bluuebunny/crossref_metadata_2025 huggingface dataset:
GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN
Authors: Jenkins B. T. , Hajra A. K.
Published: October 2006

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn‐glycerol‐3‐phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The Km for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The Km for dihydroxyacetone was 0.6 mm.

Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The Km for dihydroxyacetone was 60 μm. This cytosol fraction was also found to phosphorylate d‐glyceraldehyde and l‐glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.
A SENSITIVE ASSAY FOR DOPAMINES‐β‐HYDROXYLASE
Authors: Wise C. David
Published: October 2006

Abstract— A sensitive procedure for the determination of dopamine‐β‐hydroxylase (EC 1.14.2.1) activity in homogenates of rat brain and in rat serum is described. In the assay, the substrate, [14C]tyramine is enzymatically converted to [14C]octopamine which is then oxidized with periodate to the [l4C]p‐hydroxybenzaldehyde. The latter compound is separated by solvent extraction into ether and its radioactivity determined. A simple method has been developed for the purification and convenient storage of [14C]tyramine which results in a boiled‐blank value of about 100 d.p.m. per 106 d.p.m. of [I4C]tyramine. The low blank allows the detection of as little as 7.5 pmol of product. This makes the procedure several times more sensitive than other methods now available. The interactions of copper, N‐ethylmaleimide and p‐chloromercuriphenyl sulfonic acid with the endogenous inhibitors were also examined. The method should be generally applicable for the assay of DBH in any tissue homogenate once the appropriate copper and dilution parameters have been determined.
NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS IN NORMALS AND FOUR TYPES OF MUCOPOLYSACCHARIDOSES
Authors: Constantopoulos G. , Mccomb R. D. , Dekaban A. S.
Published: October 2006

Abstract—

Glycosaminoglycan content, composition and molecular weight distribution were determined in cerebral gray and white matter, liver and spleen from normals and 7 patients with mucopolysaccharidosis; 4 were of Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie).

There was a 3 to 4‐fold increase in glycosaminoglycan content of the brains from patients with mucopolysaccharidosis Type I, II and IIIA, but only a 40% increase in the Type V patient. Partially degraded dermatan sulfate accounted for most of the increase in Types I, II and V. Highly fragmented heparan sulfate was the major glycosaminoglycan in the brain of the Type IIIA patient and was also a sizable component in Types I and II.

Remarkably, the changes in the brain glycosaminoglycans of the Type V patient were minimal. He also was of normal intelligence
STIMULATION OF GLYCOGENOLYSIS IN RAT CAUDATE NUCLEUS SLICES BY L‐ISOPROPYLNOREPINEPHERINE, DIBUTYRYL CYCLIC AMP AND 2‐CHLOROADENOSINE
Authors: Wilkening Doug , Makman Maynard H.
Published: October 2006

Abstract—

Dopamine (DA), L‐isópropylnorepinepherine (IPNE), 2‐chloroadenosine (2‐Cl‐Ado), 3‐isobutyl, I‐methylxanthine (IBMX) and N6,O2′‐dibutyryl cyclic AMP have been investigated for their effects on glycogen levels in rat caudate nucleus slices. Incubation of slices with 1 mM‐dibutyryl cyclic AMP, or with 50 μM‐IPNE in the presence of 1mM‐IBMX, or with 5‐500 μM‐2‐Cl‐Ado reduced glycogen levels to about 50% of control. Incubation of slices with 1 mM‐IBMX alone, or with 50μM‐IPNE alone, or with 50μM‐DA either alone or in the presence of 1 mM‐IBMX was without significant effect on glycogen levels. The effect of IPNE + IBMX was completely abolished by the prior addition of 10 μM‐propranolol. The effect of 10 μM‐2‐Cl‐Ado was not effectively prevented by either 100 μM‐theophylline or 100 μM‐cordycepin. The results indicate that the β‐adrenergic adenylate cyclase in the rat caudate nucleus plays a role in the regulation of glycogen metabolism, while the DA‐stimulated adenylate cyclase is not significantly involved. The 2‐Cl‐Ado‐stimulated adenylate cyclase may be involved in the control of glycogen metabolism, but other mechanisms for the 2‐CI‐Ado action, such as interference with allosteric regulatory sites on glycogen phosphorylase, have not been ruled out.
GLYCOSYLTRANSFERASES OF RAT BRAIN THAT MAKE CEREBROSIDES: SUBSTRATE SPECIFICITY, INHIBITORS, AND ABNORMAL PRODUCTS1
Authors: Warren K. R. , Misra R. S. , Arora R. C. , Radin N. S.
Published: October 2006

Brain homogenates from young rats were assayed for their ability to synthesize cerebrosides from radioactive UDP‐galactose or UDP‐glucose and ceramide. A comparison of galactose transfer with ceramides made from different 2‐hydroxy acids showed that the shortest one tested (C7) was by far the best acceptor, while the poorest contained 18 carbon atoms; longer fatty acids were better than CIS. Glucosyltransferase, on the other hand, showed rather little chain length specificity or discrimination against hydroxy acid ceramides.

Synthetic compounds analogous in structure to ceramides were tested as inhibitors of the sugar transferases. Some were found to act as sugar acceptors themselves, particularly amides of DL‐erythro‐1‐ phenyl‐2‐amino‐1,3‐propanediol. Some amides were good inhibitors of glucosyltransferase, particularly decanoyl norephedrine, decanoyl threo‐1‐phenyl‐2‐amino‐1,3‐propanediol and decenoyl phenylalaninol. The secondary amine analogous to the first of these, N‐decyl norephedrine, was also very effective. No strong inhibitors of galactosyl transferase were found, although octanoyl D‐threo‐p‐nitrophenyla‐ minopropanediol showed promise (42% inhibition at 0.3 mM). Octanoyl phenylalaninol was nearly as good an inhibitor; the inhibition appeared only after a lag period.

It is suggested that the glucosyltransferase inhibitors might he useful in therapy of Gaucher's disease, by reducing the degradative load normally falling on glucocerebrosidase.
POTASSIUM‐INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS
Authors: Vargas Olga , DEL Carmen María , de Lorenzo Doria , Saldate María Cristina , Orrego F.
Published: October 2006

Abstract—

The release of [3H]GABA induced by elevated extracellular potassium (K)o, from thin rat brain cortex slices, has been compared with that of [3H]noradrenaline ([3H]NA), released by the same procedures, both from normal slices, and from slices pre‐treated with reserpine and nialamide, [3H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM‐(K)o released considerably more [3H]NA from normal than from drug‐treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [3H]NA from drug‐treated slices increased in proportion to (K)o, up to 36‐46 mM and then declined considerably with higher (K)o. The dependency of potassium‐induced release on the concentration of calcium in the medium, indicated that release of [3H]NA from normal slices was proportional to calcium up to 1.5‐2 mM, while that of [3H]NA from drug‐treated slices increased up to 0.5 mM‐Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM‐calcium, but no further changes were observed at higher concentrations. The calcium antagonist D‐600 inhibited high (K)o‐induced release of [3H]NA from normal slices to a greater extent than that of [3H]GABA or of [3H]NA from drug‐treated slices. These results, in which elevated (K)o‐induced release of [3H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [3H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)o as a stimulus for inducing transmitter secretions.
ACETYLTRIETHYLCHOLINE: A CHOLINERGIC FALSE TRANSMITTER IN CAT SUPERIOR CERVICAL GANGLION AND RAT CEREBRAL CORTEX
Authors: ILSON D. , COLLIER B. , BOKSA P.
Published: October 2006

Abstract—

The accumulation and metabolism of [14C]triethylcholine by cat superior cervical ganglia [rested or stimulated (20 Hz)] and by rat cerebral cortex minces was measured. In ganglia, preganglionic nerve stimulation increased the accumulation (2.4 fold) and the acetylation (5.7 fold) of triethylcholine; however the depletion of the ganglion's acetylcholine content was 9.5 times greater than the amount of acetyltriethylcholine synthesized. In the presence of eserine, neither stimulated nor rested ganglia synthesized any extra (surplus) acetyltriethylcholine. It is concluded that the rate‐limiting step in acetyltriethylcholine synthesis is the acetylation of triethylcholine by choline acetyltransferase. Subsequent preganglionic nerve stimulation of ganglia, which had been stimulated during the exposure to [14C]‐triethylcholine, caused the increased release of only acetyltriethylcholine; the release was frequency‐dependent, required the presence of Ca2+, and was blocked by increasing the ratio of Mg2+/Ca2+ in the perfusion fluid. All of the acetyltriethylcholine which had been accumulated was available for release. Rat cerebral cortex also accumulated triethylcholine and acetylated about 3% of the accumulated choline analogue. Subsequent stimulation by high K+ (46 mM)‐atropine (3 μM) caused the increased release of acetyltriethylcholine from the cortex and this release required the presence of Ca2+. Triethylcholine can therefore form a cholinergic false transmitter in the cat superior cervical ganglion and the rat cerebral cortex.
CORRELATION BETWEEN TYROSINE HYDROXYLASE ACTIVITY AND CATECHOLAMINE CONCENTRATION OR TURNOVER IN BRAIN REGIONS1
Authors: BACOPOULOS N. G. , BHATNAGAR R. K.
Published: October 2006

Abstract—

Tyrosine hydroxylase activity correlated significantly with norepinephrine concentration and turnover, when results from regions containing predominantly noradrenergic terminals were compared, and with dopamine concentration and turnover when results from regions containing predominantly dopaminergic terminals were compared. Regions containing dopamine or norepinephrine cell bodies were characterized by higher tyrosine hydroxylase activities as compared to regions containing mostly nerve terminals. Higher levels of tyrosine hydroxylase activity and transmitter turnover were observed in regions containing dopaminergic terminals than in regions containing norepinephrine terminals. These findings are consistent with the view that tyrosine hydroxylase activity is linked to rates of catecholamine utilization by neurons in the CNS.
EFFECT OF DIETARY PROTEIN ON URINARY 5‐HYDROXYINDOLEACETIC ACID LEVELS1
Authors: Nomura M. , Colmenares J. L. , Wurtman R. J.
Published: October 2006

Abstract— Among rats consuming diets containing 0%, 18%, or 40% protein (in the form of casein) for 4 consecutive days, urinary 5‐hydroxyindoleacetic acid (5‐HIAA) levels varied markedly as a function of the protein content, whether 5‐HIAA was expressed as μg/rat/day or as μg/kg body weight/day. These differences could not be attributed to 5‐hydroxyindoles in the diet; they probably reflected diet‐dependent changes in serotonin synthesis. If animals were treated concurrently with carbidopa (a drug that blocks aromatic L‐amino acid decarboxylase activity in the gut and other peripheral tissues but not in the CNS), urinary 5‐HIAA levels fell, and the effect of dietary protein on the 5‐HIAA largely disappeared. These observations indicate that serotonin synthesis in peripheral organs, as in brain, is under acute nutritional control.
THE EFFECT OF 4‐AMINO HEX‐5‐YNOIC ACID (γ‐ACETYLENIC GABA, γ‐ETHYNYL GABA) A CATALYTIC INHIBITOR OF GABA TRANSAMINASE, ON BRAIN GABA METABOLISM IN VIVO
Authors: Jung M. J. , Lippert B. , Metcalf B. W. , Schechter P. J. , Böhlen P. , Sjoerdsma A.
Published: October 2006

Abstract— 4‐Amino hex‐5‐ynoic acid (γ‐acetylenic GABA, γ‐ethynyl GABA, RM171.645), a catalytic inhibitor of GABA transaminase in vitro, induces a rapid, long‐lasting dose‐dependent decrease of GABA transaminase activity and, to a lesser extent, of glutamate decarboxylase activity in the brains of rats and mice when given by a peripheral route. The GABA concentration in whole brain increases up to 6‐fold over control values. The action of γ‐acetylenic GABA is relatively specific, as no in vivo inhibition of brain aspartate and alanine transaminase activities could be detected. Furthermore, the amount of radioactive drug bound to the protein fraction of brain homogenate is of the same order of magnitude as that of the GABA transaminase present, as calculated from total GABA transaminase activity, molecular weight and specific activity of the pure enzyme. γ‐Acetylenic GABA illustrates the usefulness of a catalytic irreversible enzyme inhibitor in altering neurotransmitter metabolism in vivo.
STRUCTURAL REQUIREMENTS FOR AMINO ACID INHIBITION OF Na+‐DEPENDENT PROLINE UPTAKE BY RAT BRAIN SYNAPTOSOMES
Authors: Raghupathy E. , Peterso N. A.
Published: October 2006

Abstract— The structural requirements for amino acid inhibition of Na+‐dependent proline uptake by rat brain synaptosomal fractions were investigated. It is shown that the amino group has to be in the α‐position to strongly inhibit proline uptake. Hydroxyamino acids are less potent inhibitors than the parent amino acids. Amino acids with net positive or negative charges on their molecules exert no effect, whereas elimination of the net charge results in compounds with profound inhibitory effects. Blocking of the carboxyl group reduces the inhibition, but does not abolish it. Since acetylation of the α‐amino group results in elimination of the inhibitory effect whereas N‐methylation does not, it is concluded that in the interaction of an amino acid with the proline transport site the positive charge on the amino group plays the most critical role.
THE SURFACE PROPERTIES OF ISOLATED RAT BRAIN MYELIN: A MICROELECTROPHORETIC STUDY
Authors: Gregson N. A.
Published: October 2006

Abstract— The surface properties of isolated rat brain myelin have been investigated by the method of particle microelectrophoresis. The mobility has been measured as a function of ionic strength and composition of the buffer, the results suggesting the presence of areas of non‐ionogenic material. Antisera against ganglioside and myelin produce a fall in mobility whereas antiproteolipid apoprotein produces a slight increase but antibasic protein and anticerebroside sera had no effect on mobility. Of the lipases studied only phospholipase D produced a clear increase in mobility. From this preliminary study some indications of the asymmetric disposition of components in the myelin sheath may be derived. The ganglioside appears to be wholly located at intraperiod line interface, along with some fraction of the galactocerebroside and phosphatidylcholine. There appears to be few amino groups, but SH groups are present.
SUITABILITY OF DIFFERENT MOLECULAR SPECIES OF 1,2‐DIACYLGLYCEROLS AS SUBSTRATES FOR DIACYLGLYCEROL KINASE IN RAT BRAIN MICROSOMES
Authors: Holub B. J. , Piekarski J.
Published: October 2006

The relative suitability of different molecular species of 1,2‐diacyl‐sn‐glycerols as substrates for the diacylglycerol kinase (ATP: 1,2‐diacyl‐sn‐glycerol phosphotransferase) in rat brain microsomes was investigated. The diacylglycerols tested were a mixture of the 1‐[3H]palmitoyl and 1‐[14C]stearoyl homologues of either the 2‐oleoyl (monoenoic), 2‐linoleoyl (dienoic), 2‐arachidonoyl (tetraenoic), or 2‐docosahexaenoyl (hexaenoic) diacylglycerols with an isotope ratio (3H/14C) approximately equal to 1.00.

At substrate concentrations of 0.125 mM and 0.60 mM, only a modest preference of the kinase for total (1‐palmitoyl plus 1‐stearoyl homologues) monoenoic over total hexaenoic species was indicated. The tetraenoic diacylglycerols gave reaction rates which were not significantly different from the monoenes, dienes, or hexaenes when the data were analyzed statistically.

No significant enzyme selectivity for either the 1‐palmitoyl or 1‐stearoyl homologues of the various 1‐saturated 2‐unsaturated diacylglycerols was apparent.

The present results, together with data on the composition of free 1,2‐diacylglycerols in brain, which reveal a preponderance of tetraenoic molecular species, suggest that the tetraenoic phosphatidic acids (mainly as 1‐stearoyl 2‐arachidonoyl species) are quite possibly the major products of diacylglycerol kinase activity in rat brain under physiological conditions.
LACK OF ENHANCEMENT OF DIMETHYLTRYPTAMINE FORMATION IN RAT BRAIN AND RABBIT LUNG IN VIVO BY METHIONINE OR S‐ADENOSYLMETHIONINE
Authors: Stramentinoli Giorgio , Baldessarini Ross J.
Published: October 2006

In vivo conversion of intracisternally administered [14C]tryptamine to [14C]N,N‐dimethyl‐tryptamine (DMT) in rat brain, even in the presence of an excess of substrate and methyl donor appeared to be insignificant, although enzymatically synthesized [14C]DMT was recovered readily after intracranial injection. Authenticity of [14C]DMT was demonstrated by cocrystallization with authentic DMT and oxalic acid to constant specific radioactivity after chromatographic separation of [14C]DMT. In rabbit lung, the apparent Km for S‐adenosylmcthionine (SAMe) (29 μM) with indolethylamine‐N‐methyltransferase was found to be close to endogenous levels of SAMe (34 μM) that are not likely to saturate the enzyme normally. Nevertheless. large doses of L‐methioninc or SAMe failed to increase the in vivo conversion of [14C]N‐methyltryptamine to [14C]DMT in this tissue. The production of [14]DMT was instead markedly irihihitrd by this treatment. possibly due to an effect of S‐adeno‐sylhomocysteine. Our results fail to support the hypothesis that psychotropic effects of methionine or SAMe are due to increased accumulations of pharmacologically active methylated indoleamines.
LEUKOCYTE PPD‐PEROXIDASE ACTIVITY WITH POLYUNSATURATED FATTY ACID HYDROPEROXIDES: NORMAL VALUES IN BATTEN'S DISEASE
Authors: Schwerer Beatrix , Bernheimer H.
Published: October 2006

Abstract— The activity of leukocyte p‐phenylenediamine (PPD)‐dependent peroxidase with respect to 3 peroxidic substrates was investigated in three patients with Batten's disease (ceroid‐lipofuscinosis) as compared with normal controls: 1. The activity of PPD‐peroxidase, using H2O2 as the peroxidic substrate, was found to be normal in our patients with Batten's disease. 2. PPD‐peroxidase was shown to be active towards arachidonic acid hydroperoxide (AAHPO) and linoleic acid hydroperoxide (LAHPO) as peroxidic substrates. No difference could be detected between patients and normals. 3. Determination of Michaelis constants with respect to H2O2, AAHPO and LAHPO in normal leukocytes revealed that PPD‐peroxidase was more active towards AAHPO (lower Km) than towards LAHPO. The same kinetic properties were found for PPD‐peroxidase in patients with ceroid‐lipofuscinosis.
THE DEGRADATION OF PEPTIDES AND AMINOACYL AMIDES BY GUINEA‐PIG BRAIN
Authors: O'Cuinn G.
Published: October 2006

Abstract— The ability of guinea‐pig brain to hydrolyse peptides and aminoacyl amides was investigated. The majority of hydrolase activity against both peptides and aminoacyl amides tested was found to reside in the 30,000 g supernatant. Starch gel electrophoretograms of the 30,000 g supernatants showed that seven of the twelve peptides tested were each hydrolysed by more than one peptide hydrolase and that each of the peptide hydrolases observed were capable of hydrolysing more than one peptide. The peptide hydrolases in the 30,000 g supernatant fraction of guinea‐pig brain were found to have very similar electrophoretic mobilities to the peptide hydrolases in the 30,000 g supernatant fraction from guinea‐pig intestinal mucosa. Only one hydrolase with activity against L‐Leu‐NH, could be detected in 30,000 g supernatant of guinea‐pig brain using starch gel electrophoresis followed by staining and its electrophoretic mobility was identical to that of one of the peptide hydrolases in the same fraction.
SEQUENCE DIVERSITY STUDIES OF RAT BRAIN RNA: EFFECTS OF ENVIRONMENTAL COMPLEXITY ON RAT BRAIN RNA DIVERSITY
Authors: Grouse Lawrence D. , Schrier Bruce K. , Bennett Edward L. , Rosenzweig Mark R. , Nelson Phillip G.
Published: October 2006

Abstract—

The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level.

Brain and liver RNA diversities were measured under double‐blind experimental conditions in three experiments with rats raised in experientially enriched (EC) or impoverished (IC) environments. Liver RNA diversity of EC animals was not different from that of IC animals. Brain total RNA of EC animals, at equivalent R0ts of 184,000‐212,000, hybridized to 10.6% of rat unique DNA (mean of 11 separate groups of rats). The average hybridization of brain RNA from 11 groups of IC animals in the same range of equivalent Rot was 8.2% of the unique DNA. The difference was statistically significant at P < 0.02. Of 10 groups of 3 littermate pairs (paired across EC and IC groups) brain RNA diversity was greater in EC animals in 8 cases. A least squares fit of the kinetics of hybridization to a pseudo first order reaction showed that, at saturation, the RNA from brains of EC animals was complementary to 16.4% of the unique DNA while that from IC animals was complementary to 9.1%. This difference was found in the least abundant class of rat brain RNA. These changes in sequence diversity reflected either an increase in the number of diverse RNA species present or an increase in the number of copies of certain RNA species in the rats raised in an enriched environment. A change in brain RNA populations of this magnitude may reflect a significant difference in brain function between EC and IC animals.
TRANSPORT OF THREONINE AND TRYPTOPHAN BY RAT BRAIN SLICES: RELATION TO OTHER AMINO ACIDS AT CONCENTRATIONS FOUND IN PLASMA1
Authors: Tews J. K. , Goodand S. S. , Harper A. E.
Published: October 2006

Abstract—

Threonine content of brain decreases in young rats fed a threonine‐limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tewset al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α‐amino‐n‐butyric acid and homoserine, less so by α‐aminoisobutyric acid, and not at all by GABA.

The complex amino acid mixtures strongly inhibited α‐aminoisobutyric acid transport by brain or liver slices but, in contrast to effects in brain, the extent of the inhibition in liver was not much affected by altering the composition of the mixture.

Tryptophan accumulation by brain slices was effectively inhibited by other large neutral amino acids in physiologically occurring concentrations. Threonine, or a mixture of serine, glycine and alanine only slightly inhibited tryptophan uptake; basic amino acids were without effect and histidine stimulated tryptophan transport slightly.

These results support the conclusion that a diet‐induced decrease in the concentration in brain of a specific amino acid may be related to increased inhibition of its transport into brain by increases in the concentrations of transport‐related, plasma amino acids.
LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U‐14C]GLUCOSE AND [3H]GLUTAMATE
Authors: Gaitonde M. K. , Harms Viki , Evans Gwyneth
Published: October 2006

Abstract—

To obtain evidence of the site of conversion of [U‐14C]glucose into glutamate and related amino acids of the brain, a mixture of [U‐14C]glucose and [3H]glutamate was injected subcutaneously into rats. [3H]Glutamate gave rise to several 3H‐labelled amino acids in rat liver and blood; only 3H‐labelled glutamate, glutamine or γ‐aminobutyrate were found in the brain. The specific radioactivity of [3H]glutamine in the brain was higher than that of [3H]glutamate indicating the entry of [3H]glutamate mainly in the ‘small glutamate compartment’.

The 14C‐labelling pattern of amino acids in the brain and liver after injection of [U‐14C]glucose was similar to that previously reported (Gaitondeet al., 1965). The specific radioactivity of [14C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U‐14C]glucose.

There was no labelling of brain protein with [3H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [14C]glutamate and [14C]aspartate in rat brain protein was observed at 5 min after the injection of [U‐14C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U‐14C]glucose in the brain.
SIMILARITIES OF β‐BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION
Authors: Sen Indira , Cooper Jack R.
Published: October 2006

Abstract— β‐Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus, has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca2+‐dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β‐bungarotoxin acts as a phospholipase A2 (phosphatide 2‐acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A2 from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy‐dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.
EFFECTS OF ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN
Authors: Millan Noemi , Murdock Larry L. , Bleier Ruth , Siegel Frank L.
Published: October 2006

Abstract—

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.
NEUROCHEMICAL ABNORMALITY IN I‐CELL DISEASE: CHEMICAL ANALYSIS AND A POSSIBLE IMPORTANCE OF BETA‐GALACTOSIDASE DEFICIENCY1
Authors: Eto Yoshikatsu , Owada Misao , Kitagawa Teruo , Kokubun Yoshiyuki , Rennert OWEN M.
Published: October 2006

Abstract—

Sphingolipid composition in both gray and white matter of a patient with I‐cell disease was normal except for the higher proportion.of GMI‐ganglioside in gray and white matter. In the patient's liver and kidney there was a significant accumulation of ceramide dihexoside and ceramide trihexoside and of sulphatide in kidney. Non‐lipid hexosamine and sialic acid concentration in brain was increased 1.2‐1.5 times above normal. Recovery of myelin from I‐cell's white matter was 80‐100%, suggesting that demyelination, if present, is minimal. Myelin lipid and myelin specific glycoprotein patterns were normal. Except for β‐galactosidase activity the activity of other brain lysosomal enzymes were within the normal range. This finding was similar to that of Hurler's syndrome. Only β‐galactosidase activity was reduced to less than 10% of normal in the patient's brain. To examine the possible metabolic significance of β‐galactosidase deficiency in I‐cell disease the physical characteristics of this enzyme, isolated from tissues from I‐cell, Hurler and control patients, were compared using isoelectric focusing, Con A‐Sepharose and Sephadex G‐150 chromatography. The isoelectric point and the binding affinity of I‐cell β‐galactosidase with Con A‐Sepharose was comparable to normal. However, the isoenzyme patterns of brain and liver I‐cell β‐galactosidase with Sephadex G‐150 gel filtration revealed decreased acid β‐galactosidase. Effects of the addition of sodium chloride on each fraction of β‐galactosidase isoenzymes isolated from I‐cell tissues were markedly different from controls, whereas the pH optimum of these enzymes were similar to normal. These enzyme characteristics in I‐cell tissues were different from normal and Hurler's syndrome. These findings suggest that β‐galactosidase deficiency in I‐cell disease is a more specific phenomenon rather than secondary inhibition as found in the mucopolysaccharidoses and thus may have an important role for the pathogenesis of brain damage and disease occurrence.
THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES
Authors: Reith M. E. A. , Schotman P. , van Zwietek B. J. , Gispen W. H.
Published: October 2006

Abstract—

The incorporation into brain slice protein of externally provided [1‐14C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two‐site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl‐tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.
PERSISTENCE OF ALTERED RNA SYNTHESIS IN RAT CEREBRAL CORTEX 12h AFTER A SINGLE ELECTROCONVULSIVE SHOCK
Authors: Wynter C. V. A.
Published: October 2006

Abstract—

The effect of electroshock (ECS) on RNA synthesis in nuclei and cytoplasm of rat cerebral cortex was examined using a double label technique by intraventricular injection of [3H] and [14C]orotate. At t h after ECS, the incorporation into nuclear RNA was 80% of the control rate and the appearance of newly synthesized RNA in the cytoplasm was only 27.6%. Analysis on composite polyacrylamide‐agarose gels of purified RNA showed that the 3H/14C ratio of each gel slice slowly increased with decreasing M.W. of the RNA. This has been interpreted as an inhibition in the rate of processing of nuclear RNA. When the nuclear RNA was subjected to denaturation with 50% dimethyl sulphoxide (DMSO) this effect was enhanced. In a similar experiment, rats were injected, treated to ECS and killed 12 h later. The overall incorporation into nuclear and cytoplasmic RNA was increased to 174%, and 137.5% respectively. Analysis on gels showed very little variation in the 3H/14C ratio of the steady state levels of nuclear RNA. They compared well with a control experiment where rats were injected with [3H] and [14C]orotate as described above but no ECS was applied to the [14C] labelled animals. However a 1 h pulse label given 11 h after ECS treatment revealed that the rate of incorporation into nuclear RNA still showed a decrease of 81% of the control. The nuclear RYA fractionated on gels clearly showed that the inhibition of the processing rate of nuclear RNA was still occurring. This effect was again magnified on denaturation of the RNA with DMSO. This suggests that ECS may disturb RNA metabolism in nervous tissue for much longer periods than previously realised.
ACTIVITY AND ISOENZYME PATTERN OF LACTATE DEHYDROGENASE IN ASTROBLASTS CULTURED FROM BRAINS OF NEWBORN MICE
Authors: Nissen Claus , Schousboe Arne
Published: October 2006

Lactate dehydrogenase activity and isoenzyme distribution was determined in primary cultures of astroblasts as a function of the culture period. The specific activity increased during this period with a peak value (1.91 ± 0.18μmol x min‐1 x mg‐1 cell protein) after 2 weeks in culture. The isoenzyme pattern changed during 3 weeks in culture towards a higher proportion of the H4 (LDH‐1) isoenzyme which is analogous to the in vivo pattern. Omission of serum with or without dBcAMP (0.5 mM) in the culture medium during the third week of culture further enhanced this prominence of the H4 isoenzyme. The specific activity (1.58 × 0.06 μmol x min‐1 x mg‐1 cell protein) of cultures grown in the presence of 0.5 mM‐dBcAMP and absence of serum was close to the activity in the adult brain.
COUPLED TRANSPORT OF GLUTAMATE AND SODIUM IN A CEREBELLAR NERVE CELL LINE
Authors: Stallcup William B. , Bulloch Karen , Baetge E. Edward
Published: October 2006

The cerebellar nerve cell line ε1 has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na+ and furthermore, 22Na+ uptake is stimulated by glutamate, indicating that glutamate uptake and Na+ uptake are coupled. Two molecules of Na + are transported for each molecule of glutamate. The Km for glutamate is found to be 5 × 10−5M in both the glutamate uptake assay and the 22Na+ uptake assay, providing additional evidence for glutamate‐Na+ coupling. Pre‐incubation with ouabain, which inhibits the Na+‐K+ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na+ gradient. Tetrodotoxin, however, has no effect on glutamate‐induced 22Na+ uptake, showing that this Na+ flux does not occur via voltage‐dependent Na+ channels. Studies on the specificity of the ε1 glutamate transport system show that it is distinct from systems that transport alanine and glycine. l‐Glutamate, d‐aspartate, l‐cysteate, and l‐cysteine sulfinate are able to utilize the transport system efficiently. d‐Glutamate, l‐homocysteate, N‐methyl‐d, l‐aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate 22Na+ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, ε1 does not have an excitatory glutamate receptor.
USE OF ARRHENIUS PLOTS OF Na‐K ATPase and ACETYLCHOLINESTERASE AS A TOOL FOR STUDYING CHANGES IN LIPID‐PROTEIN INTERACTIONS IN NEURONAL MEMBRANES DURING BRAIN DEVELOPMENT
Authors: Nemat‐Gorgani M. , Meisami E.
Published: October 2006

Abstract— The activities of Na‐K ATPase and acetylcholinesterase in the rat brain cortex were measured at different postnatal ages as a function of temperature. It was found that compared to acetylcholinesterase, Na‐K ATPase is more strongly affected by the rise in temperature and that this response is further enhanced with age. Arrhenius plots of the data were prepared and the apparent energies of activation were computed for each plot. It was observed that all plots were biphasic except that for Na‐K ATPase of the immature (5‐day‐old) brain which showed no transition temperature, with an apparent energy of activation of 15.5 kcal/mol. The enzyme from the mature brain (25‐day‐old) showed an average transition temperature of 22.6°C, with average apparent energies of activation of 15.3 and 27.2 kcal/mol above and below the transition temperature respectively. The cortex of 1‐day‐old rat showed no Na‐K ATPase activity. Arrhenius plots of acetylcholinesterase studied at ages 1, 5 and 25 days postnatally all showed transition temperatures which increased from an average of 16.1°C for 1‐day‐old to 17 and 21.5°C for 5‐ and 25‐day‐old animals respectively. The average apparent energies of activation for acetylcholinesterase below the transition temperature changed from 8.3 kcal/mole at day 1 to 8.7 and 7.2 kcal/mol at days 5 and 25, while above the transition temperature they were 4.3, 5.2 and 4.1 at days 1, 5 and 25 respectively. The results are discussed in terms of the differences and changes in the interactions of Na‐K ATPase and acetylcholinesterase with membrane lipids during the postnatal phase of brain development.
RADIOENZYMATIC ASSAY OF FEMTOMOLE CONCENTRATIONS OF DOPA IN TISSUES AND BODY FLUIDS
Authors: Zurcher G. , Prada M. Da
Published: October 2006

Abstract— A single isotope radioenzymatic procedure for the measurement of DOPA has been developed. The assay combines O‐methylation of DOPA by purified COMT using [3H]SAM as the methyl donor and subsequent purification as the DNFB derivative of 3‐O‐[methyl‐3H]DOPA. The present method is about 100 times more sensitive than currently available DOPA methods. This is due to decreased blank values and increased enzymatic conversion giving transmethylation values of 50% with tissue extracts and values of almost 100% with pure solutions. Although COMT methylates a wide variety of catechol compounds, specificity of the assay is achieved by selective extraction and purification of the final product by tlc.

The method has good inter‐assay reliability, the coefficient of variation being about 3.5%.

This ultramicromethod was used to determine the steady‐state concentrations of endogenous DOPA in minute samples of brain areas of the rat. In untreated rats brain DOPA levels varied with the mode of death; the highest levels were found in animals killed by microwave irradiation. Unconjugated DOPA was measured in microlitre aliquots of human body fluids.
GABA SYNTHESIS BY CULTURED FIBROBLASTS OBTAINED FROM PERSONS WITH HUNTINGTON'S DISEASE1
Authors: Gray Peter N. , Dana Sharon L.
Published: October 2006

Abstract— A consistent observation in particular regions of brains of persons having died with Huntington's disease (HD) is a reduction in the concentration of γ‐aminobutyric acid (GABA) and a decrease in the activity of its synthetic enzyme, glutamate decarboxylase (EC 4.1.4.15). GABA levels are also reduced in HD cerebrospinal fluids. This study suggests that skin fibroblasts obtained from persons with HD can be used to study their GABA system. A rapid and specific assay for [14C]glutamate– [14C]GABA based on Aminex A‐7 chromatography has been developed. Cell monolayers and homogenates of HD cells convert [14C]glutamate to [14C]GABA. GABA synthesis by HD cell homogenates is pyridoxal dependent and is inhibited by 1 mm‐aminooxyacetic acid. GABA synthesis by HD and control cell homogenates also show the same thermal sensitivity as rat brain GAD. When compared to non‐HD human cells the HD cells reveal disturbances in the non‐neuronal GABA metabolic pathway. Concentrated HD cell homogenates synthesize approx 3 times the amount of GABA as control cells. When diluted both extracts made similar amounts of GABA. Synthesis of GABA by HD cell homogenates is not inhibited by cysteine sulfinate. Decarboxylation of glutamate in these cells is therefore most likely due to glutamate decarboxylase and not cysteine sulfinate decarboxylase. HD cells in monolayer also synthesize 3 times the amount of GABA as compared to control cells. In addition, glutamate upake is altered in HD cells. This report indicates there may be a different pattern of enzyme regulation between HD and control cells.
AGONIST‐INDUCED AFFINITY ALTERATIONS OF A CENTRAL NERVOUS SYSTEM α‐BUNGAROTOXIN RECEPTOR1
Authors: Lukas Ronald J. , Bennett Edward L.
Published: October 2006

Abstract— The ability of cholinergic agonists to block the specific interaction of α‐bungarotoxin (α‐Bgt) with membrane‐bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of α‐Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high‐affinity form toward agonist. This change in receptor state occurs with a half‐time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of α‐Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist‐induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high‐affinity state is non‐competitive. This result suggests that there may exist discrete toxin‐binding and agonist‐binding sites on central toxin receptors.
SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA‐PIG CEREBRAL CORTEX AND SPLEEN
Authors: Thomas David Vivian
Published: October 2006

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea‐pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non‐occluded micro‐particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [3H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non‐occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6‐0.8 m‐sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub‐fractions D to G (0.4‐1.0 m‐sucrose) and the other in sub‐fraction J (1.4 m‐sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.

Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4‐0.6 m‐sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m‐sucrose).
THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA
Authors: Sarthy P. Vijay , Lam Dominic M. K.
Published: October 2006

Abstract— The uptake and release of [3H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10‐7m‐[3H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [3H]‐dopamine. (2) [3H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis‐Menten kinetics. This uptake could be explained by a single ‘high‐affinity’ mechanism with a Km of 2.61 ± 0.41 ± 10‐7m and a Vmax of 66 ± 12 ± 10‐12 mol/min/mg protein. (3) [3H]dopamine uptake was temperature‐dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca2+ or the presence of Co2+; however, more than 85, uptake was blocked in the absence of external Na+. (5) Neither 1 mm‐cyanide nor 5 mm‐iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [3H]dopamine uptake. (7) [3H]dopamine in the retina could be released by increasing the external K+ concentration. This release was Ca2+ ‐dependent and was blocked by 10mm‐Co2+ or 2Omm‐Mg2+. The amount of [3H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.