bluuebunny/crossref_metadata_2025 huggingface dataset:
METABOLISM OF GLUTAMIC ACID AND RELATED AMINO ACIDS IN THE BRAIN STUDIED WITH 14C‐LABELLED GLUCOSE, BUTYRIC ACID AND GLUTAMIC ACID IN HYPERCAPNIC RATS
Authors: Weyne J. , Van Leuven F. , Demeester G. , Leusen I.
Published: October 2006

Abstract— The influence of hypercapnia on the metabolism of glutamic acid, aspartic acid, glutamine and GABA in rat brain was studied using three different precursors. Acute hypercapnia induced a fall in the concentration of glutamic and aspartic acid, and a rise in the concentration of glutamine and GABA. Acute hypercapnia had a profound effect on the relative specific radioactivity of glutamine indicating that the excess glutamine, present in the brain in hypercapnia, was synthetized from glutamic acid in the compartment where it could become quickly labelled from butyric and glutamic acid, but not from glucose. This effect was maintained in chronic hypercapnia.
KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INHIBITION STUDIES WITH N‐(5′‐PHOSPHOPYRIDOXYL) AMINO ACIDS1
Authors: Bayón A. , Possani L. D. , Tapia R.
Published: October 2006

Abstract— Seven N‐(5′‐phosphopyridoxyl) amino acids, reduced analogs of the glutamate‐pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul‐finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non‐linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.
A MODIFIED PROCEDURE FOR ISOLATION OF ASTROCYTE‐ AND NEURON‐ENRICHED FRACTIONS FROM RAT BRAIN
Authors: Farooq Muhammad , Norton William T.
Published: October 2006

Our previously published method for isolation of neurons with extensive processes (Farooqet al., 1977) has been modified to permit the isolation of both astrocyte‐ and neuron‐enriched fractions. Rat cerebral tissue is incubated with acetylated trypsin and disrupted. The cell suspension is separated first by differential centrifugation and then by gradient centrifugation on discontinuous Ficoll gradients. The method is reproducible and is applicable equally well to immature and adult animals. The yield of astrocytes of 57% particle purity, and higher weight purity, is 4–7 × 106 cells/brain, amounting to 1.5–2.0 mg of protein. The astrocytes appear to be a mixture of fibrous and protoplasmic types. The yield of neurons of 90% particle purity is 10–14 × 106 cells/brain, amounting to 2.4–3.0 mg of protein. A total yield of neurons of 28–37 × 106 cells/brain can be obtained at 70% purity. These preparations have been characterized by light microscopy and protein, RNA and DNA content.
PURIFICATION OF MYELIN CARBONIC ANHYDRASE
Authors: Sapirstein Victor S. , Lees Marjorie B.
Published: October 2006

Abstract— A procedure has been developed for the purification of the membrane bound form of carbonic anhydrase from rat brain myelin. The procedure is rapid, requiring only two steps, and can be applied to small amounts of material. Conditions have been established whereby the enzyme can be almost quantitatively solubilized with up to a 60 fold increase in specific activity. Purification by affinity chromatography yields a preparation which is homogeneous by polyacrylamide gel electrophoresis. However, preliminary evidence suggests that activity may be reduced by the removal of lipids during chromatography and subsequent dialysis. The purified preparation is high in dicarboxylic and hydroxyl amino acids and contains only 1×2 cysteine residues. The reduction of cysteine appears to be essential for the full expression of enzymatic activity.
THE EFFECT OF BROMOCRIPTINE ON RAT STRIATAL ADENYLATE CYCLASE AND RAT BRAIN MONOAMINE METABOLISM
Authors: Markstein R. , Herrling P. L. , Asper H. R. Buurki. H. , Ruch W.
Published: October 2006

In slices and homogenate from rat brain striatum bromocriptine in marked contrast to DA. NA and apomorphine. had no stimulatory effect on adenylate cyclase activity, but antagonised the stimulatory effects of both NA and DA.

Bromocriptine (10 mg/kg s.c.) decreased the turnover of DA in striatum and limbic structures 3h after drug administration. However, an increase in the turnover of NA in the brain stem and that of 5‐HT in the cortex was observed 4h following treatment with bromocriptine. Possible modes of action of bromocriptine are discussed.
INCREASE IN THE RELATIVE RATE OF SYNTHESIS OF DOPAMINE‐β‐HYDROXYLASE IN IN THE NUCLEUS LOCUS COERULEUS ELICITED BY RESERPINE
Authors: Ross Robert A. , Joh Tong Hyub , Reis Donald J.
Published: October 2006

Abstract— Three days following a single injection of reserpine (10 mg/kg, i.p.) the activity and amount of dopamine‐β‐hydroxylase (DBH) are increased nearly 2‐fold in the noradrenergic cell bodies of the nucleus locus coeruleus of rat. To determine if this increased accumulation of DBH is due to an increased rate of enzyme synthesis, [3H]amino acids were infused into the IVth ventricle of reserpine‐and saline‐injected rats. This method was 35 times more effective than intracisternal infusion and 600 times more effective than intravenous infusion. DBH protein was isolated from the locus coeruleus by immunoprecipitation and SDS‐electrophoresis. These steps proved crucial for the complete isolation of DBH from other labelled proteins. Indeed, only 10–15% of the immunoprecipitate was finally identified as labelled DBH protein. The rate of incorporation of [3H]leucine into DBH protein of locus coeruleus was increased to 181%, of control following reserpine, whereas that into TCA‐precipitable protein was unchanged. A similar result was obtained using [3H]lysine. In contrast, the apparent half‐life of the enzyme did not change following reserpine. The relative rate of synthesis of DBH ([3H]DBH/3H‐total protein), denoting selectivity of response, was increased in the locus coeruleus of reserpine‐treated rats to 154% of control (P < 0.01). These findings indicate that increased synthesis accounts for the observed increase in DBH protein in the locus coeruleus following reserpine administration.
CALCIUM UPTAKE AND EXCHANGE IN LEG NERVES OF THE CRAB ‘LIBINIA EMARGINATA’
Authors: Bianchi C. Paul , Lakshminarayanaiah N.
Published: October 2006

Abstract—

The kinetics of washout of radioactive calcium (45Ca) from nerves of the larger legs of Libinia emarginata equilibrated in various solutions of artificial sea water (ASW) containing 1, 11, 22 or 44 mM‐Ca was followed. In every case two components were found when the period of washout was not more than 30 min. The washout of 14C labelled inulin and sucrose gave one and two components respectively; these substances were considered to exist in the extracellular regions of nerve fibers. Comparison of the total calcium of the nerves determined spectrophotometrically and by measurement of radioactivity indicated that the exchangeability of calcium in the nerves in ASW containing 44 and 22 mM‐Ca was 100%. In the case of nerves equilibrated in 11 mM‐CaASW, two groups of nerves, one (group A) containing a high concentration of Ca (13.6 μmol/g) and the other (group B) containing a low concentration (7.3 μmol/g), were detected Several factors which could account for the accumulation of Ca in nerve fibers in group A were considered. The electrolyte data relating to group A nerve fibers indicated that Ca accumulation may be due to the presence of a high concentration of sodium in those fibers. However, the exchangeability of Ca for group A was about 47%; and in group B fibers, it was 61%. The exchangeability was less than 41% in the case of nerves equilibrated in 1 mM‐CaASW. The kinetic parameters of 45Ca washout from the nerve fibers have been used to develop a model for the distribution of calcium among the different compartments of the nerve fibers.
SYNTHESIS AND METABOLISM OF l‐KYNURENINE IN RAT BRAIN
Authors: Gál E. M. , Sherman A. D.
Published: October 2006

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3‐hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma‐tography and electron‐capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3‐hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [14C]tryptophan or 1.14 nmol/g/h from [3H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [14C]tryptophan, the presence of anthranilic, 3‐hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.
THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA
Authors: Dennis S. G. C. , Clark J. B.
Published: October 2006

Abstract—

The synthesis of glutamate from 2‐oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin.

Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min−1. mg protein−1) of synaptic mitochondria (0.65 nmol. min−1. mg protein−1) when pyruvate is the precursor for 2‐oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min−1, mg protein−1) when 3 hydroxybutyrate is the precursor.

Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2‐oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5‐fold higher (5‐6nmol. min−1. mg protein−1) than glutamate synthesis from endogenously produced 2‐oxoglutarate. In the uncoupled state (or un‐coupler + oligomycin) the rate was reduced by half. (2.5‐3 nmol. min−1. mg protein−1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin).

The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH+4 addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria.

Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state.

The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.
PARTIAL PURIFICATION AND KINETICS OF γ‐GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS
Authors: Prusiner Paul E. , Prusiner Stanley B.
Published: October 2006

Abstract— γ‐Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane‐bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L‐Methionine was the best amino acid substrate and its corresponding peptides L‐methionylmethionine and L‐methionyl‐L‐serine were also good γ‐glutamyl acceptors. L‐Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ‐glutamyl acceptor than glycine, triglycine, or tetraglycine. L‐Alanylglycine was a superior acceptor to glycine, L‐alanine, or L‐alanylglycylglycine, while the D‐isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ‐glutamyl transpeptidase could catalyze the transfer of γ‐glutamyl groups to glycylglycyl‐L‐alanine and L‐alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ‐glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.
NEUTRAL AMINO ACIDS IN THE BRAIN: CHANGES IN RESPONSE TO FOOD INGESTION1
Authors: Fernstrom John D. , Faller Douglas V.
Published: October 2006

Abstract— The brain levels of each of the aromatic and branched‐chain amino acids change 2 h after fasting rats begin to consume either a carbohydrate‐fat diet or a similar diet containing 18% or 40% protein. Carbohydrate‐fat ingestion elevates the concentrations of each of the aromatic amino acids in brain, while substantially depressing those of the branched‐chain amino acids. The inclusion of protein in this diet suppresses the increases in brain aromatic amino acids and attenuates the decreases in the branched‐chain amino acids. The changes in the brain level of each neutral amino acid following the ingestion of any of these diets correlate extremely well with the effects of the diet on the serum neutral amino acid pattern, specifically on the serum concentration ratio of each neutral amino acid to the sum of the other neutral amino acids. The diet‐induced changes in the brain level of each of the amino acids also correlate surprisingly well with the calculated rate of brain influx for each amino acid.
SUBCELLULAR LOCALIZATION AND PARTIAL PURIFICATION OF A CHLORIDE DEPENDENT ANGIOTENSIN‐I CONVERTING ENZYME FROM RAT BRAIN
Authors: Benuck M. , Marks N.
Published: October 2006

Abstract— Angiotensin converting enzyme (peptidyl dipeptide hydrolase EC 3.4.15.1) was extracted from particulates of rat brain using the nonionic detergent Triton X‐100. Enzyme activity in subcellular fractions was associated with purified synaptosomes and present in the microsomal fraction, but absent in purified mitochondria and water‐shocked myelin. Partial purification was achieved by chromatography on DEAE‐cellulose and hydroxylapatite columns. The enzyme had a pH optimum of pH 7–8 and an apparent Km of 2.2 mm using hippuryl‐histidyl‐leucine as substrate; it was chloride dependent, inhibited by (Sar1‐Ala8)‐angiotensin‐II (saralasin), and, at lower concentrations, by the specific nonapeptide inhibitor SQ 20881. Associated with the purified enzyme was an aminopeptidase, cleaving N‐terminal Asp from the native substrate, which could be involved in the production of the active heptapeptide, angiotensin III (des‐Asp‐angiotensin‐II). Also present was a carboxypeptidase‐like enzyme removing C‐terminal Phe following the liberation of His‐Leu by converting enzyme, which may be involved in the inactivation of angiotensin II or III.
REGIONAL DISTRIBUTION OF MONOAMINES AND THEIR METABOLITES IN THE HUMAN BRAIN
Authors: Mackay A. V. P. , Yates C. M. , Wright A. , Hamilton P. , Davies P.
Published: October 2006

Abstract— Noradrenaline (NA), dopamine (DA). 5‐hydroxytryptamine (5‐HT), 4‐hydroxy, 3‐methoxy‐phenylethylene glycol (MHPG), homovanillic acid (HVA), 3,4‐dihydroxyphenylacetic acid (DOPAC) and 5‐hydroxyindolylacetic acid (5‐HIAA) were measured in twenty areas of post‐mortem brain from ten psychiatrically and neurologically normal patients. There was a marked difference, which did not appear to be related to sex, medication, cause of death or time between death and dissection, in amine and metabolite concentrations between brains. In the cortex, 5‐HT, MHPG, HVA. DOPAC and S‐HIAA were approximately even in their distribution; NA and DA could not be detected. In sub‐cortical areas there were clear differences in the distribution of the three amines accompanied by less marked differences in the distribution of their respective metabolites.
REGIONAL DISTRIBUTION OF HOMOCARNOSINE, HOMOCARNOSINE‐CARNOSINE SYNTHETASE AND HOMOCARNOSINASE IN HUMAN BRAIN
Authors: Kish Stephen J. , Perry Thomas L. , Hansen Shirley
Published: October 2006

Homocarnosine (HCarn) content varied over a 6‐fold range in different regions of autopsied human brain, being highest in the dentate nucleus and the inferior olive, and lowest in the caudate nucleus and mesolimbic system. HCarn content was similar in biopsied and autopsied frontal cortex. Very little if any carnosine (Carn) was present in human brain, except for the olfactory bulb, where Carn may have comprised 20% of the imidazole dipeptides present. Only HCarn was present in human CSF.

HCarn‐Carn synthetase enzyme activity in biopsy specimens of human frontal and temporal cortex was approx 10 times greater than has been reported for rat cerebral cortex. The enzyme synthesized Carn 3–5 times as rapidly as HCarn, when β‐alanine (β‐Ala) or GABA substrate concentrations were 10 MM. The synthetase was found to have an apparent Km of 1.8 mM for β‐Ala, and 8.8 mM for GABA. HCarn‐Carn synthetase activity decreases rapidly after brain death, and was not detectable in autopsied brain specimens frozen more than 6 h after patients’deaths.

Homocarnosinase activity was determined in brain, using L‐[γaminobutyryl‐1‐14C]HCarn as substrate, and measuring radioactive GABA produced by hydrolysis of HCarn at pH 7.2 in the presence of Co2+ ions. Homocarnosinase activity was similar in biopsied and autopsied human cerebral cortex, and appeared to be stable for at least 10 h after death in unfrozen brain.

Differences in the regional distribution of HCarn‐Carn synthetase and homocarnosinase activities, as well as regional differences in GABA content in human brain, do not readily account for regional differences in HCarn content, nor do they suggest a physiological role for HCarn.
REGIONAL DIFFERENCES IN LIPID COMPOSITION IN RABBIT NERVOUS TISSUE
Authors: Ishibe Takeshi , Yamamoto Akira
Published: October 2006

Lipid composition was determined for different regions of rabbit nervous tissue.

In the white matter of adult rabbit, the ratio between cholesterol, phospholipids and sphingolipids was quite constant. Among the subclasses of phospholipids, phosphatidylcholine and sphingomyelin tended to compensate for each other as constituents of myelin, as did galactosphingolipids and sphingomyelin amongst the sphingolipids. The brain was rich in phosphatidylcholine and gakactosphingolipids, while peripheral nerves (PN) were rich in sphingomyelin. The spinal cord showed a composition intermediate between the brain and PN.

The sphingolipid to phosphatidylcholine ratio seems to be useful as a myelin maturation index applicable to both CNS and PN.

The rostral part of the CNS showed a high ratio of molecular species of cerebroside with α‐hydroxy fatty acids to those with unsubstituted fatty acids (CH/CN). The caudal part of the CNS had a high concentration of cerebrosides with C24‐monoenoic fatty acids, so that there was an inverse relationship between CH/CN and C24:1/C24:0 for different regions of CNS.

The present data show that the lipid composition as well as the fatty acid composition of myelin‐specific lipids are influenced by neural differentiation and development or by neuroglial relationships.
SPIN LABEL STUDIES ON THE EFFECT OF ANESTHETICS IN SYNAPTIC MEMBRANES
Authors: Lenaz G. , Curatola G. , Mazzanti L. , Bertoli E. , Pastuszko A.
Published: October 2006

We have studied the effects of anesthetics on synaptic membranes obtained from pig brain by using stearic acid spin labels. The anesthetics used (butanol, halothane, ketamine) affect the rotational mobility of 16‐doxylstearate and the order parameter of 5‐doxylstearate. The changes in mobility of 16doxylstearate show a stronger fluidization in the membrane core than in vesicles of lipids extracted therefrom. This effect may be operationally described as a disruption of lipid‐protein interactions involving hydrophobic proteins. In fact no disordering is induced on the surface of synaptic membranes as shown by the order of Soioxylstearate, indicating a highly immobilized state of the lipids on the membrane surface. The results are discussed in view of our working hypothesis concerning the role of lipids in modulating protein conformation.
THE SUBUNIT STRUCTURE OF MAMMALIAN ACETYLCHOLINESTERASE: CATALYTIC SUBUNITS, DISSOCIATING EFFECT OF PROTEOLYSIS AND DISULPHIDE REDUCTION ON THE POLYMERIC FORMS
Authors: Vigny Marc , Bon Suzanne , Massoulié Jean , Gisiger Victor
Published: October 2006

Abstract— An analysis of the [3H]DFP‐labelled catalytic subunits of mammalian (bovine SCG) acetylcholinesterase (AChE, EC 3.1.1.7.) indicates a monomer molecular weight of 75,000. This is equivalent to the mass previously determined for the smallest active form and demonstrates that the globular, or G forms, are respectively monomeric (G1 form, 4S), dimeric (G2 form, 6.5S) and tetrameric (G4 form, 10S). In the tetrameric G4 form the catalytic chains are associated in dimers, by disulphide bonds.

The effect of reduction and proteolysis has shown that the dimeric form (G2 form, 6.5S) is readily reduced into G1, while the tetramer G4 is very stable, being only dissociated by a combination of reduction and proteolysis by high concentration of trypsin. The asymmetric forms A12 (16S), A8 (13S) and A4 (9S) are not sensitive to reduction, but are readily dissociated by low concentrations of trypsin, into each other, progressively liberating isolated tetramers. We obtained essentially identical results with AChE preparations from rat brain or superior cervical ganglion. These observations support a general model for the quaternary structure of acetylcholinesterase molecular forms.
PURIFICATION OF 2′,3′‐CYCLIC NUCLEOTIDE 3′‐PHOSPHOHYDROLASE FROM BOVINE BRAIN BY IMMUNOAFFINITY CHROMATOGRAPHY: FURTHER BIOCHEMICAL CHARACTERIZATION OF THE PROTEIN
Authors: Drummond Robert J. ,   With the technical assistance of Elizabeth B. Hamill
Published: October 2006

Abstract— The purification of small amounts of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase from bovine white matter by ion‐exchange techniques (Drummondet al., 1978) has been used to provide antigen for the production of specific rabbit antibodies to this enzyme. Specific antibody has been purified from immune serum by affinity chromatography on a column of Sepharose to which the enzyme has been attached, and the purified antibody has been coupled to cyanogen bromide‐activated Sepharose. Affinity chromatography on the immunoadsorbent effectively purifies 2′,3′‐cyclic nucleotide 3 ‐phosphohydrolase in one step from an extract of an acetone powder made from bovine white matter. This modified purification procedure has reduced the time required for purification and increased the yield of the enzyme to 57%. In SDS‐gel electrophoresis in phosphate buffer the enzyme migrates as an aggregate of about 98,000MW. When the buffer is Tris‐glycine, the apparent MW is about 44,000 and under specific conditions two proteins of only slightly different mobilities can be discerned. Within experimental error the amino acid compositions of the proteins in the two bands are indistinguishable. Peptide patterns obtained by polyacrylamide gel electrophoresis following proteolytic digestion with Straphylococcus aureus V8 protease or papain show extensive structural homology between the two proteins, but detectable differences are apparent.
PURIFICATION FROM HUMAN BRAIN AND SOME PROPERTIES OF TWO NADPH‐LINKED ALDEHYDE REDUCTASES WHICH REDUCE SUCCINIC SEMIALDEHYDE TO 4‐HYDROXYBUTYRATE
Authors: Cash Christopher D. , Maitre Michel , Mandel Paul
Published: October 2006

Abstract— Two NADPH‐linked aldehyde reductases (alcohol:NADP+oxidoreductase, EC 1.1.1.2) capable of reducing succinic semialdehyde to the anaesthetic Chydroxybutyrate have been purified from human brain to electrophoretic homogeneity. The first of these enzymes, which is typical of its category, is not specific for succinic semialdehyde and can reduce some aromatic aldehydes at a high rate. It is a monomer of molecular weight about 45,000 and is strongly inhibited by various hypnotics and anticonvulsants. The second enzyme is, in contrast, fairly specific for succinic semialdehyde. It is a dimer of molecular weight about 90,000 and is not inhibited by the hypnotics and anticonvulsants which inhibit the first enzyme. It is thus different from previously described aldehyde reductases from human brain.
NIACIN AND NIACINAMIDE TRANSPORT IN THE CENTRAL NERVOUS SYSTEM. IN VIVO STUDIES
Authors: Spector Reynold
Published: October 2006

Abstract– The concentration ol niacinamide in plasma and CSF was 0.5 and 0.7 μm respectively. The, mechanisms by which niacin and niacinamide, which are not synthesized in brain, enter brain, CSF and choroid plexus were investigated by injecting [14C]niacin or [14C]niacinamide intravenously and intraventricularly. [14C]Niacin or [14C]niacinamide, with or without unlabeled niacin or niacinamide, were infused intravenously at a constant rate into conscious rabbits. At 3 h, [14C]niacinamide, but not [14C]niacin, readily entered CSF, choroid plexus and brain. The addition of 4.1 mmol/kg niacinamide to the infusate markedly depressed the relative entry of [14C]niacinamide into choroid plexus and brain but not into CSF. After intraventricular injection, [14C]niacin was rapidly cleared from CSF and readily entered brain and choroid plexus. The addition of unlabeled niacin to the intraventricular injectate decreased the clearance of [14C]niacin from CSF and the entry of [14C]niacin into choroid plexus and brain. Unlike niacin, carrier niacinamide (82 μmol) in the injectate did not depress the extremely rapid clearance of intraventricularly injected [14C]niacinamide from CSF but did decrease the entry of [14C]niacinamide into brain. These results show that the control of entry and exit of niacinamide and niacin is the mechanism, at least in part, by which total niacin and NAD levels in brain cells are regulated. In the case of niacinamide which readily passes between CSF and plasma, the regulation of entry of niacinamide into brain cells by a high affinity accumulation system is an integral part of the homeostatic system. In the case of niacin, penetration into CSF and the extracellular space of brain from plasma as well as regulation of entry into brain cells by a saturable accumulation system are two distinct parts of the homeostatic system. In vivo, niacin that enters the central nervous system is converted to the principal plasma vitamer, niacinamide, in its free or bound forms such as NAD.
UPTAKE OF MANGANESE BY CHROMAFFIN GRANULES IN VITRO
Authors: Daniels A. J. , Johnson L. N. , Williams R. J. P.
Published: October 2006

Abstract– The distribution of metal ions in the chromaffin granule is anomalous. Calcium ions are accumulated but magnesium ions excluded. We have used the manganese ion as a convenient divalent element for following metal ion uptake because it can usefully be employed as a substitute for magnesium.

The kinetics of the manganese incorporation were followed by radio tracer technique and its transport was characterized as being non‐energy dependent and having a small thermal activation factor. The presence in the incubation medium of a membrane‐bound ATPase inhibitor (NEM) or addition of CN− or NaN3 did not affect the metal incorporation into chromaffin granules. Little endogenous magnesium exchanges for manganese when the latter is incorporated, but magnesium competitively prevented manganese uptake. The binding of manganese inside the vesicle was strong and it was not exchangeable with magnesium even in the presence of ATP.

Manganese when incorporated binds to ATP and displaces catecholamines resulting in a decrease in the CA/ATP ratio.
RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI
Authors: STOYKOVA A. S. , DABEVA M. D. , DIMOVA R. N. , HADJIOLOV A. A.
Published: October 2006

Abstract– The method of Thompson (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM‐Mg2+ in the isolating media. This technique gives a good yield of pure (85‐90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3‐fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea‐agar gel electrophoresis allows the identification of 45S, 41S, 39S, 36S, 32S and 21S pre‐rRNA components. The pattern of nucleolar pre‐rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre‐rRNA processing pathways qualitatively similar to those observed in other animal tissues.
THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE
Authors: Chee Po Yok , Dahl June L. , Fahien Leonard A.
Published: October 2006

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.
Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes
Authors: Zisapel Nava , Levi Miriam , Gozes Illana
Published: October 2006

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate‐urea polyacrylamide gel electrophoresis, by two‐dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycolbis (aminoethyl ether)‐N, N'‐tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α‐tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α‐tubulin than of the β subunit. Non‐ionic detergents such as Triton X‐100 or Nonidet P‐40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α‐tubulin is an integral vesicle membrane protein, whereas most of the β sub‐unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.
Developmental Patterns of Catechol‐O‐Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies
Authors: Goldstein D. J. , Weinshilboum R. M. , Dunnette J. H. , Creveling C. R.
Published: October 2006

Abstract: Catechol‐O‐methyltransferase (COMT) activity in the liver and kidneys of adult Fischer‐344 (F‐344) rats is only half of that in the same organs of Wistar‐Furth (W‐F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether “temporal gene” effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F‐344 rats is only 50–60% of that in the same organs of W‐F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one‐ to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F‐344 and W‐F rats.
Accumulations of Cyclic AMP in Adenine‐Labeled Cell‐free Preparations from Guinea Pig Cerebral Cortex: Role of α‐Adrenergic and H1‐Histaminergic Receptors
Authors: Daly J. W. , McNeal E. , Partington C. , Neuwirth M. , Creveling C. R.
Published: October 2006

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine‐labeled nucleotides in particulate fractions from Krebs‐Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac‐like entities, which apparently are associated with a significant portion of the tnembranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine‐labeled cyclic AMP‐generating systems by norepinephrine is by means of α‐adrenergic receptors, while activation by histamine is through H1‐ and H2‐histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via „release” of endogenous adenosine. It is proposed, based on the virtual absence of an α‐adrenergic or H1‐histaminergic response in the presence of a combination of potent adenosine and H2‐histaminergic antagonists, that α‐adrenergic and H1‐histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs‐Ringer particulate fractions, but merely facilitate activation by β‐adrenergic, H2‐histaminergic, or adenosine receptors.
Measurement of Free Glucose Turnover in Brain
Authors: Savaki H. E. , Davidsen L. , Smith C. , Sokoloff L.
Published: October 2006

A method has been developed for the measurement of the turnover rate constant or the half‐life of the free glucose content of brain. It is based on an equation derived by the mathematical analysis of a kinetic model of the equilibration of the specific activity of the free glucose in brain with that of the plasma during an infusion of radioactive glucose. The method requires the measurement of the time course of the specific activity of glucose in the arterial plasma during an intravenous infusion of radioactive glucose for a period of 1 to 4 min and the specific activity of the free glucose in brain at the termination of the infusion. The turnover rate constant, or the half‐life, is then calculated from these data by means of the operational equation of the method. The technique has been applied to conscious and anesthetized rats. In conscious rats the half‐life of the free glucose content of brain was found to be 1.6 ± 0.5 min (mean ±s.d.) when the animals were killed by decapitation and 1.2 ± 0.2 min (mean ±s.d.) when they were killed by microwave irradiation; this difference is not statistically significant. In anesthetized rats, the half‐life was found to be 2.6 ± 0.8 min (mean ±s.d.) in those killed by decapitation and 1.8 ± 0.3 min (mean ±s.d.) in those killed by microwave irradiation; this difference is statistically significant. The half‐life of the glucose content of brain was found to be significantly prolonged during anesthesia and to be significantly and positively correlated with the plasma glucose concentration (r= 0.78; p < 0.001).
Immunochemical Characterization of Bovine Brain Cathepsin D
Authors: Whitaker John N.
Published: October 2006

Antibodies to bovine brain cathepsin D were produced in rabbits and guinea pigs and utilized to characterize this enzyme immunochemically. By immunodiffusion, broad organ–but limited species–cross‐reactivity was demonstrated with antisera to the brain enzyme. On immunoelectrophoresis the immunoreactive material migrated as a single are in the beta‐gamma region. In a double antibody radioimmunoassay, which had a sensitivity for cathepsin D of 1 ng of immunoreactive enzyme per assay tube, no inhibition was produced by pepstatin, bovine myelin basic protein, or bovine basic protein peptides 1‐42, 43‐88 or 89‐169. Comparative studies of enzymatic and immunoreactive activity of bovine brain cathepsin D demonstrated a close correlation, but the immunoassay was much more sensitive in detecting the presence of enzyme protein. Immunoreactivity persisted unaltered under conditions that abolished or reduced enzymatic activity. Antibodies to brain cathepsin D inhibited the activity of the enzyme on a hemoglobin substrate at pH 5. On gel filtration of bovine brain homogenate, the immunoreactivity, measured by either radial immunodiffusion or radioimmunoassay, and enzymatic activity of cathepsin D coeluted, indicating a molecular weight of approximately 42,000. However, immunofixation of enzyme on polyacrylamide slab gels subjected to electrophoresis in the presence of sodium dodecyl sulfate demonstrated bands of immunoreactivity in purified enzyme in the molecular weight ranges of 42,000, 31,000 and 13,000 and two very faint bands in the range of 32,000‐36,000. Immunofixation also revealed antigen in 42,000 and 31,000 molecular weight species of homogenates of bovine brain, liver, spleen, and kidney.
Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles
Authors: Angelides Kimon J.
Published: October 2006

Highly purified “heavy” synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of /‐norepinephrine and was saturable with a Km for l‐norepinephrine at 3.3 μM and 1.8 mM for ATP. The ghosts also accumulated 5‐hydroxytryptamine and l‐epinephrine via an ATP‐dependent mechanism. Uptake was stereospecific for the l‐form. A functional catecholamine transporter could be solubilized by the detergent octyl‐glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l‐norepinephrine. Reserpine‐ and l‐propranolol‐sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans‐membrane pH gradient created by the proton‐translocating ATPase.
Modulation of the Distribution of Acetylcholinesterase Molecular Forms in a Murine Neuroblastoma × Sympathetic Ganglion Cell Hybrid Cell Line
Authors: Lazar Monique , Vignyt Marc
Published: October 2006

Studies were carried out on the polymorphism of acetylcholinesterase (AChE, EC 3.1.1.7) in a neuroblastoma × sympathetic ganglion cell hybrid cell line (T28) and its parental clone (N18TG2). These cells contain the tetrameric (G4, 10S), dimeric (G2, 6.5S), and monomeric (GI, 4S) forms of AChE, but not the collagen‐tailed A12 (16S) form of the sympathetic ganglion. Three variants of these forms could be distinguished on the basis of their solubility properties: (i) secreted forms which do not interact with the detergent Triton X‐100; (ii) cellular forms which may be solubilized in detergent‐free buffer and which interact reversibly with Triton X‐100; (iii) cellular forms which require detergent for solubility, and aggregate in its absence. By using a nonpenetrating inhibitor, we demonstrated that, in T28 stationary cells, the cellular G4 form is associated with the plasma membrane, whereas the G1 form is intracellular. During induction of AChE activity in T28 cells, the relative proportion of the G4 form increases, suggesting, in agreement with previous observations, that GI is a metabolic precursor of G4. The evolution of AChE molecular forms released into the culture medium closely resembles that of the cellular forms. The preferential accumulation of the G4 molecules does not simply depend on the cellular level of G1. It is favoured by culture conditions which promote morphological differentiation, but does not require the actual extension of neurites. T28 cells as well as other neuroblastoma‐derived cells appear to be useful experimental materials to investigate the regulatory mechanisms underlying the maturation of AChE globular forms.
Amino Acid Changes in a Genetic Strain of Epileptic Beagle Dogs
Authors: van Gelder N. M. , Edmonds H. L. , Hegreberg G. A. , Chatburn C. C. , Clemmons R. M. , Sylvester D. M.
Published: October 2006

A neurochemical evaluation of beagle dogs with naturally occurring spontaneous generalized convulsive seizures was performed. Amino acid profiles of serum, cerebrospinal fluid (CSF), and biopsied cerebral cortex from epileptic dogs were compared with those from seizure‐free siblings. No differences in absolute levels were noted. However, when levels were normalized as a percent of total free amino acids, the two groups differed in certain respects. Ten significant correlations between amino acid pairs appeared in epileptic dogs, but only one was seen in seizure‐free animals. Seven of these ten corretions involved glutamate or taurine. It was noted that the highly correlated amino acids (taurine, glutamate, glycine, glutamine, alanine) all utilize sodium‐dependent membrane transport processes. The sum of glutamate, aspartate, and glycine levels (competing sodium‐dependent high‐affinity systems) was significantly lower in epileptic beagles. Since this difference was noted in serum but not CSF or brain, it may indicate a diminished capacity of sodium‐dependent high‐affinity renal transport for acidic and certain small neutral amino acids.
Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5‐Hydroxytryptamine, Acetylcholine, and Glutamate
Authors: de Belleroche J. S. , Bradford H. F.
Published: October 2006

The effects of 5‐HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5‐HT inhibited (28%) synthesis of [14C]dopamine from L‐[U‐14C]tyrosine, at 10‐5M and above. This contrasts with the action of acetylcholine, which stimulated [14C]‐dopamine synthesis by 24% at 10‐4 M. Tissue levels of GABA were unaffected by either 5‐HT or acetylcholine up to concentrations of 10‐4 M. The inhibitory action of 5‐HT (5 × 10−5 M and 2 × 10−4 M) on [19C]dopamine synthesis was completely abolished by methysergide (2 × 10−6 M). Higher concentrations of methysergide (10−4 M) or cyproheptadine (10−5 M) inhibited [14C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5‐HT.

At concentrations of 2 × 10−5 M and above, 5‐HT stimulated [14C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10−6 M). Methysergide (up to 10−4 M) or cyproheptadine (2 × 10−4 M) did not reduce the 5‐HT (5 × 10−5 M)‐induced release of [14C]dopamine, but methysergide (10−4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5‐HT (2 × 10−5 M) and K+ (56 mM) on [14C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K+ (56 mM) on [14C]dopamine synthesis was not seen above the inhibitory effect of 5‐HT. Glutamate (0.1‐5 mM) did not affect [4C]dopamine release or its synthesis from L‐[U‐14C]tyrosine.

It is concluded that 5‐HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5‐HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K+ depolarisation.
Evidence for the Association of 2′,3′‐Cyclic‐Nucleotide 3′‐Phosphodiesterase with Myelin‐Related Membranes in Peripheral Nervous System
Authors: Matthieu Jean‐Marie , Costantino‐Ceccarini Elvira , Bény Michèle , Reigner Jacqueline
Published: October 2006

Abstract: In PNS, the specific activity of 2′,3′‐cyclic nucleotide 3′‐phospho–diesterase (CNP) in myelin was not enriched over the starting homogenate. Nevertheless, most of the total activity was recovered in myelin. In myelin‐deficient mutants, low CNP activities were measured in sciatic nerves. CNP specific activities were similar in myelinated and non‐myelinated nerves but in non‐nervous tissues, they were significantly lower than in nervous tissue. There was no indication for the presence of an isoenzyme of CNP in peripheral nerves. These results indicate that CNP is present in PNS myelin and preferentially localized in Schwann cell plasma membranes.
Antagonism of Benzodiazepine Binding in Brain by Antilirium, Benzyl Alcohol, and Physostigmine
Authors: Speeg K. V. , Wang S. , Avant G. R. , Berman M. L. , Schenker S.
Published: October 2006

Abstract: Antilirium®, which contains eserine (physostigmine) and benzyl alcohol, is effective in reversing diazepam‐induced sleep in man and is capable in vitro of inhibiting the binding of labeled benzodiazepine to both rat and human brain homogenates in a dose‐dependent manner. We have examined the constituents of Antilirium and report that both benzyl alcohol and eserine inhibit [3HI‐diazepam binding to rat brain in a dose‐dependent manner. A major portion of the inhibition of binding found with Antilirium is accounted for by benzyl alcohol. Scatchard analysis of inhibition of benzodiazepine binding by benzyl alcohol reveals loss of binding sites and change in equilibrium dissociation constant. Methanol, ethanol, and butanol did not inhibit benzodiazepine binding. The inhibition by benzyl alcohol may be specific since there was no inhibition of labeled ligand binding to the γ‐aminobutyric acid, opiate, muscarinic acetylcholine, or β‐adrenergic receptors. The other constituent, eserine, is a competitive inhibitor. While eserine is a more potent inhibitor at the benzodiazepine receptor than is benzyl alcohol, it is also much less specific. Eserine inhibited binding of labeled ligand to the γ‐aminobutyric acid, opiate, and muscarinic acetylcholine receptors. The inhibition of benzodiazepine binding to brain in vitro by Antilirium and its constituents, eserine and benzyl alcohol, may be the explanation, at least in part, for the reversal by Antilirium of diazepam‐induced narcosis in viva, without postulating a cholinergic mechanism for the in vivo effect.