bluuebunny/crossref_metadata_2025 huggingface dataset:
Cation‐Sensitive Neutral Endopeptidase: Isolation and Specificity of the Bovine Pituitary Enzyme
Authors: Wilk Sherwin , Orlowski Marian
Published: October 2006

A highly purified preparation of a cation‐sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS‐containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydro‐phobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH‐RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine‐containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.
Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt
Authors: Kamata Shinkichi , Okada Akira , Watanabe Takehiko , Kawashima Yasunaru , Wada Hiroshi
Published: October 2006

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.
Effect of Electroconvulsive Shock on Polysomes of Rabbit Brain, Liver, and Kidney
Authors: Popoli Maurizio , Giuditta Antonio
Published: October 2006

Abstract: Following electroshock treatment of the rabbit, polysomes have been found to disaggregate in the liver and kidney to an extent comparable with disaggregation in the cerebral cortex. After a single electroconvulsive shock, the process of polysomal disaggregation in the former two organs reaches maximum values at 10 and 60 min, but returns to control values at intermediate times. In the cerebral cortex the shift to control values observed at 20 min is not statistically significant
Enzyme‐Linked Immunoadsorbent Assays for Myelin Basic Protein and Antibodies to Myelin Basic Protein
Authors: Groome Nigel P.
Published: October 2006

Abstract: Enzyme Linked Immunoadsorbent Assays are described for Myelin Basic Protein and antibodies to this protein. Under optimal conditions 30 pg of Basic Protein and 100 pg of antibody can be detected. Both assays are simple to establish, require small volumes of sample, and are highly reproducible. All the necessary reagents are stable indefinitely.
Problems Associated with the Binding of L‐Glutamic Acid to Synaptic Membranes: Methodological Aspects
Authors: Sharif Najam A. , Roberts Peter J.
Published: October 2006

Abstract: Specific L‐[3H]glutamate binding was investigated in extensively washed synaptic membrane preparations from rat brain. Mild conditions of ultrasonication effected a significant enhancement of binding, attributable to the marked reduction in membrane vesicle size and the removal of endogenous interfering substances such as glutamate. Preincubation of freshly prepared membranes at 37°C for 30 min followed by further washing resulted in enhanced binding. Addition of supernatant from preincubated membranes effectively inhibited [3H]glutamate binding to control membranes; the possibility of the presence of an endogenous glutamate receptor inhibitor is discussed. Treatment of membranes with low concentrations of Triton X‐100, in contrast with the findings for GABA, did not produce any significant enhancement of specific glutamate binding. While binding of [3H]glutamate is almost abolished in frozen or cold‐stored membranes, lyophilisation had a remarkable effect, not only affording protection, but actually enhancing the binding properties of the synaptic membrane preparation.
The Distribution of Nerve Growth Factor in the Male Sex Organs of Mammals
Authors: Harper G. P. , Thoenen H.
Published: October 2006

Abstract: The Nerve Growth Factor (NGF) content of male sex organs of the mouse, rat, guinea pig, hamster, rabbit, human, and bull has been investigated using both a biological assay and a two‐site radioimmunoassay. The prostate glands of the rabbit and bull have been found to contain moderate levels of NGF, these being lower than the concentrations found in the guinea pig prostate and mouse submaxillary glands. The sex organs investigated of the mouse, rat, hamster, and human contained no detectable NGF activity. Genital organs, other than the prostate glands, of the guinea pig and rabbit were also devoid of NGF. The NGFs from the rabbit and bull are immunologically related to those found in the submaxillary glands of the mouse and the prostate glands of the guinea pig, but immunodiffusion and radioimmunoassay experiments show that there are also clear differences between the NGFs. The use of a two‐site radioimmunoassay, based on purified antibodies against mouse submaxillary gland NGF, for the determination of NGF levels in species other than the mouse, is described. It is essential during such applications to compensate for the fact that the NGFs from different species are sufficiently distinct that only part of the antibody population (raised against mouse NGF) is capable of recognizing NGF from species other than the mouse. The results of radioimmunoassay and biological assay determinations are in reasonable agreement, if corrections for this feature are made.
Lipid Investigation of Central and Peripheral Nervous System in Connatal Pelizaeus‐Merzbacher's Disease
Authors: Witer Brigitte , Debuch Hildegard , Klein Harold
Published: October 2006

Abstract: Brain grey and white matter of a case of Pelizaeus‐Merzbacher disease (connatal type Seitelberger) of a 19‐month‐old boy were analysed with respect to lipids. Cerebrosides and sulfatides were totally absent in the pathological brain. In comparison to control, differences in gangliosides could be detected in grey and white manner. C18:1, fatty acids were markedly reduced in the main glycerophospholipids of white matter. In sphingomyelin of cortex and white matter 90% of fatty acids were C18:0; longer chains were absent. In contrast: PNS (nervus fernoralis) lipids contained the main galactolipids. However, these a s well as all other lipid classes showed a 20% reduction compared with values obtained from nervus femoralis of an infant of the same age. The fatty acid patterns of all lipid classes were determined. The only marked deviations from normal were observed in the C24‐chains of cere‐brosides and sulfatides. The formalin‐fixed brain of an older brother (same disease) was analysed only with respect to glycolipids: neither cerebrosides nor sulfatides could be detected.
Passive Avoidance Learning Results in Region‐Specific Changes in Concentration of and Incorporation into Colchicine‐Binding Proteins in the Chick Forebrain
Authors: Mileusnic Radmila , Rose Steven P. R. , Tillson Penny
Published: October 2006

Abstract: Incorporation of [14T]leucine into trichloracetic acid‐precipitable material and tubulin‐enriched fractions, and total tubulin levels as determined by colchicine‐binding activity and retention on DE81 filter discs, were measured in various regions of the chick brain following training on a one‐trial passive avoidance task, suppression of pecking at a chromed bead as a consequence of the aversive taste of methylanthranilate. Radioactive pulse time was 0.5 h. The only brain region in which changes were found was the anterior forebrain roof, the same area in which biochemical changes in response to exposure of the birds to an imprinting stimulus have been observed previously. In the anterior forebrain roof the changes observed as a consequence of training were detectable at 0.5 and 24 h after the 10‐s training experience but not 48 h subsequently. One‐half hour after training, there were increases of the order of 20 or 30% in [14T]leucine incorporation into particulate and postmitochondrial TCA‐precipitable material and a tubulin‐enriched fraction purified as above. There were comparable increases in the total amount of colchicine‐binding activity. By 48 h, none of these increases were detectable. Subcellular fractionation of the particulate fraction showed that most of the increase of incorporation into the tubulin‐enriched fraction and in colchicine‐binding activity was present in the soluble content of the synaptosomes; there were no increases in either measure in the synaptic membrane fraction. The possible role of changed levels and turnover of tubulin in the plastic responses of the brain to learning experiences is discussed.
Changes in Neostriatal Dopamine Concentrations in Response to Levodopa Infusions
Authors: Doller Herbert J. , Connor John D.
Published: October 2006

Abstract: Levodopa was infused under various circumstances of pretreatment into the ear veins of unanesthetized rabbits. Concentrations of neostriatal dopamine formed in response to levodopa administration were determined. The aim was to characterize the temporal relationship between the concentrations of levodopa in plasma and dopamine in the neostriatum. When plasma levodopa was maintained constant by i.v. infusion, the concentration of neostriatal dopamine reached a plateau by 1 h. Increases in dopamine were proportional to the amount of precursor in plasma. The tissue half‐life of this dopamine in normal rabbits was not more than 15 min. Half‐lives of comparable duration for striatal dopamine were calculated from rabbits treated chronically with levodopa, and from rabbits with monoamine‐depleting lesions. The results show that the concentration of dopamine in rabbit neostriatum correlates closely with the concentration of levodopa in plasma. Concurrent analyses of neocortical tissues indicate that the neostriatum may not be different from other brain regions with regard to dopamine storage mechanisms. Interpretation of the results in terms of the clinical use of levodopa suggests that the durations of short‐term effects (measured in h) of the drugs are paralleled by changes in concentration of brain dopamine.
A Rapid and Specific Method for the High Resolution Purification and Characterization of Neuropeptides
Authors: Morris H. R. , Etienne A. T. , Dell A. , Albuquerque R.
Published: October 2006

Abstract: High resolution purification procedures for the separation and characterization of neuropeptides are described. The methods, which are complementary, are based on ion exchange chromatography and reverse phase high pressure liquid chromatography and are designed to be used in conjunction with radioimmunoassays and bioassays for known peptides or structural identification in the case of peptides of unknown sequence. The high pressure liquid chromatographic method is versatile, highly reproducible and applicable to picomolar quantities of peptides and it gives very high resolution between most of the neuropeptides of present‐day interest. The applicability of the method to the study of crude brain extracts is demonstrated.
Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1
Authors: McSwigan John D. , Nicol Susan E. , Gottesman I. I. , Tuason V. B. , Frey William H.
Published: October 2006

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′‐adenylylimidodiphos‐phate (MgApp(NH) p) as substrate. GTP and 5′‐ guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP‐dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the Ka for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg2+ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the Ka for free Mg2+ at the high‐affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy‐chotic drug fluphenazine blocks this shift in Ka due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg2+ and guanyl nucleotide and by increasing maximal velocity.
Muscarinic Binding Sites in Developing Normal and Mutant Mouse Cerebellum
Authors: East J. Malcolm , Dutton Gary R.
Published: October 2006

Abstract: The development of [3H]quinuclidinylbenzilate ([3H]QNB) binding to cerebellar homogenates of weaver (wv), reeler (rl), staggerer (sg) and jimpy (jp) mutants, in addition to their normal counterparts, was observed. The maximum increase in binding in normal mice occurred postnatally, during the period 5 to 15 days. [3H]QNB binding was significantly reduced in wv, rl and sg mutants (40–50% of control) but was not so markedly affected in jp (80–100% control). Binding was saturable with an apparent Kd of 0.15 nM and the affinity of [3H]QNB for its receptor was not affected during the course of development or as a result of the mutation. The presence of significant numbers of binding sites in the agranular mutants suggests that QNB binding sites are localised on cells other than the granule neurons. The possibility of synaptic reorganisation is also discussed.
A High Performance Liquid Chromatography Method for Measuring Brain Dihydroxyphenylalanine Levels and Dopamine Synthesis Rates
Authors: Tabakoff Boris , Black Richard F.
Published: October 2006

Abstract: A high performance liquid chromatography method by which one can detect as little as 45 pmol of dihydroxyphenylalanine in brain tissue is described. Dihydroxyphenylalanine was reacted with fluorescamine and was quantitated, after chromatography, by fluorescence spectroscopy. The steady state levels of dihydroxyphenylalanine in the mouse striatum were found to be below 90 pmol/g, but the dihydroxyphenylalanine levels were rapidly increased by inhibition of aromatic amino acid decarboxylase. The method described was utilized to demonstrate the effects of pharmacologic agents on dihydroxyphenylalanine synthesis in mouse striatum.
The Release of γ‐Aminobutyric Acid, Glutamate, and Acetylcholine from Striatal Slices: A Mass Fragmentographic Study
Authors: Moroni F. , Bianchi C. , Tanganelli S. , Moneti G. , Beani L.
Published: October 2006

Abstract: The release processes of endogenous Acetylcholine (ACh), γ‐aminobutyric acid (GABA), glutamate (Glu) and glutamine (GLN) were studied in superfused guinea‐pig caudatal slices. Basal ACh release remained constant for up to 2 h, while the basal release of GABA, Glu and GLN declined to half or less of its initial values after 1 h of superfusion. Electrical stimulation increased the ACh release by 700–800% and that of GABA by 80% whereas it decreased the output of Glu by 50% and failed to modify the GLN efflux. KCl (25 mM) increased the output of ACh by 400%, that of GABA by approximately 500% and decreased that of Glu by 40%. Substituting of CaCl2 by MgCl2 in the superfusion medium reduced the basal ACh release by 70% whereas no differences were observed in the basal efflux of GABA, Glu and GLN. Under these conditions, no evoked release of ACh or of GABA was detected, following electrical or KCl stimulation. Tetrodotoxin 5 × 10‐7 M decreased the basal ACh release by 60% and increased the GABA efflux by 40%. The toxin abolished the stimulus‐evoked ACh efflux but scarcely affected that of GABA. These results are consistent with a possible neurotransmitter role of ACh and GABA in the striatum and show some differences in the ionic mechanisms underlying GABA and ACh release.
Subnuclear Distribution of the S‐100 Protein Specific Binding Sites in Rat Brain
Authors: Michetti Fabrizio , Donato Rosario
Published: October 2006

Abstract: Fractionation of isolated brain nuclei previously reacted with 125I‐labelled S‐100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of 125I‐labelled S‐100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for 125I‐labelled S‐100 specific binding, both the nuclear membranes and the nucleoli were found to bind 125I‐labelled S‐100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.
Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes
Authors: Morel Nicolas , Meunier François‐Marie
Published: October 2006

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.
Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline
Authors: Weiler Molly H. , Gundersen Cameron B. , Jenden Donald J.
Published: October 2006

Abstract: Using sequential incubations in media of different K+ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic‐mass spectrometric (GC‐MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K+ and with 2 μM‐[2H9]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [2H4]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K+ solution on the metabolism during a second low or high K+ incubation of a [2H9]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [2H9]Ch metabolism versus [2H4]Ch metabolism. The most striking result we obtained was that [2H9]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K+ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.
Sex Differences in the Content of β‐Endorphin and Enkephalin‐Like Peptides in the Pituitary of Obese (ob/ob) Mice
Authors: Govoni S. , Yang H.‐Y. T.
Published: October 2006

Abstract: The β‐endorphin content in pituitary extracts of male and female obese (ob/ob) and lean (+/?) mice was determined by radioimmunoassay. The amount of β‐endorphin‐like material contained in the pituitary of 3‐month‐old ob/ob male mice is similar to that of lean male mice. In contrast, the pituitary glands of female ob/ob mice have a greater amount of β‐endorphin‐like material than lean female mice. To determine with greater precision the molecular nature of the polypeptide that accounts for the increase in β‐endorphin immunoreactivity, the various molecular forms of β‐endorphin immunoreactivity were resolved by Biogel P‐30 column chromatography. At least four peaks of immunoreactive material were detected. The first peak elutes in the void volume, and the second and the third peaks appear in the elution volumes of β‐lipotropin and β‐endorphin, respectively. That the material present in the void volume might be proopiocortin is supported by adrenocorticotropic hormone radioimmunoassay. The increased total β‐endorphin immunoreactivity in pituitary glands of ob/ob mice is accounted for mainly by β‐endorphin. The β‐endorphin content of various brain structures of ob/ob mice is similar to that of lean littermates.
Lipid Metabolism in Peripheral Nerve Cell Culture (Rich in Schwann Cells) from Normal and Trembler Mice
Authors: Bourre J. M. , Morand O. , Dumont O. , Boutry J. M. , Hauw J. J.
Published: October 2006

Abstract: A culture of peripheral nerve cells, very rich in Schwann cells, was developed from sciatic nerve. In both normal and Trembler, typical spindle‐shaped cells were seen; most of the cells were surrounded by basement membrane‐like material (predominantly in‐between adjacent cells). In Trembler cells, cultivated in the presence of labelled acetate, the fatty acids were slightly altered; phosphatidylcholine was slightly reduced and phosphatidyl‐ethanolamine increased. Sulfatides were increased four times.
Cation Dependence of Hypotaurine Uptake in Mouse Brain Slices
Authors: Kontro P. , Oja S. S.
Published: October 2006

Abstract: Mouse brain slices take up hypotaurine (2‐aminoethanesulphinic acid) from medium by means of two concentrative low‐ and high‐affinity transport systems. [35S]Hypotaurine uptake by the slices was significantly reduced in the absence of external potassium, calcium, or magnesium ions. An excess of potassium ions also inhibited hypotaurine uptake by one‐half. Uptake was almost completely abolished on removal of sodium ions. The Km constants for both low‐ and high‐affinity transport components increased in a low‐sodium medium, suggesting that sodium ions are required when hypotaurine is attached to its possible carrier sites in plasma membranes. Sodium ions also mimicked allosteric effectors of hypotaurine transport, showing positive cooperativity. More than two sodium ions may be involved in the transport of one hypotaurine molecule across the cell membrane. The calculated activation energies of transport were fairly similar in normal and sodium‐deficient media and thus sodium ions may not participate in the activation mechanisms of the transport. With respect to cation dependence, hypotaurine transport in brain slices exhibits features characteristic of neurotransmitter amino acids.
Effects of Changes in Calcium Concentration on Basal and Stimulated 32P Incorporation into Phospholipids in Rat Pineal Cells
Authors: Smith Thomas L. , Hauser George
Published: October 2006

Abstract: The Ca2+ requirement for α‐agonist stimulation of 32P incorporation into acidic phospholipids (the phosphatidylinositol effect) of dispersed pineal cells was evaluated by means of several different compounds that interfere with Ca2+ disposition. Simple omission of Ca2+ led to slight increases in basal and norepinephrine‐stimulated phosphatidyl‐CMP (CDP‐diacylglycerol) and phosphatidylglycerol labeling without affecting phosphatidylinositol labeling. In the absence of Ca2+, EGTA (200 μM) or the ionophore for divalent cations A23187 (10 μM) elicited large increases in phosphatidic acid, phosphatidyl‐CMP, and phosphatidylglycerol labeling while strongly inhibiting the phosphatidylinositol effect. The Ca2+ translocation inhibitor LaCI3 also reduced the magnitude of this effect. The phosphatidylinositol effect is, however, not induced by increased Ca2+ entry into the cytosol, since A23187 did not mimic the effect of norepinephrine. Under conditions where membrane Ca2+ was lowered, the addition of 1 mM‐inositol greatly reduced phosphatidic acid, phosphatidylglycerol, and phosphatidyl‐CMP labeling with concomitant increases in basal and norepinephrine‐stimulated phosphatidylinositol labeling approaching that observed in the presence of norepinephrine and 2.5 mM‐Ca2+. In the presence of 2.5 mM‐Ca2+, inositol had negligible effects on phosphatidylinositol labeling. It was concluded that changes in membrane Ca2+ availability and/or disposition alter phospholipid metabolism and concurrently reduce the magnitude of the phosphatidylinositol effect, perhaps by making the pool of readily available inositol in pinealocytes rate‐limiting.
Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture
Authors: Pleasure David , Hardy Mattie , Johnson Gary , Lisak Robert , Silberberg Donald
Published: October 2006

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr‐rolidone‐coated silica gel) density gradient centrifugation survived in culture on polylysine‐coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short‐term suspension culture by dual label time course and substrate concentration dependence experiments with [2‐3H]glycerol and either [1,2‐14C]ethanolamine or L‐[U‐14C]serine. Rates of incorporation of 3H from the glycerol and of 14C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM‐[1,2‐14C]ethanolamine and 4.8 mM‐[2‐3H]glycerol, rates of incorporation of 14C and 3H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, 3H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than 14C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L‐[U‐14C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. 14C from L‐[U‐14C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.
Elevated Amounts of Protein in C6 Glial Cells During the Growth‐Inhibitory Response Elicited by Dexamethasone
Authors: Grasso Robert J. , Holbrook Nikki J. , Wodzinski Steven F.
Published: October 2006

Abstract: Dexamethasone suppresses C6 glial cell proliferation in vitro. This growth‐inhibitory response is accompanied by elevated amounts of acid‐insoluble protein in the steroid‐treated cells relative to controls. These results provide additional evidence that the glucocorticoid acts to arrest C6 cell proliferation in G2.
Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins
Authors: Fu Sheung Chun , Cruz Tony F , Gurd James W
Published: October 2006

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the “PSD protein” (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [3H]fucose or [3H]N′‐acetylmannosamine. The incorporation of [3H]fucose into synaptic fractions decreased two‐ to threefold between 10 and 28 days whereas little change in the incorporation of [3H]N′‐acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with 125I‐fucose‐binding protein or labeling of sialic acid with NaIO4NaB[3H4] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with 125I‐fucose‐binding protein increased one‐ to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with 125I‐wheat germ ag‐glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.
Tissue Choline Studied Using a Simple Chemical Assay
Authors: Haubrich D. R , Gerber N , Pflueger A. B , Zweig M
Published: October 2006

Abstract: An enzymatic‐radioisotopic assay was used to measure free choline in unextracted tissue. The lowest concentration of free choline in any tissue studied was present in human cerebrospinal fluid (mean, 5.7 μM; range, 1.8–31.2 μM). A postmortem increase in concentration of free choline occurred in blood (O.2 nmol/min ml), kidney (13 nmol/min·g), and liver (22 nmol/min·g) of mice. The concentration of free choline in these tissues was estimated by extrapolation to be 5, 77, and 29 nmol/g (or ml), respectively. Several treatments were found to increase the concentration of free choline. For example, intraperitoneal administration of choline or 2‐amino‐2‐methyl‐propanol (a choline oxidase inhibitor) induced an increase in the level of choline in blood, kidneys, liver, and brain of mice, and administration of 2‐dimethylaminoethanol (deanol) caused an increase in kidney and liver choline. The level of choline in blood was increased when rats were treated orally with either antibiotics or esters of choline such as phosphorylcholine, glycerylphos‐phorylcholine, laroylcholine, or propionylcholine. The results show that the concentration of free choline may be regulated by intestinal metabolism, availability of esterified precursors, and activity of enzymes that metabolize choline.
Is Na + K ATPase a Myelin‐Associated Enzyme?
Authors: Reiss David S , Lees Marjorie B , Sapirstein Victor S
Published: October 2006

Abstract: The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter‐enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. The discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29°C) than for the microsomal fraction (21°C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions. Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10−3 M‐strophanthidin. The evidence thus indicates that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggests that this is not a significant factor and that Na + K ATPase may be a myelin‐associated enzyme.
Neutral Glycosphingolipids and Ceramide Composition of Ethylnitrosourea‐Induced Rat Neural Tumors: Accumulation of Ceramide in Tumors
Authors: Chou Kuo‐Howere , Jungalwala Firoze B.
Published: October 2006

Abstract: Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20 mg/kg body weight, in the tail vein of the mother. The neutral glycosphingolipid, sulfatide, and ceramide composition of the tumors and the normal tissues from which the tumors originated is described. The content of nonhydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) containing ceramide in all the neural tumors so far examined was significantly increased compared with the corresponding normal neural tissue. Some 8 to 18 mol% of total neutral glycolipids was as ceramide in neurinomas, oligodendrogliomas, and menin‐giomas. Lactosylceramide in normal neural tissues was about 1 mol% of the total neutral glycosphingolipids. In various neural tumors lactosylceramide increased up to 8 mol%. NFA‐ and HFA‐containing cerebrosides constitute 94–100% of the neutral glycosphingolipids in normal neural tissues. In various neural tumors the mol percent of cerebrosides was significantly reduced. A high performance liquid chromatographic method was modified to analyze simultaneously ceramides, cerebrosides, and higher neutral glycosphingolipids.
The Specific Interaction of S‐100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S‐100 and Its Binding Sites
Authors: Donato Rosario
Published: October 2006

Abstract: The dissociation of the 125I‐labelled S‐100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the 125I‐labelled S‐100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate‐bound fraction of native S‐100.
Kinetic Properties of Bovine Pineal Tryptophan‐5‐Monooxygenase Activated by an Endogenous Activating Substance
Authors: Hori Shinichiro , Ohtani Sachiko
Published: October 2006

Abstract: We previously reported that an endogenous activating substance different from bovine serum albumin, phospholipids and heparin, exists in the extract from bovine pineal glands and that this substance interacts with tryptophan‐5‐monooxygenase under reducing conditions with sulfhydryl reagents, to stimulate monooxygenase activity. The present paper reports that the activating substance is of peptide nature; that it is sensitive to trypsin‐digestion; and that it does not change the apparent Km's for substrates, L‐tryptophan and oxygen, and coenzyme, reduced biopterin or DMPH4: but that it increases the Vmax 1.5‐ to 2.3‐fold. These results suggest that an activating protein, present in some particles of the cell structure, activates tryptophan‐5‐monooxygenase under the regulation of a sulfhydryl compound. The apparent Km's for reduced biopterin and DMPH4 were 77.2μM and 294 μM, respectively. The apparent Km's for L‐tryptophan and oxygen with reduced biopterin were 15.0 μM and 4.7%, respectively: with DMPH4, they were 11.0 μM and 8.5%, respectively. Significant inhibition of both L‐tryptophan and oxygen was observed with reduced biopterin, but not with DMPH4 (at the tested concentrations of up to 0.5 MM and 20%, respectively).
[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain
Authors: Hamon M. , Mallat M. , Herbet A. , Nelson D. L. , Audinot M. , Pichat L. , Glowinski J.
Published: October 2006

Abstract: A specific binding site for [3H]metergoline characterized by a KD of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7‐dihydroxy‐tryptamine‐ and kainic acid‐induced lesions indicated that this specific binding site was localized post‐synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [3H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad‐renaline for displacing bound [3H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [3H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [3H]metergoline and [3H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7‐dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [3H]metergoline might well be the appropriate ligand for studying the characteristics of the ‘antagonist form’ of serotonin receptors postulated by Bennett and Snyder.
Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme
Authors: Lazar Mitchell A. , Truscott Roger J. W. , Raese Joachim D. , Barchas Jack D.
Published: October 2006

Abstract: Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half‐life of 15 ± 3 min at 50°C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30°C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half‐life of 5 ± 2 min at 50°C.
Acetylcholine Synthesis in the Schwann Cell and Axon in the Giant Nerve Fiber of the Squid
Authors: Heumann Rolf , Villegas Jorge , Herzfeld Daniella W.
Published: October 2006

Abstract: Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm‐free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm‐free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea, under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.
The Influence of Divalent Cations and Substrate Concentration on the Incorporation of Myo‐inositol into Phospholipids of Isolated Bovine Oligodendrocytes
Authors: Gibson A. , Brammer M. J.
Published: October 2006

Abstract: The incorporation of myo‐inositol into phosphatidylinositol by two routes (CTP‐dependent and CTP‐independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP‐dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP‐diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP‐diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP‐independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+, Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.
The Preparation of Biologically Active Messenger RNA from Human Postmortem Brain Tissue
Authors: Gilbert Jeffrey M. , Brown Beverly A. , Strocchi Paola , Bird Edward D. , Marotta Charles A.
Published: October 2006

Abstract: Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)‐cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell‐free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one‐ and two‐dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two‐dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.
Effects of Insulin on Cultured Rat Brain Cells: Stimulation of Ornithine Decarboxylase Activity
Authors: Yang Joseph W. , Raizada Mohan K. , Fellows Robert E.
Published: October 2006

Abstract: Growth‐promoting peptide hormones, including growth hormone and insulin, stimulate rat brain ornithine decarboxylase (ODC; EC 4.1.1.17) activity in vivo (Roger et al., 1974; Roger and Fellows, 1980). To determine if this is a result of a direct action on brain, we have investigated the effect of peptide hormones in primary cell cultures of brain from fetal rats of 20 days gestational age. Significant stimulation of ODC activity was observed 4 h after administration of porcine insulin and bovine growth hormone. On a molar basis, growth hormone was less potent than insulin. By contrast, glucagon, enkephalin, and angiotensin II did not stimulate ODC in this system. At 25 ng/ml, insulin stimulated ODC activity approximately threefold, with maximum stimulation of five‐ to sevenfold reached at 1 μg/ml. After a 1‐h lag, insulin‐stimulated ODC activity increased to a maximum between 5 h and 8 h and returned to basal levels by 24 h. The apparent Km of ODC, 5.66 ± 1.16μM, was not significantly altered by insulin treatment, nor was any enzyme activator found in mediating insulin actions. Additional evidence suggests that insulin stimulation of ODC activity involves both de novo synthesis of the enzyme and a prolongation of ODC half‐life by 50%. These findings, implicating insulin as a regulator of ODC activity in brain cells, suggest the possible involvement of insulin or an insulin‐like peptide in the control of growth and development of the CNS.
Cyclic AMP Generation in Medulla Oblongata and Hypothalamus of Hypertension‐Prone and ‐Resistant Rats
Authors: Kobrin I. , Ebstein R. , Belmaker R. , Ben‐Ishay D.
Published: October 2006

Abstract: Accumulation of cAMP induced by noradrenaline (NA) was higher in the medulla oblongata (MO) of hypertension‐prone (H) than in resistant (N) rats, 113 ± 3 vs. 72 ± 4 (p < 0.01). In the hypothalamus (HYP) it was higher in N than in H rats, 148 ± 8 vs. 92 ± 6 (p < 0.01). In both anterior and posterior HYP, the accumulation of cAMP was significantly higher in N than in H rats. cAMP accumulation, in the presence of increasing concentrations of phosphodiesterase inhibitor, showed significant strain differences in both MO and HYP. The observed strain differences in cAMP may be pertinent to the disparate susceptibility to hypertension of H and N rats.
Circadian Variation of Cyclic AMP in the Rat Pineal Gland
Authors: Mikuni Masahiko , Saito Yoshiro , Koyama Tsukasa , Yamashita Itaru
Published: October 2006

Abstract: This study was carried out to investigate circadian variation of cyclic AMP contents in the rat pineal glands, using the high‐energy microwave radiation technique. The pattern of cyclic AMP concentration in the pineal gland showed a distinct circadian variation, with the maximum level at 0200 and the lowest at 1400. The administration of propranolol completely blocked the dark‐induced increase in the pineal cyclic AMP level at 0200, and the administration of isoproterenol induced a threefold, rapid increase in the cyclic AMP level at 1400, although it did not change the level at 0200.