bluuebunny/crossref_metadata_2025 huggingface dataset:
Improvement in Conditions for Solubilisation and Characterisation of Brain D2 Dopamine Receptors Using Various Detergents
Authors: Wheatley Mark , Hall Jean M. , Frankham Patricia A. , Strange Philip G.
Published: December 2006

Abstract: A series of detergents of varying chemical properties has been tested for solubilisation of bovine caudate nucleus D2 dopamine receptors using [3H]spiperone binding to assay the solubilised sites. The properties of the lysophosphatidylcholine (LPC)‐ and 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulphonate (CHAPS)‐solubilised preparations are described in detail. The preparations are truly solubilised, and sucrose density gradient and gel filtration data are reported. Specific [3H]spiperone binding in the LPC‐solubilised preparation assayed at 4°C is solely to D2 dopamine re‐ceptors. If the assay temperature is raised to 25°C, the amount of specific [3H]spiperone binding is largely unchanged, but it forms a greater proportion of the total [3H]spiperone binding owing to a reduction in nonstereospecific (spirodecanone) [3H]spiperone binding at the higher temperature. The effect of raising the assay temperature is important as it enables more precise deter‐minations of specific [3H]spiperone binding to be made. Part of the specific [3H]spiperone binding at 25°C is to solubilised S2 serotonin receptors in addition to D2 dopamine receptors. Good correlations are observed between the affinities for binding of ligands to the solubilised D2 receptors and corresponding data obtained on membrane‐bound receptors. Agonist binding in LPC‐solubilised preparations is insensitive to guanine nucleotides. It is speculated that the spirodecanone sites represent, in part, proteolysed or damaged D2 dopamine, or S2 serotonin, receptors. In the CHAPS‐solubilised preparation the pharmacological profile of [3H]spiperone binding is unclear when assayed at 4°C, but in assays at 25°C a clear serotonin S2 receptor component of specific [3H]spiperone binding can be discerned. No clear D2 dopamine receptor component can be detected in this preparation under these conditions.
2‐Deoxyglucose Incorporation into Rat Brain Glycogen During Measurement of Local Cerebral Glucose Utilization by the 2‐Deoxyglucose Method
Authors: Nelson Thomas , Kaufman Elaine E. , Sokoloff Louis
Published: December 2006

Abstract: The incorporation of 14C into glycogen in rat brain has been measured under the same conditions that exist during the measurement of local cerebral glucose utilization by the autoradiographic 2‐[14C]deoxyglucose method. The results demonstrate that approximately 2% of the total 14C in brain 45 min after the pulse of 2‐[14C]deoxyglucose is contained in the glycogen portion, and, in fact, incorporated into α‐1‐4 and α‐1‐6 deoxyglucosyl linkages. When the brain is removed by dissection, as is routinely done in the course of the procedure of the 2‐[14C]deoxyglucose method to preserve the structure of the brain for autoradiography, the portion of total brain 14C contained in glycogen falls to less than 1%, presumably because of postmortem glycogenolysis which restores much of the label to deoxyglucose‐phosphates. In any case, the incorporation of the 14C into glycogen is of no consequence to the validity of the autoradiographic deoxyglucose method, not because of its small magnitude, but because 2‐[14C]deoxyglucose is incorporated into glycogen via [14C]deoxyglucose‐6‐phosphate, and the label in glycogen represents, therefore, an additional “trapped” product of deoxyglucose phosphorylation by hexokinase. With the autoradiographic 2‐[14C]deoxyglucose method, in which only total 14C concentration in the brain tissue is measured by quantitative autoradiography, it is essential that all the labeled products derived directly or indirectly from [14C]deoxyglucose phosphorylation by hexokinase be retained in the tissue; their chemical identity is of no significance.
Nigrostriatal Dopaminergic Neurons Remain Undamaged in Rats Given High Doses of l‐DOPA and Carbidopa Chronically
Authors: Perry Thomas L. , Yong Voon Wee , Ito Masatoshi , Foulks James G. , Wall Richard A. , Godin David V. , Clavier Ronald M.
Published: December 2006

Abstract: Rats were fed maximally tolerated doses of l‐3,4‐Dihydroxyphenylalanine (l‐DOPA) and carbidopa daily for 120 days in order to achieve a sustained elevation in brain dopamine levels. Some animals were also given buthionine sulfoximine, a γ‐glutamylcysteine synthetase inhibitor, in an unsuccessful effort to reduce brain glutathione contents. l‐DOPA‐ and carbidopa‐treated animals displayed no behavioral changes suggestive of nigrostriatal dopaminergic neuronal loss. When sacrificed 60 days after L‐DOPA treatment ended, all rats had normal tyrosine hydroxylase activities and dopamine contents in their striata, and cell counts were normal in the substantia nigra. It therefore seems unlikely that a model of Parkinson's disease, suitable for exploring the etiological importance of glutathione deficiency, can be produced in rats merely by administering the largest tolerable doses of l‐DOPA.
Actin‐Based Gelation of Cytoplasmic Extracts from Brain Tissue
Authors: Palmer Elizabeth , Hernandez José Manuel , Saborio José L.
Published: December 2006

Abstract: Incubation of rat brain cytoplasmic extracts under the conditions described in this paper results in the formation of three‐dimensional gels. Ultrastructurally, these gels correspond to complex supramolecular structures formed by single microfilaments and by microfilament bundles. Analysis of protein composition indicates that cytoplasmic gels are composed of actin and several associated proteins. Among the latter class of components, we have identified polypeptides with molecular weights of 55,000 (55K), 140,000 (140K), and a set of two or three polypeptides with molecular weights in the range of 235,000‐245,000 (235‐245K). The 55K and 140K components do not seem to correspond to any previously identified actin‐associated proteins, while the 235–245K polypeptides may correspond to the protein known as fodrin.
Ionic Regulation of the Binding of dl‐[3H]2‐Amino‐4‐Phosphonobutyrate to l‐Glutamate‐Sensitive Sites on Rat Brain Membranes
Authors: Butcher Steven P. , Roberts Peter J. , Collins James F.
Published: December 2006

Abstract: The effects of ions on the binding of the excitatory amino acid analogue dl‐[3H]2‐amino‐4‐phosphon‐obutyrate to l‐glutamate‐sensitive sites on rat brain synaptic membranes was investigated. The divalent cations manganese, magnesium, strontium, and particularly calcium, produced a marked enhancement in specific binding. However, this effect was manifest only in the presence of added chloride, or to a lesser extent, with bromide ions. Application of saturation analysis revealed that both chloride and calcium acted to increase the binding site density in a concentration‐dependent manner, without affecting the dissociation constant. The only other ionic species found to have a significant effect on 2‐amino‐4‐phosphonobutyrate binding was sodium, which produced an apparent reduction in site affinity, without modifying the binding site density. Although the significance of these striking ionic effects is as yet unknown, it seems feasible that chloride (and possibly also calcium) ions may serve a role in regulating the interaction of excitatory amino acids with their physiological receptors.
Activation of Amiloride‐Sensitive Sodium Transport in C6 Glioma Cells
Authors: Sapirstein Victor S. , Benos Dale J.
Published: December 2006

Abstract: We have characterized, in C6 cells, an amiloride‐sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation‐exchange system can be induced within 24–36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium‐dependent step with full activation obtained in the presence of the calcium iono‐phore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocyto‐chalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride‐sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal‐dependent step. Since diacyl‐glycerol is the presumed endogenous activator of protein kinase C., we studied the effects of dioleylglycerol. This intermediate of phosphoUpid turnover was found to increase specifically the amiloride‐sensitive sodium pathway. We feel therefore that Na+‐H+ exchange in glial cells as well as in other cells may be tightly linked to phospholipid turnover and to calcium flux and that these two processes are integral to glial cell acidification and ion homeostasis.
Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)
Authors: Nagy Agnes , Delgado‐Escueta Antonio V.
Published: December 2006

Abstract: A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (vol/vol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain.
N‐Acetyl‐Aspartyl‐Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method
Authors: Koller Kerry J. , Zaczek Robert , Coyle Joseph T.
Published: December 2006

Abstract: An isocratic HPLC method to measure endogenous N‐acetyl‐aspartyl‐glutamate (NAAG) and N‐acetyl‐aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG‐50 resin, the eluate was subject to HPLC anion‐exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10‐fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi‐lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.
Neurofilament Proteins in Cultured Chromaffin Cells
Authors: Bader M. F. , Georges E. , Mushynski W. E. , Trifaró J. M.
Published: December 2006

Abstract: Antibodies were raised against the 200‐kd, 145‐kd, and 68‐kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross‐react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti‐neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68‐kd antibody. A 70‐kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.
Effect of β‐FNA on Opiate δ Receptor Binding
Authors: Rothman Richard B. , Schumacher Uwe K. , Pert Candace B.
Published: December 2006

Abstract: (β‐FNA, the β ‐fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of δ‐receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the S receptor. In this paper we examine the effect of (β ‐FNA on the binding of the prototypic δ agonists, Leuenkephalin and d‐Ala2‐d‐Leu5‐enkephalin, its metabolically stable analogue, and show that treatment of membranes with β ‐FNA does lead to alterations in the in vitro properties of δ receptors.
Effect of the Cerebral Tryptaminergic System on the Turnover of Dopamine in the Striatum of the Rat
Authors: Fuenmayor L. D. , Bermúdez M.
Published: December 2006

Abstract: The effect of the cerebral 5‐hydroxytryptamine system on the turnover of striatal 3,4‐dihydroxyphenyl‐ethylamine (dopamine) was investigated by measuring the level of dopamine and one of its metabolites in rats depleted of cerebral 5‐hydroxytryptamine or treated with a 5‐hydroxytryptamine receptor blocker. Treatment with p‐chlorophenylalanine induced, in addition to a reduction in striatal 5‐hydroxytryptamine and 5‐hydroxyindol‐3‐ylacetic acid, an increase in the striatal concentration of dopamine, a diminution in the concentration of homovanillic acid in the same cerebral area, and a reduction in the rise of this acid after the administration of a butyrophenone derivative or tetrabenazine. Treatment with methysergide also reduced the increase of homovanillic acid induced by the butyrophenone. When probenecid was given to rats treated with p‐chlorophenylalanine, homovanillic acid failed to accutnulate, whereas the accumulation of 5‐hydroxyindol‐3‐ylacetic acid was unaffected. The decay of dopamine after α‐methyl‐p‐tyrosine administration was normal for the first 6 h but was later reduced in rats given p‐chlorophenylalanine or methy‐sergide. The results suggest that the lack of activation of 5‐hydroxytryptamine receptors leads to a reduction in the turnover of dopamine in the nigrostriatal pathway.
Ca2+, Calmodulin‐Dependent Regulation of Microtubule Formation via Phosphorylation of Microtubule‐Associated Protein 2, τ Factor, and Tubulin, and Comparison with the Cyclic AMP‐Dependent Phosphorylation
Authors: Yamamoto Hideyuki , Fukunaga Kohji , Goto Satoshi , Tanaka Etsuro , Miyamoto Eishichi
Published: December 2006

Abstract: Isolated microtubule‐associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin‐dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type‐II cyclic AMP‐dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.
The Picrotoxinin Binding Site and Its Relationship to the GABA Receptor Complex
Authors: White W. F. , Snodgrass S. R. , Dichter M. A.
Published: December 2006

Abstract: The binding characteristics of [3H]α‐dihydropicrotoxinin to the picrotoxinin binding site were investigated in membrane preparations of adult rat forebrain and living cultures of rat cerebral cortex. The binding of [3H]α‐dihydropicrotoxinin to rat forebrain was decreased by lysing, treating with Triton X‐100, and heating. Coincubation with γ‐aminobutyric acid (GABA), benzodiazepines, or alterations in the Na+ or Cl− composition of the media had no effect on the binding to the rat brain preparation. However, in the living neurons in tissue culture both GABA and diazepam significantly decreased the binding of [3H)α‐dihydropicrotoxinin. The dose‐response relationships for GABA antagonism of [3H]αdihydropicrotoxinin binding and for picrotoxinin antagonism of the GABA enhancement of [3H]flunitrazepam binding in cultured cortical neurons were also investigated. The Hill coefficients for these actions were reciprocal, suggesting that they result from complementary interactions between the binding sites for GABA and picrotoxinin. These data support the association of the picrotoxinin binding site with the postsynaptic GABA receptor complex.
Serotoninergic System in Scrapie‐Infected Hamsters
Authors: Pocchiari M. , Munson P. J. , Costa T. , Gajdusek D. C. , Gibbs C. J.
Published: December 2006

Abstract: Hamsters inoculated with scrapie virus show a dramatic hypersensitivity to serotoninergic drugs, developing a behavioral syndrome not unlike that obtained with pharmacologically induced lesions of the raphe nuclei. In an attempt to explain the state of hypersensitivity and to determine whether or not serotoninergic neurons were targets of the scrapie virus, pre‐ and postsynaptic serotoninergic sites were studied in the cerebral cortices of scrapie‐infected and sham‐inoculated hamsters, [3H]Imipramine binding and the uptake of endogenous 5‐hydroxytryptamine (5‐HT, serotonin) in synaptosomes prepared from scrapie‐inoculated animals were not different from those of controls. This suggests integrity of the serotoninergic neurons in scrapie‐infected hamsters. In contrast, affinity for the 5‐HT1 receptor (which modulates inhibitory response) was diminished whereas that for the 5‐HT2 receptor (which modulates excitatory response) was increased. This “imbalance” between the two receptors which is amplified in in vivo responses may account for the 5‐HT hypersensitivity. The alteration in the affinity of the two postsynaptic 5‐HT receptors supports the observation that scrapie virus alters cell plasma membranes.
γ‐Aminobutyric Acid Greatly Increases the In Vivo Extracellular Taurine in the Rat Hippocampus
Authors: Lerma J. , Herranz A. S. , Herreras O. , Muñoz D. , Solís J. M. , del Río R. M. , Delgado J. M. R.
Published: December 2006

Abstract: The effects of γ‐aminobutyric acid (GABA) on the extracellular levels of taurine and on excitability in the dentate gyrus were studied in anesthetized rats by the dialytrode technique. The dentate gyrus was perfused by means of a dialytrode with Krebs‐Ringer‐bicarbonate or GAB A solutions. Amino acid contents in perfusates and dentate field potentials evoked by electrical stimulation of the perforant pathway were evaluated. GAB A drastically elevated the levels of extracellular taurine in a dose‐dependent manner, decreasing the amplitude of the population spike. This result indicates that GABA stimulates taurine release, probably by a counter‐transport process. It is suggested that in physiological conditions an increase in extracellular taurine may be produced by synaptically released GABA.
A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase
Authors: Brenner Talma , Lisak Robert P. , Rostami Abdolmohammad , McMorris F. Arthur , Silberberg Donald H.
Published: December 2006

Abstract: Monoclonal antibody against 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid‐phase radioimmunoassay, immunoblot of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS‐PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin‐specific enzyme in CNS myelin and in cells responsible for myelin production.
Uptake and Release of Tryptophan and Serotonin: An HPLC Method to Study the Flux of Endogenous 5‐Hydroxyindoles Through Synaptosomes
Authors: Wolf William A. , Kuhn Donald M.
Published: December 2006

Abstract: An HPLC assay with fluorometric detection has been developed that is sensitive enough to measure simultaneously endogenous levels of tryptophan, serotonin (5‐hydroxytryptamine, or 5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) inside synaptosomes as well as that released into the incubation medium. Using this assay, we have observed that tryptophan is rapidly taken up by synaptosomes and turned over to 5‐HIAA without a concurrent release of 5‐HT. Exogenous 5‐HT is also rapidly taken up, and, within 20–30 min, 80% of the 5‐HT is deaminated. Veratridine induces release of both tryptophan and 5‐HT from synaptosomes. Changes in the disposition of exogenous tryptophan or 5‐HT can be completely accounted for by uptake or by stoichiometric changes in metabolites. This assay method should be valuable in the study of 5‐HT pools and in the determination of from which pool 5‐HT release occurs.
Involvement of Calcium in the Regulation of Serotonin N‐Acetyltransferase in Retina
Authors: Iuvone P. Michael , Besharse Joseph C.
Published: December 2006

Abstract: The possible involvement of calcium in the regulation of retinal serotonin N‐acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in defined medium. Omitting CaCl2 from the culture medium completely inhibited the dark‐dependent increase of NAT activity at night. Approximately 10−4‐10−3M free Ca2+ was found to be required for the maximal increase of NAT activity in the dark. Other divalent cations—Ba2+, Sr2+, and Mn2+—did not substitute for Ca2+. Antagonists of voltage‐sensitive calcium channels, including nifedipine, methoxyverapamil (D600), Co2+, and Mg2+, were found to be effective inhibitors of the dark‐dependent increase of retinal NAT activity. Trifluoperazine also decreased retinal NAT activity. These studies indicate that the increase of retinal NAT activity in the dark is mediated by a specific Ca2+‐dependent process and that Ca2+ influx through voltage‐sensitive calcium channels is involved.
Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation
Authors: Liles W. Conrad , Nathanson Neil M.
Published: December 2006

Abstract: Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E‐115, a murine neuroblastoma cell line. At a concentration of 0.35 μg/ml, tunicamycin inhibited macromolecular incorporation of [3H]mannose by 75–80%, whereas incorporation of [3H] leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter‐binding assay using [3H]quinuclidinyl benzilate ([3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol‐induced down‐regulation. The rate of mAChR degradation (control t1/212–14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [3H]QNB (a membrane‐permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [3H] N‐methylscopolamine ([3H]NMS, a membrane‐impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane.
Surface Glycosyltransferase Activities During Development of Neuronal Cell Cultures
Authors: Matsui Yoshiki , Lombard Dominique , Massarelli Raphaël , Mandel Paul , Dreyfus Henri
Published: December 2006

Abstract: Neurons in culture obtained from dissociated cerebral hemispheres of 8‐day‐old chick embryos showed measurable activities of galactosyl‐, fucosyl‐, and sialyl‐transferases at the external surface of their plasma membrane. Important changes in these activities were observed during cell proliferation and maturation, in particular the surface fucosyltransferase activity, and/or the amount of intraceilular fucosylated acceptors increased during synaptogenesis, between 3 and 5 days in culture (d.i.c.). A sodium dodecyl sulfate radioelectrophoretic analysis of the fucosylated neuronal acceptors labelled with [14C]fucose showed, during synaptogenesis, the high labelling of two protein bands of 116 and 50 × 103 daltons. The fucosylation of glycoconjugates occurred preferentially, in neurons, upon glycoproteins whereas in glial cell cultures glycolipids were more fucosylated. The reasons for such a difference are not yet understood but the results suggest that the surface fucosyltransferase activity and fucosylated proteins in particular may play a role during the synaptogenesis of neurons in culture.
Distribution of γ‐Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct
Authors: Murashima Yoshiya L. , Kato Takahiko
Published: December 2006

Abstract: An enzymatic microassay method for glutamate decarboxylase (GAD) and γ‐aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20‐μm thick freeze‐dried sections (0.2–1.5 μg dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10‐fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.
Recovery of Altered Fatty Acid Composition Induced by a Diet Devoid of n‐3 Fatty Acids in Myelin, Synaptosomes, Mitochondria, and Microsomes of Developing Rat Brain
Authors: Youyou A. , Durand G. , Pascal G. , Piciotti M. , Dumont O. , Bourre J. M.
Published: December 2006

Abstract: Rats were fed a semisynthetic diet containing either sunflower oil or soya oil. Half the litter fed with sunflower oil diet was changed to a soya oil diet when the pups were 15 days old (during active myelination). Fatty acid analysis was then performed on subcellular fractions of the animals fed (a) soya oil, (b) sunflower oil, and (c) soya oil replacing sunflower oil from the 15th day, to determine the speed of the recovery. All material from animals fed sunflower oil showed an important reduction in docosahexaenoic acid (22:6 n‐3), compensated by an increase in docosapentaenoic acid (22:5 n‐6), whereas arachidonic acid (20:4 n‐6) was not affected. In all fractions examined, when sunflower oil was replaced by soya oil in 15‐day‐old pups the recovery started from the very first day but lasted more than 2 months (this recovery was determined by the increase of 22:6 n‐3 up to the normal value and decrease of the 22:5 n‐6). In addition a delay was found for myelin recovery, starting only from the 25th day.
Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer‐Type Dementia
Authors: Shimohama Shun , Taniguchi Takashi , Fujiwara Motohatsu , Kameyama Masakuni
Published: December 2006

Abstract: Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer‐type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (–)‐[3H]nicotine and [3H]quinuclidinyl benzilate ([3H]QNB), respectively. Compared with the controls, (–)‐[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.
Regional and Subcellular Calmodulin Content of Rat Brain
Authors: Hoskins B. , Burton C. K. , Liu D. D. , Porter A. B. , Ho I. K.
Published: December 2006

Abstract: Calmodulin contents of cortex, cerebellum, striatum, diencephalon, and medulla + pons and of sub‐cellular fractions of each region were determined by radioimmunoassay. The diencephalon had the highest level of calmodulin (48.87 ± 4.56 μg/mg protein), whereas medulla + pons had the lowest level (8.01 ± 0.84 μg/mg protein). In all brain regions, the mitochondrial fraction was richest in calmodulin (from 71 to 227 μg/mg protein) whereas other areas contained from 6 to 66 μg/mg protein.
Altered Colchicine‐Binding Capacity in Chick Brain Regions: Relationship to Intensity or Information Content of Visual Input
Authors: Bondy S. C. , Hall D. L.
Published: December 2006

Abstract: The binding of [3H]colchicine to postmitochondrial supernatant fractions of chick brain has been studied, absorbing colchicine‐bearing proteins on DEAE‐cellulose filter discs. This was assayed at several times after unilateral enucleation of day‐old chicks. Binding was unaltered in optic lobes or anterior dorsal forebrain regions contralateral to the removed eye, relative to the corresponding regions contralateral to and thus directly or secondarily innervated by the intact eye. Colchicine binding was also assayed after training chicks to suppress pecking at a metallic bead coated with aversive‐tasting methylanthranilate. At 4 but not 24 h after the one‐trial training, binding was selectively elevated in the anterior dorsal forebrain roof. Results are taken to imply that alterations in tubulin content of chick brain may reflect stress‐related changes, possibly mediated by systemic endocrine flux, rather than a more localized learning experience.
Phorbol 12,13‐Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat
Authors: Wang Maoyin , Cahill Anne L. , Perlman Robert L.
Published: December 2006

Abstract: Phorbol 12, 13‐dibutyrate (PDBu) increased the production of 3,4‐dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half‐maximal at a concentration of approximately 0.1 μM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+‐free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8‐Br‐cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 μM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.
Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties
Authors: Filla A. , De Michele G. , Morra V. Brescia , Palma V. , Di Lauro A. , Di Geronimo G. , Campanella G.
Published: December 2006

Abstract: Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25°C and it was 10‐fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5‐fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With α‐ketoglutarate (α‐KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Kmα‐KG= 1.9 × 10−3 M, KmNADH= 0.21 × 10−3 M, and KmNH4+= 28 × 10−3 M, and in leukocytes they were Kmα‐KG= 1.7 × 10−3 M, KmNADH= 0.24 × 10−3 M, and KmNH4+= 28 × 10−3 M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.
Effect of Phencyclidine on the Metabolism of Individual Brain Proteins
Authors: Koul Omanand , March James E. , Sarma Raqhupathy , Griffiths Cynthia , Deutsch Dale G.
Published: December 2006

Abstract: The effect of phencyclidine on the metabolism of a selected number of rat brain proteins was determined using two‐dimensional gel electrophoresis and quantitative fluorography. When rats were injected with phencyclidine, modulation of individual protein metabolism occurred in the pituitary and cortex. That is, a few proteins showed increased and others decreased incorporation of [35S]methionine, whereas total protein metabolism was unaltered. In contrast, in vitro treatment of brain tissue with phencyclidine inhibited incorporation of ra‐diolabel into all proteins by — 50%, as shown by quantitative fluorography of individual proteins.
Unusual Phylogenetic Conservation of the N‐Terminal Amino Acid Sequence of the Central Nervous System‐Specific Membrane Glycoprotein F3‐87‐8 (CNSgp130)
Authors: Flanagan Brian F. , Teplow David B. , Dreyer William J. , Fabre John W.
Published: December 2006

Abstract: CNSgp 130 is a CNS‐specific membrane glycoprotein abundantly expressed throughout the mature mammalian CNS. The molecule is recognised by the mouse monoclonal antibody F3‐87‐8, which reacts with a determinant of CNSgp 130 common to all mammals tested to date. Rat and human CNSgp 130 were purified by a combination of F3‐87‐8 monoclonal antibody affinity and gel permeation chromatography, and the N‐terminal amino acid sequence was determined by gas‐phase sequencing techniques. The results show a remarkable conservation of the N terminus of the CNSgp 130 polypeptide between rats and humans, with complete identity of the first 20 amino acid residues. There was an unusually high and phylogenetically conserved number of cysteines in this region. The sequence showed no homology to other known sequences and should prove useful in precisely identifying the relationship of CNSgp 130 to other CNS membrane molecules.
Specific Binding of Glycosylated β‐Galactosidase to Bovine Brain Synaptic Membrane
Authors: Naoi M. , Nagatsu T.
Published: December 2006

Abstract: The binding of a series of glycosylated β‐ga‐lactosidases to a fraction rich in synaptic membrane of bovine brain was examined. β‐Galactosidase modified with p‐aminophenyl β‐D‐galactopyranoside (β‐D‐Gal β‐gal) was found the most effective in binding to synaptic membrane, followed by that modified with β‐D‐glucopyranoside, whereas the enzyme modified with p‐aminophenyl derivatives of α‐D‐galactopyranoside, α‐D‐glucopyranoside, and α‐ and β‐L‐fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4°C and the optimal pH was approximately 4.0. The binding of β‐D‐Gal β‐gal was inhibited by free p‐aminophenyl β‐D‐galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 ± 35 nM and 27.5 ± 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.
Heterogeneity of Rat Neurofilament Polypeptides Revealed by a Monoclonal Antibody
Authors: Vitadello M. , Triban C. , Fabris M. , Gorio A. , Schiaffino S.
Published: December 2006

Abstract: A monoclonal antibody obtained from mice immunized with a crude neurofilament preparation from newborn rat brain revealed the existence of heterogeneity of the 200,000‐ and 150,000‐dalton neurofilament polypeptides. On immunoblot the monoclonal antibody iC8 reacted with both the 200,000‐ and 150,000‐dalton components in the CNS, but only with the 150,000‐dalton. polypeptide in sciatic nerve preparations. In addition, the 150,000‐dalton polypeptide appeared as a single band in the sciatic nerve, whereas in the CNS a doublet was labeled by iC8. In contrast a second monoclonal antibody (3H5) reacted with the 200,000‐dalton peptide and a single 150,000‐dalton component in both the central and peripheral nervous system preparations. The differences revealed by iC8 were probably not due to phosphorylation, as the pattern of antibody binding in immunoblots was not changed by pretreatment with alkaline phosphatase. The findings suggest that different isoforms of neurofilament polypeptides are present in the nervous system.
Assignment of A and B Types of Monoamine Oxidase in NCB20 Hybrid Cells to Those of the Parental Cells by Peptide Mapping
Authors: Nakano Tamotsu , Saito Shigeru , Higashida Haruhiro , Kojima Kohichi , Nagatsu Toshiharu
Published: December 2006

Abstract: The structures of [3H]pargyline‐labeled, flavin‐containing polypeptides of monoamine oxidase (MAO) from hybrid NCB20 cells, and their parental cells, A/J mouse brain cells and Chinese hamster brain cells, were analyzed and compared by using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and limited proteolysis and one‐dimensional peptide mapping in SDS gels. After preincubation of mitocnondrial preparations with deprenyl or clorgyline, the flavin‐containing polypeptide of type A or type B MAO was selectively labeled with [3H]pargyline. SDS‐PAGE of [3H]pargyline‐labeled mitochondrial samples revealed that the polypeptide with apparent Mr of 62,000 was associated with type A activity in the three types of cells, and that the polypeptide with apparent Mr of 61,000 or 58,000 was associated with type B activity in Chinese hamster brain cells and NCB20 cells or A/J mouse brain cells, respectively. Chymotrypsin digestion of the [3H]pargyline‐labeled polypeptides and the peptide mapping in SDS gels from A/J mouse and Chinese hamster brain cells produced identical map patterns between the two type A MAOs, almost the same map patterns (with the exception of one additional peptide fragment) between the two type B MAOs, and different map patterns between type A and type B MAOs. The results of identical treatments of the [3H]pargyline‐labeled polypeptides of MAOs in NCB20 cells showed that type A and type B MAO in NCB20 cells were similar to type A MAO of A/J mouse and Chinese hamster brain cells and to type B MAO of Chinese hamster brain cells. We previously reported that the activity of type A MAO in NCB20 cells coincided with the activity of MAO in N18TG‐2 neuroblastoma cells which originate from A/J mouse and express solely type A MAO activity. Previous karyological studies indicated that most chromosomes of NCB20 cells derived from A/J mouse and that chromosome X, which may contain type A MAO of Chinese hamster origin, could not be recognized in NCB20 cells. The present study and these previous findings indicate that type A and type B MAOs in NCB20 cells may originate from A/J mouse and Chinese hamster, respectively, and that type A and type B MAO activities may be associated with distinct enzyme molecules.
Changes in Some Myelin Protein Markers and in Cytoskeletal Components During Wallerian Degeneration of Mouse Sciatic Nerve
Authors: de Néchaud B. , Gumpel M. , Bourre J. M.
Published: December 2006

Abstract: After transection of the mouse sciatic nerve, the sequence of events occurring in the distal degenerating segment was followed by the biochemical changes related to the cytoskeletal components and to the myelin protein markers. The components of the intermediate filaments and of the microtubules undergo early changes. Within 3 days, the neurofilament triplet and the peripherin disappear whereas many peptides bearing the antigenic determinant common to all classes of intermediate filaments accumulate. Several of them persist after 1 month. The tubulin pattern changes from a high level of microheterogeneity—reflecting mostly the axonal contribution—to a lower level displayed by the predominant Schwann cells. A decrease in the amount of the myelin markers is also observed. However, a month after transection, immunoreactive basic protein is still present in the degenerated segment homogenate.
Uptake of γ‐Aminobutyric Acid by Brain Tissue Preparations: A Reevaluation
Authors: Wood J. D. , Sidhu H. S.
Published: December 2006

Abstract: The kinetic constants Km and Vmax for the uptake of γ‐aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie‐Hofstee plots and computer analysis. The Km values were much greater in 0.1‐mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the “Km level.” Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 μM, 43 μM, and 3.9 mM, respectively. In contrast, only two uptake systems for d‐aspartate were detected, with km values of 1.8 μM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.
Identification and Characterization of Neuron‐Specific and Developmentally Regulated Gene Transcripts in the Chick Embryo Spinal Cord
Authors: Dickson John G. , Prentice Howard M. , Kenimer James G. , Walsh Frank S.
Published: December 2006

Abstract: Clones corresponding to neuron‐specific and developmentally regulated messenger RNA species in the chick have been isolated from a complementary DNA library prepared using polyadenylated RNA from 7‐day embryonic spinal cord. The library was initially screened by differential complementary DNA hybridization procedures for clones identifying polyadenylated RNAs present in embryonic spinal cord but absent from or at low abundance in liver tissue. A high proportion of selected recombinant plasmids were found to identify different RNA species which, although present in 14‐day embryonic spinal cord, could not be detected in a corresponding region of the developing chick CNS that is devoid of neuronal cell bodies, the optic nerve. The neuron‐specific assignment of these mRNAs within the developing neuroectoderm was confirmed using bulk‐isolated neuronal and glial‐enriched cell fractions from 7‐day embryonic spinal cord. In addition, several distinctive patterns of developmentally regulated expression of neuron‐specific messenger RNA species have been observed in the chick spinal cord. The studies lay a foundation for detailed examination of the regional and temporal distribution and control of neuronal gene expression in the chick spinal cord during embryogenesis.