bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of Brain Enolase Isozymes with an Enzyme Immunoassay at the Level of Single Neurons
Authors: Kato Kanefusa , Suzuki Fujiko , Semba Reiji
Published: October 2006

Abstract Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (αα, αγ, and γγ forms) were prepared by use of β‐d‐galactosidase from Escherichia coli as label and the purified rabbit antibodies to αα and γγ enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen‐coupled Sepharose. Sandwich‐type immunoassay systems with the galactosidase‐labeled antibody Fab’fragments and the antibody F(abapos;)2‐immobilized polystyrene beads could determine amounts as small as 1 amol (10−18 mol) of each isozyme. Purkinje cell bodies picked up from the bulk‐separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the γγ but also the αγ and αα enolases at a level of amol per cell body, although the concentration of γγ was the highest. Immunohistochemical experiments on the cerebellum with the peroxidase‐labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both α and γ subunits of the enolase were stained intensely in axons.
Comparative Effects of Chronic Haloperidol and Sulpiride Treatment on Nigral and Striatal GABA Content
Authors: Nicoletti Ferdinando , Patti Francesco , Condorelil Daniele Filippo , Rampello Liborio , Giammona Giovanni , Giorgio Rosa‐Maria Di , Canonico Pier Luigi , Scapagnini Umberto
Published: October 2006

Nigral and striatal GABA contents were assayed in male rats treated chronically with haloperidol or sulpiride, two dopamine‐receptor blocking agents that have different neuropharmacological spectra in regard to their biochemical, behavioural, and clinical properties. No great difference was observed between the chronic effects of haloperidol and sulpiride on nigral and striatal GABA content. However, low doses (30 μg/kg, intraperitoneally) of the dopamine‐receptor agonist apomorphine, injected 12 h after the discontinuation of chronic haloperidol or chronic sulpiride treatment, induced opposite changes in nigral GABA levels suggesting the existence of a different “status” of the dopamine receptors during the 12 h‐period following the withdrawal of haloperidol or sulpiride.
Intrauterine Growth Retardation (Malnutrition by Vascular Ligation) Induces Modifications in Fatty Acid Composition of Neurons and Oligodendrocytes
Authors: Morand O. , Chanez C. , Masson M. , Dumont O. , Flexor M. A. , Baumann N. , Bourre J. M.
Published: October 2006

Intrauterine growth retardation (IUGR) induced by ligation of one uterine artery on day 17 of pregnancy in the rat lead to major abnormalities in the fatty acid content of neurons and oligodendrocytes but not in astrocytes. In neurons from IUGR rats, monounsaturated fatty acids were decreased; in the polyunsaturated series, ω‐3 fatty acids were increased and Ω‐6 fatty acids were decreased. In oligodendrocytes, monounsaturated fatty acids were also decreased, but the modifications in polyunsaturated fatty acids were the opposite of those in neurons: Ω‐3 being decreased and w‐6 increased. Although the animals received a normal diet after birth, the alterations were still present in adulthood. In addition, fatty acid composition of brain cells is a very indicative criterion of brain maturation.
Influence of Prenatal Hypoxia on Brain Development: Effects on Body Weight, Brain Weight, DNA, Protein, Acetylcholinesterase, 3‐Quinuclidinyl Benzilate Binding, and In Vivo Incorporation of [14C]Lysine into Subcellular Fractions
Authors: Gross Johann , Burgoyne Robert D. , Rose Steven P. R.
Published: October 2006

Abstract: The influence of prenatal hypoxia on subsequent brain development in the young rat was investigated by examining body and brain weight, cerebral cortex wet weight, protein and DNA concentrations, acetylcholinesterase (AChE) activity, 3‐quinuclidinyl benzilate (QNB)‐binding levels, the relative amounts of protein in various subcellular fractions, and the in vivo incorporation of [14C]lysine into the protein of homogenate and subcellular fractions. Exposure of pregnant females to a mild hypoxia (9.1% Os, 10 h per day for the 9‐11 days preceding birth) resulted in a reduced body weight in the pups at days 1 and 5 after birth; total cortical DNA was reduced but brain weight and protein content were unaffected, leading to a higher protein/DNA ratio in prenatally hypoxic pups. By 10 days of age these differences between prenatally hypoxic and control animals were no longer apparent. There were no differences between prenatally hypoxic and control animals in AChE and QNB binding per milligram cortex protein. The relative amount of synaptic membrane protein from the cerebral cortex was reduced at day 1 in prenatally hypoxic animals and the synaptic membrane fraction showed a higher level of incorporation of [14C]lysine on days 1, 5, and 10. The developmental profile of [14C]lysine incorporation showed a peak on day 10 which was higher in prenatally hypoxic rats. By 46 days after birth little difference could be found between prenatally hypoxic and control animals.
On the Role of Long‐Chain Aldehydes in Mammalian Plasmalogen Biosynthesis
Authors: Reddy Padala V. , Schmid Harald H. O.
Published: October 2006

Abstract: [1‐3H, 1‐14C]Palmitaldehyde(3H:14C= 15) was injected intracerebrally to 18‐day‐old rats and incorporation of radioactivity into brain lipids was followed over a 24‐h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower 3H:14C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium‐free alcohols derived from 14C‐labeled fatty acids. Virtually no 3H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.
Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons
Authors: Wenthold R. J. , McGarvey M. L.
Published: October 2006

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two‐dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two‐dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [3H]fucose as a precursor or by separating [35S]methionine‐labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.
Neuroblastoma × Glioma Hybrid Cells Synthesize Enkephalin‐Like Opioid Peptides
Authors: Glaser Thomas , Hübner Karin , Hamprecht Bernd
Published: October 2006

Abstract: Partially purified extracts from neuroblastoma × glioma hybrid cells 108CC15 inhibit, like opioids, the prostaglandin E1‐evoked formation of cyclic AMP in a dose‐dependent manner in the same hybrid cells. The inhibition is prevented by the opioid antagonist naloxone. In addition, the same extract competes with [3H]naloxone and [3H]Leu‐enkephalin for binding to opioid receptors of hybrid cell membranes and to a specific antiserum, respectively. The opioid activity in the extracts is destroyed by carboxypeptidase A and leucine aminopeptidase, but not by trypsin. Further purification of the extracts by HPLC, TLC, or high‐voltage paper electrophoresis reveals in each case two active fractions which behave like Met‐ and Leu‐enkephalin. The Metenkephalinlike, but not the Leuenkephalin‐like, fraction is inactivated by treatment with BrCN. Dimethylaminonaphtylsulfonyl (dansyl) derivatives of Met‐ and Leu‐enkephalin correspond to [3H]dansyl derivatives of Met‐ and Leu‐enkephalin‐like substances from hybrid cells. Three to four times as much Met‐ertkephalin‐like as Leu‐enkephalin‐like material is present in the extract. The overall concentration of opioid peptides in the hybrid cells varies between 0.03 and 1.0 pmol Leu‐enkephalin equivalents per mg protein. The amount of opioids in the hybrid cells is strongly dependent on the cell density. The findings suggest that neuroblastoma × glioma hybrid cells contain opioid peptides that are very similar, if not identical, to Met‐ and Leu‐enkephalin. Opioid activity can also be detected in other neuronal cell lines and even in glioma cells.
Effects of N6‐Methyladenosine on the Synthesis of Phenylethanolamine N‐Methyltransferase in Cultured Explants of Rat Adrenal Medulla
Authors: Burke William J. , Joh Tong H.
Published: October 2006

Abstract: Explants of adrenal medullae were cultured in defined media for up to 48 h, during which time the tissue remained histologically intact. Addition of N6‐methyladenosine to the medium led to a diminution in the activity of phenylethanolamine N‐methyltransferase (EC 2.1.1.28) in the tissue. The enzyme activity was inversely proportional to the concentration N6‐methyl‐adenosine in the culture medium. The extent of loss of phenylethanolamine N‐methyltransferase, as measured by immunochemical titration, corresponded to the degree of loss in enzyme activity under the same conditions. Furthermore, the decreased amount of enzyme protein was due to a decrease in the rate of synthesis of phenylethanolamine N‐methyltransferase. Neither adenosine nor several methylated nucleosides, including 7‐methylguanosine, N2‐methylguanosine, and 5‐methylcytosine, had an effect on the enzyme. Two other adrenal medullary enzymes, monoamine oxidase (EC 1.4.3.4) and acid phosphatase (EC 3.1.3.2), were not affected by addition of N6‐methyladenosine to the medium. The results are consistent with the view that this effect of N‐methyladenosine on the concentration of phenylethanolamine N‐methyltransferase is due to an inhibition of its biosynthesis rather than to an alteration of its rate of degradation.
Met‐Enkephalin [Arg6,Phe7] Immunoreactivity in Bovine Caudate and Bovine Adrenal Medulla
Authors: Boarder Michael R. , Lockfeld Alexandre J. , Barchas Jack D.
Published: October 2006

Abstract: A radioimmunoassay specific for the COOH‐terminus of Met‐enkephalin [Arg6,Phe7] and a separate assay specific for the COOH‐terminus of Met‐enkephalin are described. Immunoreactivity by these two assays was compared in bovine caudate and bovine chromaffin granule preparation after Sephadex G75 chromatography in 50% acetic acid. When the assays were applied to the chromatography fractions of the bovine caudate extract, the majority of the immunoreactivity was found in the fractions corresponding to the heptapeptide and the pentapeptide respectively. When the chromaffin granule chromatography fractions were assayed, both of the radioimmunoassays showed that most reactivity was in several peaks in the larger molecular weight fractions. The major peak for the Met‐enkephalin [Arg6,Phe7] assay had an apparent molecular weight of 2800, while with the Met‐enkephalin assay the dominant peak of immunoreactivity had an apparent molecular weight of 10,000. The presence of authentic Met‐enkephalin [Arg6,Phe7] in both caudate and chromaffin granule extracts was confirmed by reverse‐phase chromatography of the previously sized fractions. It appears then that the processing of precursors of opioid peptides is directed, in the caudate, to the synthesis and storage of the enkephalins and of Met‐enkephalin [Arg6,Phe7]; in the adrenal medulla the major products of precursor processing are a variety of polypeptides of larger sizes.
Galactosyltransferase Defects in Reeler Mouse Brains
Authors: Shur Barry D.
Published: October 2006

Abstract: Galactosyltransferase activities were examined in the cerebellum, cerebral cortex, and brain stem of reeler and wild‐type mice. Galactosyl‐transferase assays were optimal for all required substrates, linear with incubation time, and proportional to protein concentration. In brain areas affected by the reeler mutation (i.e., cerebral cortex and cerebellum), galactosylation of both endogenous and exogenous glycoprotein acceptors was greatly reduced in reeler relative to controls. On the other hand, glycosylation of endogenous glycolipids was low, and equal between reeler and wild‐type. Galactosyl‐transferase activities were similar, though not identical, in reeler and wild‐type brain stems, which are phenotypically normal in reeler mice. Glucosyl‐transferase, β‐galactosidase, β‐N‐acetylglucosaminidase, acid phosphatase, and lactate dehydrogenase specific activities were all unaffected in reeler cerebella, while galactosyltransferase activity was 52% of control. Inhibition of either UDPgalactose hydrolysis or β‐galactosidase had no effect on galactosyltransferase activity. The spectrum of galactosyltransferase deficiencies in reeler suggests that this enzyme is associated with the development of young granule cells.
Acyl‐CoA: sn‐Glycerol‐3‐Phosphate O‐Acyltransferase in Rat Brain Mitochondria and Microsomes
Authors: Fitzpatrick Susan M. , Sorresso Giovanna , Haldar Dipak
Published: October 2006

Abstract: The subcellular distribution of acyl‐CoA: sn‐glycerol‐3‐phosphate O‐acyltransferase between brain mitochondria and microsomes was investigated. The activities associated with purified rat brain mitochondrial and microsomal preparations could be distinguished by differences in their acyl‐CoA specificity, products of acylation, and sensitivity to N‐ethylmaleimide, trypsin, acetone, and polymyxin B. It was concluded that both brain mitochondria and microsomes possess the acyltransferase.
Buprenorphine: High‐Affinity Binding to Dorsal Spinal Cord
Authors: Villiger John W. , Taylor Kenneth M.
Published: October 2006

Abstract: The binding of the mixed opiate agonist‐antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H‐ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H‐ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high‐affinity (KD= 0.12 nM) binding site in addition to the high‐affinity site (KD= 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and d‐Ala2, Met5‐enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.
Monoamine Oxidases A and B as Components of a Membrane Complex
Authors: White Helen L. , Stine Deborah K.
Published: October 2006

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane‐bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high‐speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A2, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.
Highly Activatable Adenylate Cyclase in [2‐3H]Adenine‐Prelabeled Vesicles Prepared from Guinea Pig Cerebral Cortex by a Simplified Procedure
Authors: Psychoyos Stacy , Dove Jane , Strowbridge Ben , Nusynowitz Irving
Published: October 2006

Abstract: An improved procedure utilizing a simple fractionation technique is described for the preparation and use of [2‐3H]adenine‐prelabeled vesicles from guinea pig cerebral cortex containing highly responsive adenylate cyclase activity. Adenosine consistently increased activity 1500‐2000%, contrasted with activations of 200‐300% previously reported by other investigators. Adenosine at 5 p M was more active in our system than at 20 times this concentration in studies by other investigators, increasing activity by 580‐840%. Experimental conditions were explored, and Ca2+ was found to be necessary during tissue homogenization, but not during subsequent vesicle incubation. However, neither the higher Ca2+ concentration used by us (2.5 mM) nor the method of tissue homogenization could adequately explain the high activity of our preparations. The size of the incubation vessel was critical for both low basal activity and high activity in the presence of adenosine. Our preparations were similar to others in that combinations of neurohormones, which included histamine and epinephrine, elicited higher activations than individual neurohormones. Inspection of our vesicle preparations by scanning electron microscopy also showed them to be compatible with previously described preparations.
Adenylate Cyclases in Vertebrate Retina: Enzymatic Characteristics in Normal and Dystrophic Mouse Retina
Authors: Ferrendelli J. A. , Campau K. M. , De Vries G. W.
Published: October 2006

Abstract: Adenylate cyclase activity and the effects of various activators and inhibitors of this enzyme were measured in retinas from normal mice (C57BL/6J) and congenic animals with photoreceptor dystrophy. In normal retina, approximately 250 μM‐ATP was required for half‐maximal stimulation of the enzyme. Activity was supported by Mg2+ and Mn2+, but Ca2+ was ineffective. The enzyme was inhibited by EGTA and stimulated by 5′‐guanylylimidodiphosphate (GPP(NH)P), dopamine, and NaF. The stimulatory effects of GPP(NH)P and dopamine were greater in the presence of EGTA. Examination of microdissected normal retinas revealed that the inner (neural) retina had adenylate cyclase activity four times that of the photoreceptor cell layers, and that EGTA inhibited activity in the inner retina, but had no effect in the outer retina. In dystrophic retinas basal enzyme activity was 60% higher than that in normal retina. The enzyme in this tissue was stimulated by EGTA, GPP(NH)P, and dopamine, and their effects were additive. These results indicate that adenylate cyclase activity in vertebrate retina is under complex regulation by substrate, divalent cations, guanine nucleotides, dopamine, and perhaps calmodulin. In addition, the data demonstrate that adenylate cyclase is not evenly distributed in the retina and that it is regulated differently in the inner and outer retina. Finally, the present results indicate that regulation of this enzyme in dystrophic retina may be qualitatively and quantitatively different from that in normal retina.
Myelin Basic Protein Deposition in the Optic and Sciatic Nerves of Dysmyelinating Mutants Quaking, Jimpy, Trembler, MLD, and Shiverer During Development
Authors: Jacque C. , Delassalle A. , Raoul M. , Baumann N.
Published: October 2006

Abstract: An ontogenetic survey of the basic protein of myelin, common to both central and peripheral nervous systems, was carried out on normal C57B1 and five dysmyelinating mutant mice. Myelin basic protein (MBP) was quantified by radioimmunoassay in the optic and sciatic nerves of mice from birth to adult stages, giving special attention to the premyelinating and early myeli‐nation periods. In the optic nerves of normal mice, MBP was already detectable at birth but the active period of myelin deposition was shown to occur after day 10 postnatal. The timing and rate of accumulation of MBP were normal in Trembler. In contrast, they were abnormal in the other mutants. In the quaking mouse, the active period of MBP deposition was delayed, and its final concentration represented no more than 12% of normal in the adult. No active period of MBP deposition was observed in the other mutants. In the jimpy mouse, a slow accumulation of MBP resulted in a final concentration reaching 2% of the normal value at 25 days. In mid and shiverer mice, the MBP was hardly detectable. In the sciatic nerves of normal mice, the active period of MBP deposition occurred between days 3 and 12 postnatal. No substantial changes occurred in the period of 2 months‐2 years. Deposition of MBP was normal in jimpy. In quaking, it was not only delayed but also reduced, leading to a concentration as low as 40% of normal adult. In mid and shiverer PNS, MBP concentrations were very low but noticeably higher than in the optic nerve. In the sciatic nerve of Trembler a myelination‐demyelination process was strongly suggested by MBP determination. After a delayed but active period of deposition from days 5–11 postnatal, the MBP content was found to decline within a few days and remained almost undetectable until old age.
Ascorbate‐Induced Lipid Peroxidation and Inhibition of [3H]Spiroperidol Binding in Neostriatal Membrane Preparations
Authors: Heikkila Richard E. , Cabbat Felicitas S.
Published: October 2006

Abstract: Sodium ascorbate caused an increased lipid peroxidation and a large decrement in [3H]spiroperidol binding in a rat neostriatal membrane preparation (preparation C). Both effects were greater at intermediate (0.05 and 0.5 mM) than at higher or lower ascorbate concentrations. In contrast, in another neostriatal membrane preparation (preparation A), there was no loss of [3H]spiroperidol binding and only a small increase in lipid peroxidation caused by ascorbate. However, both the ascorbate‐induced increase in lipid peroxidation and loss of [3H]spiroperidol binding were greatly enhanced in preparation A by the addition of iron salts. In experiments designed to explore reasons for these apparent discrepancies, we discovered that the method of tissue preparation was a critical factor. The ascorbate effects were consistently greater in a tissue preparation which was originally homogenized in an isotonic sucrose medium and centrifuged, and the cell debris discarded (as was done in preparation C), than in one in which the tissue was homogenized in a hypotonic medium and in which no low‐speed centrifugation was done (as was done in preparation A). In other experiments, of several cations tested, only ferrous and ferric potentiated the above‐described effects of ascorbate. Some ascorbic acid derivatives (e.g., isoascorbic acid) had properties similar to those of ascorbic acid, whereas several reducing agents could, in the presence of added iron salts, cause both a lipid peroxidation and a loss of [3H]spiroperidoI binding. However, the other reducing agents tested (e.g., dithiothreitol) were iess potent in both regards than was ascorbate. In other experiments, several chelating agents (e.g., EDTA) or commonly used inhibitors of lipid peroxidation (e.g., propyl gallate) were able to counteract the effects of ascorbate on lipid peroxidation and on [3H]spiroperidol binding.
Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine
Authors: Racké Kurt , Muscholl Erich
Published: October 2006

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [3H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [3H]Dopamine represented 70% and [3H]noradrenaline 30% of the [3H]catecholamines. Desipramine (1 μM) prevented the formation of [3H]noradrenaline without affecting the storage of [3H]dopamine. Nomifensine (10 μM) blocked the storage of [3H] dopamine and [3H]noradrenaline. Thus, in the NIL, [3H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [3H] dopamine is converted to [3H]noradrenaline, indicating a significant dopamine β‐hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [3H]dopamine can be achieved in the presence of desipramine.
Studies on [3H]Diazepam and [3H]Ethyl‐β‐Carboline Carboxylate Binding to Rat Brain In Vivo. I. Regional Variations in Displacement
Authors: Minchin M. C. W. , Nutt D. J.
Published: October 2006

Abstract: The binding of [3H]diazepam and [3H]ethyl‐β‐carboline carboxylate (β‐CCE) to rat brain membranes has been studied following injection of the ligand via a tail vein. “Ex vivo” binding was avoided by homogenising the tissue in an excess of unlabelled ligand. The dissociation rate constant for [3H]diazepam and [3H]β‐CCE was approximately 0.46 min−1 at 0°C. Displacement of [3H]diazepam by β‐CCE in vivo showed regional variation: the dose of β‐CCE required to inhibit 50% of [3H]diazepam binding in the cerebellum was one quarter of that required in the cortex, hippocampus, or striatum. However, when diazepam was used to displace [3H]β‐CCE in vivo the converse occurred: the dose needed for 50% inhibition in the cerebellum was more than four times that required in the other three regions. These findings support suggestions from in vitro experiments that two receptors exist with different affinities for benzodiazepines and β‐carbolines. The benzodiazepine receptor antagonist Ro 15–1788 did not differentiate between the two receptor subtypes.
Effect of Electroconvulsive Shock on Muscarinic Cholinergic Receptors in Rat Cerebral Cortex and Hippocampus
Authors: Lerer Bernard , Stanley Michael , Demetriou Sàndra , Gershon Samuel
Published: October 2006

Abstract: Single electroconvulsive shock (ECS) induced no change in [3H]quinuclidinyl benzilate ([3H]QNB) binding to muscarinic cholinergic receptors in rat cortex and hippocampus. ECS administered once daily for 7 days induced a significant reduction in [3H]QNB binding in both brain areas. Concurrent ECS reversed the significant increase in cortical [3H]QNB binding induced by chronic atropine administration. These findings may have relevance to the antidepressant or amnestic effects of electroconvulsive therapy.
Detergent Effects on the Phosphatidylinositol‐Specific Phospholipase C in Rat Brain Synaptosomes
Authors: Manning Robert , Sun Grace Y.
Published: October 2006

Abstract: In the presence of Ca2 + (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol‐specific phospholipase C (PI‐PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2 + (3.5 mM) system, synaptosomal PI‐PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2 + system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent‐induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2 + and the taurocholate + Ca2 ++ EDTA systems was strongly inhibited by divalent metal ions such as Zn2 +, Cu2 +, Pb2 +, and Fe2+, whereas Mg2 + and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2 + system was responsive to enzyme inhibition by membrane‐perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.
Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice
Authors: Weise Michael J. , Greenfield Seymour , Brostoff Steven W. , Hogan Edward L.
Published: October 2006

Abstract: Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age‐related changes in the relative amounts of large (P1) and small (Pr) basic proteins were found. In control animals, the ratio Pr/P1 increased with age, a change similar to that observed for the large (B1) and small (Bs) CNS myelin basic proteins of adult mice. Pr/P1 also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.
Characterization of Axonally Transported Glycoproteins in Regenerating Garfish Olfactory Nerve
Authors: Cole Gregory J. , Elam John S.
Published: October 2006

Abstract: This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase‐generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration‐related increase in the proportion of [3H]glucosamine label in lower‐molecular‐weight Con A‐binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose‐rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A‐binding glycopeptides are enriched in membrane subfractions, particularly in a high‐density fraction that morphologically resembles isolated cell surface coat. Regeneration‐related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport‐labeled glycoproteins disclosed growth‐correlated increases in radioactivity associated with 180–200K, 105–115K, and 80–90K components, while a 150–160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50–140K range.
Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization
Authors: Grossman Steven H.
Published: October 2006

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13‐fold and 4‐fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the KD for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate‐induced conformational change. Creatine kinase labeled with two molecules of 5′‐iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle‐type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme‐MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 ‐ (4 ‐ maleimidylphenyl) ‐ 7 ‐ diethylamino ‐ 4 ‐ methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.
Characteristic Constituents of Glycolipids from Frog Brain and Sciatic Nerve
Authors: Saito Shintaro , Tamai Yoichi
Published: October 2006

Abstract: Cerebroside, sulfatide, monoglycosyl glyceride, and ester cerebroside were isolated from frog brain and sciatic nerve, and their distribution and chemical constituents were determined. The long‐chain base compositions of cerebroside, sulfatide, and ester cerebroside were unique in the presence of branched‐base components (5–15% of the total bases) and in the abundance of saturated dihydroxy base components (15–45% of the total). The amount of branched long‐chain bases was greater in sciatic nerve than in brain. The hexose composition of the glycolipids consisted entirely of galactose except for brain cerebroside, in which a small amount of glucose was detected. Monogalactosyl glyceride consisted of the diacyl and alkylacyl forms, in a molar ratio of 81:19 for brain and 62:38 for sciatic nerve. The fatty acid com position of glycosphingolipids was characterized by the predominance of hydroxy and nonhydroxy 24:1 acids, and the concentration of 24:0 was extremely low. The proportion of unsaturated fatty acids accounted for 80% of the total. Major fatty acids of monogalactosyl glyceride were palmitic, oleic, stearic, and palmitoleic acids; the highest concentration was that of palmitic acid. Ester cerebroside was separated into three subfractions mainly on the basis of the proportion of hydroxy and nonhydroxy components in the amide‐linked fatty acids.
Polypeptides in Frog and Rat: Evolutionary Changes in Rapidly Transported and Abundant Nerve Proteins
Authors: Perry G. W. , Wilson David L.
Published: October 2006

Abstract: Dorsal root ganglion (DRG) neurons from rat and frog were labeled in vitro with [35S]methionine, and the newly synthesized, rapidly transported proteins were collected at ligatures on the sciatic nerves. The proteins were extracted and separated by two‐dimensional polyacrylamide gel electrophoresis. Exposure of x‐ray film to dried gels allowed comparison of the labeled, rapidly transported proteins from frog and rat. The gel staining patterns of abundant proteins in the sciatic nerves were also compared. Triolets of gels were examined: one gel from frog, one from rat, and one from frog plus rat combined. Among the transported proteins, some (including A2, A17 and/or A18, B6, B14a‐i, C1, C22, and some members of Ala‐i and B3a‐g) co‐migrated on the gels, suggesting that these proteins have been well conserved during evolution. The gel staining patterns of abundant proteins in the sciatic nerves also show some similarities: two forms of actin, serum albumin, and α‐ and β‐tubulin are each in identical positions on the frog and rat gels. Other sciatic nerve and rapidly transported proteins had similar, but not identical, positions on the gels. A number of the rat and frog proteins had no obvious counterpart. We have calculated the magnitude of expected changes in charge and molecular weight of proteins due to accumulation of point mutations during evolution. We conclude that many of the differences between rat and frog protein patterns on the two‐dimensional gels could be the result of such point mutations, but we cannot rule out radical changes in polypeptide sequence or abundance between frog and rat for some of these proteins.
Structural and Metabolic Relationships Between Goldfish Brain Glycoproteins Participating in Functional Plasticity of the Central Nervous System
Authors: Schmidt Rupert , Shashoua Victor E.
Published: October 2006

Abstract: Ependymins β and γ (MW 32,000 and 26,000 daltons) are two secreted goldfish brain glycoproteins that exhibit a specifically enhanced turnover rate when the animals successfully acquire a new pattern of swimming behaviour. Both proteins are bound identically to concanavalin A and can be isolated from brain extracellular fluid and from brain cytoplasm by lectin affinity chromatography. Radioimmunoassay data, using purified 125I‐ labeled ependymins and antisera directed against ependymin β or ependymin γ, show complete cross‐reactivity between the two proteins. It is demonstrated by Scatchardplot analysis that the antisera recognize identical immunological determinants in both proteins. The amino acid composition of the ependymins is similar, and several identical polypeptide fragments are obtained after limited proteolysis with Staphylococcus aureus protease. The proteins are capable of forming complexes of the compositions γ2, βγ, and β2. A protease present in the extracellular fluid of goldfish brain promotes proteolysis of ependymin β to ependymin γ. The finding that ependymin γ is physiologically derived from ependymin β suggests the possibility that ependymin β might exert its biological function during consolidation of new behavioural patterns via smaller polypeptide fragments.
Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+/Calmodulin‐ and cAMP‐Dependent Protein Kinases
Authors: Moskowitz Nathan , Glassman Adam , Ores Christine , Schook William , Puszkin Saul
Published: October 2006

Abstract: Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5‐15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity‐purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin‐dependent protein kinase. Affinity‐purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin‐dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP‐dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.
Studies on Polyphosphoinositides in Developing Rat Brain
Authors: Uma S. , Ramakrishnan C. V.
Published: October 2006

Polyphosphoinositides in rat brain exist in two forms: the metabolically active form that is readily attacked by the polyphosphoinositide phosphohydrolases, and the inert form that is attacked by the enzymes at a slower rate. The two pools continue to increase even during the postweaning period, suggesting a role in glial as well as myelin development apart from their role in neurons.
A General Model of the P2 Protein of Peripheral Nervous System Myelin Based on Secondary Structure Predictions, Tertiary Folding Principles, and Experimental Observations
Authors: Martenson Russell E.
Published: October 2006

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable α‐helix, β‐structure, β‐turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high β‐sheet content and virtual absence of α‐helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the β‐structure over the α‐helical conformation. In order to define those β‐sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted β‐strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential β‐strands in the predicted secondary structure were then ordered into β‐sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a “Greek key”β‐barrel, consisting of eight antiparallel β‐strands with a two‐stranded ribbon of antiparallel β‐structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative β‐strand orderings yield three specific models having different interstrand connections across the barrel ends.
Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain
Authors: Eells Janis Thane , Spector Reynold
Published: October 2006

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20‐40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [14C]cytidine diphosphate (CDP) to [14C]deoxy‐cytidine diphosphate with subsequent hydrolysis and separation of the product ([14C]deoxycytidine) from substrate ([14C]cytidine) by Dowex‐1‐borate ion‐exchange chro‐matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10‐12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10‐12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the Km for CDP. The Km for CDP in brain of newborn and adult rats was 80‐90 μM. In 10‐ to 12‐week‐old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.
Selective Effects of Neonatal Hypothyroidism on Monoamine Oxidase Activities in the Rat Brain
Authors: Vaccari Andrea , Biassoni Roberto , Timiras Paola S.
Published: October 2006

Hypothyroidism of mild intensity was obtained with prenatal and neonatal submission of Long‐Evans rats to an iodide‐rich diet. Chronic daily administration of methimazole to iodide‐supplemented Long‐Evans pups or to iodine‐deprived Charles‐River rats through the first 29–30 days of age provoked severe hypothyroidism. Monoamine oxidase type A (MAO‐A) and not type B (MAO‐B) activity was consistently, although slightly (by approximately 20%), increased in the hypothyroid brain. Triiodothyronine (T3)‐induced hyperthyroidism did not affect MAO activity. Replacement therapy with T3 did not normalize MAO‐A activity in hypothyroidism. Methimazole displayed a competitive and reversible in vitro inhibition of MAO‐A but not MAO‐B activity. Although this effect was obtained at concentrations far higher than those estimated to reach the brain after a single injection of the goiterogen, the occurrence of accumulation processes in the metabolism‐deficient hypothyroid neonate rs cannot be excluded. Thus, MAO‐A activity might be either directly depressed during the goiterogenic treatment, or increased as the result of some kind of rebound effect after interruption of methimazole administration.
Glycosylation as a Criterion for Defining Subpopulations of Fast‐Transported Proteins
Authors: Stone George C. , Hammerschlag Richard
Published: October 2006

The role carbohydrate residues may play in the sorting of newly synthesized fast‐transported proteins during the initiation of fast axonal transport has been examined by identifying individual fast‐transported glycoproteins that contain either or both fucose and galactose. [3H]Fucose or [3H]galactose was incorporated together with [35S]methionine in vitro in bullfrog dorsal root ganglia. Fast‐transported proteins that accumulated proximal to a ligature on the spinal nerve were separated via two‐dimensional gel electrophoresis, and 92 gel spots were analyzed quantitatively for the presence of 35S and 3H. Of these spots, 56 (61%) contained either or both fucose and galactose. Glycomoieties were generally associated with families of charged spots whose isoelectric points could be altered with neuraminidase treatment. Single spots tended to be unglycosylated and were unaffected by neuraminidase. The prevalence of glycoproteins was considerably greater in the higher‐molecular weight range. Of the 55 spots analyzed with molecular weight greater than approximately 35,000 daltons, 89% were glycosylated, whereas only 19% of the 37 spots with lower molecular weight contained sugar moieties. When considered in light of previous studies in which similar Subpopulations have been described, the current findings suggest that the presence or absence of glycomoieties may represent another criterion by which proteins are sorted during the initiation of fast axonal transport.