bluuebunny/crossref_metadata_2025 huggingface dataset:
Haloperidol and Cerebral Metabolism in the Conscious Rat: Relation to Pharmacokinetics
Authors: Pizzolato Gilberto , Soncrant Timothy T. , Rapoport Stanley I.
Published: December 2006

Abstract: The time course and distribution of alterations in cerebral metabolic activity after haloperidol administration were evaluated in relation to the pharmacokinetics of haloperidol and the topography of the dopaminergic system in the brain. Local cerebral glucose utilization was measured, using the 2‐deoxyglucose technique, in awake rats after i.p. administration of the dopamine antagonist haloperidol (0.5 or 1 mg/kg). Haloperidol significantly reduced glucose utilization in 60% of 59 brain regions examined, but produced a large increase in the lateral habenula. The regional distribution of changes in glucose utilization was not closely related to the known anatomy of the brain dopaminergic system. The time course of the effect of haloperidol on cerebral metabolism was different for the two doses studied (0.5 and 1 mg/kg), and was not simply related to estimated brain concentrations of haloperidol. However, a linear relation between the metabolic effect and the time‐integrated brain concentration was demonstrated. These results show that haloperidol has an effect on CNS metabolic activity that is more widespread than would be predicted from the topography of the dopaminergic system; this may be due to indirect propagation of the primary effects of haloperidol. The metabolic response to haloperidol depends on brain concentration and duration of exposure to the drug.
Bovine Nucleus Caudatus Acetylcholinesterase: Active Site Determination and Investigation of a Dimeric Form Obtained by Selective Proteolysis
Authors: Landauer P. , Ruess K.‐P. , Liefländer M.
Published: December 2006

Abstract: The number of catalytic subunits of purified bovine nucleus caudatus acetylcholinesterase (E.C. 3.1.1.7) has been determined by active site labelling with [3H]diisopropyl fluorophosphate ([3H]DFP). The 10.5 S, 16 S, and 20 S forms were estimated to contain two, four, and six active sites, respectively, per molecule. A 4.8 S form, which showed a weak amphiphile‐dependent activity behavior, was obtained by selective proteolytic digestion with pronase. The inability of the purified 4.8 S form to aggregate after detergent removal, and the molecular mass in the range of 130‐165 kD under nondenaturating conditions, indicate that this form is a dimeric form, lacking those hydrophobic regions responsible for aggregation.
Characterization of Cholecystokinin from the Human Brain
Authors: Miller Laurence J. , Jardine Ian , Weissman Edward , Go Vay Liang W. , Speicher David
Published: December 2006

Abstract: Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin‐octapeptide, with the sequence of Asp‐Tyr(SO3)‐Met‐Gly‐Trp‐Met‐Asp‐Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin‐octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed‐phase; and that they possess similar biological activities.
Colchicine Neurotoxicity Demonstrates the Cholinergic Projection from the Supracommissural Septum to the Habenula and the Nucleus Interpeduncularis in the Rat
Authors: Fonnum Frode , Contestabile Antonio
Published: December 2006

Abstract: Colchicine injections in the supracommissural septum of the rat caused degeneration of several neurons in the nucleus triangularis septi and the nucleus septo‐fimbrialis. The lesions resulted in significant decreases of choline acetyltransferase in the habenula (–34%) and in the nucleus interpeduncularis (–36%), thus demonstrating the existence of a major cholinergic projection to these nuclei from the supracommissural septum. A large fall in choline acetyltransferase was also noticed in the dorsal hippocampus as a consequence of colchicine damage to the fimbria‐fornix fibers crossing the injected area.
An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions
Authors: Kranzler J. D. , Jones E. E. , MacLusky N. J. , Sakamoto H. , Naftolin F.
Published: December 2006

Abstract: An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in micro‐dissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate‐median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.
Regional Accumulation of Calcium in Postischemic Rat Brain
Authors: Dienel Gerald A.
Published: December 2006

Abstract: The regional concentrations of intravenously injected 45Ca and total calcium were measured in rat brain during recovery from transient occlusion of the four major arteries to the brain. 45Ca was injected at intervals after ischemia, and the regional distribution of 45Ca was estimated by autoradiography. The 45Ca appeared to enter the brain via the choroid plexus, labeling the paraventricular tissue at 1 h after the injection. Control brains had more 45Ca in the gray matter compared to fiber‐rich areas at 5 and 24 h, but within these regions the optical density was nearly uniform. The accumulation and retention of 45Ca in postischemic brain were selective and time‐dependent. The regional pattern of 45Ca uptake correlated with the temporal progression of ischemic cell change. Infarction and preischemic hyperglycemia increased morphological damage, and increased the extent and distribution of 45Ca accumulation. The rise in total calcium concentration appeared to be biphasic in irreversibly damaged tissue.
Regional Studies of Changes in Brain Fatty Acids Following Experimental Ischaemia and Reperfusion in the Gerbil
Authors: Bhakoo Kishore K. , Crockard H. Alan , Lascelles Peter T.
Published: December 2006

Abstract: Regional studies of brain phospholipid metabolism were carried out during a period of ischaemia induced in the gerbil by bilateral carotid occlusion for 60 min. The associated changes in free fatty acids (FFAs) during this period and following recirculation for up to 180 min were noted. Following ischaemia there was a generalised rise in the levels of all FFAs with no selective release of either the unsaturated (arachidonic and doco‐sahexaenoic) or saturated (palmitic and stearic) fatty acids. There were no observed differences between the brain regions studied, which is in contrast to previously reported observations for prostaglandins. There was also no indication of any specific phospholipid fraction being involved in FFA release. This would indicate that the release of FFAs from phospholipids is a nonspecific event, probably due to the action of hydrolytic lipases. Restoration of the circulation resulted in a short, sharp increase (within 5 min) in all FFAs, but in contrast to the observations during ischaemia alone there was a relatively larger rise in the unsaturated FFAs as compared to the saturated FFAs. Following this increase there was a gradual general decline in all FFA levels until 180 min of reperfusion. Since there was no preferential depletion of unsaturated FFAs during reperfusion, when free radical attack is considered to be at its maximum, it is our opinion that free radical peroxidation is unlikely to explain the pathology described in our model.
Radioactive Choline Uptake in the Isolated Rat Phrenic Nerve‐Hemidiaphragm Preparation. A Biochemical and Autoradiographic Study
Authors: Veldsema‐Currie R. D. , van Marle J. , Langemeijer M. W. E. , Lind A. , van Weeren‐Kramer J.
Published: December 2006

Abstract: When hemidiaphragms are stimulated via the phrenic nerve in the presence of 10 μM radioactive choline (Ch), the rate of radioactive Ch uptake in the end‐plate‐rich area (EPA) is greater than that in the endplate‐poor muscle (M). Ch uptake in the EPA is temperature‐dependent, with a Q10 of 2.9 and an activation energy of 19.5 kcal/mol. It is inhibited in a Na+‐depleted medium, in the absence of Ca2+, and by 10–20 μM hemicholinium‐3 (HC‐3) and it is not inhibited by α‐bungarotoxin even when the muscle is completely paralyzed. In the absence of stimulation the rate of uptake in the EPA is slightly, but not significantly, greater than in M. Using autoradiography, we find an enhanced amount of isotope in the nerve terminals and their immediate vicinities compared with the muscle fibres, in both stimulated and unstimulated hemidiaphragms. There is no enhanced uptake of isotope into the nerve terminals in stimulated tissues in the presence of 26 μM HC‐3. The uptake of isotope into the muscle is not altered by any of these treatments. There is a positive correlation between the initial rate of radioactive Ch uptake in the EPA and the amount of isotope in the nerve terminals (the mean corrected grain density above the nerve terminals). Without correcting for the large amount of diffusion that occurs, the ratio of the grain density above the synapses to that above the muscle fibres is 1.66 in tissue stimulated at 1 Hz, 1.04 in stimulated tissues in the presence of 26 μM HC‐3, and 1.31 in unstimulated tissues. Thus the increase in the rate of radioactive Ch uptake in the EPA of diaphragms caused by stimulation is probably due to an enhanced uptake of isotope into the nerve terminals and not into the muscle fibres. This rate is directly proportional to the stimulation frequency (up to 2 Hz), and the slope of the line is equal to 0.84 pmol radioactive Ch/impulse/whole diaphragm.
Biosynthesis of Triglyceride and Other Fatty Acyl Esters by Developing Rat Brain
Authors: Bishop James E. , Hajra Amiya K.
Published: December 2006

Abstract: The biosynthesis of triglyceride from 1,2‐diglyceride and long‐chain acyl coenzyme A (CoA) was studied in developing rat brain. Diglyceride acyltransferase activity was highest in a microsomal fraction, had a neutral pH optimum, and was stimulated by MgCl2. Palmitoyl CoA and oleoyl CoA served equally well as acyl donors. The enzyme catalyzed the acylation of both endogenous diglyceride and several naturally occurring and synthetic exogenous diglycerides. In addition, short‐chain primary and secondary alcohols were found to be acylated under these conditions. A second acylation system, active at low pH, was found to catalyze esterification of ethanol and cholesterol, but not diglyceride, with free fatty acid. These results demonstrate that brain has the capacity to acylate a wide variety of physiological and nonphysiological hydroxyl compounds.
Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins
Authors: Hikita T. , Bader M. F. , Trifaró J. M.
Published: December 2006

Abstract: Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane‐containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane‐bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I‐calmodulin indicated the presence of high‐affinity binding sites (KD= 1.3 × 10−8M; Bmax= 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I‐calmodulin followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin‐binding poly‐peptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus‐secretion coupling.
Glutamyl‐Taurine Is the Predominant Synaptic Taurine Peptide
Authors: Marnela Kirsi‐Marja , Morris Howard R. , Panico Maria , Timonen Maritta , Lähdesmäki Pekka
Published: December 2006

Abstract: Several taurine‐containing peptides have been identified from trichloracetic acid extracts of synaptosomes and their subcellular vesicles prepared from calf brain. These peptides contain aspartic and glutamic acids, serine and taurine, and are often present in an N‐acetylated form. The peptides were isolated as single spots by TLC. Glutamyl‐taurine was found to be the predominant structure when analyzed by fast atom bombardment (FAB) mass spectrometry.
Ovine Pineal Indoles: Effects of l‐Tryptophan or l‐5‐Hydroxytryptophan Administration
Authors: Sugden David , Namboodiri M. A. A. , Klein David C. , Grady Richard K. , Mefford Ivan N.
Published: December 2006

Abstract: l.‐5‐Hydroxytryptophan (l‐5‐HTP) (20 or 200 mg/kg i.p.) but not l‐tryptophan (500 mg/kg i.p.) loading substantially increases serum melatonin in sheep. In the present study we examined the effects of these compounds on pineal serotonin and six serotonin metabolites. l‐Tryptophan failed to increase 5‐hydroxytryptamine (5‐HT; serotonin) or any of its metabolites despite a fivefold increase in pineal tryptophan. In contrast, l‐5‐HTP loading produced a marked increase in pineal 5‐HT and its metabolites, including N‐acetylserotonin (NAS) and melatonin, indicating that an increased synthesis of melatonin is responsible for the increased serum melatonin concentration after loading with this precursor. No change in pineal indoleamine N‐acetyltransferase (NAT) activity was seen. These results are consistent with the suggestion that, during daytime in the sheep, 5‐HT availability may limit the production of melatonin.
Reconstitution of Calmodulin‐Sensitive Adenylate Cyclase from Bovine Brain with Phosphatidylcholine Liposomes
Authors: Yeager Robert E. , Heideman Warren , Olwin Bradley B. , Keller Charles H. , Schmidt John W. , Shattuck Rebecca L. , Storm Daniel R.
Published: December 2006

Abstract: A partially purified calmodulin (CaM)‐sensitive adenylate cyclase from bovine cerebral cortex was reconstituted with a series of phosphatidylcholine liposomes having variable fatty acid composition. The enzyme was successfully associated with dimyristoyl, dipalmitoyl, distearoyl, and dioleoylphosphatidylcholine liposomes. The specific activity of the enzyme in the various liposomes varied over a 4.6‐fold range indicating some degree of specificity for fatty acid composition. The adenylate cyclase‐liposome preparation retained sensitivity to both CaM and 5′‐guanylylimidodiphosphate (GppNHp). Arrhenius plots of enzyme activity in the four different liposome preparations all exhibited a pronounced discontinuity at 30°C ± 2, even though the bulk‐phase thermal transition points for the liposomes varied from −20 to 54°C. Fluorescence anisotropy studies of reconstituted liposome systems illustrated that incorporation of protein did not alter the normal‐phase transition point of these lipids. Since Arrhenius plots of the enzyme in Lubrol PX, prior to reconstitution with lipids, were strictly linear, it is concluded that the breaks at 30°C may be the effect of a local enzyme‐phospholipid environment. It appears that this adenylate cyclase is not particularly sensitive to phase transitions of the bulk lipid phase. The phospholipid reconstituted enzyme system appears suitable for examination of the influence of lipids on the CaM‐sensitive adenylate cyclase.
Elemental Composition and Water Content of Neuron and Glial Cells in the Central Nervous System of the North American Medicinal Leech (Macrobdella decora)
Authors: Saubermann Albert J. , Scheid Vicky L.
Published: December 2006

Abstract: Elemental (Na, P, S, Cl, K, Ca, Mg) composition and water content of neurons and glial cells of the leech (Macrobdella decora) were determined by x‐ray microanalysis of frozen hydrated and dried section techniques. Results are reported as elemental mass fractions (mass/mass) and water content as percent mass. Specific cell compartments and cell types had distinct elemental patterns and water content which suggests that chemical composition of specific cell types is unique and may represent an expression of cell differentiation analogous to morphological specialization. Water content of cells was also cell specific and ranged from 55% (neurons) to 90% (vacuolated zone of glial cells), K and Na were present in concentrations greater than predicted by ion‐selective microelectrode measurements, indicating that not all the K and Na were simultaneously accessible to such electrodes.
Comparison of Three 18F‐Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography
Authors: Arnett Carroll D. , Shiue Chyng‐Yann , Wolf Alfred P. , Fowler Joanna S. , Logan Jean , Watanabe Masazumi
Published: December 2006

Abstract: The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine‐18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)‐butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4‐h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8‐h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.
A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5‐Hydroxytryptamine‐1A Sites in Rat Brain
Authors: Ransom Richard W. , Asarch Kenneth B. , Shih Jean C.
Published: December 2006

Abstract: The inhibition of [3H]5‐hydroxytryptamine ([3H]5‐HT) binding in rat brain by 1‐[2‐(3‐bromoacetamidophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5‐HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5‐HT‐1A site) and 19 μM (5‐HT‐1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5‐HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high‐affinity sites for each compound represented about 35% of the specific [3H]5‐HT binding. In the presence of 1 μMspiperone, a concentration that saturates the 5‐HT‐1A sites while having a minimal effect on 5‐HT‐1B sites, BrAcTFMPP displaced [3H]5‐HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5‐HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single‐site model with a KD of 21 μM for spiperone. In corpus striatum, 5‐HT‐1A sites, as defined with spiperone, represented 15% of the specific [3H]5‐HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5‐HT‐2 receptors. BrAcTFMPP (KD= 41 nM) had an 80‐fold lower affinity for these sites than spiperone (KD= 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5‐HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5‐HT‐1A sites versus both 5‐HT‐1B and 5‐HT‐2 sites.
Purification and Kinetic Mechanism of Human Brain Soluble Catechol‐O‐Methyltransferase
Authors: Jeffery Douglas R. , Roth Jerome A.
Published: December 2006

Abstract: The soluble form of human brain catechol‐O‐methyltransferase (EC 2.1.1.6, COMT) has been purified approximately 4,000‐fold from a 250,000 ×g supernatant solution. The purified enzyme exhibits a molecular weight near 27,500 and a pI value equal to approximately pH 5.0. Initial velocity and product inhibition studies are consistent with an ordered reaction mechanism for soluble COMT. Tropolone, a dead‐end inhibitor, exhibited a competitive pattern of inhibition when dopamine (DA) was the varied substrate and an uncompetitive pattern when S‐adenosyl‐l‐methionine (SAM) was the varied substrate. These observations strongly suggest that the soluble form of COMT from human brain catalyzes the O‐methylation of cathecholamines via an ordered reaction mechanism in which SAM is the leading substrate. Since the membrane‐bound form of COMT catalyzes the O‐methylation of catecholamines through an identical reaction mechanism, these data provide further evidence that two forms of COMT, while being localized in distinct subcellular compartments, are quite similar in their molecular structure.
Identiflcation of a Proenkephalin Precursor in Striatal Tissue
Authors: Beaumont Ann , Metters Kathleen M. , Rossier Jean , Hughes John
Published: December 2006

Abstract: Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin‐containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin‐containing intermediates do not accumulate. In this investigation Sephacryl‐300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin‐like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)‐enkephalin, (Leu5)‐enkephalin, and (Met5)‐enkephalin‐Arg6‐Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)‐enkephalin‐Arg6‐Phe7 constituted the C‐terminal seven residues of this molecule.
Comparison of Proteins Involved with Cyclic AMP Metabolism Between Synaptic Membrane and Postsynaptic Density Preparations Isolated from Canine Cerebral Cortex and Cerebellum
Authors: Aoki Chiye , Carlin Richard K. , Siekevitz Philip
Published: December 2006

Abstract: Synaptic membrane and postsynaptic density (PSD) fractions isolated from canine cerebral cortex and cerebellum were assayed for the following proteins: adenylate cyclase and phosphodiesterase (PDE) activities against cyclic AMP and cyclic GMP, the regulatory subunit of the cyclic AMP‐dependent protein kinase, and the substrate proteins for this kinase. The results were expressed on the basis of both the protein content of the fractions and the number of synapses in the synaptic membrane fractions. The number of synapses on a constant protein content basis was about three times higher in the cerebral cortex synaptic membrane fraction than in the comparable cerebellar fraction. Adenylate cyclase activity was from 3.4 to 5.6 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content but only slightly higher based on synapse counts. PSD fractions had no adenylate cyclase activity. The cyclic AMP‐PDE activity was from 17 to 27 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content, and about five times higher based on synapse counts. By doing PDE histochemistry at the electron microscopy level it was found that all the cerebral cortex PSDs in the isolated fraction contained PDE activity, none being found associated with the broken‐up material in the fraction. The amount of the regulatory subunit of the cyclic AMP‐dependent protein kinase was about equal in the two fractions based on protein, but about one‐third lower in cerebral cortex fraction than in cerebellar fractions. In the cerebral cortex membrane fraction the primary substrate for the cyclic AMP‐dependent protein kinase is synapsin I, with much lower amounts in the cerebellar membrane fraction. The PSD fraction from the two sources also showed these differences in synapsin I content. In the cerebellar membrane fraction, the primary substrate for the enzyme is a 245,000 Mr protein not found in the cerebral cortex membrane fraction. The findings that the turnover of cyclic AMP is much higher in cerebral cortex synapses than in cerebellar synapses, and that differences are found between the cerebral cortex and cerebellum with regard to the substrate proteins for the cyclic AMP‐dependent protein kinase indicate a divergence in the effect of cyclic AMP between cerebral cortex and cerebellar synapses.
Evidence for the Presence of a Ganglioside Transfer Protein in Brain
Authors: Gammon CM. , Ledeen R. W.
Published: December 2006

Abstract: An extract from rat brain has been shown to catalyze the transfer of ganglioside GM1 from sonicated vesicles to erythrocyte ghosts. It also enhanced the transfer of GM1 to a crude neuronal membrane preparation, whereas myelin took up only a very limited amount. The transfer activity was heat‐labile. Similar transfer activities were found in extracts from bovine gray and white matter, that of the former being comparable to rat brain whereas the latter was greater per milligram protein.
Purification of Astroprotein (Astrocyte‐Specific Cerebroprotein) by Reversed‐Phase C‐1 HPLC
Authors: Morimoto Kazuyoshi , Hashimoto Tadao , Maeda Yasutaka , Hayakawa Toru , Tagawa Kunio , Mogami Heitaro
Published: December 2006

Abstract: A method for purification of astroprotein (an astrocyte‐specific cerebroprotein) with HPLC is described. A linear gradient from 30 to 70% acetonitrile in 0.1% trifluoroacetic acid (pH 2.2) was applied to the re‐versed‐phase C‐l (particle size 10 μm) column. Cerebroproteins from the crude extract from human glioma were clearly separated by this procedure. Highly purified astroprotein was homogeneous by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and has immunoreactivity to antiserum against astroprotein. Reversed‐phase C‐1 HPLC offers advantages over previously available preparative techniques in the higher purity and better separation time of the products.
Glycoprotein Properties of Muscarinic Acetylcholine Receptors from Bovine Cerebral Cortex
Authors: Rauh James J. , Lambert Mary P. , Cho Nam J. , Chin Hemin , Klein William L.
Published: December 2006

Abstract: Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin‐solubilized receptors were representative of the entire membrane‐bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin‐aga‐rose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N‐acetylneuraminic acid residues reduced binding to wheat germ agglutinin‐agarose by 40%; further treatment with endo‐glycosidases D and H, to remove all N‐linked carbohydrate, decreased binding by a total of 67%. Removal of N‐acetylneuraminic acid residues had no effect on agonist binding properties of the membrane‐bound receptors. The carbohydrate‐specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X‐100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pi of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate‐urea‐polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N‐linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.
Monoclonal Antibodies to Human Myelin Basic Protein
Authors: Chou Chi‐Hsin Jen , Cox Alberta A. , Fritz Robert B. , Wood John G. , Kibler Robert F.
Published: December 2006

Abstract: SJL/J and (SJL × PL) F1 hybrid mice were immunized with intact human myelin basic protein (MBP) or the three major peptic fragments of MBP, residues 1–38, 39–89, and 90–170. Immune spleen cells were fused with mouse myeloma P3 × 63Ag8 (NS1) cells in the presence of polyethylene glycol. Hybridoma supernatant culture fluids were screened for antibody to MBP by a solidphase radioimmunoassay (RIA). The specificity of the monoclonal antibody (mAb) was characterized by RIA using the three major MBP peptic fragments and subfragments as well as MBP and MBP fragments of different species with known amino acid sequence differences. Six MBP mAbs were generated, one of them IgM isotype and the remainder IgG isotypes. One mAb each reacted against regions of residues 22–38, 39–69, 70–89, 90–116, and two reacted against residues 118–157. Immunoblots also showed that the five IgG mAbs were reactive against MBP and the peptic fragment of MBP containing the epitope. Immunohistochemical studies showed the IgG mAbs specifically stained myelinated fiber tracts in human brain tissue.
Local Cerebral Glucose Utilisation Following Indoleamine‐ and Piperazine‐Containing 5‐Hydroxytryptamine Agonists
Authors: Grome John J. , Harper A. Murray
Published: December 2006

Abstract: Substances with varying structural components have been shown to have 5‐hydroxytryptamine (5‐HT)‐like properties in the CNS. In this study, putative 5‐HT agonists with indoleamine moeities—lysergic acid diethylamide (LSD) and 5‐methoxy‐N,N‐dimethyltryptamine (5‐MeODMT)—and with piperazine moeities—quipazine (Quip) and 6‐chloro‐2‐(1‐piperazinyl)pyrazine (6‐CPP) were administered to rats. Local cerebral glucose utilisation was measured using the [14C]2‐deoxyglucose autoradiographic technique. It was found that in most cerebral structures, these substances produced dose‐dependent reductions in glucose utilisation. However, Quip and 6‐CPP increased glucose utilisation in specific areas of the diencephalon (e.g., nucleus reuniens) and produced a biphasic effect in some but not all extrapyramidal structures (e.g., ventromedial caudate nucleus). No such increases in local cerebral glucose utilisation were measured following LSD or 5‐MeODMT administration. These results indicate that although similarities exist between the effects of indoleamine‐ and piperazine‐containing 5‐HT agonists on local cerebral glucose utilisation there are also significant differences in the overall patterns of response produced.
4‐Methylumbelliferyl Lipase in Human and Mouse Brain: A Possible Localization in Myelin
Authors: Tada Y. , Sekiguchi S. , Ito F. , Eto Y.
Published: December 2006

Abstract: 4‐Methylumbelliferyl (4‐MU) lipase activity in human and mouse brain, measured with 4‐MU palmitate, was characterized with respect to effects of pH and detergents, and subcellular and myelin localization. Purified myelin isolated by Norton's procedure [J. Neurochem. 21, 749–757 (1983)] contained higher specific activity of 4‐MU lipase, particularly in alkaline pH. Myelin lipase activity was markedly affected by the addition of different types of detergents, the amount of detergents added, and substrate. The optimal pH in myelin was bimodal—pH 4.5 and up to 8.0, respectively. These data indicate that myelin possesses 4‐MU lipase activity at alkaline pH, with lower levels at acidic pH.
Acute Effects of Soman, Sarin, and Tabun on Microsomal and Cytosolic Components of the Calmodulin System in Rat Striatum
Authors: Hoskins B. , Liu D. D. , Ho I. K.
Published: December 2006

Abstract: Two hours after administration of Soman (120 μg/kg, s.c.), Sarin (150 μg/kg, s.c.), or Tabun (240 μg/kg, s.c.), microsomes and cytosol were prepared from rat striata. Microsomal and cytosolic calmodulin (CaM) levels, microsomal adenylate and guanylate cyclase activities, protein kinase activities, and Ca2++ Mg2+‐ATPase activities were determined while cytosolic phosphodiesterase (PDE) activities were determined. CaM levels in both cell fractions were significantly increased by Soman and Sarin. Cyclic AMP‐PDE and adenylate cyclase activities were decreased by Soman and Sarin. All three agents decreased activities of cyclic GMP‐PDE and guanylate cyclase. Sarin and Tabun administration caused significant increases in microsomal protein kinase activity and none of the agents affected activity of divalent cation ATPases. The intensity of effects of the three organophosphates roughly paralleled their observed neurotoxic potencies. The results indicate that components of the CaM system are implicated as either causative or adaptive changes induced by these agents.
β2‐Adrenergic Receptors on Peripheral Nerves
Authors: Schreurs Jolanda , Seelig Tina , Schulman Howard
Published: December 2006

Abstract: We report that peripheral nerves have a functional adenylate cyclase‐coupled β‐adrenergic receptor. The pharmacological specificity of this receptor is shown to be of the β2 subtype. Two peripheral nerves, the sciatic from the frog and rat and the vagus from the rat, responded to β2‐agonists with 10–50‐fold increases in intracellular cyclic AMP level. This increase was inhibited by the β‐adrenergic antagonist propranolol. In contrast, a central nerve tract, the corpus callosum, responded to isoproterenol with only a minimal one‐ to twofold increase in cyclic AMP level. These studies demonstrate that peripheral nerves have β2‐adrenergic receptors that are responsive to exogenously applied catecholamines and suggest a role for these ligands in the previously described modulation of axonal conduction.
Selective Extraction of the DM‐20 Brain Proteolipid
Authors: Skalidis G. , Trifilieff E. , Luu B.
Published: December 2006

Abstract: Brain DM‐20 proteolipid was previously shown to be structurally different from the myelin major proteo‐lipid (MMPL). In an attempt to set up a large‐scale purification of DM‐20, we studied extraction of brain proteo‐lipids with mixtures of methylene chloride containing up to 80% methanol. The CH2Cl2‐CH3OH (3:7, vol/vol) mixture is highly selective for the extraction of bovine brain DM‐20 compared to MMPL. Purified DM‐20 was obtained after chromatography of the extract on methylated Sephadex.
Incorporation of Tritiated Galactose into Galactocerebroside by Cultured Rat Oligodendrocytes: Effects of Cyclic Adenosine 3′,5′‐Monophosphate Analogues
Authors: Pleasure David , Parris Judy , Stern Janet , Grinspan Judith , Kim Seung U.
Published: December 2006

Abstract: Cells dissociated from the forebrains of 21‐day‐old rats were enriched in oligodendroglia by Percoll gradient centrifugation, seeded on polylysine‐coated surfaces, and cultured in a serum‐containing medium. Incorporation by the cultures of tritium from d‐[3H]galactose into the galactosyl residue of galactocerebroside (galC) increased in an almost linear fashion for 48 h with 1–8 μCi of d‐[3H]galactose (30 mCi/μmol) per milliliter medium. Treatment for 2 days (day 1–3 after seeding) with 10−4M or 10−3M dibutyryl cyclic adenosine 3′,5′‐monophosphate (db cyclic AMP) or 10−4M 8‐bromo cyclic AMP stimulated galC radiolabelling. Incorporation of d‐[3H]galactose into galC during a terminal 48‐h radiolabelling period was not stimulated when the cells were continuously treated with these cyclic AMP analogues for 8 rather than 2 days.
Differential Roles of Raphe Nuclei in the Regulation of Dopamine β‐Hydroxylase in the Adrenal Gland of the Rat
Authors: Lima L. , Sourkes T. L. , Dubrovsky B.
Published: December 2006

Abstract: The effect of reserpine on the activity of dopamine β‐hydroxylase (DBH) in the adrenal gland of the rat was determined following electrolytic lesion of the dorsal raphe nucleus (DRN) or medial raphe nucleus (MRN). In sham‐operated rats, as well as in those with a lesion of the DRN, there was no significant modification of the action of reserpine on this enzyme. However, a lesion of MRN potentiated the inducing action of the drug. A specific role of MRN in the serotonergic regulation of adrenal DBH is suggested by this work.
Isolation of Postsynaptic Densities from Day‐Old Chicken Brain
Authors: Murakami Kentaro , Gordon‐Weeks Phillip R. , Rose Steven P. R.
Published: December 2006

Abstract: Synaptic plasma membranes from chicken brain were used to isolate a postsynaptic density (PSD) fraction using an aqueous two‐phase polymer system and the detergent n‐octyl glucoside. The protein and glyco‐protein composition and the morphology of the day‐old chicken brain PSD fraction were compared with a PSD fraction isolated from 12‐week‐old chicken brain. The PSD fraction from day‐old chicken brain contained predominantly PSDs although, like the fraction from 12‐week‐old chicken, there was some membrane contamination. The major polypeptides in the day‐old chicken fraction resolved by polyacrylamide gel electrophoresis comigrated with α‐ and β‐tubulin (Mr 57,000 and 55,000) and actin (Mr 45,000). The major PSD polypeptide (mPSDp) of 12‐week‐old chicken forebrain, which has a molecular weight of 52,000 was not a major component in day‐old chicken. A polypeptide of molecular weight 63,000 was also far more prominent in the 12‐week‐old chicken PSD fraction whereas the reverse was true for a polypeptide of 31,000. Day‐old chicken brain PSDs contained at least 14 concanavalin A‐binding glycoproteins of high (>85,000) molecular weight, the two most prominent having molecular weights of 170,000 and 180,000. In contrast to the polypeptide composition, the glycoprotein pattern of day‐old chicken PSDs was very similar to that of the 12‐week‐old bird. Intraperitoneally injected [3H]fucose was incorporated into the glycoproteins of synaptic plasma membranes and PSDs from day‐old chickens. In the synaptic plasma membranes, maximum specific radioactivity relative to homogenate (2.4) was reached after a 24‐h pulse, whereas in the PSDs, relative specific radioactivity continued to rise up to 72 hr after injection to reach a value of 3 times that of the homogenate.
Expression of Myelin Proteolipid Protein and Basic Protein in Normal and Dysmyelinating Mutant Mice
Authors: Sorg Barbara J. A. , Agrawal Daya , Agrawal Harish C. , Campagnoni Anthony T.
Published: December 2006

Abstract: Expression of myelin proteins was studied in the brains of 21‐day‐old normal mice and three dysmyelinating mutants—jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell‐free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21‐day‐old quaking brain homogenates showed a reduction in the steady‐state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21‐day jimpy brain homogenates revealed no appreciable steady‐state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.
Different Subcellular Localization of Muscarinic and Serotonin (S2) Receptors in Human, Dog, and Rat Brain
Authors: Luabeya M. K. , Maloteaux J. M. , De Roe C. , Trouet A. , Laduron P. M.
Published: December 2006

Abstract: Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two‐step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain.
H2 Histamine Receptors on the Epithelial Cells of Choroid Plexus
Authors: Crook Richard B. , Farber Michael B. , Prusiner Stanley B.
Published: December 2006

Abstract: A major site of cerebrospinal fluid production in vertebrates is the choroid plexus. The epithelial cells of the choroid plexus accumulate intracellular cyclic AMP in response to several effectors, including histamine. Since histamine is known to regulate fluid secretion in the stomach via H2 histamine receptors, we asked whether H2 receptors might also be present on epithelial cells of bovine choroid plexus. Using agonists and antagonists of histamine, we show that an agonist and antagonist pair specific for the H2 subtype were clearly more effective than an H1 agonist and antagonist pair in mimicking or inhibiting histamine stimulation of cellular cyclic AMP. Analysis by Schild plot allowed assignment of an apparent dissociation constant to the H2 antagonist metiamide which was 34‐fold lower than that of its H1 counterpart, diphenhydramine. These results indicate that epithelial cells of the choroid plexus possess H2 histamine receptors.
A Role for Transglutaminase in Neurotransmitter Release by Rat Brain Synaptosomes
Authors: Pastuszko Anna , Wilson David F. , Erecińska Maria
Published: December 2006

Abstract: Rat brain synaptosomes exhibit calcium‐dependent transglutaminase activity. This activity, measured in detergent‐treated or sonicated preparations, was six‐ to sevenfold lower than that in the liver. The synaptosomal transglutaminase was inhibited by various amines and α‐difluoromethylornithine, compounds known to inhibit activity of this enzyme in other tissues. The inhibitors of transglutaminase induced release of catecholamines, but not of γ‐aminobutyric acid, from synaptosomes both under basal and K+‐stimulated conditions. The concentrations of the agents that caused stimulation of catecholamine release were approximately the same as those that inhibited the activity of transglutaminase. Stimulation of release was largely reduced by the withdrawal of calcium from the incubation medium. Inhibitors of transglutaminase had little effect either on the uptakes of neurotransmitters or the amounts of deaminated products of catecholamine degradation released into the medium. It is suggested that a synaptosomal transglutaminase is involved in suppressing vesicular release of catecholamines by resting (nondepolarized) neurons and that this action may also be a part of negative feedback control which prevents excessive transmitter release at the synapse during increased neuronal activity.