bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly
Authors: Poduslo Joseph F.
Published: October 2006

Abstract: Schwann cell biosynthesis of the major myelin glycoprotein, P0, was investigated in the crush‐injured adult rat sciatic nerve, where there is myelin assembly, and in the permanently transected nerve, where there is no myelin assembly. Endoneurial fractions from desheathed rat sciatic nerves distal to the crush were compared with similar fractions from the permanently transected nerves at 7, 14, 21, 28, and 35 days after injury. The Schwann cell expression of this asparagine‐linked glycoprotein was evaluated after sodium dodecyl sulfatepore gradient electrophoresis by Coomassie Blue and silver stain and by autoradiography after direct overlay of radioiodinated lectins [wheat germ agglutinin, gorse agglutinin, and concanavalin A (Con A)]. As evaluated by these parameters, the concentration of P0 after crush decreased and subsequently increased as a function of time after injury, corresponding to the events of demyelination and remyelination. After permanent transection, the Po concentration decreased following the same time course found after crush. At subsequent time points, P0 could not be detected with Coomassie Blue stain, silver stain, or wheat germ agglutinin. Both gorse agglutinin and Con A, however, showed binding to P0. Radioactive precursor incorporation studies with [3H]fucose or [3H] mannose into endoneurial slices at 35 days posttransection revealed active oligosaccharide processing of P0 glycoprotein by Schwann cells in this permanent transection model. Compared with other Schwann cell glycoproteins in the transected nerve, the highest level of incorporation of [3H]mannose was found in P0 which accounted for 42.7% of the incorporated label. In contrast, incorporation of [3H]mannose into endoneurial slices at 35 days after crush accounted for only 13.3% in Po. In addition, higher levels of Con A binding were observed in P0 in the transected nerve compared with the contralateral control or the crushed nerve. Both the [3H]fucose incorporation and gorse agglutinin binding to P0 in the transected nerve suggest posttranslational processing of this glycoprotein in the Golgi apparatus; however, the absence of wheat germ agglutinin binding, the high level of mannose incorporation, and the high level of binding by Con A imply that additional processing steps are required prior to its assembly into myelin.
Enhancement of Hypophysectomy‐Induced Dopamine Receptor Hypersensitivity in Male Rats by Chronic Haloperidol Administration
Authors: Gordon John H. , Diamond Bruce I.
Published: October 2006

Abstract: It has been reported that hypophysectomy (HYPOX) would antagonize the development of a neuroleptic‐induced dopamine receptor hypersensitivity, and suggested that the neuroleptic‐induced dopamine receptor hypersensitivity may be mediated by the neuroleptic‐induced hyperprolactinemia. Conversely, we and others have reported on the ability of HYPOX animals to develop a neuroleptic‐induced dopamine receptor hypersensitivity. The present study was undertaken to define the possible role(s) of prolactin in the modulation of striatal dopamine receptor sensitivity. The data from these studies indicate: that HYPOX alone will result in the development of a striatal dopamine receptor hypersensitivity; that the HYPOX‐induced dopamine receptor hypersensitivity could be increased by the chronic administration and withdrawal of haloperidol; that administration of prolactin to HYPOX rats would partially antagonize the development of the neuroleptic‐induced dopamine receptor hypersensitivity; and that the administration of prolactin alone had minimal effects on the apomorphine‐induced behavior or neurochemistry of the HYPOX animals. These results suggest that the neuroleptics do not require the presence of a pituitary secretion (specifically, prolactin) to induce a striatal dopamine receptor hypersensitivity; however, they do indicate that a pituitary secretion, perhaps prolactin, may have the ability to modulate striatal dopamine sensitivity.
Effects of Chlorpromazine on the Activities of Antioxidant Enzymes and Lipid Peroxidation in the Various Regions of Aging Rat Brain
Authors: Roy Deodutta , Pathak Deena Nath , Singh Rameshwar
Published: October 2006

Abstract: In this work, the effect of chronic intraperitoneal administration of chlorpromazine (5 and 10 mg/kg) on the antioxidant enzymes superoxide dismutase (SOD), catalase (CA), glutathione reductase (GR), and glutathione peroxidase (GP); lipid peroxidation; and lipofuscin accumulation in the brains of rats ages 6, 9, and 12 months was studied. Chlorpromazine increased the activities of SOD, GR, and GP in particulate fraction from cerebrum, cerebellum, and brain stem in a dose‐dependent manner. While GR and SOD associated with soluble fraction increased, GP associated with soluble fraction was not affected. CA did not change after chlorpromazine administration in any regions of the brain of rats from all age groups. Chlorpromazine, thus, had a somewhat different action on antioxidant enzymes in different subcellular fractions. Chlorpromazine inhibited lipid peroxidation, both in vivo and in vitro, and it also inhibited accumulation of lipid peroxidation fluorescent products (lipofuscin), which was studied histochemically and biochemically as well. The data indicate that chlorpromazine inhibition of lipid peroxidation and of accumulation of lipofuscin can result from elevation of the activity of brain antioxidant enzymes.
On the Metabolism of [3H]Noradrenaline in Different Compartments of Rat Brain with Respect to the Role of Catechol‐O‐Methyltransferase
Authors: Köster G. , Goege E. , Breuer H.
Published: October 2006

Abstract: Rats were pretreated with either reserpine or desmethylimipramine, either alone or in combination with tropolone. At either 10 min or 1 h after the intraventricular injection of [3H]noradrenaline, in several brain regions the complete metabolic patterns were determined: normetanephrine; the glycol metabolites (methylated and nonmethylated) and their sulfate conjugates; and the acidic metabolites (methylated and nonmethylated). A reserpine‐induced increase in the turnover of [3H]noradrenaline caused a transient increase of the catechol glycol followed by elevated levels of the two glycol sulfates. The stimulated [3H]noradrenaline turnover if achieved by desmethylimipramine caused a transient increase of normetanephrine and initially lowered values of catechol glycols (both free and sulfated), which were followed by elevated levels. Drug‐pretreated rats compensated for the inhibition of catechol‐O‐methyltransferase by tropolone in different ways: Reserpine caused an early increase of the catechol glycol beyond the measurements in other treatment groups, whereas desmethylimipramine increased the nonmethylated carboxylic acid and glycol sulfates rather slowly to levels beyond those of other groups. The results support the existence of two compartments with a fast metabolism (an intraneuronal monoamine oxidase compartment and an extraneuronal catechol‐O‐methyltransferase compartment). In addition, there seems to exist another extraneuronal space with a slow, monoamine oxidase‐dependent noradrenaline turnover.
Proton Nuclear Magnetic Resonance Spectroscopy of Rabbit Brain Homogenate
Authors: Middlehurst Carl R. , Beilharz Georg R. , Hunt Glenn E. , Kuchel Philip W. , Johnson Gordon F. S.
Published: October 2006

Abstract: Proton nuclear magnetic resonance (1H NMR) spectroscopy in conjunction with the inversion‐recovery spin‐echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.
Solubilization and Characterization of Striatal Dopamine Receptors
Authors: Lew J. Y. , Goldstein M.
Published: October 2006

Abstract: Dopamine receptor binding proteins were sol‐ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio ‐ 2 ‐ hydroxy ‐ 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [3H]spiroperidol ([3H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO‐solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga‐tion at 100,000 ×g for 2 h. The CHAPSO‐solubilized dopamine receptor proteins, as well as the prelabeled [3H]Spi‐receptor protein complex, bind specifically to wheat germ agglutinin (WGA)‐agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO‐solubilized, prelabeled [3H]Spi‐receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA‐agarose‐bound [3H]Spi‐receptor protein complex with digitonin (CHAPSO‐digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [3H]Spi‐receptor complex in the CHAPSO‐digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO‐solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [3H]Spi to CHAPSO‐solubilized dopamine receptor proteins are similar to those for membrane‐bound proteins. Chronic treatment with haloperidol increases the Bmax, and does not change the KD for [3H]Spi in the CHAPSO‐solubilized and in the membrane‐bound preparations. Thus, the CHAPSO‐solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane‐bound dopamine receptors.
Trifluoperazine Stimulates Acetylcholine Receptor Synthesis in Cultured Chick Myotubes
Authors: Schneider Mark , Shieh Bih‐Hwa , Pezzementi Leo , Schmidt Jakob
Published: October 2006

Abstract: Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I‐α‐bungarotoxin. At drug concentrations of 5 to 10 × 10−6M, receptor appearance rate was significantly enhanced while receptor half‐life, cellular protein, net protein synthesis rate, and acetyl‐cholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 × 10−5M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KC1. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D‐600, phenothiazines may stimulate receptor synthesis by blocking a voltage‐gated calcium channel.
Effect of Intravenous Administration of D‐Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell‐Free System Derived from Brain
Authors: Cosgrove James W. , Brown Ian R.
Published: October 2006

Abstract: An initiating cell‐free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D‐lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation‐dependent amino acid incorporation in the brain cell‐free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met‐tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell‐free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.
Binding of Chlorpromazine to S‐100 Protein
Authors: Donato Rosario
Published: October 2006

Abstract: Chlorpromazine (CPZ) induces in S‐100 conformational changes resulting in the exposure of titratable SH groups of the protein to the solvent. This effect is even greater in the presence of Mg2+± Ca2+. S‐100 possesses binding sites for CPZ. The binding of CPZ to 3 μM S‐100 is half‐saturated by 0.18 μM CPZ in the presence of Mg2+ plus Ca2+ and by 0.24 μM CPZ in the presence of Mg2+ plus EGTA. The extent of the binding is greater in the presence of Ca2+ than in the presence of EGTA, especially at low CPZ concentrations.
Clonal Variability of PC12 Pheochromocytoma Cells with Respect to Catecholamine Biosynthesis
Authors: Koike Tatsuro , Takashima Akihiko
Published: October 2006

Abstract: Clonal variants of PC12 cells with respect to catecholamine biosynthesis were isolated, and the catecholamine content was measured by high performance liquid chromatography with electrochemical detection. The dopamine content of 13 subclones, which were selected and isolated in tyrosine‐free medium, was substantially higher than the control level: 0.91 ± 0.10 nmol/mg protein (mean ± SEM; n = 3). In contrast, the noradrenaline content showed a marked heterogeneity: only two subclones contained noradrenaline levels similar to or higher than the control level: 0.40 ± 0.05 (n = 5). The rest of them contained below the level of 0.20, and only negligible amounts of noradrenaline were found in four subclones. Thus, the noradrenaline‐to‐dopamine ratio varied widely between 0.003:1 and 0.53:1. This divergence of the noradrenaline content appears to be related to differing levels of dopamine β‐monooxygenase activity. The administration of ascorbate to the medium alone, however, did not restore the level of noradrenaline to the normal level in a subclone. Heterogeneity of the response to applied glucocorticoid was also demonstrated.
Guanyl Nucleotide and Divalent Cation Regulation of Cortical S2 Serotonin Receptors
Authors: Battaglia George , Shannon Michael , Titeler Milt
Published: October 2006

Abstract: Computer‐assisted quantitative analysis of radioligand binding to rat cortical S2 serotonin receptors indicates the existence of two affinity states of the same receptor population. Monophasic antagonist competition curves for [3H]ketanserin‐labelled sites suggest a uniform population of receptors with one affinity state for antagonists. Biphasic competition curves of agonists suggest that agonists discriminate high‐and low‐agonist‐affinity forms of the S2 receptors. The affinities of agonists for the high‐and low‐affinity states, and the apparent percentages of high agonist‐affinity forms varies with different agonists. The guanine nucleotides GTP and guanyl‐5′‐imido‐diphosphate [Gpp(NH)p], as well as divalent cations, modulate the proportion of the sites with high affinity for agonists as evidenced by their ability to shift the agonist competition curves for [3H]ketanserin‐labelled S2 receptors. GTP and Gpp(NH)p effects appear to be agonist‐specific, as they do not affect antagonist competition for [3H]ketanserin‐labelled S2 receptors, or [3H]ketanserin binding to S2 receptors. ATP and ADP have little or no effect on the binding properties of S2serotonin receptors, whereas GDP is less potent than GTP. The presence of these specific nucleotide effects are the first evidence suggesting involvement of a guanine nucleotide‐binding protein in the mechanism of agonist interaction with the S, serotonin receptor. In general, the binding properties of [3H]ketanserin‐labelled S2 serotonin receptors strongly resemble those of adenylate‐cyclase coupled receptors such as the β‐adrenergic, the α2‐receptor, and the D‐2 dopamine receptor. This may indicate the S2 serotonin receptor is coupled to adenylate cyclase activity, through a GTP binding protein.
Identification of an Acidic Dipeptide, β‐Aspartylglycine, in the CNS of Aplysia
Authors: McCaman M. W. , Stetzler J.
Published: October 2006

Abstract: A novel dipeptide, (β‐aspartylglycine (β‐DG), has been isolated from tissues of the marine gastropod mollusc Aplysia californica. This compound was detected only in Aplysia and not in other molluscs, such as Helix or Mercenaria, or in lobster or frog. Among the Aplysia tissues, the highest levels of β‐DG were in nervous tissue and in the reproductive tract. β‐DG was assayed by HPLC as the o‐phthaldialdehyde derivative and found to be present in all individual, identified neurons at a concentration of approximately 40 pmol/μg protein. The peptide was identified as β‐DG by gas chromatography‐mass spectrometry (GCMS) using trimethylsilyl derivatives prepared before and after acid hydrolysis. It was further characterized as the β‐isomer by TLC, including Rf, atypical blue‐gray color with ninhydrin, and a violet color with Cu2+‐ninhydrin. A fractionation scheme is described whereby acid‐soluble tissue constituents can be divided into acidic, neutral, and basic components using mini ion‐exchange columns. This partial purification prior to TLC analysis was necessary to remove compounds that interfered with the isolation of β‐DG.
Evidence for Feedback Regulation of Glutamate Decarboxylase by γ‐Aminobutyric Acid
Authors: Porter Terence G. , Martin David L.
Published: October 2006

Abstract: Although feedback control mechanisms for regulating the synthesis of various neurotransmitters have been demonstrated no such mechanism has been described for γ‐aminobutyric acid (GABA) in mammalian brain. Physiological concentrations of GABA inactivated glutamate decarboxylase, the enzyme responsible for GABA synthesis, by converting it to apoenzyme. This inactivation was opposed by the cofactor, pyridoxal 5′‐phosphate (pyridoxal‐P), and was promoted by ATP. GABA also competitively inhibited the enzyme, and the Ki for inhibition was essentially the same as the concentration of GABA giving the half‐maximal rate of inactivation (16 mM). These results provide a mechanism for direct feedback control of presynaptic GABA synthesis and provide further support for the regulation of glutamate decarboxylase in vivo by a cycle of inactivation and reactivation.
Purification of the Chick Eye Ciliary Neuronotrophic Factor
Authors: Barbin Gilles , Manthorpe Marston , Varon Silvio
Published: October 2006

Abstract: Dissociated 8‐day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2½‐day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion‐exchange chromatography, membrane ultrafiltration‐concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate‐polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10–20 μg of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 × 106 trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24‐h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8‐day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.
Increase of Choline Acetyltransferase by Colchicine in Primary Cell Cultures of Spinal Cord
Authors: Ishida Isao , Deguchi Takeo
Published: October 2006

Abstract: Colchicine (5–10 μM) increased choline ace‐tyltransferase (ChAT) activity 5–10‐fold and suppressed acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) activities to 30% and 50%, respectively, of the levels of control cells in mouse spinal cord cells cultured for several days. The synthesis of radiolaheled acetylcholine (ACh) from [14C]choline was also enhanced 4.6‐fold, although the uptake of [14C]choline into cells was decreased to 80% of control level. Neither the incorporation of [3H]Ieucine into protein nor the total amount of protein was increased by colchicine. Vinblastine also increased ChAT activity while cytochalasin B was not effective. Immunochemical titration study revealed that the increase of ChAT activity by colchicine was due to the accumulation of ChAT molecules. Co‐culture of spinalcord cells with skeletal muscle markedly stimulated ChAT activity, and the addition of colchicine to the co‐ cultures showed greater than additive effect. These observations indicate that colchicine increases ChAT molecules in a specific manner, that the stimulatory effect of colchicine on ChAT activity is possibly mediated via the interaction with microtubules, and that the increase of ChAT activity is based on a mechanism different from that of co‐cultures with skeletal muscle cells.
Regional Distribution of Prostaglandins D2, E2, and F2α and Related Enzymes in Postmortem Human Brain
Authors: Ogorochi Toshiya , Narumiya Shuh , Mizuno Noboru , Yamashita Kouwa , Miyazaki Hiroshi , Hayaishi Osamu
Published: October 2006

Abstract: The presence of prostaglandins D2, E2, and F2α was demonstrated and their contents measured in various regions of postmortem human brain, pineal body, and pituitary by using specific radioimmunoassays and gas chromatography‐mass spectrometry. The three prostaglandins were widely distributed in similar concentrations ranging from several hundred pg/g wet weight to about 40 ng/g wet weight. Prostaglandins D2 and E2 showed consistent and similar regional distributions in all six brains tested; amounts were high in pineal body, pituitary, olfactory bulb, and hypothalamus. On the other hand, prostaglandin F2α was distributed more evenly. Prosta‐ glandin D synthetase and prostaglandin E synthetase activities were found in cerebrum homogenate from a single subject and were recovered from the 100,000 ×g supernatant. The presence of 1 mM glutathione, reduced form, markedly stimulated the activity of prostaglandin E synthetase, but did not affect prostaglandin D synthetase activity. Activity of 15‐hydroxyprostaglandin dehydrogenase was found in the cerebrum homogenate and was partially purified. This enzyme required NADP as a cofactor and copurified with prostaglandin E 9‐ketoreductase.
Regional Development of Glutamate Dehydrogenase in the at Brain
Authors: Leong S. F. , Clark J. B.
Published: October 2006

Abstract: The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (>2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons > midbrain = hypothalamus > cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90‐day‐old adult value, but not in other regions. The enzyme‐specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90‐day‐old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.
Effects of Denervation and Axotomy on Nervous System‐Specific Protein, Ornithine Decarboxylase, and Other Enzyme Activities in the Superior Cervical Sympathetic Ganglion of the Rat
Authors: Ando Masato , Miwa Masato , Kato Kanefusa , Nagata Yutaka
Published: October 2006

Abstract: The time courses of changes of three enolase isozymes (αα, αγ, and γγ), S‐100 protein, 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase), ornithine decarboxylase (ODC), β‐galactosidase, and glucose‐6‐phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1‐2 weeks after the operations. Among enolase isozymes in the SCG, neuron‐specific γγ‐enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic αα‐enolase and the αγ‐hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S‐100 protein, an astrocyte‐specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin‐associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation. Ornithine decarboxylase, a rate‐limiting enzyme catalyzing polyamine biosynthesis, which participates in the regulation of growth and differentiation of the cells, increased significantly within 2–4 days after axotomy and then decreased to a subcontrol level after the next week; the enzyme activity changed only little after denervation. Axotomy caused a gradual decrease of a neuronal marker enzyme, β‐galactosidase activity, but produced an increase of G6PDH, an enzyme essential in lipid biosynthesis for myelin formation.
Synthesis of Gluco‐ and Galactocerebrosides in Bovine Neurones and Oligodendroglia
Authors: Brammer M. J.
Published: October 2006

Abstract: Oligodendroglial and neuronal perikarya have been isolated from fresh and frozen bovine brain and used to investigate the synthesis of cerebrosides from UDP‐hexoses and ceramides. The radioactive cerebrosides produced have been identified by TLC of the intact lipids on borate‐silica gel and by a combination of acid hydrolysis and ion‐exchange chromatography of the liberated hexoses. Incubation of either neuronal or oligodendroglial homogenates with UDP‐galactose (UDP‐gal) and mixed ceramides (hydroxy plus nonhydroxy fatty acids) leads to the synthesis of hydroxy fatty acid galactocere‐broside. This lipid is also formed by both neuronal and oligodendroglial homogenates if the UDP‐gal is replaced by UDP‐glucose (UDP‐glu). Formation of glucocerebroside has been observed only in the presence of neuronal homogenates and UDP‐glu. Electron microscopic examination of the isolated cell preparations has shown that, although the oligodendroglia are relatively pure, the neurones are contaminated with oligodendroglia, which may account for some of the hydroxy fatty acid galactocere‐broside synthesis. When unlabelled UDP‐gal is included in the incubations containing cell homogenates, mixed ceramides, and labelled UDP‐glu, galactocerebroside formation is greatly reduced, but there is comparatively little effect on glucocerebroside synthesis. In addition, experiments using D‐[14C]galactose 1‐phosphate and UDP d‐[6‐3H]glucose simultaneously have shown that the rate of formation of tritiated cerebroside is much greater than that of the 14C‐labelled compound. For these reasons it is suggested that galactocerebroside is formed from UDP‐glu and ceramide by epimerisation of the UDP hexose to UDP‐gal rather than by synthesis of UDP‐gal from galactose 1‐phosphate and UDP‐glu.
Sulfhydryl Groups in Receptor Binding of Thyrotropin‐Releasing Hormone to Rat Amygdala
Authors: Sharif Najam A. , Burt David R.
Published: October 2006

Abstract: Binding of [3H]‐[3‐Me‐His2]thyrotropin‐releasing hormone ([3H]MeTRH) to TRH receptors in rat amygdala was decreased by sulfhydryl reagents in a time‐, temperature‐, and concentration‐dependent manner. A pronounced reduction in receptor density, with little or no change in binding affinity, was apparent following disulfide bond reduction by dithiothreitol (DTT), alkylation of thiol groups by N‐ethylmaleimide (NEM), and their oxidation by 5,5′‐dithiobis (2‐nitrobenzoic acid). Heavy metals (Cd2+, Hg2+), which complex with reactive ‐SH residues, also potently inhibited binding. The pharmacological specificity of residual [3H]MeTRH binding in chemically modified amygdala membranes was the same as that in control preparations. Sequential exposure to thiol reagents, in the presence or absence of cations, revealed possible additive effects. Pretreatment of membranes with TRH (10‐‐8‐‐10‐‐6M), and its continued presence during modification, afforded protection against DTT and NEM. These results indicate the possible importance of thiol groups in the maintenance of TRH receptor conformation.
Effects of Hypoglycemia on Rat Brain Polyribosome Sedimentation Pattern
Authors: Ulovec Z. , Narancsik P. , Gamulin S.
Published: October 2006

Abstract: Insulin‐induced hypoglycemia provokes polyribosome disaggregation and accumulation of monomeric ribosomes in the brain of rats with hypoglycemic paresis and coma. The extent of brain polyribosome disaggregation depends on the decrease of blood glucose concentration, and in comatose animals on the duration of hypoglycemia. Cycloheximide prevents the disaggregation of brain polyribosomes induced by hypoglycemia, indicating that hypoglycemia affects brain protein synthesis, decreasing the rate of initiation relative to the rate of elongation of polypeptide chain synthesis.
Effects of Two Convulsant β‐Carboline Derivatives, DMCM and β‐CCM, on Regional Neurotransmitter Amino Acid Levels and on In Vitro D‐[3H]Aspartate Release in Rodents
Authors: Chapman Astrid G. , Cheetham Sharon C. , Hart Geneen P. , Meldrum Brian S. , Westerberg Eva
Published: October 2006

Abstract: Clonic seizures were induced in Swiss or DBA/2 mice by methyl‐6‐7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), 0.048 mmol/kg i.p., or by methyl‐β‐carboline‐3‐carboxylate (β‐CCM), 0.044 mmol/kg i.p. Measurement of regional brain (cortex, hippocampus, striatum, and cerebellum) amino acid levels after 15 min of seizure activity showed increases in γ‐aminobutyric acid (GABA) (in all regions after β‐CCM, and in cortex and hippocampus after DMCM), and an increase in glycine in the striatum after β‐CCM. Aspartate levels fell (in cortex and hippocampus) after DMCM, but were unchanged in all regions after β‐CCM. Glutamate levels fell in cortex after β‐CCM and in striatum after DMCM. Pretreatment with the excitatory amino acid receptor antagonist, 2‐amino‐7‐phosphonoheptanoic acid, 0.5 mmol/kg i.p., 45 min prior to the β‐carboline, significantly increased the ED50 for DMCM‐induced clonic seizures (4.68 μmol/kg vs. 9.39 μmol/kg). Similar pretreatment did not significantly alter the ED50 for β‐CCM (4.22 μmol/kg vs. 6.6 μmol/kg). Pretreatment with 2‐amino‐7‐phosphonoheptanoic acid, 1.0 mmol/kg, blocked the increase in GABA content produced by DMCM but not the fall in cortical aspartate content. Potassium‐induced release of preloaded D‐[3H]aspartate from rat cortical or hippocampal minislices was enhanced in the presence of DMCM (100 μM). In contrast, stimulated release of D‐[3H]aspartate (from cortex or hippocampus) was not altered in the presence of β‐CCM (100 μM). Although DMCM and β‐CCM are both considered to induce convulsion by acting at the GABA‐benzodiazepine receptor complex, the convulsions differ in several pharmacological and biochemical respects. It is suggested that enhanced release of excitatory amino acid neurotransmitters plays a more important role in seizures induced by DMCM.
Polymorphism of Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain
Authors: Zakut Haim , Matzkel Avi , Schejter Eduardo , Avni Adi , Soreq Hermona
Published: October 2006

Abstract: The molecular forms and membrane association of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and pseudocholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) were determined in the presence of protease inhibitors in dissected regions of developing human fetal brain, as compared with parallel areas from mature brain. All areas contained substantial cholinesterase activities, of which acetylcholinesterase accounted for almost all the activity. Two major forms of acetylcholinesterase activity, sedimenting at 10–11S and 4–5S, respectively, were detected on sucrose gradients and possessed similar catalytic properties, as judged by their individual Km values toward [3H]acetylcholine (ca. 4 × 10−4M). The ratio between these forms varied by up to four‐ to fivefold, both between different areas and within particular areas at various developmental stages, but reached similar values (about 5:2) in all areas of mature brain. Acetylcholinesterase activity was ca. 35–50% lowsalt‐soluble and 45–65% detergent‐soluble in various developmental stages and brain areas, with an increase during development of the detergent‐soluble fraction of the light form. In contrast, pseudocholinesterase activity was mostly low‐salt‐soluble and sedimented as one component of 10–11S in all areas and developmental stages. Our findings suggest noncoordinate regulation of brain acetylcholinesterase and pseudocholinesterase, and indicate that the expression of acetylcholinesterase forms within embryonic brain areas depends both on cell type composition and on development.
Molecular Weight of Human Brain Neutral Sphingomyelinase Determined In Situ by the Radiation Inactivation Method
Authors: Levade Thierry , Potier Michel , Salvayre Robert , Douste‐Blazy Louis
Published: October 2006

Abstract: The radiation inactivation method was used to determine the molecular weight of membrane‐bound neutral sphingomyelinase from normal human brain. Inactivation curves showed a molecular mass of 167,000 ± 32,000. Molecular weights of two control enzymes, β‐Nacetylglucosaminidase and nonspecific β‐glucosidase, determined by the same procedure, were consistent with previous reports.
Irreversible Inhibition of Calcium Uptake in Synaptosomes by Quinacrine Mustard: Relationship to Labeling Sites of Quinacrine Mustard
Authors: Ohta Akira , Baba Akemichi , Iwata Heitaroh
Published: October 2006

Abstract: The sites of interaction of quinacrine with synaptic membranes were labeled with quinacrine mustard. Quinacrine mustard had an inhibitory effect on depolarization‐induced calcium uptake by synaptosomes similar to that of quinacrine. The inhibition of depolarization induced calcium uptake by quinacrine was reduced by 70% after washing, whereas that by quinacrine mustard was not affected. Fluorescence electrophoretograms of the quinacrine mustard‐treated synaptic membranes showed that quinacrine mustard specifically labeled two proteins, with corresponding molecular weights of about 37,000 and 32,000.
Neurological Mutation Characterized by Dysmyelination in NCTR‐Balb/C Mouse with Lysosomal Lipid Storage Disease
Authors: Weintraub H. , Abramovici A. , Sandbank U. , Pentchev P. G. , Brady R. O. , Sekine M. , Suzuki A. , Sela B.
Published: October 2006

Abstract: Morphological and biochemical studies were performed on the CNS of neurologically affected NCTR Balb/C mouse. Histological and electron microscopic techniques demonstrated severe myelin deficiency in the affected brains. Neither the presence of lipid‐containing macrophages nor reactive gliosis was apparent. Analysis of myelin‐associated lipids and proteins revealed prominent depletion of galactocerebroside, sulfatide, and proteolipid proteins. In contrast to the scarcity of myelin specific constituents a marked accumulation of GM2 and GM3 gangliosides and several neutral glycolipids, i.e., glucocerebroside, lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide were found in affected CNS. These abnormalities were already apparent in 12‐day‐old pups as well as in 65‐day‐old mice. A significant deficit in the proportion of long‐chain fatty acids (C24), notable in both normal and α‐hydroxy acids of cerebrosides from affected white matter, was measured. The lack of reactive gliosis, the observed depletion of galactocerebroside and sulfatide at the early age of 12 days, and the relative decrease in long‐chain fatty acids in affected CNS strongly suggest a defect in myelinogenesis in this mutant rather than a secondary process of myelin breakdown.
Norepinephrine Turnover in the Hypothalamus of Adult Male Rats: Alteration of Circadian Patterns by Semistarvation
Authors: Schweiger Ulrich , Warnhoff Michael , Pirke Karl‐Martin
Published: October 2006

Abstract: Norepinephrine (NE) turnover, as estimated by 3‐methoxy‐4‐hydroxyphenylethyleneglycol concentration, was studied in the mediobasal hypothalamus of control and semistarved adult male rats at eight time points of a 24‐h period. The marked circadian periodicity of NE turnover with a peak in the dark phase in control rats is completely suppressed in semistarved rats. The average 24‐h concentration of the NE precursor tyrosine in brain and of tyrosine flow into brain (calculated from plasma amino acid concentrations) is reduced in semi starved rats, but both brain tyrosine and tyrosine flow show continuing circadian fluctuations.
Alkenylhydrolase: A Microsomal Enzyme Activity in Rat Brain
Authors: Gunawan Johannes , Debuch Hildegard
Published: October 2006

Abstract: Rat brain microsomes have the capacity to liberate radioactive free aldehydes from 1‐[1‐14C]alk‐1′‐enyl‐sn‐glycero‐3‐phosphoethanolamine (lysoplasmalogen). Glycerophosphoethanolamine was found using 1‐alk‐1′‐enyl‐sn‐glycero‐3‐phospho‐[3H]ethanolamine. The ratio of both products released by lysoplasmalogenase action was 1:1. Another enzymic activity could be demonstrated, which hydrolyzes lysoplasmalogen at the hydrophilic part of the molecule, a lysophospholipid phospho diesterase. Thus, 1‐[1‐14C]alk‐1′‐enylglycerol was detected as well as [3H]ethanolamine, again in a molar ratio, from the respective labeled substrates. This enzyme possesses nearly the same affinity toward the substrate as lysoplasmalogenase. Whereas the lysophospholipid phos phodiesterase is totally inhibited in the presence of NaF or EDTA, lysoplasmalogenase activity is not affected by these reagents. 1‐[1‐‘14C]Alk‐l’‐enylglycerol acts also as substrate for lysoplasmalogenase, which liberates radioactive aldehydes at the same rate as from lysoplasmal ogen. Because the apparent Km and Vmax values are nearly identical for both substrates, the enzyme activities are inhibited in the same way, and the pH optimum is about 7.2 in both cases, it is concluded that both substrates were attacked by the same enzyme. The enzyme does not differentiate between a substrate substituted at the sn‐3 position of glycerol and one that is not. It requires only a free OH group at the sn‐2 position. Phosphoethanolamine phosphatase activity was also determined under our experimental conditions.
Human Brain 6‐Phosphogluconate Dehydrogenase: Purification and Kinetic Properties
Authors: Weisz Karen S. , Schofield Philip J. , Edwards Michael R.
Published: October 2006

Abstract: 6‐Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6‐phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6‐phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6‐phosphogluconate also providing considerable protection against loss of activity caused by p‐chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6‐phosphogluconate. Product inhibition by NADPH was noncompetitive when 6‐phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5‐phosphate was noncompetitive when both 6‐phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5‐phosphate are released from the enzyme in random order with NADPH as the last product released.
Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain
Authors: Sheu Kwan‐Fu Rex , Lai James C. K. , Kim Young Tai , Dorante Gary , Bagg Jennifer
Published: October 2006

Abstract: Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme‐linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the α peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the αE1, βE1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar. Our results suggest that variations of PDHC activity among different brain regions represent quantitative variations in the amount of PDHC protein.
Some Long‐Lasting Neurochemical Efafects of Prenatal or Early Postnatal Exposure to Diazepam
Authors: Frieder Brina , Grimm Veronika E.
Published: October 2006

Abstract: Changes in the uptake of various neurotransmitters were measured in the frontal cortex and hippocampus of male and female rats that were exposed to diazepam through the placenta or through the mother's milk during the prenatal or early postnatal period of rapid brain development. Earlier studies from our laboratory showed that early diazepam exposure has long‐lasting behavioral consequences. The present results show that prenatally diazepam‐exposed rat pups show significant reduction in choline uptake in the frontal cortex at 10 days of age. At 60 days of age, both pre‐ and postnatally exposed males, but not females, show significant differences from controls in terms of choline uptake, whereas postnatally exposed females whose behavior was shown previously to be profoundly affected by the diazepam exposure showed significant increase in γ‐aminobutyric acid (GABA) uptake in the hippocampus and reduction of 5‐hydroxytryptamine (5HT) uptake in the cortex at 60 days of age.
Effect of Glutamate Analogues on Brain Tumor Cell Lines
Authors: Campbell Graham LeM. , Bartel Roxanne , Freidman Henry S. , Bigner Darell D.
Published: October 2006

Abstract: Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.
Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products
Authors: Law Mona J. , Deibler Gladys E. , Martenson Russell E. , Krutzsch Henry C.
Published: October 2006

Abstract: Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion‐exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy‐terminal analyses with carboxypeptidases A, B, and Y and amino‐terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89‐Asn90, Lys133‐Ser134, and Lys153‐Leu154 bonds. Cleavages also occurred at the Arg31‐His32, Lys53‐Agr54, and Arg25‐His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high‐voltage electrophoresis‐TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9‐His10, Lys13‐Tyr14, Lys103‐Gly104, Lys137 Gly138, Lys140‐Gly141, and Arg160‐Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44‐Phe45 bond. Instead it cleaves at the Arg31‐His32 and Lys53‐Arg54 bonds, thus preserving the entire hydrophobic sequence Ile‐Leu‐Asp‐Ser‐Ile‐Gly‐Arg‐Phe‐Phe as well as short sequences to either side.
Cell Survival Characteristics and Choline Acetyltransferase Activity in Motor Neurone‐Enriched Cultures from Chick Embryo Spinal Cord
Authors: Flanigan Thomas P. , Dickson John G. , Walsh Frank S.
Published: October 2006

Abstract: The effect of muscle extract on cell survival and choline acetyltransferase (ChAT) activity in cultures of enriched cholinergic neurones from 7‐day chick embryo spinal cord was examined. When neurones were grown on hydrated collagen gels, considerable cell survival and ChAT activity were obtained even in the absence of tissue extract. These parameters were stimulated twofold in the presence of skeletal muscle extract but not liver or skin extracts. The cholinergic neurotrophic activity was found to be heat‐ and trypsin‐sensitive, non‐dialysable, and to act in the virtual absence of glial cells. These data are consistent with a retrogradely acting motor neurone trophic activity.