bluuebunny/crossref_metadata_2025 huggingface dataset:
CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS
Authors: Zimmerman A. W. , Quarles R. H. , Webster H. de F. , Matthieu Jean‐Marie , Brady R. O.
Published: October 2006

—Myelin preparations from the whole brains of 16‐day‐old rats and from cortical regions and brainstem, respectively, of 40‐day‐old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16‐day‐old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16‐day‐old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one‐third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16‐day‐old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16‐day‐old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40‐day‐old rats was 4‐fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16‐day‐old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin‐related membranes, although the heavy fraction from 16‐day‐old rats also may be substantially contaminated with membranes which are unrelated to myelin.
THE INFLUENCE OF SODIUM, POTASSIUM AND LANTHANUM ON AMINO ACID RELEASE FROM SPINAL–MEDULLARY SYNAPTOSOMES
Authors: Osborne R. H. , Bradford H. F.
Published: October 2006

Abstract— The effects of electrical pulses and raised potassium on glycolysis and the release of the putative transmitter amino acids glutamate, aspartate, glycine and GABA from spinal medullary synaptosomes is studied and compared with effects on glutamine, alanine and serine. The actions of ouabain, p‐hydroxymercuribenzoate (p‐HMB), lanthanum chloride and lowered sodium levels on the release of all the amino acids is examined. All agents caused a change in the pattern of release of the amino acids, mostly, producing an increase, but only lanthanum, p‐HMB and lowered sodium prevented the stimulus‐induced release.
ANALYSIS OF THE PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE BY CORTICAL SLICES FROM RAT BRAIN WITH THE AID OF TWO DIFFERENT LABELLED PRECURSORS
Authors: Molenaar P. C. , Polak R. L.
Published: October 2006

—The release of newly synthesized acetylcholine (ACh) by cortical slices from rat brain in the presence of 25 mm‐KCl was studied. The slices were incubated for 5 min in a medium containing both [2‐14C]pyruvate and choline labelled with 3 deuterium atoms (choline‐d3) in order to label at the same time the acetyl moiety and the choline moiety of ACh. The non‐labelled ACh and the ACh‐d3 were measured by pyrolysis‐gas chromatography/mass spectrometry, and the [I4C]ACh by liquid scintillation counting. It was found that the newly formed [4C]ACh as well as the newly formed ACh‐d3 had a more than 2.5 times greater probability of being released than the preformed non‐labelled ACh. These findings strongly suggest that it is not simply the ACh synthesized immediately inside the nerve ending membrane from incoming undiluted labelled choline, which is preferentially released, but that all newly formed ACh has a greater probability of being released than preformed ACh. No preferential release of newly formed ACh was observed when the incubation medium contained 5.6 mm‐pyruvate instead of 10 mm‐glucose + 0.6 mm‐pyruvate. The cause of this difference remains unexplained.
REDUCTION OF γ‐AMINOBUTYRIC ACID LEVEL IN THE CAT CEREBRAL CORTEX BY AFFERENT ELECTRICAL STIMULATION
Authors: Lichtshtcin D. , Dobkin J.
Published: October 2006

—Electrical stimulation for 30 s of one brachial plexus in cat (afferent electrical stimulation = AES) produced a 20% decrease in GABA level of the stimulated (contralateral) cerebral cortex as compared to the non‐stimulated (ipsilateral) cortex in the same animal. This change in GABA was reversed within a few seconds after cessation of stimulation. Inhibition of GABA catabolism by aminooxyacetic acid elevated considerably the cortical level of GABA but failed to prevent lowering GABA by AES. When AES was performed in preconvulsive condition induced by administration of picrotoxin, the decrease in GABA was negligible, while similar treatment with pentylenetetrazol had no influence on the decrease in GABA produced by AES. The observed lowering cortical GABA by AES is interpreted as being associated with some mechanism of the inhibitory transmitter inactivation.
SODIUM‐DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE
Authors: Simon J. R. , Atweh S. , Kuhar M. J.
Published: October 2006

Abstract—

The sodium‐dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50‐80% reduction in sodium‐dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal‐hippocampal or habenuleinterpeduncular tracts by lesions (10 min‐1 h) also caused a similar, large reduction in sodium‐dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively.

We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal‐hippocampal tract. Stimulation (40 Hz) for 10‐15 min completely reversed the depression in sodium‐dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal.

Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium‐de‐pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration.

We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium‐dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake.

We examined the conversion of [3H]choline to [3H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60‐70% of the total sodium‐dependent tritium content was present as [3H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake.

A kinetic analysis of sodium‐dependent high affinity choline uptake was performed after all treatments. We found changes in Vmax, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium‐dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium‐dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.
INHIBITION OF THE UPTAKE OF GABA AND RELATED AMINO ACIDS IN RAT BRAIN SLICES BY THE OPTICAL ISOMERS OF NIPECOTIC ACID
Authors: Johnston G. A. R. , Krogsgaard‐Larsen P. , Stephanwn A. L. , Twitchin B.
Published: October 2006

Abstract—

R(‐)‐Nipecotic acid was a more potent inhibitor than the S(+)‐isomer of the uptake of GABA, (+)‐nipecotic acid, and β‐alanine in rat brain slices. (‐)‐Nipecotic acid was an order of magnitude more potent as an inhibitor of GABA uptake than as an inhibitor of β‐alanine uptake, whereas the (+)‐isomer was less selective. (–)‐Nipecotic acid was a weak inhibitor of L‐proline uptake and of rat brain acetylcholinesterase activity.

Kinetic studies showed that both isomers of nipecotic acid were competitive inhibitors of GABA uptake when added at the same time as GABA, but non‐competitive inhibitors when preincubated with the tissue for 15 min before addition of GABA. The apparent slope inhibition constants, which were not influenced by preincubation, indicated that (–)‐nipecotic acid has an affinity for the carrier some 5 times higher than that for (+)‐nipecotic acid. (–)‐Nipecotic acid stimulated the release of preloaded radioactive GABA from rat brain slices. These observations indicate that (–)‐nipecotic acid is a substrate‐competitive inhibitor of GABA which combines with the GABA carrier and is taken up.

(−)‐Nipecotic acid and (+)‐2,4‐diaminobutyric acid, on the basis of their absolute structures and inhibition kinetics, are proposed to interact in a similar way with the GABA transport system.
THE UPTAKE, METABOLISM AND RELEASE OF HOMOCHOLINE: STUDIES WITH RAT BRAIN SYNAPTOSOMES AND CAT SUPERIOR CERVICAL GANGLION
Authors: COLLIER B. , LOVAT S. , ILSON D. , BARKER L. A. , MITTAG T. W.
Published: October 2006

Abstract—

The accumulation of [3H]homocholine (3‐trimethylamino‐propan‐1‐01) by isolated synaptosomes prepared from rat brain was resolved kinetically into a high (KT= 3.0 μM) and a low (KT= 14.5 μM) affinity system. Although homocholine was not acetylated by solubilized choline acetyltransferase, 64% of the homocholine accumulated by intact synaptosomes via the high affinity uptake process was acetylated. Homocholine was also acetylated in the superior cervical ganglion of the cat, and the amount of acetylhomocholine formed was increased (12‐fold) by preganglionic nerve stimulation. In ganglia, acetylhomocholine was available for release by preganglionic nerve impulses, and its release was Ca2+‐dependent, It is concluded that homocholine can form a cholinergic false transmitter, and that the substrate specificity of choline acetyltransferase in vitro might be different from that in situ.
METABOLIC CHANGES ASSOCIATED WITH ISO‐OSMOTIC REGULATION IN BRAIN CORTEX SLICES
Authors: Fishman R. A. , Reiner M. , Chan P. H.
Published: October 2006

Abstract— The metabolism of single, first cortical rat brain slices was studied in response to incubation in media of various osmolalities. There was an inverse relationship between osmolality and the magnitude of the increase in tissue water content, and a direct relationship between osmolality and inulin space. Brain sodium varied directly with the media sodium, but hyperosmolal sucrose and glucose resulted in a drop in brain sodium. Brain potassium was constant in hyperosmolal sodium media, but it fell in hypo‐osmolal media and hyperosmolal sucrose and glucose media.

Hypo‐osmolality depressed the oxidation of [14C]glucose to 14CO2 whereas hyperosmolality obtained with sodium, sucrose, or glucose stimulated glucose oxidation. Lactate production was enhanced only by sodium hyperosmolality. Hypo‐osmolality caused a 70% drop in phosphocreatine and a minor decrease in energy charge potential, analagous to the effects of hypoxia. Hyperosmolality had no effect on energy metabolism.

The total amino acid nitrogen released into the media was suppressed by hypo‐osmolality but was increased by hyperosmolal incubation. Hyperosmolality also increased production of ammonia fourfold.

The time course of the change in tissue osmolality and ion content following incubation in hyperosmolal sodium media showed the tissue osmolality reached the media osmolality within 5 min, but it took 30 min for the tissue sodium to reach equilibrium with the media sodium. This indicates that unidentified or‘idiogenic osmoles’are induced transiently by hyperosmolality associated with changes in amino acid and ammonia metabolism.
PROPERTIES OF MEMBRANE‐BOUND DOPAMINES‐HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA
Authors: Aunis Dominique , Bouclier Martine , Pescheloche M. , Mandel P.
Published: October 2006

Abstract— Properties of membrane‐bound and soluble dopamine‐β‐hydroxylase were studied. Both enzyme forms have identical affinities for tyramine as the substrate. Arrhenius plots of the membrane‐bound activity displayed a discontinuity at 29°C, the activation energy changing from 20,500 cal/mol below 29°C to 9500 cal/mol above 29°C. The soluble enzyme, like the purified enzyme, did not show discontinuities in Arrhenius plots, the activation energies being 18,500 cal/mol and 16,500 cal/mol respectively. The membrane‐bound enzyme showed a discontinuity in the pKm for tyramine versus reciprocal temperature plot, with a transition at 29°C, whereas the soluble enzyme failed to show such transition.

The membrane‐bound dopamine‐β‐hydroxylase is solubilized by Triton X‐100 as well as by lysolecithin. Of lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce solubilization of membrane‐bound dopamines β‐hydroxylase. The 29°C‐transition was not removed by treatment either with lysolecithin or with Triton X‐100 used at concentration of up to 2%. This could indicate a solubilization of dopamine‐β‐hydroxylase with an associated lipid moiety which cannot be dispersed from the enzyme molecule and which still affects the activation energy and Michaelis constant for tyramine.

Results are discussed in terms of the relationship between lipid organization and enzyme activities; the significance of the solubilization by lysolecithin during exocytosis is considered in terms of the exocytosis process and fate of the chromaffin granule.
KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT BRAIN MITOCHONDRIA
Authors: Minn A. , Gayet J.
Published: October 2006

Abstract— The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non‐synaptosomal mitochondria, either on a glutamate‐hydroxyl antiporter or on a glutamate‐aspartate antiporter.

An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N‐ethylmaleimide. Swelling of the mitochondria in iso‐osmotic NH4+‐l‐glutamate was inhibited in the presence of avenaciolide and N‐ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino‐oxyacetic acid had no effect on the glutamate‐hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double‐beam spectrophotometry, and this reduction was inhibited on the one hand by N‐ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of l‐[14C]glutamate into brain mitochondria was performed by using the centrifugation‐stop procedure. This penetration followed saturation kinetics, with a mean apparent Km of 1.56 MM at pH 7.4 and at 20°C, the value of Knax was 4.34 nmol per min per mg protein in the same conditions. IV‐Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non‐competitive with a Ki of 0.7 Mm ‐at pH 7.4 and at 20°C. The entry of glutamate was pH‐dependent and it increased 2‐fold in the pH range of 7.4 to 6.4. A temperature‐dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated EA of 12.8 kCal per mol of glutamate and a Q10 of 2.16. The activity of γ‐glutamyl transpeptidase was practically absent in these rat brain mitochondria.

Oxidation of extramitochondrial NADH by the‘malate‐aspartate shuttle’reconstituted in vitro was followed in rat brain non‐synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the ‘shuttle’ did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or γ‐aminobutyrate did not replace glutamate efficiently. A high inhibition of the‘malate‐aspartate shuttle’occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of n‐ethylmaleimide, glisoxepide or n‐butylmalonate.

Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.
DIURNAL RHYTHM AND TURNOVER OF TRYPTOPHAN HYDROXYLASE IN THE PINEAL GLAND OF THE RAT
Authors: Sitaram B. R. , Lees G. J.
Published: October 2006

Tryptophan hydroxylase in the pineal gland of the rat was found to undergo a diurnal rhythm in activity with an elevated activity at night. The rhythm was abolished in constant light. Cycloheximide (15 mg/kg, i.p.), administered both at night and during the day, caused a rapid decay in activity suggesting that tryptophan hydroxylase was subject to a rapid turnover in vivo. The primary site of control appeared to be at the level of translation since actinomycin D had no effect. Some relevant properties of the enzyme were studied. Thiol‐containing compounds were shown to substantially protect pineal tryptophan hydroxylase from inactivation at 0°C but provided little protection at higher temperatures. The inactivation process appeared to be independent of oxygen. The activity of the enzyme, lost after ageing at 0°C. could be recovered by incubation with dithiothreitol under anaerobic conditions. Fresh enzyme, or enzyme inactivated at 37°C could not be activated by this process. A re‐examination of the action of p‐chlorophenylalanine (PCPA) on pineal tryptophan hydroxylase revealed that an irreversible inactivation occurred within 6h (25% of initial activity) followed by a recovery within 24 h. The rapid turnover of the enzyme is the probable reason for the failure of previous studies to observe an irreversible inhibition of this enzyme by PCPA.
THE EFFECTS OF 5‐HYDROXYTRYPTOPHAN AND 5‐HYDROXYTRYPTAMINE ON DOPAMINE SYNTHESIS AND RELEASE IN RAT BRAIN STRIATAL SYNAPTOSOMES1
Authors: Andrews David W. , Patrick Robert L. , Barchas Jack D.
Published: October 2006

Abstract—

The effects of 5‐hydroxytryptophan (5‐HTP) and serotonin (5‐HT) on dopamine synthesis and release in rat brain striatal synaptosomes have been examined and compared to the effects of tyramine and dopamine. Serotonin inhibited dopamine synthesis from tyrosine, with 25% inhibition occurring at 3 μM‐5‐HT and 60% inhibition at 200 μM. Dopamine synthesis from DOPA was also inhibited by 5‐HT, with 30% inhibition occurring at 200 μ. At 200 μM‐5‐HTP, dopamine synthesis from both tyrosine and DOPA was inhibited about 70%. When just the tyrosine hydroxylation step was measured in the intact synaptosome, 5‐HT, 5‐HTP, tyramine and dopamine all caused significant inhibition, but only dopamine inhibited soluble tyrosine hydroxylase [L‐tyrosine 3‐monooxygenase; L‐tyrosine, tetrahydropteridine oxygen oxidoreductase (3‐hydroxylating); EC 1.14.16.2] prepared from lysed synaptosomes. Particulate tyrosine hydroxylase was not inhibited by 10 μM‐5‐HT, but was about 20% inhibited by 200 μM‐5‐HT and 5‐HTP. At 200 μM both 5‐HT and 5‐HTP stimulated endogenous dopamine release. These experiments suggest that exposure of dopaminergic neurons to 5‐HT or 5‐HTP leads to an inhibition of dopamine synthesis, mediated in part by an intraneuronal displacement of dopamine from vesicle storage sites, leading to an increase in dopamine‐induced feedback inhibition of tyrosine hydroxylase, and in part by a direct inhibition of DOPA decarboxylation.
MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME
Authors: Malthe‐Sosrenssen D. , Lea T. , Fonnum F. , Eskeland T.
Published: October 2006

Abstract—

Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25–30 μmol ACh formed per min and mg protein. Disc electrophoresis at pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC‐34 whilst a high molecular weight fraction was occasionally observed by gelfiltration on Sephadex G‐200. The enzyme was, however, separated into two molecular forms (A and B) on CM‐Sephadex chromatography. Both molecular forms had the same S220w but differed in heat stability and affinity for acetyl‐CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl‐CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.
REGIONAL AND SUBCELLULAR DISTRIBUTION OF HOMOCARNOSINE–CARNOSINE SYNTHETASE IN THE CENTRAL NERVOUS SYSTEM OF RATS
Authors: Ng R. H. , Marshall F. D.
Published: October 2006

Abstract—

Homocarnosine–carnosine synthetase and carnosinase were assayed in homogenates, 100,000 g supernatants, and ammonium sulfate fractions of the supernatants from nine regions of the central nervous system (CNS), as well as subcellular fractions of whole brains. The enzymes were detected in all CNS regions tested, with olfactory bulbs having the highest activities of both enzymes. In the subcellular fractions, the synthetase was found mainly in the cell‐sap; carnosinase was detected in all fractions, the highest activity being in the mitochondria. The synthetases from olfactory bulbs, cerebellum and spinal cord have similar Km's for β‐alanine and GABA.
MEASUREMENTS OF IN VIVO RATES OF PROTEIN SYNTHESIS IN BRAIN, SPINAL CORD, HEART AND LIVER OF YOUNG VERSUS ADULT RATS, INTACT VERSUS HYPOPHYSECTOMIZED RATS
Authors: Reith M. E. A. , Schotman P. , Gispen W. H.
Published: October 2006

Abstract— In vivo protein synthesis rates in rats were estimated by single i.p. injections of large quantities of [1‐14C]valine. This method gives reliable estimates of the precursor specific activity and average protein synthesis rates. In the brain, spinal cord, heart and liver, the average rates for adults were 0.65, 0.42, 0.49 and 1.92% replacement of protein‐bound amino acid per h. In the brain and liver of 10‐day olds the average rates were estimated to be 1.46 and 3.12% per h respectively. Hypophysectomy decreased synthesis rates by 25% or more in all tissues studied. The disadvantages of the method are that applying large amounts of valine i.p. appeared to constitute a stress and that the valine solution required for injection was hypertonic, causing withdrawal of body fluids of the animal.
MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS
Authors: Marotta Charles A. , Strocchi Paola , Gilbert Jeffrey M.
Published: October 2006

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two‐dimensional gels and one‐dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S1 and S2, with similar DNase I affinity were also isolated. S11 and S2 have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S1 and S2 is 3‐fOld greater in synaptosomes when compared to similar non‐synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I‐actin complex.
DEVELOPMENTAL CHANGES OF ENZYMES ASSOCIATED WITH ENERGY METABOLISM AND THE SYNTHESIS OF SOME NEUROTRANSMITTERS IN DISCRETE AREAS OF HUMAN NEOCORTEX
Authors: Diebler M. F. , Farkas‐Bargeton E. , Wehrlé R.
Published: October 2006

Abstract—

Developmental changes in pyruvate kinase (PK), lactate dehydrogenase (LDH), α‐glycerophosphate dehydrogenase (α‐GPD), glucose‐6‐phosphate dehydrogenase (G6P), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), choline acetyltransferase (CAT), glutamate decarboxylase (GAD), activities were measured in early autopsy material in the following areas of human neocortex: area 4 (motor cortex), area 17 (visual cortex), area 40 (gyrus supramarginalis, associative cortex). Changes with age were analysed from 8 fetal weeks to adult age. The important points emerging from this study are: 1. Enzymes associated with glycolytic pathways show a high activity in early fetal period, decline through to the end of the active phase of neurogenesis and then, rise continuously to the end of the first year of life. 2. G6P, an enzyme associated with the oxidative segment of the pentose phosphate pathway, shows a high activity at 8 fetal weeks and, gradually declines through to the end of the active phase of neurogenesis; it then either does not change significantly (motor cortex) or increases slightly. 3. Enzymes related to the tricarboxylic pathway have a low level of activity throughout the first half of gestation, and then rise markedly during the last fetal months and the first year after birth. SDH increase is of much higher magnitude (× 10) than that observed for glycolytic enzymes (×4). For the enzymes of oxidative metabolism, motor cortex is the most advanced area, while associative cortex matures more slowly. 4. CAT activity at 8 fetal weeks is high in visual cortex and declines to the fifth month. After that time, there is a continuous rise until the age of 11 years. Although the time pattern in reaching the adult value is different in motor and associative cortex, there exists a continuous increase from fetal onset to adult level in both areas. Developmental changes in GAD activity are very unusual. The development of activity lags behind that of CAT and commences after birth. After a steady rise in the first year of life, the activity decreases after this age.
PROPERTIES OF A PUTATIVE MUSCARINIC CHOLTNERGIC RECEPTOR FROM DROSOPHILA MELANOGASTER
Authors: Haim N. , Nahum S. , Dudai Y.
Published: October 2006

Abstract—

The powerful muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) specifically binds to homogenates of Drosophila melanogaster head at a level of 65 ± 6 fmol/mg protein, with an apparent dissociation constant of 0.15–0.7 nM. The half‐life of the ligand‐receptor complex at 25°C is 30–40 min. Binding is inhibited by low concentrations of muscarinic ligands but not by low concentrations of nicotinic ligands, anticholinesterases or non‐cholinergic drugs. Binding‐sites are membrane bound and are inactivated by trypsin and by Triton X‐100. Part of the activity (<20%) is released into a high speed supernatant by 2 M‐NaCI. The gene coding for the putative muscarinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase
RABBIT SCIATIC NERVE FASCICLE AND‘ENDONEURIAL’PREPARATIONS FOR IN VITRO STUDIES OF PERIPHERAL NERVE GLUCOSE METABOLISM
Authors: Greene Douglas A. , Winegrad Albert I. , Carpentier Jean‐Louis , Brown Mark J. , Fukuma Michio , Orci Lelio
Published: October 2006

Abstract— Suitable preparations for in vitro studies of the composite glucose and energy metabolism of peripheral nerve axons and Schwann cells have not been available. Methods are described for the preparation and incubation of a defined segment of a rabbit sciatic nerve fascicle, free of epineurial contamination, but with an intact perineurial membrane; removing the perineurium provides in addition an‘endoneurial’preparation. Conditions were selected for incubating each preparation with glucose that maintained stable P‐creatine and ATP concentrations and a stable rate of O2 uptake; under these conditions the preparations retained an unaltered EM appearance during a 2‐h incubation. Glucose diffusion into the endoneurial compartment of the fascicle is restricted, possibly by the perineurial membrane, and a higher medium glucose concentration (20 mM) was required to maintain a steady state of energy metabolism in this preparation than in the‘endoneurial’preparation, which was incubated with 5 mwglucose. The‘endoneurial’preparation required 0.50 mm‐myoinositol in the medium to prevent a decrease in tissue free myoinositol and a slow decrease in O2 uptake, which occurred when it was omitted. Under the incubation conditions selected the glucose concentrations in the‘endoneurial’preparation and in the endoneurial compartment of the fascicle were reasonably similar, and the preparations had similar rates of respiration, similar estimated rates of glucose utilization, and similar relative rates of respiration and lactate production. The preparations derive the major fraction of their energy requirements from respiration. Their rates of O2 uptake are 60% higher than the previous indirect estimate of O2 uptake in whole rabbit tibial nerve in situ. Constant rates of incorporation of 14C from [U‐14C]glucose into CO2 and total lipid were observed in the‘endoneurial’preparation after a 15‐min equilibration period. The preparations reported provide suitable tools for in vitro studies of peripheral nerve metabolism not previously available.
PARTIAL PURIFICATION OF DROSOPHILA GLUTAMATE DECARBOXYLASE1
Authors: Chude Obi , Roberts Eugene , Wu Jang‐Yen
Published: October 2006

Abstract— A 600‐fold purification of l‐glutamatc decarboxylasc from Drosophila melanogaster Oregon R Wild Type has been achieved. The purification procedures involve the initial homogenization of whole flies in dilute potassium phosphate buffer containing dithiothreitol and phenylmethyl sulfonyl fluoride as protectors, followed by a series of column chromatography with hydroxylapatite, Sephadex G‐150 and DEAE‐Sephadex. The purified enzyme has an apparent Km of 11 mm for l‐glutamate and requires a 40mm‐K+ for maximum activity.

The purified enzyme shows only 1 pH optimum around pH 7.5, while crude preparations of the inset display 2 pH optima, pH 4.8–5.2. and 7.5. The significance and possible application of this study are also discussed.
STIMULATORY EFFECT OF l‐GLUTAMATE AND RELATED AMINO ACIDS ON [3H]DOPAMINE RELEASE FROM RAT STRIATUM: AN IN VITRO MODEL FOR GLUTAMATE ACTIONS
Authors: Roberts P. J. , Anderson Susan D.
Published: October 2006

Abstract— The effects of l‐glutamate and a number of structural analogues on the spontaneous release of [3H]dopamine from slices of rat striatum were examined. Glutamate, and other excitatory amino acids produced a marked stimulation of [3H]DA release which was Ca2+‐dependent and unaffected by either procaine or tetrodotoxin. The glutamate‐stimulated release was abolished in kainate‐lesioned striatum. The action of glutamate was effectively antagonised by glutamamate diethylester and 2‐amino‐4‐phosphonobutyric acid, but only weakly by l‐methionine‐dl‐sulfoximine. Other proposed amino acid antagonists were inactive. The likely site of the releasing action of l‐glutamate on presynaptic sites on nigro‐striatal DA terminals is discussed.
EFFECT OF CONCUSSIVE HEAD INJURY ON CENTRAL CATECHOLAMINE LEVELS AND SYNTHESIS RATES IN RAT BRAIN REGIONS 1
Authors: Huger F. , Patrick G.
Published: October 2006

—Unanesthetized male Sprague‐Dawley rats were subjected to head injury using a unique acceleration‐deceleration model designed to mimic the most prevalent form of clinical head injury. Endogenous tyrosine, dopamine and norepinephrine were determined fluorometrically and catecholamine synthesis rates were determined by a radioisotopic method. These values were determined in four brain regions: cortex‐striatum, midbrain‐hypothalamus, medulla‐pons, and cerebellum, and were performed at 5 min, 15 min, 1 h, and 2h post‐trauma. Dopamine levels were elevated in the medulla pons region at 5 min after trauma and in the midbrain‐hypothalamus at the 15 min and 1 h time periods. This increase in dopamine levels may reflect disruption of dopamine beta hydroxylase activity. Norepinephrine synthesis rate in the cerebellum was elevated at 2 h after trauma. Changes in the other parameters were observed but appeared to be related to stress, and the effect of stress in this model is discussed.
ACTH‐SENSITIVE PROTEIN KINASE FROM RAT BRAIN MEMBRANES
Authors: Zwiers H. , Tonnaer J. , Wiegant V. M. , ScHotman P. , Gispen W. H.
Published: October 2006

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B‐50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X‐100 in 75 mM‐KCl solubilized 15% of the total B‐50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH1–24. The rate of endogenous phosphorylation of protein band B‐50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B‐50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE‐cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH‐sensitive B‐50 protein kinase.
Axonal Transport of Neuropeptides in the Cervical Vagus Nerve of the Rat
Authors: Gilbert R. F. T. , Emson P. C. , Fahrenkrug J. , Lee C. M. , Penmant E. , Wass J.
Published: October 2006

Abstract: Accumulations of the neuropeptides substance P (SP), somatostatin (ST) and vasoactive intestinal polypeptide (VIP) proximal to a crush in the cervical vagus nerve of the rat have been measured using sensitive radioim‐munoassays. Each of the peptides was rapidly transported towards the peripheral terminals of vagal afferent fibres, with average rates of flow ranging from 0.8 to 2.7 mm h−1. In the rabbit vagus nerve, SP was transported with an average rate of 4 mm h−1, which is more than double the rate for this peptide in the rat. Double crush experiments in rabbit vagus nerves indicated that the rapidly transported proportion of the total content of SP in the nerve free was about 34%. From this, the rate of transport of SP in the rapidly transported pool in the rabbit vagus nerve can be calculated to be 12 mm h−1 (280 mm day−1). Since such double crush experiments were not possible in the rat, it is not clear whether the different average rates of transport of SP in the rat and the rabbit reflect real differences in the rate of rapid transport in the two species. In common with rapid axonal transport of other neurotransmitters, the transport of SP and ST in the rat vagus nerve was blocked by colchicine, a drug that disrupts microtubules.
Oxidative Metabolism of 4‐Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched‐chain Fatty Acid
Authors: Cunningham J. , Clarke D. D. , Nicklas W. J.
Published: October 2006

Abstract: The oxidation of 4‐aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched‐chain fatty acids 2‐methyl‐2‐ethyl caproate (MEC) and 2, 2‐dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O2 uptake, was determined in medium containing either 5 or 100 mM‐[K+]. The apparent Km for GABA was 1.16 ± 0.19 mM and the Vmax in state 3 was 23.8 ± 5.5 ng‐atoms O2. min−1. mg protein−1 in 5 mM‐[K+]. In a medium with 100 mM‐[K+] the apparent Km was 1.11 ± 0.17 mM and Vmax was 47.4 ± 5.7 ng‐atoms O2. min−1. mg protein−1. The Km for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM‐[K+], respectively. For DMV, the Ki was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM‐[K+] medium, respectively. The O2 uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K2] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The Vmax for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched‐chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.
Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity
Authors: Williams Stuart K. , Gillis John F. , Matthews† Maureen A. , Wagnert Roger C. , Bitensky Mark W.
Published: October 2006

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5′‐nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and ‐γ‐glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.
Uptake of l‐[35S]Cystine by Isolated Rat Brain Capillaries
Authors: Hwang Shing Mei , Weiss Steven , Segal Stanton
Published: October 2006

Adult rat brain capillaries were isolated by a simplified procedure and showed an enrichment of the marker enzyme, γ‐glutamyltranspeptidase. The uptake of [35S]cystine at 37°C by this preparation can be divided into two components, a sodium‐ and energy‐dependent transport process for the free amino acid pool, with an apparent Km of 36 μm, and a binding process, with an apparent Km of 1.13 mm. Chemical analysis of the amino acid pool indicates that cystine is the major form of intracapillary 35S. Cystine transport was not inhibited by lysine, but glycine, α‐methylaminoisobutyric acid and β‐2‐aminobicyclo‐[2,2,1]‐heptane‐2‐carboxylic acid were inhibitory to a small extent.
Transient Formation of Superoxide Radicals in Polyunsaturated Fatty Acid‐Induced Brain Swelling
Authors: Chan Pak Hoo , Fishman Robert A.
Published: October 2006

The involvement of superoxide free radicals and lipid peroxidation in brain swelling induced by free fatty acids has been studied in brain slices and homogenates. The polyunsaturated fatty acids linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), and docosahexaenoic acid (22:6) caused brain swelling concomitant with increases in superoxide and membrane lipid peroxidation. Palmitic acid (16:0) and oleic acid (18:1) had no such effect. Furthermore, superoxide formation was stimulated by NADPH and scavenged by the addition of exogenous superoxide dismutase in cortical slice homogenates. These in vitro data support the hypothesis that both superoxide radicals and lipid peroxidation are involved in the mechanism of polyunsaturated fatty acid‐induced brain edema.
Regional Distribution of Glutathione Peroxidase in the Adult Rat Brain
Authors: Brannan Timothy S. , Maker Howard S. , Weiss Cipora , Cohen Gerald
Published: October 2006

Glutathione peroxidase activity was measured in 10 areas of perfused adult rat brain with the use of a fluorometric assay coupled to NADPH oxidation. The caudate‐putamen and the substantia nigra had the highest activities. Cortical areas and several nuclear areas had somewhat lower activity. Activity was lowest in a white matter structure (corpus callosum). High activity of glutathione peroxidase may be related to the need to reduce hydrogen peroxide arising in the course of monoamine metabolism.
Polarographic Measurements of Spontaneous and Mitochondria‐Promoted Oxidation of 5,6‐and 5,7‐Dihydroxytryptamine
Authors: Klemm H. P. , Baumgarten H. G. , Schlossberger H. G.
Published: October 2006

Abstract: Spontaneous oxygen consumption by 5,6‐ and 5,7‐DHT (dihydroxytryptamine), related indoleethylamines, and 6‐hydroxydopamine and oxygen consumption by these compounds in the presence of rat liver mitochondria were measured by the polarographic oxygen electrode technique. 5,6‐ and 5,7‐DHT react with oxygen at very different rates (2.7 nmol O2/min and 33.4 nmol O2/min, respectively) when incubated in buffer, pH 7.2, at a concentration of 1 mm and with different kínetic characteristics. While the oxidation of 5,7‐DHT obeys a reaction of second‐order type, the oxidation of 5,6‐DHT is more complex and characterized by autocatalytic promotion. Coloured quinoidal oxidation products appeared during the degradation of both indoleamines. Glutathione, ascorbate, dithiothreitol, cysteine, albumin, and superoxide dismutase partially prevented 5,6‐ and 5,7‐DHT from oxidative destruction. Catalase saved oxygen only in the case of 5,6‐DHT by recycling of O2 released from near‐stoichiometrically formed H2O2 during oxidation of 5,6‐DHT: 5,7‐DHT did not generate H2O2 in measurable amounts. Oxygen consumption rates of 5,6‐ and 5,7‐DHT were enhanced after addition of rat liver mitochondria to the incubation medium; this resulted in an accelerated formation of quinoidal products. This stimulatory effect on the oxidation rates of both 5,6‐ and 5,7‐DHT was blocked by cyanide, but not rotenone, and was abolished by boiling of the mitochondria fraction. The observed increase in oxygen consumption in the presence of mitochondria was found not to be influenced by monoamine oxidase‐dependent deamination of 5,6‐ and 5,7‐DHT. It is postulated that 5,6‐ and 5,7‐DHT are capable of participating in the electron transfer of the mitochondrial respiration chain beyond complex III. Results obtained in determinations of ADP:0 ratios in respiratory control experiments exclude a possible interference of 5,6‐DHT, 5,7‐DHT, and 6‐OH‐DA with phosphorylating sites. During the activated state of respiration, no signs of electron transfer inhibition by 5,6‐ and 5,7‐DHT were detectable. A comparison and evaluation of the autoxidation rates of various hydroxylated indoleethylamines, of their affinity to the 5‐HT transport sites, and their neurotoxic potency in vivo reveals that interaction of these compounds with oxygen at restricted reaction velocity is a prerequisite for efficient toxicity in monoaminergic neurons following active accumulation in these neurons via the high‐affinity uptake systems.
Preparation and Retrograde Axonal Transport of an Antiviral Drug/Horseradish Peroxidase Conjugate
Authors: Haschke Richard H. , Ordronneau Jacqueline M. , Buntt Ann H.
Published: October 2006

Abstract: Recurrent Herpes simplex virus infections of the cornea are currently treated with antiviral drugs administered locally during periods of active infection. We have examined the feasibility of an alternate approach to treating recurrent infections, that of delivering an antiviral drug by axonal transport to cell somata in the trigeminal ganglion where the latent virus is thought to reside. We have coupled an antiviral drug to horseradish peroxidase (HRP), a protein which is readily transported retrogradely in corneal sensory axons to ganglion cell somata in the trigeminal ganglion. The antiviral drug 5‐iodo‐5′‐amino‐2′,5′–dideoxyuridine (AIDU) was synthesized with 125I and coupled to horseradish peroxidase by Schiff base formation between the 5’ amino group and aldehydes generated on the carbohydrate of the HRP following short periodate oxidation. The useful ratio of AIDU:HRP was 2–9; higher ratios resulted in an insoluble product. Sodium dodecylsulfate (SDS) acrylamide gel electrophoresis of the drug‐protein conjugate revealed considerable aggregation and the isoelectric point of HRP was changed from 8.1 to 4.4–5.8 following the coupling procedure. Despite evidence that the protein conformation was considerably altered, the specific enzymatic activity of the final product was 58% of native HRP and the drug‐protein conjugate was still strongly transported retrogradely. Retrograde transport of this conjugate was demonstrated by autoradiography of the trigeminal ganglion neurons 24 h after corneal injection of the [125I]AIDU/HRP.
2′, 3′‐Cyclic Nucleotide 3′‐Phosphohydrolase and Lipids of Myelin from Multiple Sclerosis and Normal Brains
Authors: Göpfert Ernst , Pytlik Susanne , Debuch Hildegard
Published: October 2006

Abstract: A study of purified myelin samples from normal‐appearing white matter of 10 multiple sclerosis (MS) brains was undertaken and the results were compared with 10 age‐matched control brains. Statistical evaluations were carried out with Student's r‐test for differences. In pathological samples the yield of myelin came to only two‐thirds of the corresponding controls. Enzyme assays of the 2′, 3′‐cyclic 3′‐phosphohydrolase revealed an obviously significant reduction of specific activity to one‐half in MS myelins. In myelin the contents of protein, lipid classes as cholesterol, glycolipids and phospholipids did not differ significantly. No cholesterol esters or any lysophospholipid were detectable either in MS or in controls. Within the individual phospholipids the main components were in the same order, while a significant decrease of the acidic representatives and of sphingomyelin occurred. Analysis of the fatty acid pattern of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the aldehydes from the last, revealed quite similar values with no significant differences, except C22: 4 fatty acid in the PE fraction and C20: 1 fatty acid in PS, which were reduced in MS myelin samples.