolic rate for glucose (94.6 × 3.6 μmol/100 g/min) more than accounted for overall cerebral oxygen consumption, and yielded an oxygen:glucose ratio (mol:mol) of 5.1. Thus, as in adult animals, PaCO2 is an important regulator of cerebral blood flow in puppies, and glucose is the major substrate for oxidative energy production in the immature brain. The oxidation of ketone bodies by the newborn dog brain accounts for not more than 6% of the in vivo cerebral oxygen consumption.
THE LABELLING OF NERVE ENDING PHOSPHOLIPIDS IN GUINEA‐PIG BRAIN IN VIVO AND THE EFFECT OF ELECTRICAL STIMULATION ON PHOSPHATIDYLINOSITOL METABOLISM IN PRELABELLED SYNAPTOSOMES
Authors: Pickard M. R. , Hawtoorne J. N.
Published: October 2006

Abstract—

The intracerebral injection of 32Pi into guinea‐pig cortex resulted in a steady rate of incorporation into all phospholipids over a 20 h period. The specific radioactivities of phosphatidate and phos‐phatidylinositol in synaptosomes prepared from cortex prelabelled, in vivo, were at a maximum after 2 h and the respective activities were 3–8 times higher than in whole cortex. This peak in labelling corresponded with the maximum specific activity of the brain ATP. No similar differential labelling pattern was observed for phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine.

Electrical stimulation of the prelabelled synaptosomes produced a rapid drop in the specific activity of phosphatidylinositol and phosphatidate and an increase in the specific activity of CDP‐diacylglycerol. The specific activity of synaptosomal ATP was not affected.

Study of the subsynaptosomal fractions obtained after osmotic rupture of the synaptosomes revealed that the most highly labelled phosphatidylinositol was in the synaptic vesicle fraction (D) and the most active phosphatidate was in a ‘microsomal’ fraction (E). Electrical stimulation caused a loss of phosphatidylinositol radioactivity from fraction D and a loss of phosphatidate radioactivity from fraction E. The specific activity of these lipids in other fractions was not affected.

A possible role for presynaptic phosphatidylinositol is suggested.
ENTRY OF β‐OXALYL‐l‐α,β‐DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN INTO THE CENTRAL NERVOUS SYSTEM OF THE ADULT RAT, CHICK AND THE RHESUS MONKEY
Authors: Rao S. L. N.
Published: October 2006

Abstract— A comparative study of the tissue disposition of the L. sativus neurotoxin has been done using [3H]0DAP with special reference to the CNS in normal and Diamox treated (acidotic) adult rhesus monkeys and also in the day old chick, a species susceptible to the neurotoxin, and the adult rat which is refractory to it. The neurotoxin was detected in the CNS of the acidotic monkey and also in the normal monkey in nearly the same quantity. The neurotoxin was largely localised in the lumbosacral region of the spinal cord. The amount of radioactive ODAP in the CNS following its intraperitoneal injection was twice that seen following its intravenous injection.

The concentrations of the neurotoxin 90 min after injection in the CNS of the adult rat and the day old chick were almost the same and increased 2 fold by 24 h. No radioactive metabolite of ODAP could be detected in either the tissues or the urine of the different species. Species (and age) differences in susceptibility to the L. sativus neurotoxin is thus independent of its entry into the CNS.
STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS
Authors: Laduron P. M. , Wimp M. Ver , Leysen J. E.
Published: October 2006

Abstract—

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non‐specific binding sites.

The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course.

Kinetic experiments with [3H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum.

A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum.

The subcellular distribution pattern revealed a marked enrichment of [3H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [3H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.
OXIDIZED AND REDUCED GLUTATHIONE IN THE RAT BRAIN UNDER NORMOXIC AND HYPOXIC CONDITIONS
Authors: Folbergrová Jaroslava , Rehncrona Stig , Siesjö Bo K.
Published: October 2006

In order to test the proposition that hypoxia leads to a change in the concentration ratio of reduced (GSH) and oxidized (GSSG) glutathione in the brain, enzymatic, fluorometric assays were worked out for measuring GSH and GSSG. In lightly anaesthetized and immobilized rats. GSH concentrations in the cerebral cortex and the cerebellum were close to 2 μmol.g‐1 while a slightly lower concentration (approx 1.4μmol.g‐1) was found in the brain stem. In order to avoid artefactual oxidation of GSH during sample preparation for GSSG determination the tissue was extracted with trichloroacetic acid, following alkylation of SH groups with N‐ethylmaleimide. With these precautions GSSG concentrations were approx 0.7% of the corresponding GSH concentrations. However. the results indicated that the true GSSG concentrations may be even lower. During hypoxia there was neither a decrease in GSH nor an increase in GSSG concentrations in cortical tissue or cisternal CSF.
IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6‐ AND 5,7‐DIHYDROXYTRYPTAMINE12
Authors: Klemm H.‐P. , Baumgarten H. G. , Schlossberger H. G.
Published: October 2006

[14C]5,6‐Dihydroxytryptamine ([14C] 5,6‐DHT) and [14C]5,7‐dihydroxytryptamine ([14C]5,7‐DHT) were deaminated to toluene‐isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons‐medulla oblongata. [14C]5,6‐Dihydroxyindole acetic acid ([14C]5.6‐DHIAA) and [14C]5,7‐dihydroxyindole acetic acid ([14C]5,7‐DHIAA) were detected as MAO metabolites by TLC besides non‐identified components. The conversion of [14C]5,6‐DHT and [14C]5,7‐DHT obeyed, at least initially, Michaelis‐Menten kinetics (Km 5,7‐DHT: 0.5 × 10−3M; Km 5,6‐DHT: 1.25 × 10−3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7‐ and 5,6‐DHT seemed to be preferred substrates of MAO A.

Incubation of rat brain homogenates with [14C]5,6‐DHT and [14C]5,7‐DHT or with the MAO metabolites [14C]5,6‐DHIAA and [14C]5,7‐DHIAA caused a time‐dependent break‐down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components.

5,6‐DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6‐DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L‐ascorbic acid. Reduction of [O2] in the incubation medium slightly attenuated the inactivation of MAO by 5,6‐DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6‐DHT. The results suggest that oxidation products of 5,6‐DHT, e.g. its corresponding o‐quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6‐DHT.

The lack of inactivation of MAO by 5,7‐DHT in vitro correlated with a low degree of radioactivity binding (from [14C]5,7‐DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l‐ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7‐DHT.
A RAPID AND SIMPLE METHOD FOR THE DETERMINATION OF PICOGRAM LEVELS OF SEROTONIN IN BRAIN TISSUE USING LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION
Authors: Ponzio F. , Jonsson G.
Published: October 2006

A rapid and simple technique using solvent extraction and high pressure liquid chromatography with electrochemical detection has been developed for the determination of serotonin in small brain tissue samples (1‐20 mg). The method has a reasonably good specificity and a very low experimental error (less than 3%s.e., calculated from six samples processed and analysed from the same brain homogenate). The recovery of authentic 5‐HT added is 80‐90%. The 5‐HT levels of rat whole brain was found with the present technique to be 690 ± 17.5 ng/g and of mouse neocortex 304 ± 16 ng/g. Monoamine oxidase inhibition with pargyline (2 h) increased 5‐HT levels in mouse neocortex to 194 ± 15% (N = 5) of control, while reserpine depleted 5‐HT to 13 ± 4% of control. The method has a sensitivity level of about 20 pg (0.1 pmol) per brain sample.
EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY
Authors: Gonzalez C. , Kwok Y. , Gibb J. , Fidone S.
Published: October 2006

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1‐3 h in a low O2 atmosphere. Basal TH levels were 5‐6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty‐eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O2 in N2 for two 30‐min periods (20‐min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O2 in N2 for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.
PURIFICATION OF L‐GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN
Authors: Thomas Su Y. Y. , Wu J.‐Y. , Lam Dominic M. K.
Published: October 2006

—L‐Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS‐polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD‐anti‐GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti‐GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD‐anti‐GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.
Association of Newly Synthesized Tubulin with Brain Microsomal Membranes
Authors: Soifer David , Czosnek Henryk
Published: October 2006

Tubulin has been found to be synthesized on both membrane‐bound and free polyribosomes prepared from brain. Cell‐free studies indicate that tubulin made on rough microsomes is incorporated into the endoplasmic reticulum membrane as it is synthesized. This tubulin remains associated with the membrane after sedimentation and washing. The tubulin is not removed from the membrane after stripping ribosomes from the membranes in KCI‐puromycin, followed by repeated washing by either sedimentation or flotation in 0.5 M‐KCI. The membrane tubulin is partially susceptible to proteolysis by trypsin and chymotrypsin: /3‐tubulin is more accessible to the proteases than is a‐tubulin. Nonionic detergents extract mostly /3‐tubulin from the microsomal membrane. Newly synthesized tubulin which has been extracted from microsomal membranes in 0.5% Nonidet P‐40, coassembles and disassembles with carrier microtubule protein. The insertion of newly synthesized tubulin into endoplasmic reticulum membrane may be the first step in the incorporation of tubulin into the plasma membrane.
Blood–Brain Glucose Transport in the Conscious Rat: Comparison of the Intravenous and Intracarotid Injection Methods
Authors: Gjedde Albert , Rasmussen Mette
Published: October 2006

Abstract: The unidirectional transfer of d‐glucose from blood to parietal cortex tissue of the brain of awake rats was measured by single intravenous injection of tracer glucose, as well as by single intracarotid injection according to the method of Oldendorf. The maximal unidirectional blood–brain glucose transfer rate (Tmax) was 407 μ mol (100 g)–1 min–1 when measured by intravenous injection, and 352 μ mol (100 g)–1 min–1 when measured by intracarotid injection. The half–saturation constants (Km) were 7.8 mm and 16.8 HIM, respectively. The comparison shows that the two methods give similar results when cerebral perfusion is assessed accurately.
Effects of Chronic Striatal Kainate Lesions on Some Dopaminergic Parameters and Enkephalin Immunoreactive Neurons in the Basal Ganglia
Authors: Schwarcz Robert , Fuxe Kjell , Hökfelt Tomas , Terenius Lars , Goldstein Menek
Published: October 2006

Abstract: The chronic effects of kainate‐induced lesions of the neostriatum have been evaluated in rats 12 months following the injection of kainic acid. Light microscopical analysis revealed marked disappearance of nerve cells in the neostriatum, with some cells remaining within the medial and lateral zone of the neostriatum and in the most ventral part. The rest of the markedly atrophied neostriatum was mainly made up of densely packed myelinated nerve bundles. Tyrosine hydroxylase immunoreactivity was used as a marker for dopamine neurons and revealed that tyrosine hydroxylase immunoreactive nerve terminals remained between the axon bundles in the striatum and that tyrosine hydroxylase immunoreactive nerve cell bodies in the substantia nigra seemed intact. Studies on enkephalin immunoreactive neurons revealed a marked disappearance of such nerve cells and nerve terminals within the neostriatum. Neurochemical analysis showed a clearcut reduction in the number of dopamine receptors as evaluated by studies on both [3H]spiperone binding and on [3H]ADTN binding. Dopamine levels remained unchanged while choline acetyltransferase activity was reduced significantly. Taken together, the present findings demonstrate that the chronically kainate lesioned striatum is characterized by a substantial loss of enkephalin immunoreactive and cholinergic nerve cells and a marked reduction in the number of dopamine receptors. These findings are discussed in relation to neurochemical and therapeutic aspects of Huntington's disease.
White Matter Proteins in Multiple Sclerosis
Authors: Newcombe J. , Cuzner M. L. , Röyttä M. , Frey H.
Published: October 2006

Abstract: The SDS‐soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non‐myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high‐molecular‐weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non‐myelin protein were also a consistent feature of the normal‐appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.
Effects of Polyamines on Calcium‐Dependent Rat Brain Phosphatidylinositol‐Phosphodiesterase
Authors: Eichberg J. , Zetusky W. J. , Bell M. E. , Cavanagh E.
Published: October 2006

Abstract: The effect of polyamines on the ability of calcium‐dependent soluble rat brain phosphatidylinositol‐phosphodiesterase to hydrolyze dispersed phosphatidylinositol was examined. Putrescine and cadaverine stimulated activity at all concentrations tested. In contrast, spermine and spermidine stimulated the reaction slightly at low concentrations but caused progressively greater inhibition as their levels were further increased. Phosphatidylinositol hydrolysis was inhibited by several multivalent cations, especially lanthanum and manganese. Spermidine partially replaced the calcium requirement of the enzyme. The possibility that polyamines may play a role in the regulation in vivo of phosphatidylinositol‐phosphodiesterase is discussed.
A Sensitive Method for Characterization of Oligoclonal Immunoglobulins in Unconcentrated Cerebrospinal Fluid
Authors: Lasne Y. , Benzerara O. , Chazot G. , Creyssel R.
Published: October 2006

Abstract: Although isoelectric focusing has been used to demonstrate the presence of oligoclonal IgG in the CSF, the technique has not allowed detection of oligoclonal IgG in unconcentrated CSF. A new technique is reported, by which unconcentrated CSF is separated by isoelectric focusing, and the IgG bands are then detected by radioimmunofixation. Samples as small as 20 μl may be used.
[3H]Propyl β‐Carboline‐3‐Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass
Authors: Braestrup Claus , Nielsen Mogens
Published: October 2006

Abstract: Ethyl β‐carboline‐β‐carboxylate (β‐CCE) is a mixed‐type inhibitor of [3H]flunitrazepam ([3H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [3H]Propyl β‐carboline‐3‐carboxylate ([3H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [3H]PrCC was used as radioligand. However, we observed that [3H]PrCC labelled only 57% of [3H]FNM binding sites in rat hippocampus (Bmax values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [3H]FNM binding was inhibited by β‐CCE indicate that β‐CCE and some other β‐carboline‐3‐carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [3H]PrCC. We conclude that [3H]PrCC at low concentration (0.3–0.4 × 10‐9 M) labels a subclass of benzodiazepine receptors, BZ1, while another class, BZ2 receptors, are not labelled by [3H]PrCC when filtration assays are used. By parallel determinations of the proportion between [3H]FNM and [3H]PrCC binding we calculated the percentage of BZ1 receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.
Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices
Authors: Doležal Vladimir , Tuček Stanislav
Published: October 2006

Abstract: Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5‐14C]citrate, [1‐14C‐acetyl]carnitine, [1‐14C]acetate, [2‐14C]pyruvate, or [U‐14C]glucose. The synthesis of acetyl‐labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2‐14C]pyruvate > [U‐14C]glucose > [1‐14C‐acetyl]carnitine > [1,5‐14C]citrate > [1‐14C]acetate. The utilization of [1,5‐14C]citrate and [1‐14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of and 14C‐labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5‐14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcar‐nitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brain; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl‐CoA to the extramitochondrial space in cerebral cholinergic neurons.
Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Whole Retina Studies
Authors: Dokas Linda A. , Kohsaka Shinichi , Burrell Harry R. , Agranoff Bernard W.
Published: October 2006

Abstract: Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid‐soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5′‐deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post‐crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.
Norepinephrine Metabolism in Man Using Deuterium Labelling: The Conversion of 4‐Hydroxy‐3‐Methoxyphenylglycol to 4‐Hydroxy‐3‐Methoxymandelic Acid
Authors: Mårdh Göran , Sjöquist Birgitta , ÄnggÅrd Erik
Published: October 2006

Abstract: 4‐Hydroxy‐3‐methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study the disposition of peripherally administered HMPG. Five healthy men were given an intravenous pulse dose of 4.3 μmol of labelled HMPG and subsequent plasma and urine levels of endogenous and labelled HMPG as well as those of 4‐hydroxy‐3‐methoxymandelic acid (HMMA, VMA) were determined by gas chromatography‐mass spectrometry, using selected ion detection. Approximately 40% of the injected amount of deuterium‐labelled HMPG was recovered in the urine as HMMA and another 40% was eliminated as HMPG conjugates. Thus, the HMPG formed from norepinephrine either in the central or peripheral nervous system undergoes both conjugation and extensive oxidation.
Impaired Synthesis of Acetylcholine by Mild Hypoxic Hypoxia or Nitrous Oxide
Authors: Gibson Gary E. , Duffy Thomas E.
Published: October 2006

The effect of mild hypoxic hypoxia on brain metabolism and acetylcholine synthesis was studied in awake, restrained rats. Since many studies of hypoxia are done with animals anesthetized with nitrous oxide (N2O), the effects of N2O were evaluated. N2O (70%) increased the cerebral cortical blood flow by 33% and the cortical metabolic rate of oxygen by 26%. In addition, the synthesis of acetylcholine in N2O‐anesthetized animals, measured with [U‐14C]glucose and [1‐2H2,2‐2H2]choline, decreased by 45 and 53%, respectively. Consequently, mild hypoxia was studied in unanesthetized rats. Control rats breathing 30% O2 (partial pressure of oxygen, Pao2= 120 mm Hg) were compared with rats exposed to 15% O2 (Pao2= 57 mm Hg) or 10% O2 (Pao2= 42 mm Hg). The synthesis of acetylcholine, measured with [U‐14C]glucose, was decreased by 35 and 54% with 15% O2 and 10% O2 respectively; acetylcholine synthesis, measured with [1‐2H2,2‐2H2]choline, was decreased by 50 and 68% with 15% O2 and 10% O2 respectively. Animals breathing either 15% or 10% O2 had normal cerebral metabolic rates of oxygen but had increased brain lactates and increased cortical blood flows compared with animals breathing 30% O2. These results show that even mild hypoxic hypoxia impairs acetylcholine synthesis, which in turn may account for the early symptoms of brain dysfunction associated with hypoxia.
Microvessels Isolated from Rat Brain: Localization of Astrocyte Processes by Immunohistochemical Techniques
Authors: White Fredric P. , Dutton Gary R. , Norenberg Michael D.
Published: October 2006

Microvascular networks were isolated from rat telencephalon by density gradient centrifugation. The vessels prepared were characterized morphologically at the light and electron microscope level and immunohistochemically by the localization of glutamine synthetase and glial fibrillary acidic protein, two proteins found almost exclusively in astrocytes. The vast majority of the vessels prepared contained more than just endothelial cells surrounded by a basement membrane. Many arterioles were found still retaining their smooth muscle cells. Pericytes were found in association with most of the venules and many of the capillaries. Astrocyte processes remained attached to most of the microvessels. These results show that vessels prepared from rat brain still maintain most of their complex intercellular contacts and must be viewed as a heterogeneous network of cells.
Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism
Authors: Cahill Anne L. , Ehret Charles F.
Published: October 2006

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate‐limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α‐methyl‐p‐tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of “time of day” as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.
Protein Synthesis and Axonal Transport During Nerve Regeneration
Authors: Perry G. W. , Wilson David L.
Published: October 2006

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [35S]‐methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two‐dimensional polyacryl‐amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.
Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools
Authors: Levi Giulio , Gallo Vittorio
Published: October 2006

Abstract: The aim of the present paper was to determine whether the release of glutamate from putative “glutamergic” terminals in the cerebellum is influenced by γ‐aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+‐dependent way; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released in a Ca2+‐dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors.

GABA and muscimol at low concentrations (2‐20 μM) potentiated the depolarization‐induced release of D‐[3H]aspartate (a glutamate analogue which labels the glutamate “reuptake pool”) from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D‐[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin‐sensitive manner, the depolarization‐evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of “glutamergic” nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.