bluuebunny/crossref_metadata_2025 huggingface dataset:
[3H]5‐Hydroxytryptamine Binding Sites: Species and Tissue Variation
Authors: Schnellmann R. G. , Waters S. J. , Nelson D. L.
Published: October 2006

Abstract: The characteristics of spiperone inhibition of [3H]5‐hydroxytryptamine ([3H]5‐HT; [3H]serotonin) binding were examined in dorsal (DH) and ventral (VH) hippocampus, corpus striatum (CS) or caudate nucleus (CN), and frontal cortex (FC) in the rabbit, guinea pig, and cat. Some of the properties of spiperone inhibition of [3H]5‐HT binding in these species were similar to the properties previously found in the rat. Spiperone was significantly more potent in DH, VH, and FC than in CS or CN. It produced shallow or biphasic inhibition curves, resulting in Hill slopes of less than 1.0. Nonlinear regression analysis of the data showed that the inhibition curves fit a two‐site binding model significantly better than a one‐site model in each brain region. The dissociation constants of spiperone for the high‐affinity binding site (KH) for all the tissues and species, except cat FC and rabbit DH, were very close to those previously found in the rat (2‐13 nM). However, the dissociation constants for the low‐affinity binding site (KL) were different from those in the rat in all species and tissues examined, except cat FC and CS. The present data are consistent with the concept of multiple 5‐HT1 binding sites and suggest the presence of at least two, and perhaps as many as three, groups of sites in the mammalian brain.
Nerve Growth Factor Stimulates Phospholipid Methylation in Target Ganglionic Neurons Independently of the Cyclic AMP and Sodium Pump Responses
Authors: Skaper Stephen D. , Varon Silvio
Published: October 2006

Abstract: Suspensions of neurons prepared from embryonic day 12 (E12) chick sympathetic ganglia were incubated with [methyl‐3H]methionine in the absence of nerve growth factor (NGF). Presentation of the factor for different periods of time resulted in an approximate threefold stimulation of radioactivity incorporated into total phospholipid, followed by a rapid decline thereafter. Both the magnitude and the time of the response were dependent on the NGF concentration used. Also examined were possible relationships of phospholipid methylation to two other short‐latency responses to NGF, i.e., control of the Na+,K+‐pump and elevation of cyclic AMP content. Incubation of E12 sympathetic neurons with known transmethylase inhibitors (shown to be active in the present system) failed to prevent reactivation of the Na+,K+‐pump in response to NGF administration. E16 sympathetic neurons and E15 sensory neurons, which do not depend on exogenous NGF for control of their Na+,K+‐pump, still show a stimulation of phospholipid methylation when challenged with the factor. Blockage of the pump with ouabain also fails to prevent a methylation response. Thus, the pump and methylation responses to NGF occur independently of each other. Intact E8 chick dorsal root ganglia, but not E12 sympathetic ganglia, display a rapid and transient rise in their cyclic AMP content when presented with NGF. At a concentration of 10 biological units/ml, NGF elicits a peak of phospholipid methylation at 4 min, and a peak of cyclic AMP at 10 min. Methylation inhibitors prevent the methylation response, but not that of cyclic AMP. Dibutyryl cyclic AMP, which is able to cause a cyclic AMP elevation similar to NGF, failed to produce any stimulation of phospholipid methylation. These findings are discussed in terms of the role of short‐latency responses in the mode of action of NGF.
Glutamate Decarboxylase Activities in Single Vertebrate Neurons
Authors: Murashima Yoshiya L. , Kato Takahiko
Published: October 2006

Abstract: An enzymatic microassay method for glutamate decarboxylase (GAD) and γ‐aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze‐dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000‐350,000‐fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four‐ to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.
Effects of Ethanol In Vitro and In Vivo on the Release of Endogenous Catecholamines from Specific Regions of Rat Brain
Authors: Holman R. B. , Snape B. M.
Published: October 2006

Abstract: Blocks of tissue from the hypothalamus, olfactory bulb, or striatum of rats were incubated in vitro to study the basal and potassium‐stimulated release of endogenous catecholamines. When ethanol (100–250 mM) was added to these preparations in vitro, no changes in release were observed. When ethanol (3.0 g · kg−1) was injected intraperitoneally in vivo, however, and 3,4‐dihydroxyphenylethylamine (DA, dopamine) release was measured in vitro at various times after drug administration, significant increases in the basal release and decreases in the potassium‐stimulated release were observed in striatum and olfactory bulb. In striatum, these changes showed a more rapid onset and a longer duration than in olfactory bulb. In both brain regions, DA release did not differ from controls at 4–6 h after the ethanol injection, although blood ethanol concentrations remained elevated. This may imply the tissue's acquisition of acute functional tolerance to the drug. Similar increases and decreases in the basal and the potassium‐induced release of DA from striatal tissues were also found at 1 h after injection of a lower dose of ethanol (1.0 g · kg−1). In terms of behavior, this lower dose of ethanol produced only mild intoxication and ataxia, in contrast to the loss of righting reflex following the higher dose.
Polymorphism of Pseudocholinesterase in Torpedo marmorata Tissues: Comparative Study of the Catalytic and Molecular Properties of this Enzyme with Acetylcholinesterase
Authors: Toutant J. P. , Massoulié J. , Bon S.
Published: October 2006

Abstract: We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10−5M eserine) that differs from acetylcholinesterase (AChE, EC 3.1.1.7) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10−5M BW 284C51, but rapidly inactivated by 10−8M diisopropyl‐fluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 × 10−3M AcSCh. (d) It does not cross‐react with anti‐AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the “nonspecific” or pseudocholinesterase (pseudo‐ChE, EC 3.1.1.8) that exists in other species, although it is closer to “true” AChE than classic pseudo‐ChE in several respects. Thus, it shows the highest Vmax with acetyl‐, and not propionyl‐ or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo‐ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo‐ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo‐ ChE exists in several molecular forms, including collagen‐tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo‐ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X‐100 within days. In addition, it is converted, on storage of the extracts, into more rapidly sedimenting, Triton X‐100‐resistant forms (G2 and G4). A G4 form exists in spinal cord and plasma. Collagen‐tailed forms, A12 and A8, were characterized in high‐salt extracts from spinal cord and heart ventricle. Pseudo‐ChE molecular forms sediment slightly slower than the corresponding AChE forms. The two polymorphic enzyme systems, AChE and pseudo‐ChE, therefore arose as early as elasmobranchs during the evolution of vertebrates.
Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes
Authors: Freysz L. , Binaglia L. , Dreyfus H. , Massarelli R. , Golly F. , Porcellati G.
Published: October 2006

Abstract: The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base‐exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP‐[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base‐exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.
Amphiphilic Detergent‐Soluble Acetylcholinesterase from Torpedo marmorata: Characterization and Conversion by Proteolysis to a Hydrophilic Form
Authors: Stieger S. , Brodbeck U.
Published: October 2006

The membrane‐bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X‐100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent‐soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X‐100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent‐solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent‐soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.
Identification of Pig Brain Aldehyde Reductases with the High‐Km Aldehyde Reductase, the Low‐Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase
Authors: Cromlish James A. , Flynn T. Geoffrey
Published: October 2006

Abstract: Four NADPH‐dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high‐Km aldehyde reductase, glucuronate reductase, and tissue‐specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low‐Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin‐9‐ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of γ‐hydroxybutyrate.
Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates
Authors: Tai F. L. , Smith Ross , Bernard C. C. A. , Hearn M. W. T.
Published: October 2006

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed‐type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T‐cell‐mediated response to this antigen and substantial cross‐reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.
Choline Uptake and Metabolism in Affinity‐Purified Cholinergic Nerve Terminals from Rat Brain
Authors: Richardson Peter J.
Published: October 2006

Abstract: Cholinergic nerve terminals were affinity purified from rat caudate nucleus. These terminals possessed both high‐(KT= 2.7 μM) and low‐ (KT= 58 μM) affinity uptake mechanisms for exogenous [3H]choline. The proportion of [3H]choline acetylated was reduced from 75 to 30% under conditions of anoxia and hypoglycaemia, whereas the phosphorylation of choline increased from 4 to 52%. Choline phosphorylation was also increased when the terminals were preloaded with choline. The affinity‐purified terminals were shown to release acetylcholine in a Ca2+‐dependent manner on depolarization. The relationship between choline acetylation and phosphorylation in the cholinergic nerve terminal is discussed.
Enhancement of Histamine H1‐Receptor Agonist Activity by 1,4‐Dithiothreitol in Guinea‐Pig Cerebellum and Cerebral Cortex
Authors: Donaldson J. , Hill S. J.
Published: October 2006

Abstract The disulphide bond‐reducing agent 1,4‐dithio‐threitol (1 mM) produced a marked potentiation of hista‐mine‐stimulated accumulation of [3H]inositol phosphates in lithium‐treated slices of guinea‐pig cerebellum and cerebral cortex. This was seen as a parallel shift of the concentration‐response curve for histamine to lower agonist concentrations, with no significant effect on the maximal response or Hill coefficient. Dithiothreitol similarly potentiated the augmentation of adenosine‐stimulated cyclic AMP accumulation elicited by histamine in guinea‐pig cerebral cortex. Studies with partial agonists suggested that this potentiating effect was associated with an increase in agonist efficacy rather than a change in agonist binding affinity. Thus, dithiothreitol increased the maximal accumulation of [3H]inositol phosphates produced by both 2‐pyridylethylamine and 2‐methylhistamine, which appeared to act as partial agonists in guinea‐pig cerebral cortex. Dithiothreitol similarly increased the maximal extent of the augmentation of adenosine‐stimulated accumulation of cyclic AMP produced by 2‐methylhistamine. The site of action of dithiothreitol is not known; however, a comparison of the effect of dithiothreitol on muscarinic and histamine H1‐receptor‐mediated phosphoinositide responses in guinea‐pig cerebral cortex suggests that it is before the stage at which the receptor‐effector pathways are shared by these two receptor systems.
Occurrence and Sequence Complexity of Polyadenylated RNA in Squid Axoplasm
Authors: Capano Carla Perrone , Giuditta Antonio , Castigli Emilia , Kaplan Barry B.
Published: October 2006

Abstract: Axoplasmic RNA from the giant axon of the squid (Loligo pealii)comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/μg of total RNA) suggests that the poly(A)+ RNA population makes up ∼0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments < 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower‐reacting component (moderately abundant and rare sequences). The latter component accounts for ∼56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of ∼2.7 kilobases and 3.1 × 102 kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents ∼0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.
Regulation of Dopamine Release from Interplexiform Cell Processes in the Outer Plexiform Layer of the Carp Retina
Authors: O'Connor Patricia M. , Zucker Charles L. , Dowling John E.
Published: October 2006

Abstract: The 7‐aminobutyric acid (GABA) antagonists bicuculline and picrotoxin stimulate a four‐ to fivefold increase in endogenous dopamine release from isolated intact carp retina. The release evoked by these agents is Ca2+ dependent, a finding suggesting a vesicular release. Using light microscopic autoradiography, we have localized the sites of dopamine release to the dopaminergic interplexiform cell processes of the outer plexiform layer, which synapse onto horizontal cells. Our findings support previous suggestions that the dopaminergic interplexiform cells receive GABAergic inhibitory input and that the effects of GABA antagonists on horizontal cells are mediated by dopamine release from the interplexiform cells.
Effect of Ganglion Blockade on Cerebrospinal Fluid Norepinephrine
Authors: Goldstein David S. , Zimlichman Reuven , Kelly Gerald D. , Stull Robin , Bacher John D. , Keiser Harry R.
Published: October 2006

Abstract: The source of norepinephrine (NE) in CSF has been unclear. It has been suggested that CSF NE indicates central neural noradrenergic tone and is determined differently from plasma NE. If CSF NE depended specifically on NE release in the CNS, then interference with ganglionic neurotransmission would be expected to decrease plasma NE but not CSF NE. Hypotension caused by ganglionic blockade might be expected to increase CSF NE reflexively. We infused the ganglion blocker, trimethaphan, intravenously into anesthetized dogs and measured the effects on mean arterial blood pressure (MAP) and on cisterna magna CSF levels of NE. The results were compared with those obtained on administration of saline, clonidine (2 μg/kg), yohimbine (0.25 mg/kg), or nitroprusside and with those obtained when hypotension during ganglion blockade was prevented by concurrent treatment with phenylephrine. Trimethaphan decreased MAP by 40%, arterial NE by 64%, and CSF NE by 61%. Nitroprusside administered intravenously to produce the same 40% depressor response increased arterial NE by 612% and CSF NE by 155%. Prevention of ganglion blockade‐induced hypotension using phenylephrine did not prevent the decrease in CSF NE caused by trimethaphan, and when phenylephrine was discontinued, the resulting hypotension was not associated with increases in CSF NE. The similar decreases in plasma NE and CSF NE during ganglionic blockade, and the abolition of reflexive increases in CSF NE during hypotension in ganglion‐blocked subjects, cast doubt on the hypothesis that CSF NE indicates central noradrenergic tone and are consistent instead with at least partial derivation of CSF NE from postganglionic sympathetic nerve endings.
Synapsin I Is Associated with Cholinergic Nerve Terminals in the Electric Organs of Torpedo, Electrophorus, and Malapterurus and Copurifies with Torpedo Synaptic Vesicles
Authors: Volknandt W. , Naito S. , Ueda T. , Zimmermann H.
Published: October 2006

Using an affinity‐purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate‐immunofluorescence and using α‐bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I‐like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl‐1000. Our results suggest that synapsin I‐like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin I.
Two Monoclonal Antibodies Recognize Alzheimer's Neurofibrillary Tangles, Neurofilament, and Microtubule‐Associated Proteins
Authors: Ksiezak‐Reding H. , Yen S.‐H.
Published: October 2006

Abstract: Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain mi‐crotubule preparations. Both antibodies bind to the 200‐ki‐lodalton (kd) (NF‐H) and 160‐kd (NF‐M) but not to the 68‐kd (NF‐L) NF triplet proteins. They also bind to high‐molecular‐weight microtubule‐associated proteins (MAPs) and t. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing elec‐troblotted NF proteins with Escherichia coli alkaline phos‐phatase completely blocks AD 10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with α‐chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD 10 and AB18 in NF‐H is ∼ 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF‐M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60‐kd polypeptide adjacent to the α‐helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.
Characterization of [3H]Mazindol Binding in Rat Brain: Sodium‐Sensitive Binding Correlates with the Anorectic Potencies of Phenylethylamines
Authors: Angel Itzchak , Luu My‐Do , Paul Steven M.
Published: October 2006

Abstract: Saturable low‐affinity binding sites for [3H]mazin‐dol have been demonstrated in crude synaptosomal membranes from rat brain using both a centrifugation and a fil‐tion assay. Studies on the regional distribution of these binding sites revealed that the hypothalamus and brainstem had the highest density of sites. Kinetic analysis of the binding of [3H]mazindol to hypothalamic membranes demonstrated a single class of noninteracting binding sites with an apparent affinity constant (KD) of 10.2 ± 0.7 μM and maximal number of binding sites (Bmax) of 786 ± 94 pmol/mg of protein. Specific [3H]mazindol binding was rapidly reversible, temperature sensitive, labile to pretreatment with proteolytic enzymes, and inhibited by physiological concentrations of sodium. In most peripheral tissues, such as the liver and kidney, very low levels of binding were observed; however, the adrenal gland had a relatively high density of sites. The potency of a series of anorectic drugs in inhibiting specific [3H]mazindol binding to hypothalamic membranes was highly correlated with their anorectic potencies in rats, but not with their motor stimulatory effects. These results suggest the presence of a specific drug recognition site in the hypothalamus that may mediate the anorectic activity of mazindol and related phenylethylamines.
d‐Aspartate in Human Brain
Authors: Man Eugene H. , Fisher George H. , Payan Iris L. , Cadilla‐Perezrios Rodolfo , Garcia Nancy M. , Chemburkar Radhika , Arends Georgine , Frey William H.
Published: October 2006

Abstract: The presence of the biologically uncommon D‐aspartic acid (D‐aspartate) in hu manbrain white matter has been previously reported. The earlier study has now been expanded to include D/L‐aspartate ratios from 67 normal brains. The data show that the D‐aspartate content increases rapidly from 1 year to ∼35 years of age, levels off in middle age, and then appears to decrease somewhat. The D‐aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L‐aspartateratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D‐aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.
Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat
Authors: Wenthold R. J. , Altschuler R. A. , Skaggs K. K. , Reeks K. A.
Published: October 2006

Abstract: The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity‐purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross‐reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat‐ and Vibratome‐cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.
Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain
Authors: Haikala Heimo
Published: October 2006

Abstract: A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4‐dihy‐droxyphenylacetic acid, homovanillic acid, 3‐methoxytyra‐mine (3‐MT), noradrenaline, 3‐methoxy‐4‐hydroxyphenyl‐ethyleneglycol (MOPEG), 5‐hydroxytryptamine (5‐HT), and 5‐hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40%; wt/wt) and graphite powder (60%; wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24–67%; (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20–60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8–16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed‐phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G‐10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HC1O4, and the excess of HC1O4 was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer. Adjustment of the pH was necessary so that elution of 5‐HT from the Sephadex G‐10 columns in the same fraction with 3‐MT was avoided. If these compounds were in the same solution, their peaks would overlap on HPLC. MOPEG sulfate was purified by diethylaminoethyl‐Sephadex A‐25 (anion exchange resin) from the first fraction collected from the Sephadex G‐10 columns. The contents of the compounds under investigation in nine brain parts agreed with those found by other investigators.
Insulin Enhances Development of Functional Voltage‐Dependent Ca2+ Channels in Aneurally Cultured Human Muscle
Authors: Desnuelle Claude , Askanas Valerie , Engel W. King
Published: October 2006

Abstract: Voltage‐dependent Ca2+ channels were studied by the binding of the potent Ca2+ channel antagonist PN200‐MO and by the K+‐induced 45Ca2+ uptake in human muscle cultured aneurally in the presence of insulin, fibroblast growth factor, and epidermal growth factor, added in combination or individually. Compared to the muscle grown in medium without growth factors, 14–15 days of treatment with insulin (10 μg/rnI) alone or in combination with two other growth factors caused a 3.4‐and 3.8‐fold increase per culture dish in the number of PN200–110 binding sites, respectively. There was no change in the affinity of the ligand‐receptor complex. Under the same conditions, there was also a fourfold increase of the K+‐in‐duced 45Ca2+ uptake in cultured human muscle. Neither fibroblast growth factor nor epidermal growth facto alone influenced PN200–110 binding sites. Our study demonstrates that insulin enhances the development of functional voltage‐dependent Ca2+ channels in cultured human muscle.
Developmental Expression of the Myelin Proteolipid Protein and Basic Protein mRNAs in Normal and Dysmyelinating Mutant Mice
Authors: Sorg Barbara A. , Smith Matthew M. , Campagnoni Anthony T.
Published: October 2006

Abstract: Expression of the myelin proteolipid protein (PLP) was examined in the nuclei and polysomes of 12–27‐day‐old quaking, jimpy, and shiverer mouse brains and in 2–27‐day‐oId normal brains and compared with expression of the myelin basic proteins (MBPs). Northern blots showed the presence of multiple mouse PLP RNAs, the developmental expression of which coincided with my‐elination. Two major mouse PLP RNAs, 3.5 and 2.6 kilo‐bases in length, were observed in both cytoplasmic polyri‐bosomes and nuclei, and, in addition, a larger 4.6‐kilobase PLP RNA was observed in nuclei. Quantitative measurements with slot blot analyses showed that the levels of PLP and MBP RNAs peaked simultaneously at 18 days in nuclei but that maximal levels of PLP RNA lagged behind MBP RNA by several days in the polysomes. The developmental expression of both major classes of myelin protein mRNAs was affected in all three mutants. In shiverer brains, the levels of PLP mRNA in polysomes and nuclei were only 30–55% of control levels after 15 days. Thus, the deletion off a portion of the MBP gene appeared to have a major effect on the expression of the PLP gene in this mutant. In jimpy mice, where the mutation has been shown to involve the PLP gene, expression of MBP mRNA was also severely reduced, to <25% of control values. In quaking brains, the expression of each gene followed its own developmental course, different from each other and different from the normal mouse. The extent to which the expression of PLP and MBP was affected by the quaking mutation depended on the age at which it was examined.
Isolation and Immunochemical Study of a Soluble Cerebellar Lectin Delineating Its Structure and Function
Authors: Zanetta J. P. , Meyer A. , Kuchler S. , Vincendon G.
Published: October 2006

Abstract: A procedure of sequential extractions of cerebellar tissue was set up, which allowed specific solubilization of endogenous lectins by mannose. Two cerebellar soluble lectins, CSL1 (Mr= 33,000) and CSL2 (Mr= 31,500), were isolated. They appeared to consist of structurally and immunologically related polypeptides chains. By immunoaffinity, another minor component (Mr= 45,000) was isolated. Immunological studies suggested that the minor component is the precursor of the two other, i.e., CSL1 and CSL2, subunits. CSL1 (mainly lysosomal) possesses an additional peptide compared with CSL2 (mainly cytoplasmic and extracellular), which seems to be implicated in the signal for secretion and release.
Ca2+/Calmodulin Distinguishes Between Guanyl‐5′‐yl‐Imidodiphosphate‐and Opiate‐Mediated Inhibition of Rat Striatal Adenylate Cyclase
Authors: Ahlijanian Michael K. , Halford Marianne K. , Cooper Dermot M. F.
Published: October 2006

Abstract: The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl‐5′‐yl‐imidodiphos‐phate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p‐mediated inhibition accurately reflected hormone‐mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p‐mediated inhibition was dependent on the presence of Ca2+/CaM at 24°C; the inhibition was independent of Ca2+/CaM at 18°C; and inhibition could not be detected in the presence, or absence, of Ca2+/ CaM at 30°C. In contrast, naloxone‐reversible, morphine‐induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+dose‐response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p‐and morphine‐induced inhibition. Because Gpp(NH)p‐mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18°C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24°C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p‐and morphine‐induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p‐mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone‐mediated inhibition is Ca2+/CaM independent under all conditions. Further, this study shows that, under certain conditions, Gpp(NH)p‐mediated inhibition of adenylate cyclase does not mimic the inhibition of adenylate cyclase produced by a complete interaction between the inhibitory hormone receptor, the guanine nucleotide‐regulatory component that mediates inhibition, and the catalytic unit of the adenylate cyclase complex in striatal plasma membranes.
Comparison of the Effects of Forskolin and Dibutyryl Cyclic AMP in Neuroblastoma Cells: Evidence that Some of the Actions of Dibutyryl Cyclic AMP Are Mediated by Butyrate
Authors: Yusta Bernardo , Ortiz‐Caro Javier , Pascual Angel , Aranda Ana
Published: October 2006

Abstract: We have compared the effects of forskolin, N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (dibutyryl cyclic AMP, Bt2‐cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2‐cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose‐dependent inhibition of cell proliferation, but the effect of Bt2‐cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2‐cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2‐cAMP than in untreated cells or in forskolin‐treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2‐cAMP produced histone acetylation, a well‐known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2‐cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1°, a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1° in the cells treated with Bt2‐cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2‐cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.
Amphiphilic and Nonamphiphilic Forms of Torpedo Cholinesterases: II. Electrophoretic Variants and Phosphatidylinositol Phospholipase C‐Sensitive and ‐Insensitive Forms
Authors: Bon Suzanne , Toutant Jean‐Pierre , Méflah Khaled , Massoulié Jean
Published: October 2006

Abstract: We report an electrophoretic analysis of the hy‐drophobic properties of the globular forms of acetylcholin‐esterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. In charge‐shift electrophoresis, the rate of electrophoretic migration of globular amphiphilic forms (Ga) is increased at least twofold when the anionic detergent deoxycholate is added to Triton X‐100, whereas that of globular nonamphiphilic forms (Gna) is not modified. The G2a forms of the first class, as defined by their aggregation properties, are converted to nonamphiphilic derivatives by phosphatidylinositol phospholipase C (PI‐PLC) and human serum phospholipase D (PLD). AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which also exists in very small quantities in detergent‐solubilized extracts of electric lobes and spinal cord. They present different electrophoretic mobilities, so that each of these tissues contains a distinct “electromorph,” or two in the case of electric organs. The G2a forms of the second class (AChE in plasma, BuChE in heart), as well as G4a forms of AChE and BuChE, are insensitive to PI‐PLC and PLD but may be converted to nonamphiphilic derivatives by Pronase.
Noradrenaline Antagonizes and Ouabain Potentiates the Effects of iV‐Methyl‐D‐Aspartate on Rat Cerebellar Cyclic GMP Production
Authors: Carter C. J. , Gueugnon J. , Scatton B.
Published: October 2006

Abstract: Noradrenaline potently antagonizes the effects of N‐methyl‐D‐aspartate (NMDA) (80 μM) on cyclic GMP production in immature rat cerebellar slices in vitro (IC50 = 0.6 μM). The effect is stereospecific (D‐noradrenaline, IC50 = 100 μM), and also observed with adrenaline (IC50= 0.5 μM) and isoprenaline (IC50= 1.2 μM). The α1‐adrenoceptor agonists methoxamine or phenylephrine or the mixed α/α2 agonists oxymetazoline or xylometazoline (100 μM) do not block the effects of NMDA, but the α2‐adrenoceptor agonist clonidine is weakly active (IC50 = 200 μM). Salbutamol and terbutaline were also inactive except at high concentrations (300 μM), as were a number of other catechol and phenylethylamine derivatives. The antagonistic effects of noradrenaline on the NMDA response were insensitive to phentolamine, atenolol, or propranolol (up to 100 μM), but were blocked by the α2 antagonist idazoxan (1–10 μM). The Na+,K+‐ATPase inhibitor ouabain (0.1–10 μM) markedly potentiates the effects of NMDA in this model, and also antagonizes and reverses the ability of noradrealine (10 μM) to block the effects of NMDA. The results suggest that noradrenaline and Na+,K+‐ATPase activity have potent modulatory effects on the NMDA response.
Agonist and Antagonist Binding of Muscarinic Acetylcholine Receptors Purified from Porcine Brain: Interconversion of High‐ and Low‐Affinity Sites by Sulfhydryl Reagents
Authors: Berstein Gabriel , Haga Kazuko , Haga Tatsuya , Ichiyama Arata
Published: October 2006

Abstract: The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer‐assisted nonlinear regression analysis. The displacements by antagonists fitted a single‐site model. In contrast, the displacements by agonists did not fit the single‐site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H‐sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H‐sites did not result from interaction between receptors and GTP‐binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H‐sites for carbachol. Preincubation of the preparation with 5,5′‐dithiobis(2‐nitrobenzoic acid) resulted in an increase in the proportion of H‐sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide‐reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands. In particular, molecules carrying (an) intact, probably intramolecular, disulfide bond(s) show high affinity for agonists, whereas those carrying the reduced state of the relevant thiol group(s) show low affinity for agonists.
Morphological and Neurochemical Effects of Diazepam and Phenobarbital on Selective Culture of Neurons from Fetal Rat Brain
Authors: Daval Jean‐Luc , De Vasconcelos Anne Pereira , Lartaud Isabelle
Published: October 2006

The responses to diazepam (DZ) and phenobarbital (PhB) were studied in enriched neuronal primary cultures from rat embryo hemispheres. Cells were grown in chemically defined medium and the drugs were added for 3 days to cultures, at pharmacologically active concentrations. Following exposure to DZ or to PhB, morphological changes, such as less prominent neuronal processes, were observed in neurons. It was also shown that each drug reduced the specific uptake of 2‐deoxy‐D‐glucose by the cells and interfered with protein and RNA metabolism. It was concluded that both DZ and PhB might affect, at least transiently, the normal growth of neurons in culture.
Differentiation‐Dependent Sialylation of Individual Neural Cell Adhesion Molecule Polypeptides During Postnatal Development
Authors: Breen Kieran C. , Regan Ciaran M.
Published: October 2006

The postnatal sialylation of individual neural cell adhesion molecule (N‐CAM) polypeptides by a develop‐mentally regulated sialyltransferase in Golgi‐enriched fractions isolated from rat brain is described. The 120‐kilodal‐ton polypeptide of N‐CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140‐ and 180‐kilodalton N‐CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N‐CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated “embryonic” form of N‐CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The “embryonic” form of N‐CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N‐CAM was virtually undetectable in the immunoblots of postnatal material.