bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism and Axonal Transport of Polyamines in a Single Identified Neuron of Aplysia californica
Authors: Kremzner Leon T. , Ambron Richard T.
Published: October 2006

Abstract: The metabolism of polyamines was investigated by injecting purified [3H]putrescine directly into the soma of the giant neuron R2 of Aplysia. Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [3H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10‐6M, metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10−4M, relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S‐adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of 3H‐glycoproteins, with the finding that while the acid‐soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.
Acid Sphingomyelinase of Human Brain: Purification to Homogeneity
Authors: Yamanaka Tatsuhiro , Suzuki Kunihiko
Published: October 2006

Abstract: Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X‐100, followed by sequential chromatography on Concanavalin A‐Sepharose, octyl‐Sepharose, hydroxylapatite, DEAE‐cellulose, red A‐agarose, Sephadex G‐200, and DEAE‐cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000‐fold from the starting homogenate with a 1% yield. Specific activity of up to 800 μmol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X‐100. Triton X‐100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton‐free enzyme showed the same Km and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X‐100, in that removal of the detergent made the enzyme more heat labile. The Km of purified enzyme for sphingomyelin was 36 μM. It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and α‐mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis[p‐nitrophenyll‐ and bis[4‐methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.
Separation of Neurofilaments from ATPase Activity by Precipitation with Anti‐Neurofilament Antibodies
Authors: Glicksman Marcie A. , Willard Mark
Published: October 2006

Abstract: The possibility that neurofilaments could be involved in the transduction of chemical and mechanical energy in axons led us to investigate whether neurofilament proteins can hydrolyze ATP. We fractionated neurofilaments from rabbit spinal cord and found that preparations highly enriched for neurofilament proteins hydrolyzed ATP at a substantial rate (as high as 0.4 μmol/min/mg protein). However, the ATPase activity was neither inhibited by anti‐neurofilament antibody, nor was it precipitated by the antibody under circumstances that precipitated most of the neurofilament polypeptides. We conclude that neurofilament proteins do not hydrolyze ATP at a significant rate under the conditions of our assay; if hydrolysis of ATP is a physiological function of neurofilaments, additional factors are required.
Chronic Alcohol Consumption Increases Cyclo (His‐Pro)‐Like Immunoreactivity in the Rat Brain
Authors: Mori Masatomo , Prasad Chandan , Wilber John F.
Published: October 2006

Abstract: Administration of histidyl‐proline diketopiperazine (cyclo (HisPro)) to rats attenuates ethanol‐induced sleep. To understand the role played by cyclo (His‐Pro) in the pathophysiology of prolonged alcohol consumption, we have measured the distribution of this peptide in brains of control and alcohol‐treated rats. The data show that prolonged alcohol consumption increases the concentration of cyclo (His‐Pro) in hypothalamic as well as extrahypothalamic brain. These changes may reflect a physiologic adaptation of the brain during alcohol consumption.
Melittin Stimulates Incorporation and Degradation of Sphingomyelin in Synaptosomal Plasma Membranes
Authors: Pellkofer R. , Marsh D. , Hoffmann‐Bleihauer P. , Sandhoff K.
Published: October 2006

Abstract: Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane‐bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20‐fold. Melittin in concentrations as high as 100 μM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxy1 derivative of SPM, respectively. In the concentration range 100‐1000 μM. melittin was observed to rigidify SYM. The incorporation of SPM.erivatives into the lipid bilayer of SYM.as demonstrated by ESR measurements. Melittin enhanced the uptake of SPM‐derivatives into SYM.
Specific High‐Affinity Binding of L‐[3H]Aspartate to Rat Brain Membranes
Authors: Di Lauro Andrea , Meek James L. , Costa Erminio
Published: October 2006

Abstract: The binding of L‐[3H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na‐independent binding was saturable, reversible, and optimal at 20°C and at pHs in the neutral range. The dissociation constant (Kd) at 20°C was about 200 nM. This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na‐independent L‐[3H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex > ponsmedulla > spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na‐independent binding in this region. The Na‐dependent binding of L‐[3H]‐aspartate (Kd= 484 nM) was strongly inhibited by D‐aspartate, L‐glutamate, D,L‐aspartate β‐hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex > cerebellum = pons‐medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.
Cysteine Sulfinic Acid in the Central Nervous System: Specific Binding of [35S]Cysteic Acid to Cortical Synaptic Membranes‐An Investigation of Possible Binding Sites for Cysteine Sulfinic Acid
Authors: Iwata Heitaroh , Yamagami Satoru , Baba Akemichi
Published: October 2006

Abstract: Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L‐[35S]cysteic acid as a ligand. Two specific binding systems of [35S] cystec acid were found, one Na+‐dependent and the other Na+‐independent. The Na+‐independent specific binding of [35S]Cysteic acid was saturable, with a Kd of 474 nM and Bmax of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37°C. Treatment of the membranes with proteases, concanavalin A, or Triton X‐100 markedly reduced the binding. Of various compounds related to cysteic acid, L‐cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na+‐independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.
Cysteine Sulfinic Acid in the Central Nervous System: Antagonistic Effect of Taurine on Cysteine Sulfinic Acid‐Stimulated Formation of Cyclic AMP in Guinea Pig Hippocampal Slices
Authors: Baba Akemichi , Lee Eibai , Tatsuno Tohru , Iwata Heitaroh
Published: October 2006

Abstract: The stimulatory effect of cysteine sulfinic acid on cyclic AMP formation was examined in slices from three different regions of guinea pig brain. The inhibitory effect of taurine on the stimulated formation of cyclic AMP was also studied. Cysteine sulfinic acid (1–10 mM) greatly increased the cyclic AMP level in striatal, cortical, and especially hippocampal slices. In hippocampal slices, taurine (0.1‐30 mM) markedly lowered the increase of cyclic AMP induced by cysteine sulfinic acid, but not that induced by glutamate or aspartate. In this region, taurine also reduced the stimulatory effects on cyclic AMP formation of adenosine, norepinephrine, and histamine, but not of depolarizing agents. It did not, however, inhibit the effects of any of these stimulants in cortical slices. These results suggest that sulfur‐containing amino acids, such as cysteine sulfinic acid and taurine, regulate the cyclic AMP level in the hippocampus.
The Distribution of Radioligand Binding Activity and 5′‐Nucleotidase Activity in Bovine Caudate Nucleus Subcellular Fractions
Authors: Withy Raymond M. , Mayer R. John , Strange Philip G.
Published: October 2006

Abstract: The distribution of 5′‐nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5′‐nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.
Incorporation of [1‐14C]Palmitic Acid into Neutral Lipids and Phospholipids of Rat Cerebral Cortex In Vitro
Authors: Mizobuchi Masato , Morisaki Nobuhiro , Matsuoka Nobuo , Saito Yasushi , Kumagai Akira
Published: October 2006

Abstract: Incorporation of [1‐14C]palmitic acid into neutral lipids and phospholipids of rat cerebral cortex was examined in vitro in normal Krebs‐Ringer bicarbonate buffer containing 3% (wthol) albumin and 0.75 mM palmitic acid. Under standard assay conditions, radioactivity in the triacylglycerol fraction increased rapidly during the first 30 min, and then decreased after 60 min, with corresponding increase in radioactivity in phosphatidyl choline, phosphatidyl ethanolamine, and a fraction of phosphatidyl inositol plus phosphatidyl serine. Diacylglycerol was shown to be an intermediate metabolite. Radioactivity increased in triacylglycerol, and decreased in phosphatidyl choline and phosphatidyl ethanolamine throughout incubation under NZ gas. In the fraction of phosphatidyl inositol plus phosphatidyl serine, radioactivity decreased after 30 min during incubation under N, gas. A possible acylation‐deacylation cycle, in which triacylglycerol could be a source of free fatty acids for phospholipids, is discussed.
Neurotransmitter Receptor Ligand Binding and Enzyme Regional Distribution in the Pigeon Visual System
Authors: Vischer A. , Cuenód M. , Henke H.
Published: October 2006

Abstract: The relative importance of acetylcholine, dopamine, endogenous opiates, γ‐aminobutyric acid (GABA), glutamate, glycine, noradrenaline, and serotonin as transmitters in the pigeon visual system was estimated by measuring the activity of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and aromatic amino acid decarboxylase (AAD) as well as the binding of dihydroalprenolol, etorphine, kainic acid, muscimol, serotonin, spiroperidol, strychnine, and quinuclidinyl benzilate (QNB) in the tectum opticum, nucleus rotundus, ectostriatum, dorsolateral thalamus, and hyperstriatum (Wulst). As a nonvisual reference structure, the paleostriatal complex was included in the examination. The regional distribution of most of these parameters was very similar to data reported in the mammalian CNS supporting the hypothesis that the avian tectofugal and thalamofugal visual systems are homologous to the mammalian tecto‐thalamo‐cortical and retinogeniculo‐striate pathways, respectively. On the basis of the low values of their parameters, some transmitters can be excluded as significant contributors in a number of structures. As a hypothesis for further investigations, the presence of cholinergic and serotoninergic systems in the Wulst, possibly originating in the dorsolateral thalamus and nucleus raphe, respectively, and of glycinergic and dopaminergic terminals in the paleostriatal complex is proposed.
Characterization of Postmortem Human Brain Proteins by Two‐Dimensional Gel Electrophoresis
Authors: Kosik Kenneth S. , Gilbert Jeffrey M. , Selkoe Dennis J. , Strocchi Paola
Published: October 2006

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two‐dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie‐blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two‐dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two‐dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.
Solubilization by CHAPS Detergent of Barbiturate‐Enhanced Benzodiazepine‐GABA Receptor Complex
Authors: Stephenson F. Anne , Olsen Richard W.
Published: October 2006

Abstract: Barbiturates enhance the binding of [3H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, Kd, for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration‐dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [3H]Flunitrazepam binding activity is stable with storage at 4°C., but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly (t1/2= 48 h). [3H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS‐solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.
Rat Brain α‐Mannosidase: Purification, Properties, and Interaction with Its Antibodies
Authors: Zanetta J. P. , Meyer A. , Dontenwill M. , Basset P. , Vincendon G.
Published: October 2006

Abstract: α‐d‐Mannosidase (EC 3.2.1.24.) was purified to homogeneity from adult rat brain. The enzyme, of apparent molecular weight 397,000, appears to be formed of subunits of molecular weight 120,000 made of two protomers (62,000) bound by disulfide bridges. Isoelectric focusing gives two bands, of pi 5.40 and 5.15. Both isoenzymes seem to have the same pH curve (a small peak of activity at pH 4.5 and a maximum of activity around pH 6.0). These two isoenzymes are immunologically related.
Glycosylation of Proteins in Developing Human Brain
Authors: Korpeinen Tarja , Mononen I. , Krusius T. , Järnefelt J.
Published: October 2006

Abstract: The carbohydrate content of human brain glycoproteins was studied during development from the age of 12 fetal weeks to 8 postnatal months. The concentration of all the glycoprotein monosaccharides increased with age. The total amount of glycoprotein monosaccharides per lipid‐free dry tissue increased by about 150% from the end of the third fetal month to the time of delivery. The increase leveled off around term, and only minor increase occurred after birth. The adult level was reached by the fifth postnatal month.
In Vivo Incorporation of [32P]Orthophosphate into Myelin Basic Protein of Developing Rabbit Brain: Its Location in Components 3 and 5 and in a New Protein Tentatively Identified as Basic Protein Component 7
Authors: Agrawal Harish C. , Martenson Russell E. , Agrawal Daya
Published: October 2006

Abstract: Myelin was isolated from the brains of 16‐day‐old rabbits that had received intracerebral injections of [32P]orthophosphate 24 h earlier. The basic protein was extracted and examined by polyacrylamide gel electrophoresis both at low and at high pH. At low pH a single band corresponding to the basic protein contained all of the covalently bound protein radioactivity. At high pH, three bands of radioactivity corresponding to basic protein components 3, 5, and 7 were observed. Estimation of the specific radioactivities of the components in conjunction with their high pH electrophoretic mobilities indicated that components 3, 5, and 7 contained one, two, and three phosphate groups per molecule of protein, respectively.
The Complete Amino Acid Sequence of Human P2 Protein
Authors: Suzuki Masaru , Kitamura Kunio , Sakamoto Yasushi , Uyemura Keiichi
Published: October 2006

Abstract: The complete amino acid sequence of P2 protein from human peripheral nerve myelin was determined from nine staphylococcal protease peptides and four cyanogen bromide peptides. Human P2 protein is composed of 131 amino acids and has a molecular weight of 14,818. Compared to bovine P2 protein, there are replacements at nine positions (human↔bovine): 18(Asp↔Glu), 39(Thr↔Arg), 56(Thr↔Pro), 83(Ile↔Thr), 87(Gln↔Ala), 96(Arg↔Lys), 100(Lys↔Asn), 115 (Ala↔Val), and 121(Gly↔Asp).
Postnatal Developmental Changes in Fucosyltransferase Activity in Rat Brain
Authors: Léon Monique , Broquet Pierre , Morélis Renée , Louisot Pierre
Published: October 2006

Abstract: GDP‐fucose:asialofetuin fucosyltransferase activity was studied during the postnatal development of rat brain. The enzymatic activity was very low during the first days of life and reached a maximum level around 21 days. This increase in enzymatic activity was characterized by two periods of rapid change. A rapid increase occurred between 3 and 7 days after birth, followed by a slow increase from 7 to 17 days, then a new rapid change from 17 to 21 days. Stimulation of the enzymatic activity by Triton X‐100 increased with age. The developmental profiles of GDP‐fucose pyrophosphatase and fucosidase did not change during this period.
Cerebral Oxidative Metabolism and Blood Flow During Acute Hypoglycemia and Recovery in Unanesthetized Rats
Authors: Ghajar J. B. G. , Plum F. , Duffy T. E.
Published: October 2006

Abstract: Progressive neurological depression leading to coma was produced in unanesthetized rats at a constant level of hypoglycemia induced by insulin. High‐energy phosphate concentrations in brain remained normal during hypoglycemic lethargy, but ATP declined by 6% during stupor and by 40% during coma that was characterized by an isoelectric EEG. Cerebral blood flow (CBF) remained normal during hypoglycemia whereas the cerebral metabolic rates for oxygen (CMRo2) and glucose (CMRglucose) decreased by 45 and 73%, respectively, indicating oxidation of nonglucose fuels. A plot of CMRo2 and CMRglucose versus plasma glucose indicated increasing oxidation of alternate substrates (elevated CMRo2/CMRglucose) at plasma glucose concentrations below 2.5 mm. The cerebral uptake of β‐hydroxybutyrate increased during hypoglycemic stupor and its complete oxidation could account for the CMRo2 in excess of glucose utilization. Brain ammonia, a byproduct of amino acid metabolism, reached a level during hypoglycemic coma sufficient to produce coma in normoglycemic animals. The rate and degree of recovery after glucose administration depended on the duration of hypoglycemia and the pretreatment neurological state of the animal. Following 10 min of glucose infusion, ATP levels that were modestly depressed in stuporous rats recovered fully, paralleling the animals' apparently full neurological recovery. Rats that had been in hypoglycemic coma for 1 min or less fully recovered high‐energy phosphate concentrations in brain. However, when normalization of plasma glucose was delayed for more than 1 min of coma, the CMRo2 remained depressed, CBF decreased to 40% of control, and high‐energy substrates failed to normalize. In keeping with the depression of oxidative metabolism and blood flow, neurological function and the EEG remained abnormal even after 1 h of glucose infusion. The findings suggest that irreversible brain injury may develop within the first minutes of hypoglycemic coma.
Simultaneous Determination of Serotonin, 5‐Hydroxyindoleacetic Acid, 3,4‐Dihydroxyphenylacetic Acid and Homovanillic Acid by High Performance Liquid Chromatography with Electrochemical Detection
Authors: Sperk Günther
Published: October 2006

Abstract: A rapid and highly sensitive procedure for simultaneous determination of serotonin, 5‐hydroxyindoleacetic acid, 3,4‐dihydroxyphenylacetic acid and homovanillic acid is described. After precipitation of proteins with perchloric acid the samples are applied directly to a high performance liquid chromatograph, with electrochemical detection. As little as 20 pg of serotonin, 5‐hydroxyindoleacetic acid, and 3,4‐dihydroxyphenylacetic acid and 200 pg of homovanillic acid can be detected. One chromatographic run requires less than 10 min.
Effects of Anticonvulsants on the Formation of γ‐Hydroxybutyrate from γ‐Aminobutyrate in Rat Brain
Authors: Whittle Susan R. , Turner Anthony J.
Published: October 2006

Abstract: The conversion of γ‐aminobutyrate (GABA) via succinic semialdehyde to γ‐hydroxybutyrate has been examined in rat brain homogenates. A number of anticonvulsants, including sodium valproate and phenobarbitone, inhibited this metabolic pathway. These results are interpreted in the light of the characteristics of aldehyde reductases known to reduce succinic semialdehyde.
Brain Maturation Following Administration of Phenobarbital, Phenytoin, and Sodium Valproate to Developing Rats or to Their Dams: Effects on Synthesis of Brain Myelin and Other Subcellular Membrane Proteins
Authors: Patsalos Philip N. , Wiggins Richard C.
Published: October 2006

Abstract: The anticonvulsant drugs phenobarbital, phenytoin, sodium valproate, and phenytoin‐sodium valproate in combination were administered daily to (a) pregnant rats starting on the 5th day after conception, and continued through 17 days postpartum, or (b) to developing rats between 3 and 17 days of age. Each drug was prepared in water and administered at either a therapeutic dose (TD), three times therapeutic dose (3TD), or 9TD. Drug administration had no discernible effect on litter size or sex ratio in the offspring; however, phenobarbital administration to dams caused small but significant reductions in birth weights. Body weights of developing rats treated with anticonvulsant drugs either via dams or directly by intraperitoneal injection lagged behind controls. At 20–24 days of age the brain weights of the offspring of phenobarbital (9TD)‐exposed dams lagged control weights by 5% whereas brain weights in the offspring of the other treated groups were indistinguishable from controls. In contrast, administration of phenobarbital directly to developing rats caused no significant brain weight deficits whereas significant deficits were observed with phenytoin (9TD), sodium valproate (9TD), and phenytoin‐sodium valproate (9TD) in combination. At 20–24 days of age the relative incorporation of radioactive leucine into purified myelin and crude nuclear proteins of drug‐treated rats or the offspring of drug‐treated dams was reduced by 10–20% in all cases. Dose‐related differences were not observed however, and the effects of phenytoin and sodium valproate in combination approximated those of phenytoin administered alone.
Density‐Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells
Authors: Schengrund C.‐L. , Repman M. A.
Published: October 2006

Abstract: Density‐dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)‐susceptible sialyl residues, and membrane‐ associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN‐releasable sialic acid was observed with increasing cell density. GM3 was the major ganglioside component of preconfluent S20Y cells, whereas GDIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN‐susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.
Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro
Authors: Shitara Nobuyuki , McKeever Paul E. , Smith Barry H. , Pleasants Rogers E. , Banks Maurice A. , Kornblith Paul L.
Published: October 2006

Abstract: With [3H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one‐third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.
An Amphiphile‐Dependent Form of Human Brain Caudate Nucleus Acetylcholinesterase: Purification and Properties
Authors: Sørensen K. , Gentinetta R. , Brodbeck U.
Published: October 2006

Abstract: Different forms of acetylcholinesterase (AChE), EC 3.1.1.7, were demonstrated in human brain caudate nucleus. One form was solubilized at high ionic strength, the other with Triton X‐100. The detergent‐extractable form was purified to homogeneity by affinity chromatography. This form of AChE is amphiphile‐dependent; i.e., it was active only in the presence of amphiphiles (detergents or lipids). Further, the enzyme was shown to bind detergents and to interact hydrophobically with Phenyl‐Sepharose. In the presence of detergents the enzyme is a tetramer (subunit molecular weight, 78,000) which aggregates on the removal of detergents. Human brain AChE showed a reaction of identity with human erythrocyte AChE in crossed‐line immunoelectrophoresis. The high‐salt‐soluble brain enzyme did not cross‐react with the erythrocyte enzyme. The two classes of AChE seem not to be related, as they show no common antigenic determinant.
Inhibition of [3H]Diazepam and [3H]3‐Carboethoxy‐β‐Carboline Binding by Irazepine: Evidence for Multiple “Domains” of the Benzodiazepine Receptor
Authors: Skolnick Phil , Schweri Margaret , Kutter Eberhard , Williams Evan , Paul Steven
Published: October 2006

Abstract: The binding of [3H]diazepam and [3H]3‐carboethoxy‐β‐carboline was examined in rat brain synaptosomal membranes treated with irazepine, an alkylating benzodiazepine. Under incubation conditions that resulted in a 25–33% reduction in the Bmax of [3H]diazepam binding, only modest (<8.5%) reductions in the Bmax of [3H]3‐carboethoxy‐β‐carboline were observed. The differential effects of irazepine on the binding of these two compounds may be explained by the presence of multiple areas or “domains” on the benzodiazepine receptor.
Analysis by Transfer Electrophoresis of Reactivity of IgG with Brain Proteins in Multiple Sclerosis
Authors: Newcombe J. , Glynn P. , Cuzner M. L.
Published: October 2006

Abstract: Central nervous system proteins, immobilized on cellulose nitrate sheets, can be identified by incubation with specific antisera. Using this technique, no disease‐related antibodies against CNS proteins were detected in the cerebrospinal fluid, brain extracts, or sera from cases of multiple sclerosis, although a positive response was seen in sera from animals with experimental autoimmune encephalomyelitis.
Isolation, Characterization, and Synthesis of AlphaFetoprotein from Neonatal Rat Brain
Authors: Mujoo Kalpana , Ali Masarrat , Sahib Maharaj K.
Published: October 2006

Abstract: Monospecific anti‐rat serum alpha‐fetoprotein (AFP) IgG was coupled to cyanogen bromide‐activated Sepharose‐4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto‐neonatal rat brain. The purified AFP was immunologically and electrophoreti‐cally similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto‐neonatal rat serum. The AFP was observed to bind estradiol with Ka= 5.8 × 108M−1 and 1.3 × 108M−1 by dextran‐coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto‐neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.
Myelin‐Associated Glycoprotein: Electron Microscopic Immunocytochemical Localization in Compact Developing and Adult Central Nervous System Myelin
Authors: Webster Henry de F. , Palkovits Christina G. , Stoner Gerald L. , Favilla Jacqueline T. , Frail Donald E. , Braun Peter E.
Published: October 2006

Abstract: Light microscopic immunocytochemical studies have shown that myelin‐associated glycoprotein (MAG) is localized in myelin of the developing CNS; but in the adult, MAG appears to be restricted to periaxonal regions of myelinated fibers. To extend these observations, we embedded optic nerves of 15‐day‐old rats, adult rats, and an adult human in epon after aldehyde and osmium tetroxide fixation. After 5% H2O2 pretreatment, thin sections were immunostained with 1:250–1:5,000 rabbit antiserum to rat CNS MAG according to the avidin‐biotin‐peroxidase complex (ABC) method. Dense deposits of reaction product covered compact myelin in both developing and adult optic nerves. When we used 1:500, 1:1,000, and 1:2,000 anti‐MAG, less intense immunostaining of myelin was found. We also obtained the same localization in compact myelin with the peroxidaseantiperoxidase (PAP) method. With 1:250 anti‐MAG, dense deposits of reaction product were not observed on axolemmal membranes or on oligodendroglial membranes located periaxonally and paranodally. In thin sections of adult human optic nerve, anti‐MAG also stained compact myelin intensely. When thin sections of rat and human optic nerves were treated with preimmune or absorbed serum, no immunostaining was observed. Immunoblot tests showed that our MAG antisera did not react with any non‐MAG myelin proteins. In contrast with earlier light microscopic data, this study shows that MAG localization does not change during CNS development; both developing and adult compact myelin sheaths contain MAG. As many biochemical studies also show that MAG is present in compact myelin, we suggest that this 100,000 dalton glycoprotein now be called myelin glycoprotein (MGP) instead of MAG.
Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain
Authors: Margolis Renée K. , Salton Stephen R. J. , Margolis Richard U.
Published: October 2006

Abstract: We have examined the oligosaccharide structure of a major Mr= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross‐reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3‐ to 5‐fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF‐inducible large external, or NILE, glycoprotein. Tri‐ and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N‐acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high‐mannose oligosaccharides. Sequential lectin‐agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris, together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri‐ and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α‐linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross‐reactive species present in brain (Mr= 205,000) is apparently due to the greater size of the PC12 cell tri‐ and tetraantennary complex oligosaccharides.
Endogenous Substrates for Protein Carboxyl Methyltransferase in Cytosolic Fractions of Bovine Brain
Authors: Aswad Dana W. , Deight Elizabeth A.
Published: October 2006

Abstract: A method of polyacrylamide gel electrophoresis utilizing the discontinuous pH‐stacking gel format, the cationic detergent cetylpyridinium chloride, and an acidic buffer system has been applied to detection of specific substrates for protein carboxyl methyltransferase (PCM, EC 2.1.1.24) in cytosol fractions of bovine cerebral cortex. This electrophoresis system produces a high‐resolution separation of proteins while preventing spontaneous hydrolysis of protein carboxyl methyl esters. Separation occurs largely on the basis of molecular weight. By running polyacrylamide gels at 4°C or 25°C, it was possible to demonstrate that any specific methyl‐accepting protein is modified to form a labile methyl ester rather than the more stable N‐derivative. Using this system, we have found that partially purified fractions of PCM contain a variety of endogenous methyl‐accepting proteins. The apparent specificity of these substrates varies widely; some apparently abundant proteins show little or no methylation, while other apparently less abundant proteins exhibit a relatively high degree of methylation. One protein, with an apparent Mr of 46,000, exhibited an exceptional degree of methylation. Two distinct classes of protein carboxyl methyl esters could be distinguished by their differing susceptibility to nonenzymatic hydrolysis. The possible relevance of our findings to the recent suggestion that PCM specifically methylates abnormal d‐aspartyl residues in age‐racemized proteins is considered.
Myelin Membrane from Adrenoleukodystrophy Brain White Matter—Biochemical Properties
Authors: Brown Frank R. , Chen Winston W. , Kirschner Daniel A. , Frayer Karen L. , Powers James M. , Moser Ann B. , Moser Hugo W.
Published: October 2006

Abstract: Adrenoleukodystrophy (ALD) is an X‐linked progressive neurological disorder characterized by the accumulation of saturated very‐long‐chain fatty acids (C24 to C30) in lipids, especially cholesterol esters of tne brain white matter and adrenal cortex. In the present study we have investigated the localization of accumulated cholesterol esters in brain white matter. During isolation of purified myelin membrane from regions of active demyelination, significant enrichment in cholesterol ester was found in two fractions, mainly in a low‐density floating fraction and to a lesser degree in the purified myelin preparation. The fatty acid composition of cholesterol esters from both the ALD floating and myelin fractions was enriched approximately 10‐fold in saturated very‐long‐chain fatty acids (≥C24) compared with control preparations.
Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue
Authors: Monroe Philip J. , Smith David J.
Published: October 2006

Abstract: High‐affinity [3H]5‐hydroxytryptamine ([3H]5‐HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [3H]5‐HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [3H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high‐affinity binding appears to be to multiple sites, since displacement studies using 2 nM [3H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (nH): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [3H]5‐HT, since unlabeled 5‐HT did not discriminate between them. Thus, the high‐affinity [3H]5‐HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5‐HTIA, 5‐HTIB). In addition to the multiple high‐affinity spinal cord binding sites, a low‐affinity [3H]5‐HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [3H]5‐HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high‐and low‐affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [3H]5‐HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [3H]5‐HT in the spinal cord is associated with the 5‐HT uptake carrier, since fluoxetine, an inhibitor of 5‐HT uptake, does not alter binding characteristics. In addition, a 5‐HT binding site analogous to the site designated 5‐HT, was not apparent in the spinal cord. Ketanse‐rin and cyproheptadine, drugs that are highly selective for 5‐HT, sites, did not displace [3H]5‐HT from spinal tissue, and [3H]spiperone, a radioligand that binds with high affinity to 5‐HT2 sites, did not exhibit saturable binding in the tissue. Thus, the 5‐HT2 binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.
Properties of [3H]Prazosin‐Labeled α1Adrenergic Receptors in Rat Brain and Porcine Neurointermediate Lobe Tissue
Authors: Battaglia George , Shannon Michael , Borgundvaag Bjug , Titeler Milt
Published: October 2006

Abstract: [3H]Prazosin binding to α1 receptors in homogenates of rat prefrontal cortical tissue and porcine pituitary neurointermediate lobe tissue was investigated. Competition curves produced by coincubating adrenergic agonists and antagonists with 0.5 nM [3H]prazosin and tissue revealed some anomalous binding properties. In the brain and pituitary tissue, agonist competition curves produced “shallow” slopes, with Hill coefficients significantly lower than unity. The IC50 of the agonists epinephrine, norepinephrine, and clonidine for inhibition of 0.5 nM [3H]prazosin binding were significantly lower in the porcine pituitary than in the rat brain. Most antagonists, such as prazosin, chlorpromazine, and piperoxan, produced “steep” competition curves with Hill coefficients close to unity, with two notable exceptions. WB‐4101 and phentolamine produced competition curves with Hill coefficients significantly less than unity in the rat brain preparation. Ketanserin, an antagonist, displayed a sevenfold higher affinity for the a, sites in the pituitary tissue than in the brain tissue. These anomalies in the binding results may indicate the presence of an endogenous modulatory factor affecting agonist and antagonist affinities for the a, receptor.
Monoclonal Antibodies Against Vasoactive Intestinal Polypeptide: Studies of Structure and Related Antigens
Authors: Gozes Illana , Milner Robert J. , Liu Fu Tong , Johnson Eric , Battenberg Elena L. F. , Katz David H. , Bloom Floyd E.
Published: October 2006

Abstract: Hybridomas secreting monoclonal anti‐vaso‐active intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro. The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme‐linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti‐mouse imunnoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.
Structure of Somatostatin Isolated from Bovine Retina
Authors: Marshak David W. , Reeve Joseph R. , Shively John E. , Hawke David , Takami Mimi S. , Yamada Tadataka
Published: October 2006

Abstract: Somatostatin‐like immunoreactivity (SLI) from bovine retina was purified and its structure determined. Retinal tissue (1868 g) extracted with 3% acetic acid yielded 18.6 nmol SLI. This peptide was purified by chromatography on an affinity column made with anti‐somatostatin antiserum, a reverse‐phase C‐18 HPLC column, and three sequential applications on a reverse‐phase phenyl HPLC column. The peptide was purified 103,000‐fold from the initial extract with an overall yield of 14.4%. Amino acid sequence determination by an automatic Edman degradation technique revealed the sequence to be as follows: Ser ‐ Ala ‐ Asn ‐ Ser ‐ Asn ‐ Pro ‐ Ala ‐ Met ‐ Ala ‐ Pro ‐ Arg ‐ Glu ‐ Arg ‐ Lys ‐ Ala ‐ Gly ‐ (Cys) ‐ Lys ‐ Asn ‐ Phe ‐ Phe ‐ Trp ‐ Lys ‐ Thr ‐ (Phe, Thr, Ser, Cys). The apparent identity of this peptide with somatostatin octacosapeptide (S28) purified from other mammalian tissue indicates the phylogenetic conservation of its structure and facilitates the use of the retina as a model system for studying the neurotransmitter function of somatostatin.
Age‐Related Changes in Aldehyde Dehydrogenase Activity of Rat Brain, Liver, and Heart
Authors: Danh Hung Cao , Benedetti Margherita Strolin , Dostert Philippe
Published: October 2006

Abstract: Aldehyde dehydrogenase (ALDH) activity was measured in brains, livers, and hearts of 23–26‐month‐old and 3‐month‐old rats. A significant increase of ALDH activity was found in whole brain of old rats with both acetaldehyde (39%) and propionylaldehyde (15%) used as substrates. In different brain areas of old rats, with acetaldehyde used as substrate, a significant increase of ALDH activity was found in striatum (30–50%) and cerebral cortex (37%). However, no significant difference in ALDH activity was found in livers and hearts of young and old rats. Preliminary experiments showed a significant increase of aldehyde reductase activity (52%) with p‐nitrobenzaldehyde used as substrate in whole brain of old rats compared with young rats. The present work indicates that an increase of ALDH activity in brain of old rats may be an adaptive phenomenon.
Studies on the Osmotic Disruption and Resealing of Synaptosomes
Authors: Adam‐Vizi V. , Marchbanks R. M.
Published: October 2006

Abstract: The release of lactate dehydrogenase and K+ when synaptosomes are exposed to resuspension in media of various osmolarity has been investigated in order to measure their disruption. Even when resuspended in distilled water a significant percentage (10–20%) of lactate dehydrogenase and K+ remains unreleased. The particles containing these substances sediment to the same density as synaptosomes. Synaptosomes retaining their internal organlles after hypoosmotic treatment can be seen in electron micrographs. Resealing of disrupted synaptosomes was measured by the inclusion of [14C]sucrose. The resealing is spontaneous, essentially complete (80–90%) within 20 min and not noticeably affected by temperature, pH, or the addition of fusogen. The synaptosome preparation after hypoosmotic disruption will therefore contain some undisrupted synaptosomes with some or all of their complement of cytoplasmic constituents, as well as resealed synaptosomes. The retention of the ability of the hypoosmotically treated preparation to convert [14C]choline to [14C]acetylcholine is demonstrated as an example of the disproportionate effect these undisrupted particles have on its properties.