bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of Six Synaptic Membrane Antigens in Subcellular Fractions of Rat Brain Cortex
Authors: Mahadik S. P. , Korenovsky A. , Ciccarone V. , Rapport M. M.
Published: October 2006

Abstract: The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62K, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigens were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.
Characterization of γ‐Aminobutyric Acid Binding Sites on Crude Synaptic Membranes
Authors: Kurioka Susumu , Hayata Hideo , Matsuda Makoto
Published: October 2006

Abstract: [3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X‐100 [membranes (TX)] involved two classes of GABA binding sites (KD= 38.7 and 78.0 nM) in the absence of Na+, but the high‐affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD= 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high‐affinity class of GABA receptor.
Substantia Nigra γ‐Aminobutyric Acid Receptors in Huntington's Disease
Authors: Cross Alan J. , Waddington John L.
Published: October 2006

Abstract: The specific binding of [3H]γ‐aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high‐affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.
The Distribution of Estrone Sulphatase, Dehydroepiandrosterone Sulphatase, and Arylsulphatase C in the Primate (Macaca radiata) Brain and Pituitary
Authors: Lakshmi S. , Balasubramanian A. S.
Published: October 2006

Abstract: Estrone sulphatase, arylsulphatase C and dehydroepiandrosterone sulphatase were measured in the pituitary, hypothalamus‐preoptic area, amygdala, hippocampus, midbrain, septum, frontal cortex and occipital cortex of monkey (Macaca radiata) brain. All the regions showed measurable activities of all three enzymes. In all the animals tested, either the midbrain or hypothalamus‐preoptic area showed the greatest activity of all three enzymes. In particular, estrone sulphatase showed the highest specific activity in either of the above two regions in all animals. Subcellular distribution studies in the hypothalamus preoptic area showed a similarity in the distribution profile between arylsulphatase C and estrone sulphatase and a significant difference of dehydroepiandrosterone sulphatase from the others.
α‐Hydroxylation of Fatty Acids in Brain: Characterization of Heat‐Labile Factor
Authors: Singh Inderjit , Kishimoto Yasuo
Published: October 2006

Abstract: The particulate fraction, heat‐labile factor, heat‐stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (α‐hydroxylignoceric acid). The heat‐labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD‐Agarose affinity chromatography. The specific activity of the heat‐labile factor was increased 105‐fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS‐polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat‐labile factor was incubated with 1 mM‐N‐ethylmaleimide. This inhibition was prevented by preincubating the heat‐labile factor with 1 mM‐NADH. These observations indicate that the heat‐labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.
On the Specificity of 125I‐α‐Bungarotoxin Binding to Axonal Membranes
Authors: Jumblatt J. E. , Marquis J. K. , Mautner H. G.
Published: October 2006

Abstract: 125I‐α‐Bungarotoxin (α‐BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α‐BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible (KDapp± 1.7 ± 0.32 × 10‐7 M; Bmax± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM‐Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca2+ and Mg2+) in the medium. 125I‐α‐BGT binding could be inhibited by cholinergic drugs (atropine ≅d‐tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4‐aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α‐BGT binding sites in other tissues are discussed.
Enzymic Lipid Peroxidation in the Microsomal Fraction of Rat Brain
Authors: Player T. J. , Horton A. A.
Published: October 2006

Abstract: An enzymic lipid peroxidation system has been demonstrated in the microsomal fraction of rat brain and the requirements and optimal conditions for assay determined. The involvement of NADPH‐cytochrome c reductase was demonstrated in vesicles reconstituted with lipids extracted from the brain microsomal fraction. Further characterization of the system made use of substances shown to inhibit the liver microsomal system. α‐Tocopherol was shown to be an effective inhibitor of lipid peroxidation in the brain microsomal system, whereas Na2SO3 had no effect, which is indicative that free radical transfer occurs only in the hydrophobic regions. Neither superoxide dismutase nor catalase inhibited lipid peroxidation. The implications of an NADPH‐cytochrome c reductase‐dependent lipid peroxidation system that is not linked to a drug hydroxylation system and appears to differ from the liver microsomal system in a number of other ways are discussed.
Interaction Between the Oxygen and Tryptophan Dependence of Synaptosomal Tryptophan Hydroxylase
Authors: Katz Ira R.
Published: October 2006

Abstract: The substrate dependence of tryptophan hydroxylase activity in rat striatal synaptosomes was examined. The Km for tryptophan in air is 8‐13 μM, comparable to the concentration present in cerebrospinal fluid. The reaction is inhibited by amino acids with large nonpolar side chains. For leucine the inhibition appears competitive; it results from a decrease in the steady state levels of intrasynaptosomal tryptophan. The oxygen Km at 10 μM‐tryptophan is 3‐4 mm Hg, which is increased when the reaction is assayed with low levels of tryptophan in the medium, and in the presence of amino acids such as leucine. Similarly, the tryptophan Km is increased at low oxygen tensions; at 9 mm Hg of oxygen it is 23 μM. These interactions between tryptophan and oxygen dependence of the reaction are discussed in terms of likely physiological significance and implications for the pharmacological use of tryptophan.
Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata
Authors: Diebler M. F. , Morot‐Gaudry Y.
Published: October 2006

Abstract: The ability of cholinergic vesicles to incorporate acetylcholine (ACh) was studied using highly purified synaptic vesicles from Torpedo electric organ. Depleted vesicles were capable of rapidly taking up exogenous ACh. Evidence that this represented true incorporation was that labelled ACh comigrated with vesicular ATP on gel filtration and that vesicle‐associated ACh was protected against enzymatic hydrolysis and was releasable under hypoosmotic conditions. The total amount of ACh incorporated depended on the ACh concentration up to 100 MM. A sudden fall in the external ACh concentration did not cause leakage of the ACh incorporated in vitro. Preliminary results indicated that retention of ACh inside the vesicle was pH‐dependent. Choline was also taken up by vesicles, but the time pattern strongly suggested that it was not being retained. The magnitude of ACh incorporation was estimated with respect to the intravesicular space.
Ethanol Alters Kinetic Characteristics and Function of Striatal Morphine Receptors
Authors: Tabakoff Boris , Urwyler Stephan , Hoffman Paula L.
Published: October 2006

Abstract: Morphine was shown to promote dopamine (DA) synthesis and release in mouse striatum, but mice rendered tolerant and dependent on ethanol were found to be less responsive to morphine's effects on striatal DA metabolism than control animals. Ethanol feeding also produced a change in the affinity of striatal “opiate” receptors for [3H]dihydromorphine, and these ethanol‐induced receptor changes may be responsible for the altered biological effect of morphine.
Alteration of Dopamine Synthesis in Rat Striatum Subsequent to Selective Type A Monoamine Oxidase Inhibition
Authors: Schoepp Darryle D. , Azzaro Albert J.
Published: October 2006

Abstract: Pretreatment of rat striatal slices with the selective type A monoamine oxidase (MAO) inhibitor clorgyline was found to produce significant inhibition of dopamine (DA) synthesis. DA synthesis was reduced by nearly 50%, but not until more than 90% of the type A enzyme was inhibited. In contrast, complete inhibition of the type B MAO following deprenyl treatment had no effect. It is suggested that interneuronal accumulation of DA following inhibition of type A MAO leads to feedback inhibition at the rate‐limiting step in DA biosynthesis, tyrosine hydroxylation. These results are also consistent with the presence of a type A MAO within DA‐containing neurons and provide evidence of a regulatory role for type A MAO in the synthesis of brain DA.
Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum
Authors: Slevin J. T. , Coyle J. T.
Published: October 2006

Abstract: The development of the specific binding sites for l‐[3H]glutamic acid (KD= 370 nM) and for [3H]kainic acid (KD= 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.
Inhibition of the Synthesis of Acetylcholine in Rat Brain Slices by (−)‐Hydroxycitrate and Citrate
Authors: Tuček Stanislav , Doležal Vladimír , Sullivan Ann C
Published: October 2006

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM‐NaCl, 5 mM‐KCl, 1.2 mM‐MgCl2, 1.2 mM‐NaH2PO4, 25 mM‐NaHCO3, 0.2 mM‐choline chloride, 0.058 mM‐paraoxon, 1 mM‐EGTA, and oxidizable substrates. (−)‐Hydroxycitrate, a specific inhibitor of ATP‐citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5‐14C]citrate by 82–86%, but that from [U‐14C]glucose by only 33%, from [2‐14C]pyruvate by 24% and from [1‐14C‐acetyl]carnitine by 8%; the production of 14CO2 from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM‐ and 5 mM‐citrate diminished it by 43% and 66%, respectively; the production of from [U‐14C]glucose and from [1‐14C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca2+ and decreases the supply of mitochondrial acetyl‐CoA to the cytosol. The results with (−)‐hydroxycitrate indicate that the cleavage of citrate by ATP‐citrate lyase is not responsible for the supply of more than about one‐third of the acetyl‐CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)‐hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP‐citrate lyase.
Cholesterol‐Esterifying Enzymes in Developing Rat Brain
Authors: Jagannatha H. M , Sastry P. S
Published: October 2006

Abstract: A cholesterol‐esterifying enzyme which incorporates exogenous fatty acids into cholesterol esters in the presence of ATP and coenzyme A was demonstrated in 15‐day‐old rat brain. This enzyme was maximally active at pH 7.4 and distinct from the cholesterol‐esterifying enzyme reported earlier (Eto and Suzuki, 1971), which has a pH optimum at 5.2 and does not require cofactors. Properties of the two enzymes have been compared. Both the enzymes showed negligible esterification with acetate and were maximally active with oleic acid. The pH 5.2 enzyme esterified desmosterol, lanosterol and cholesterol at about the same rate, while the pH 7.4 enzyme was only 50% as active with lanosterol as it was with cholesterol and desmosterol. Phosphatidyl serine stimulated the pH 5.2 enzyme but not the pH 7.4 enzyme. Phosphatidyl choline and sodium taurocholate showed no effect on either of the enzymes. Both the enzymes were associated with particulate fractions, but the pH 7.4 enzyme was localized more in the microsomes. Purified myelin showed 2.6‐fold and 1.5‐fold higher specific activities of pH 5.2 and 7.4 enzymes respectively, when compared with homogenate. About 7–10% of total activity of both the enzymes was associated with purified myelin. Brain stem and spinal cord showed higher specific activity of pH 5.2 enzyme than cerebral cortex and cerebellum, while pH 7.4 enzyme specific activity was higher in cerebellum and brain stem than in cerebral cortex and spinal cord. Microsomal pH 7.4 activity showed progressive increase prior to the active period of myelination, reaching a maximum on the 15th day after birth and declined to 20% of the peak activity by 30 days. In contrast, pH 5.2 enzyme reached maximum activity about the 6th day after birth and remained at this level well into adulthood. In 15‐day‐old rat brain, pH 7.4 enzyme had five to six times higher specific activity than pH 5.2 enzyme, while in adults the activities were equal. The pH 7.4 enzyme showed a threefold higher specific activity than pH 5.2 enzyme in myelin from 15‐day‐old rats, but in adults the reverse was true.
A Rapid and Simple Method for the Determination of Picogram Levels of 3‐Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection
Authors: Ponzio F , Achilli G , Algeri S
Published: October 2006

Abstract: A rapid and simple technique using solvent extraction, ion‐pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3‐methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3‐methoxytyramine added to the samples is 45–50%. 3‐Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3‐methoxytyramine levels in rat striata, while catechol‐O‐methyltransferase inhibition with 3′,4′‐dihydroxy‐2 methylpropiophenone completely depleted 3‐methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D‐amphetamine were also examined. The 3‐methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.
A Radioimmunoassay for Ependymins β and γ: Two Goldfish Brain Proteins Involved in Behavioral Plasticity
Authors: Schmidt Rupert , Shashoua Victor E
Published: October 2006

Abstract: A radioimmunoassay (RIA) using 125I‐labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since 125I‐β‐antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera becomes possible.
Free Sterols of the Rabbit Optic Nerve and Cerebral White Matter During Ontogenic Development
Authors: Adamczewska‐Goncerzewicz Z , Trzebny W
Published: October 2006

Abstract: Free sterol composition of the developing rabbit optic nerve was compared with that of the homologous cerebral white matter at corresponding stages of ontogeny. The sterols were detected and identified by means of combined gas‐chromatography and mass spectrometry. The following free sterols were found in both the optic nerve and cerebral white matter: cholesterol, desmosterol, lanosterol, two dimethylsterols, which are probably 4,4‐dimethyl‐5α‐cholest‐8,24‐diene‐3β‐ol, with a molecular weight of 412, and 4α,14α‐dirnethyl‐5α‐cholest‐7‐ene‐3β‐ol, with a molecular weight of 414 and probably cholestene, with a molecular weight of 368. The sterol spectrum of the developing optic nerve differed not only from that of the mature nerve but also from that of age‐matched white matter of the rabbit brain. The tri‐ and dimethyl‐sterols, detected for the first time in the rabbit optic nerve and cerebral white matter, are natural components of the developing nervous tissue but they were not found in the mature nerve nor in cerebral white matter.
Synthesis of Cytoskeletal Proteins in Bulk‐Isolated Neuronal Perikarya Takashi Nakayama
Authors: Nakayama Takashi
Published: October 2006

Abstract: Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L‐[35S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000 g. Newly synthesised polypeptides in each fraction were resolved by SDS‐gel and two‐dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α1‐, α2 and β‐tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two‐dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two‐dimensional gel electrophoresis and also by one‐dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikarya in vitro yielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesised in situ. The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikarya in vitro, but to disproportionately weaker extents compared with the 68,000 component.
Effect of α‐Methylphenylalanine and Phenylalanine on Brain Polyribosomes and Protein Synthesis
Authors: Binek Patricia A , Johnson Terry C , Kelly Chester J
Published: October 2006

Abstract: A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor α‐methyl‐D, L‐phenylalanine (0.43 mg/g body wt). The presence of α‐methylphenylalanine in newborn mice inhibited 65–70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40‐fold in both plasma and brain following injection of α‐methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of α‐methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.
The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake
Authors: Richardson P. J , Whittaker V. P
Published: October 2006

Abstract: The Na+ and K+ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na+ (290 MM) and low internal K+ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na+, 96 mM; K+, 81 mM); 0.1 mM‐ouabain abolished these gradients. The trans‐membrane Na+ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na+ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain‐sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.
Light‐Stimulated Release of Taurine from Retinas of Kainic Acid‐Treated Chicks
Authors: Pasantes‐Morales H , Quesada O , Cárabez A
Published: October 2006

Abstract: The light‐stimulated release of [3H]taurine from chick retina was studied in chicks intraocularly injected with kainic acid (60 nmol). This treatment produced a loss of more than 80% of the inner nuclear and the inner synaptic layers, sparing the outer retinal layers. Concomitantly, the treatment produced a marked decrease of endogenous GABA and glycine but not of taurine. The activity of glutamate decarboxylase was also markedly decreased in the kainic acid‐treated retinas. The release of [3H]taurine, either spontaneous or stimulated by light, was unaffected by the treatment. These results suggest that the light‐stimulated efflux of taurine occurs from the retinal layers which are not affected by the kainic acid treatment.
Presence of Radiolabelled Metabolites in Release Studies Using [3H]γ‐Aminobutyric Acid
Authors: Gardner Colin R , Richards Mary H
Published: October 2006

Abstract: Most studies on γ‐aminobutyric acid (GABA) release from nervous tissue have been conducted using radiolabelled GABA in the presence of aminooxyacetic acid (AOAA) to inhibit GABA: 2‐oxoglutarate aminotransferase (GABA‐T) to prevent conversion of labelled GABA to labeled catabolites. Here we present data showing that even in the presence of 10 μM‐AOAA the spontaneous release of tritium from rat cortical synaptosomes prelabelled with 2,3‐[3H]GABA is mainly in the form of tritiated water but that the increase in tritium release in the presence of unlabelled GABA or high potassium‐ion concentrations is in the form of authentic [3H]GABA. Interpretation of results should take these facts into account.
Adenosine Transport into Guinea‐pig Synaptosomes
Authors: Barberis C. , Minn A. , Gayet J.
Published: October 2006

Abstract: Kinetics for transport of adenosine into guinea‐pig neocortex synaptosomes were studied by incubating them with [14C]adenosine for up to 30 s. The apparent Km value of the high‐affinity transport system for adenosine was 21.1 μM and the Vmax value was 257.3 pmol/min/mg protein. The transport system was inhibited by both compounds structurally related (compounds 554 and 555) and unrelated (dipyridamole) to adenosine. Because electrically stimulated synaptosomes release up to 1.5% of the adenosine derivative content per min, the physiological significance of adenosine uptake is discussed as a possible mechanism to compensate for the loss of adenine nucleotides from synaptosomes preparations.
Effects of Selective Alkylphosphorylation of Propionylcholinesterase on the Regeneration of Acetylcholinesterase in the Aqueous Soluble Fraction of Superior Cervical Ganglia of the Rat Following Sarin
Authors: Koelle Winifred Angenent , Rickard Kathleen Kitto , Koelle George B.
Published: October 2006

Abstract: The rates of regeneration of acetylcholinesterase (AChE) and propionylcholinesterase (PrChE) in the supernatants of aqueous homogenates of rat superior cervical ganglia, centrifuged at 100,000 g for 90 min, were determined at 1,3, 6, and 16 h following their inactivation (>90%) by administration of sarin, 2.0μmol/kg i.v. Values were compared with those in animals in which the PrChE was continually suppressed by the repeated, fractional administration of iso‐OMPA, in a total dose of 10 or 20 μmol/kg i.p. These doses of iso‐OMPA alone produced 96–99% inactivation of PrChE with no detectable effect on AChE. Significant suppression of AChE regeneration by iso‐OMPA administration was noted only at 6 h; in contrast with earlier findings in the cat, administration of iso‐OMPA alone caused no significant increase in ganglionic AChE activity.
UDP‐Galactose Hydrolysis in Brain and Its Effect on Cerebroside Synthesis
Authors: Satomi Daisaku , Kishimoto Yasuo
Published: October 2006

Abstract: The enzymatic hydrolysis of UDP‐galactose in rat and calf brain was studied. The hydrolysis occurs in two steps: The first is the conversion of UDP‐galactose to galactose‐1‐phosphate catalyzed by nucleotide pyrophosphatase (EC 3.6.1.9), and the second is the conversion of the latter to free galactose by alkaline phosphatase (EC 3.1.3.1). The overall conversion has a pH optimum of 9.0, but there is considerable activity at pH 7.4, which is the optimum for UDP‐galactose:ceramide galactosyltransferase in the synthesis of cerebrosides. Preparations from cytosol from calf brain cerebellum or stem that were enriched in UDP‐galactose hydrolytic activity inhibit cerebroside synthesis under conditions optimal for the synthesis. Microsome‐rich and nuclear debris fractions contain the highest apparent specific activity among the subcellular fractions studied. Hydrolysis of UDP‐galactose occurs in all areas of brain, brainstem having the highest activity. The apparent specific activity in jimpy mouse brain homogenate is nearly twice as high as in the control brain homogenate.
Characterization of Enkephalin Release from Rat Striatum
Authors: Lindberg Iris , Dahl June L.
Published: October 2006

Abstract: Using antisera specific for methionine‐ and leucine‐enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium‐stimulated release; tyr‐arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short‐term incubation periods used in these experiments.
Polypeptide Hormones and Chromatin‐Associated Proteins Act as Acceptors for Cholera Toxin‐Catalyzed ADP‐Ribosylation
Authors: Trepel J. B. , Chuang D.‐M. , Neff N. H.
Published: October 2006

Abstract: Cholera toxin catalyzed the ADP‐ribosylation of the pituitary protein hormones thyrotropin (TSH), lutropin (LH), follitropin (FSH), human chorionic gonadotropin (hCG). and corticotropin (ACTH)1–24, and ADP‐ribosylation of the basic proteins histone subfraction H1 and protamine. Casein and phosvitin, acidic nuclear proteins, did not act as acceptors for toxin‐catalyzed ADP‐ribosylation. The isolated TSH A and B subunits were tested for their ADP‐ribose acceptor activity. The TSH A subunit showed fourfold greater ADP‐ribose acceptor activity than the TSH B subunit. The ADP‐ribose acceptor protein protamine was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis following incubation with cholera toxin under ADP‐ribosylating conditions. [3H]ADP‐ribose incorporated into protein from [3H]NAD migrated with the acceptor protein protamine. In the absence of added acceptor protein, the [3H]ADP‐ribose incorporated into protein migrated with the A1 fragment of cholera toxin. Cholera toxin A and B subunits were isolated and tested for their ability to catalyze the transfer of ADP‐ribose to protamine. The cholera toxin A subunit showed 50‐fold greater ADP‐ribosyltransferase activity than the B subunit. Our data indicate that a variety of adenohypophyseal hormones and regulatory proteins act as acceptors for toxin‐catalyzed ADP‐ribosylation. These studies may help in understanding the role of endogenous ADP‐ribosyltransferases and the physiological effects of this modification of protein.
Rapid Incorporation In Vivo of Intracerebrally Injected 32Pi into Polyphosphoinositides of Three Subfractions of Rat Brain Myelin
Authors: Deshmukh D. S. , Kuizon S. , Bear W. D. , Brockerhoff H.
Published: October 2006

Abstract: At intervals ranging from 1 to 10 min after injection of 32Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho‐inositides (PPI) was then determined. There was rapid incorporation of 32Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated 32Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos‐phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di‐ (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged 32Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.
Myelin Gangliosides: An Unusual Pattern in the Avian Central Nervous System
Authors: Cochran Frederick B. , Yu Robert K. , Ando Susumo , Ledeen Robert W.
Published: October 2006

Abstract: Gangliosides were isolated from purified myelin obtained from brain and spinal cord of mature chickens and pigeons. Total concentrations were approximately two‐ to fivefold greater than for previously reported mammalian species, and their patterns also differed in containing significantly more sialosylgalactosylceramide (GM4). The latter comprised one‐third to one‐fourth of total myelin ganglioside, approximately equivalent to GM1 (II3NeuNAc‐GgOse4Cer). As in mammals, GM4 of avian CNS appeared to be localized in myelin. Fatty acids of this ganglioside included both the hydroxy‐ and unsubstituted types. and, long‐chain bases were almost entirely C18. Ganglioside GM1 split into two closely migrating bands on TLC, the slower of which resembled mammalian GM1 in having stearate as the main fatty acid with a measurable amount (10%) of C20‐sphingosine; the faster band had predominantly longer‐chain fatty acids and very little C20‐sphingosine.
A Model System for the Study of the Release of Luteinizing Hormone‐Releasing Hormone from Isolated Storage Granules
Authors: Burrows G. Howard , Porter John C. , Barnea Ayalla
Published: October 2006

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone‐releasing hormone (LH‐RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH‐RH‐containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH‐RH affinity columns. The LH‐RH associated with the granules passes freely through the columns, whereas the LH‐RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH‐RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH‐RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH‐RH. Incubation of granules at pH 7.8 in the presence of 160 mM‐KC1 resulted in the release from the granules of 14% of the stored LH‐RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH‐RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH‐RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH‐RH are stable under defined biochemical conditions, and LH‐RH release from these granules is stimulated by ions and MgATP.
Alterations in Norepinephrine and Dopamine Content in Selected Brain Areas of Guinea Pigs Adapted to Simulated High Altitude
Authors: Light K. E. , Shetlar D. , Strahlendorf J. C. , Hughes M. J. , Barnes C. D.
Published: October 2006

Abstract: The alterations in brain content of norepinephrine (NE) and dopamine (DA) were studied in guinea pigs adapted to simulated high altitude (hypobaric hypoxia) equivalent to 5500 meters. The animals were adapted for 46 days over a period of 82 days to a pressure of 375 mm Hg. The animals were then killed and the following brain parts dissected: cerebellum, neocortex, caudate head/basal forebrain, diencephalons/rhinencephalon, and brain stem. NE and DA content were analyzed by high pressure liquid chromatography with electrochemical detection by a technique described. Results showed a significant increase of NE and DA in neocortex; a significant increase of DA but not NE in caudate/basal forebrain, and a significant decrease of NE and not DA in diencephalons/rhinencephalon.
Metabolite Alterations in the Genetically Spastic Mouse
Authors: Stewart James A.
Published: October 2006

Abstract: The concentration of soluble amino acids in the brains of homozygous spastic mice was measured. It was found that brain arginine levels were reduced by 3% and urea concentration was 50% higher in affected mice. The activity of the brain and liver enzymes for arginine synthesis were similar in spastics and in controls. However, in spastic mice the activity of brain arginase is elevated. This increased arginase activity accounts for the decreased arginine concentration in the mutant mice.
In Vitro Modulation by Avermectin B1a of the GABA/Benzodiazepine Receptor Complex of Rat Cerebellum
Authors: Supavilai Porntip , Karobath Manfred
Published: October 2006

Abstract: Avermectin B1a, a macrocyclic lactone anthelmintic agent, causes a concentration‐dependent increase of [3H]flunitrazepam binding to membranes from rat cerebellum by increasing the affinity and the number of binding sites. This effect appears to be independent of the concentration of chloride ions. The effects of avermectin B1a occur with high affinity (EC50= 70 nM), and they persist after washing of the membranes with drug‐free buffer. Pretreatment of the membranes with Triton X‐100 completely abolishes the action of avermectin B1a. GABA and the GABA‐mimetic compounds piperidine‐4‐sulfonic acid and THIP diminish the effects of avermectin B1a on benzodiazepine receptor binding in a bicuculline‐methiodide‐sensitive mode. In addition, the stimulation of [3H]flunitrazepam binding by avermectin B1a is decreased by the pyrazolopyridines etazolate and cartazolate. These observations suggest that avermectin B1a stimulates benzodiazepine receptor binding by acting on a modulatory site which 1s independent of the GABA recognition site and of the drug receptor for the pyrazolopyridines, but which is in functional interaction with these sites.
Postmortem Stability of Brain 3‐Methoxy‐4‐Hydroxyphenylethyleneglycol and 3,4‐Dihydroxyphenylethyleneglycol in the Rat and Mouse
Authors: Warsh Jerry J. , Godse Damodar D. , Li Peter P. , Cheung Siu W.
Published: October 2006

Abstract: To assess the postmortem stability of brain 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) and 3,4‐dihydroxyphenylethyleneglycol (DHPG) levels, groups of rats and mice were killed by cervical dislocation and left at either 21° or 4°C for intervals of up to 24 h until removal and freezing of whole brain. Whole brain free and total MHPG and DHPG levels were determined simultaneously by gas chromatography‐mass fragmentography (GC‐MF). By 2 h after death, statistically significant decrements occurred in rat brain free DHPG (20%), total MHPG (21%), and total DHPG (11%) at 4°C, but free MHPG increased significantly (50%) compared with controls. At 21°C, rat brain total MHPG increased compared with controls at 2 h (15%) but decreased at 4 h (15%) and 8 h (15%), whereas free MHPG levels were increased at these times. Although brain total and conjugated DHPG levels showed little change, free DHPG levels were reduced at all times. In mouse brain no significant changes occurred in free MHPG and DHPG by 24 h at 4°C. At 21°C, mouse brain DHPG levels decreased whereas MHPG concentrations increased over the 8‐h period of study. These findings demonstrate the occurrence of significant postmortem time‐ and temperature‐dependent changes in brain MHPG and DHPG concentrations and indicate caution in the interpretation of changes in these metabolites in studies employing human postmortem brain tissue.
Alterations of Acetylcholine and Choline Metabolism in Mammalian Preparations Treated with β‐Bungarotoxin
Authors: Gundersen Cameron B. , Jenden Donald J.
Published: October 2006

Abstract: We have studied the effects of β‐bungarotoxin on acetylcholine and choline metabolism in central and peripheral cholinergic preparations using a gas chromatographic‐mass spectrometric assay for acetylcholine and choline. In contrast with previous reports, β‐bungarotoxin did not inhibit the high‐affinity uptake of labeled choline or the synthesis of acetylcholine in rat brain synaptosomal fractions. However, the toxin did cause a significant increase of medium choline when it was incubated with synaptosomal fractions. This increase of endogenous choline in the medium may account for the previously reported inhibition of choline uptake because of a dilution of the specific activity of the labeled choline in the medium. Several experiments are reported in which a further characterization was made of the effect of β‐bungarotoxin on medium choline. β‐Bungarotoxin was also shown to cause a large increase of acetylcholine release from rat brain minces and a depletion of the acetylcholine content of minces. A similar phenomenon was found in diaphragm preparations that were exposed continuously to β‐bungarotoxin. However, diaphragms that were treated for only 30 min with toxin showed the previously reported increase of acetylcholine content. β‐Bungarotoxin did not have any measurable effect on acetylcholine turnover in smooth muscle preparations from guinea pig ileum. These results help to explain certain inconsistencies in the literature regarding the action of β‐bungarotoxin.
The Inactivation of γ‐Aminobutyric Acid Transaminase in Dissociated Neuronal Cultures from Spinal Cord
Authors: Rando Robert R. , Bangerter Faan Wen , Farb David H.
Published: October 2006

Abstract: It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter γ‐aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase‐dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10−6 M‐gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long‐term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.
Properties of a CDP‐Diglyceride Hydrolase from Guinea Pig Brain
Authors: Rittenhouse Harry G. , Seguin Edward B. , Fisher Stephen K. , Agranoff Bernard W.
Published: October 2006

Abstract: Enzymatic hydrolysis of the pyrophosphate bond of CDP‐diglyceride (CDP‐DG), previously shown to occur in bacteria, is demonstrable in mammalian tissues. Activity was enriched in a lysosomal fraction obtained from guinea pig cerebral cortex and was purified 92‐fold relative to the homogenate by a combination of XM‐300 ultrafiltration and DEAE‐cellulose column chromatography. When incubated with CDP‐dipalmitin, the purified enzyme produced stoichiometric amounts of CMP and phosphatidate. dCDP‐DG served as a substrate, while ADP‐DG was an inhibitor, as were 5′‐AMP and 5′‐dAMP. CDP‐DG hydrolysis was not affected by the presence of excess amounts of CDP‐choline, CDP‐glycerol, sodium pyrophosphate, or cyclic 3′,5′‐AMP.
Effect of Estrogen on Denervated Muscle
Authors: Max Stephen R.
Published: October 2006

Abstract: The rate of increase of glucose 6‐phosphate dehydrogenase activity in denervated rat extensor digitorum longus muscle shows sexual dimorphism. This phenomenon is further investigated in this report by assessing the effects of ovariectomy, hypophysectomy, hormone replacement therapy, and treatment with an estrogen antagonist, MER‐25. The data demonstrate that physiologic doses of estrogens enhance the rate and extent of the increase in glucose 6‐phosphate dehydrogenase activity after denervation. The data further indicate that aromatization of androgens may be a significant source of estrogen involved in hormonal modulation of the neural control of glucose 6‐phosphate dehydrogenase and other processes in muscle. Furthermore, choline acetyltransferase activity, a marker for the neuromuscular synapse, decreased in rat extensor digitorum longus muscles after denervation, but was unaffected by ovariectomy.
Apparent Co‐Sequestration of Immunoreactive Corticotropin, α‐Melanot opin, and γ‐Lipotropin in Hypothalamic Granules
Authors: Barnea Ayalla , Cho Gloria , Porter John C.
Published: October 2006

Abstract: In the present study, the question of whether immunoreactive α‐melanotropin (α‐MSH1), corticotropin (ACTH1), and β‐melanotropin (β‐MSH1) are co‐sequestered in hypothalamic granules of adult male rats was addressed. When a 900 ×g supernatant fluid prepared from a hypothalamic homogenate was fractionated on continuous sucrose density gradients under non‐equilibrium Conditions, two populations of particles containing α‐MSH1, ACTH1, or β‐MSH1 Were observed. However, when fractionated under equilibrium conditions, the two populations of particles containing α‐MSH1 ACTH1, or β‐MSH1 were recovered as a single band. This sedimentation characteristic indicates that the particles containing a given peptide differ in size but are similar in density. In their sedimentation, the small particles containing α‐MSH1, ACTH1, and β‐MSH1 are indistinguishable from granules containing α‐MSH1, whereas the large particles containing α‐MSH1 (ACTH1, and β‐MSH1 are indistinguishable from synaptosomes containing α‐MSH1, β‐MSH1 had an apparent molecular weight (M.W.) of about 5,000, which is similar to that of γ‐lipotropin. ACTH1 was comprised of three species of molecules: big (M.W. ≥ 10,000), 5.7K (M.W. ≌ 5,700), and 4.5K (M.W. ≌ 4,500). Big ACTH was the predominant and 5.7K ACTH the minor component of ACTH1 present in granules as well as in synaptosomes. These results are suggestive that α‐MSH, ACTH and its precursors, and γ‐lipotropin are co‐sequestered in hypothalamic granules.