bluuebunny/crossref_metadata_2025 huggingface dataset:
Sidedness of Phospholipid Synthesis on Brain Membranes
Authors: Butler Madeline , Morell Pierre
Published: October 2006

Abstract: We have investigated the localization of the site of incorporation and the subsequent equilibration of newly synthesized phospholipids in brain membranes. Rats were injected intracranially with [3H]glycerol; the animals were killed at varying times afterwards, and microsomal fractions were isolated from the brains. In some cases, microsomes were subfractionated on sucrose gradients. Initially, most of the radioactive phosphatidylethanolamine appeared in a pool that reacted with the impermeable reagent trinitrobenzene sulfonic acid (TNBS). This probe presumably modified only the lipid on the outer face of microsomal vesicles (which may, in large part, consist of pinched‐off endoplasmic reticulum). At 5 min after injection, the specific radioactivity of the TNBS‐modified phosphatidylethanolamine (cytoplasmic face) was four times that of the unmodified (luminal or inner face) phosphatidylethanolamine. With time, the ratio of the specific activities in the modified and unmodified pools of phosphatidylethanolamine approached 1.0, with kinetics that suggested a half‐time on the order of 30 min form vivo conversion of the TNBS‐accessible to the ‐inaccessible pool. This equilibration in specific activities could be the result of either translocation of phospholipids across endoplasmic reticulum membranes or conversion with time of initially labeled endoplasmic reticulum to other membranous organelles which form randomly oriented vesicles upon homogenization. A similar experimental design, using phospholipase C to hydrolyze outer face phospholipids preferentially, verified this conclusion for phosphatidylethanolamine and yielded similar results for phosphatidylcholine. Control studies measuring radioactive sucrose permeability indicated that neither TNBS nor phospholipase C treatment significantly disrupted microsomal vesicles under the conditions used.
Postnatal Changes in the Activity Ratio of Specific and Nonspecific Cholinesterases from Neuronal Perikarya
Authors: Ochoa Enrique L. M. , Brusco Alicia , Idoyaga‐Vargas Victor P. , Carminatti Hector
Published: October 2006

Abstract: A soluble fraction from rat brain neuronal perikarya was shown to contain both the specific and nonspecific forms of the enzyme acetylcholines‐terase (EC's 3.1.1.7. and 3.1.1.8., respectively). The ratio of the enzyme activities varied along the course of brain development: the nonspecific form being predominant from 1 to 15 days of age and the specific one showing the pattern of rising activity from day 15 onward. We suggest a possible relationship between this changing in cholinesterase activities and the establishment of synapses within the rat cerebral cortex.
Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat
Authors: Edwards J. A. , Brimijoin S.
Published: October 2006

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague‐Dawley, Fischer‐344, and Wistar‐Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague‐Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100‐fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer‐344 or Wistar‐Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue‐specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague‐Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body‐wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar‐Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.
Purification, Properties, and Immunohistochemical Localisation of Human Brain 14‐3‐3 Protein
Authors: Boston Paul F. , Jackson Peter , Kynoch Pamela A. M. , Thompson R. J.
Published: October 2006

Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14‐3‐3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14‐3‐3‐2 protein, and a slower minor component, termed 14‐3‐3‐1 protein, which consists of approximately 12% of the total protein. Both 14‐3‐3‐1 and 14‐3‐3‐2 have a native molecular weight of approximately 67,000. 14‐3‐3‐2 appears to have the subunit composition (αβ; 14‐3‐3‐1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β’ subunits show some common peptides. Immunoperoxidase labelling shows that 14‐3‐3 is localised in neurones in the human cerebral cortex. 14‐3‐3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide‐dependent protein kinase, or purine nucleoside phosphorylase activity. 14‐3‐3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin‐associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.
Glial Cells Identified by Anti‐α‐Albumin (Anti‐GFA) in Human Pineal Gland
Authors: Lowenthal A. , Flament‐Durand †J. , Karcher D. , Noppe M. , Brion J. P.
Published: October 2006

Abstract: α‐Albumin, a CNS specific protein, identical to GFA protein and specific for glial cells, has been found in the human pineal gland using histoimmunological and quantitative methods. The significance of its presence in the pineal gland is discussed.
H‐Pro‐[3H]Leu‐Gly‐NH2: Uptake and Metabolism in Rat Brain
Authors: de Wildt D. , Verhoef J. , Witter A.
Published: October 2006

Abstract: The uptake and metabolism of H‐Pro‐[3H]Leu‐Gly‐NH2 ([3H]PLG) in rat brain was investigated by reverse‐phase paired‐ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal‐cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial‐synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7–7.5. The major labeled metabolite was [3H]leucine, whereas 3H‐labeled Leu‐Gly‐NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2‐terminus and that the dipeptide intermediate H‐Leu‐Gly‐NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.
Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins
Authors: Bigbee John W. , Eng Lawrence F.
Published: October 2006

Abstract: Intermediate filament (IF) proteins from rat spinal cord were analyzed by two‐dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA‐dependent reticulocyte lysate system stimulated with 16‐day‐old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two‐dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post‐translational processing or experimental artifact.
Purification and Characterization of Bovine Brain 5′‐Nucleotidase
Authors: Montero Josefa Mallol , Fes Jorge Bozal
Published: October 2006

Abstract: The 5′‐nucleotidase located in the cytoplasmic fraction of bovine brain cortex was purified to electrophoretic homogeneity. The molecular weight was 134,000 daltons in the presence of sodium deoxycholate, whereas the enzyme formed high molecular weight aggregates in the absence of detergent. The purified enzyme showed the same kinetic and electrophoretic behaviour as the enzyme present in the original cytoplasmic fraction, and the presence of surfactants did not change the Km and Vm, values. The nucleotidase from this source was a phosphohydrolase of 5′‐mononucleotides acting on the deoxyribonucleotides and ribonucleotides of purines and pyrimidines. 5′‐IMP was the preferred substrate; the optimum pH was 7.5. The study of the influence of the temperature on the initial reaction rates allowed calculation of the δEa, and δHo values. The variation of Vm and Km with a change in pH suggests the existence of a sulfhydryl group and an imidazole group in the enzymesubstrate complex.
Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina
Authors: Watling Keith J. , Dowling John E.
Published: October 2006

Abstract: The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose‐related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 μM. Porcine histidine isoleucine‐containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 μM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 μM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone‐releasing hormone, α‐melanocyte‐stimulating hormone, cholecystokinin octapeptide26–33, gastrin‐releasing peptide, thyrotropin‐releasing hormone, and VIP10–28 were totally inactive. The response to 10 μM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors. These data are discussed in terms of the presence, localization, and possible function of a putative VIP‐ergic innervation in the carp retina.
Cell Cycle‐Dependent Expression of Specific Opiate Binding with Variable Coupling to Adenylate Cyclase in a Neurotumor Hybrid Cell Line NG108–15
Authors: Scheideler Mark A. , Lockney Michael W. , Dawson Glyn
Published: October 2006

Abstract: Monolayer cultures of neuroblastoma × glioma hybrid (clonal) cell line NG108–15, synchronized by the isoleucine/glutamine deprivation method, showed maximal expression of opiate binding sites at the same point in the cell cycle at which prostaglandin E, (PGE1) had a maximum stimulatory effect on cyclic AMP synthesis. However, the capacity of enkephalin [D‐Ala2D‐Leu5] to block the stimulation of cyclic AMP synthesis by PGE1 was not related to the number of opiate receptors expressed. The Ki for the inhibition of cyclic AMP synthesis by opioid peptides increased substantially during the period of the cell cycle at which maximal expression of opiate binding sites occurred, making the effective level of inhibition of adenylate cyclase activity by 0.1 μM enkephalin [D‐Ala2D‐Leu5] the same through the cell cycle. Data are presented to suggest that enkephalin receptor coupling to adenylate cyclase, via a GTP‐binding protein, is maximal during G1 phase (which may approximate the state of the differentiated neuron) and minimal during S + G2 phase, just prior to cell division, when many receptors are uncoupled.
Glycosylation‐Dependent Regulation of Opiate (Enkephalin) Receptors in Neurotumor Cells
Authors: McLawhon Ronald W. , Cermak Daniel , Ellory J. Clive , Dawson Glyn
Published: October 2006

Abstract: Electron inactivation analysis revealed that the opiate (enkephalin) binding site in neurotumor cell lines NG108–15 and NCB‐20 had an apparent target size of 200,000 daltons. Expression of functional opiate receptors in neurotumor cells appeared to require glycosylation, as treatment of such cells with tunicamycin (TM; under conditions where de novo glycosylation of asparagine residues in protein was reduced by 80%, but overall protein and DNA synthesis were inhibited by <10%) resulted in the loss of 50% of the opiate binding sites. The loss of binding sites could not be prevented by addition of protease inhibitors to cell cultures, but binding sites were partially restored 48–60 h after removal of the TM. In addition, the number of enkephalin binding sites in TM‐treated cells was also restored to near‐normal levels by addition of physiological concentrations (1–10 mM) of manganese ions to the in vitro receptor binding incubation mixture. TM treatment resulted in receptor supersensitivity to manganese ions for both opiate agonists and antagonists, no change in the sodium effect for either agonists or antagonists, and subsensitivity to GTP for both agonists and antagonists. However, opiate binding to cell membranes was not substantially inhibited by either neuraminidase treatment or short‐term incubation with lectins such as wheat germ agglutinin, ricin, or concanavalin A. Thus, the data suggest that oligosaccharide units are not directly involved in opiate receptor‐ligand interactions, but protein glycosylation is required for functional expression of receptors.
Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor
Authors: Herron G. Scott , Schimerlik Michael I.
Published: October 2006

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase‐treated and untreated receptor suggest that high‐affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N‐acetylglucosamine or its β(1 → 4)‐linked oligomers.
Structure of the Mannose‐Rich Oligosaccharide Chains of Concanavalin A‐Binding Glycopeptides Derived from Beef Brain Glycoproteins
Authors: Somawardhana Chandrasiri W. , Brunngraber Eric G.
Published: October 2006

Abstract: A neutral, mannose‐rich, concanavalin A (Con A)‐binding glycopeptide fraction was obtained by proteolytic digestion of defatted beef brain tissue. Hydrazinolysis followed by gel filtration of the reaction products provided three oligosaccharides. A portion of each oligosaccharide was treated by exhaustive digestion with α‐mannosidase. Another portion was subjected to selective acetolysis of Manαl‐6Man linkages, providing two fragments that were recovered by gel filtration. The structure of the intact oligosaccharides, as well as the fragments obtained by selective acetolysis and enzymatic treatment, were resolved by gas‐liquid chromatographic‐mass spectrometric analysis. The structures of the three oligosaccharides were: (a) Manαl‐2Manαl‐6(Manαl ‐3)Manαl‐6(Manαl‐2Manαl‐2Manαl 3)Manβ1‐4‐N‐acetylglucosamine (GlcNAc)β ‐4N‐ acetylglucosaminitol (GlcOLNAc); (b) Manαl ‐2Manαl ‐6(Manαl ‐3)Manαl‐6(Manαl‐2Manαl‐3)‐Manβ1‐4GlcNAcβl ‐4GlcOLNAc; and (c) Manαl ‐6(Manαl‐3) Manαl ‐ 6(Manαl ‐ 3)Manβl ‐4GlcNAc‐βl ‐ 4GlcOLNAc. These structures account for 15–20% of the glycoprotein‐carbohydrate of whole beef brain and most of the oligosaccharides that demonstrate a high affinity for Con A. In view of the large number of Con A‐binding glycoproteins in brain tissue, it appears that many of these different glycoproteins must contain structurally identical oligosaccharides.
Enhancement of Diazepam and γ‐Aminobutyric Acid Binding by (+) Etomidate and Pentobarbital
Authors: Thyagarajan R. , Ramanjaneyulu R. , Ticku M. K.
Published: October 2006

Abstract: (+) Etomidate and pentobarbital enhance [3H]diazepam and [3H]γ‐aminobutyric acid ([3H]GABA) binding to cerebral cortex membranes. Both (+)etomidate and pentobarbital increase the affinity of [3H]diazepam for its binding sites. In contrast, they increase the Bmax of both the high‐ and low‐affinity GABA receptor sites. The enhancement of [3H]diazepam and [3H]GABA by (+)etomidate and pentobarbital is blocked by GABA antagonists. These results indicate that hypnotic drugs such as (+)etomidate and pentobarbital, which are not structurally related, modulate diazepam and GABA binding sites via similar mechanisms.
Possible Involvement of Humoral Regulation in the Effects of Elevated Cerebral 4‐Aminobutyric Acid Levels on the Polyamine Metabolism in Brain
Authors: Lapinjoki Seppo P. , Pulkka Anitta E. , Laitinen Seppo I. , Pajunen Antti E. I.
Published: October 2006

Abstract: It has been reported in several recent studies that the manipulation of cerebral 4‐aminobutyric acid (GABA) level results in unexpected changes in the cerebral polyamine metabolism in vivo. The mechanisms behind these interactions have remained unknown. The present results show that the changes in polyamine metabolism are not limited to the brain, but are observable also in the liver, which served as a peripheral reference tissue. Different types of responses in the activities of the poiyamine‐synthesizing enzymes, ornithine decarboxylase and adenosylmethionine decarboxylase, were observed after increasing the cerebral GABA concentration of mice with varying doses of two GABA transaminase inhibitors, gabaculine and ethanolamine‐O‐sulphate. The time course of the significant changes in the enzyme activities showed significant correlation between the brain and liver. The possibility of direct effects of the drugs on liver was excluded by injecting them intracerebroventricularly, and by performing control experiments with equal doses given peripherally. It is concluded that the observed changes in the polyamine metabolism of liver are produced through centrally mediated humoral regulation, and that the corresponding changes in the brain are obviously due to the same factor or factors, since they are significantly correlated to the changes in liver.
Solubilization of Brain Benzodiazepine Receptors with a Zwitterionic Detergent: Optimal Preservation of Their Functional Interaction with the GABA Receptors
Authors: Mernoff Stephen T. , Cherwinski Holly M. , Becker John W. , de Blas Angel L.
Published: October 2006

Abstract: Rat brain benzodiazepine receptors have been solubilized by means of the zwitterionic detergent CHAPS under conditions in which the GABA stimulation of [3H]flunitrazepam binding to the benzodiazepine receptors is maintained intact. This stimulation is partially or totally abolished when using other conventional deter gents.
Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat
Authors: Cahill Anne L. , Perlman Robert L.
Published: October 2006

Abstract: Adenylate cyclase activity in cell‐free homogenates of the rat superior cervical ganglion (SCG) was assayed under a variety of experimental conditions. Adenylate cyclase activity was decreased by approximately one‐half when 1 mM EGTA was included in the homogenization buffer and assay mixture, indicating the presence of a Ca2+‐sensitive adenylate cyclase in the ganglion. In the presence of EGTA, basal adenylate cyclase activity in homogenates of the SCG was 12.9 ± 0.6 pmol cyclic AMP/ganglion/10 min. Enzyme activity was stimulated three‐ to fourfold by 10 mM NaF or 10 mM MnCl2, Both GTP and its nonhydrolyzable analog guanylylimidodiphosphate (GppNHp) stimulated adenylate cyclase in a concentration‐dependent manner over the range of 0.1–10.0 μM. Stimulation by GppNHp was five to six times greater than that produced by GTP at all concentrations tested. Decentralization of the ganglion had no effect on basal or stimulated adenylate cyclase activity. Receptor‐linked stimulation of adenylate cyclase was not obtained with any of the following: isoproterenol, epi‐nephrine, histamine, dopamine, prostaglandin E2, or va‐soactive intestinal peptide. Thus the receptor‐linked regulation of adenylate cyclase activity appears to be lost in homogenates of the ganglion.
Subcellular Localization of Bioactive Cholecystokinin‐Like Peptides in the Rat Cerebral Cortex
Authors: Varró Andrea , Lonovics J. , Hajnal F. , Somogyi J.
Published: October 2006

Abstract: The subcellular distribution of cholecystokinin (CCK), especially its exact localization within the synaptosome, was studied in the rat cerebral cortex. Highest CCK‐like bioactivity was measured in the synaptic membrane fractions, paralleling the distribution of (Na++ K+)‐dependent ATPase. In the synaptic vesicles, which were characterized by high acetylcholine content and by the absence of (Na++ K+)‐ATPase, only minimal quantities of CCK were detected.
[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores
Authors: Schoffelmeer Anton N. M. , Mulder Arie H.
Published: October 2006

Abstract: Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10‐120 mM K+) or to veratrine (1‐25 μM). In the absence of extracellular Ca2+ veratrine, in contrast to K+‐depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 μM). The Ca2+ antagonist Cd2+ (50 μM), which strongly reduced K+‐induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 μM), known to inhibit Ca2+‐entry into mitochondria, enhanced veratrine‐induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine‐induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulussecretion coupling process in central noradrenergic nerve terminals.
3′,5′‐Cyclic Adenosine Monophosphate‐ and Ca2+‐Calmodulin‐Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II
Authors: Treiman Marek , Weber Wolfgang , Gratzl Manfred
Published: October 2006

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle‐bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg2+ and Zn,2+ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP‐dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.
Blood‐Brain Barrier Transport of Basic Amino Acids Is Selectively Inhibited at Low pH
Authors: Oldendorf William H. , Crane Paul D. , Braun Leon D. , Wade Lester A. , Diamond Fared M.
Published: October 2006

Abstract: The transport of amino acids across the blood‐brain barrier was measured with the single‐pass carotid injection method. The pH of the injected bolus varied between 4.5 and 8.5. Arginine and lysine uptakes were inhibited 24% at pH 5.5 and 59% at pH 4.5. The uptakes of 2‐aminobicyclo (2,2,1) heptane‐2‐carboxylic acid and phenylalanine were unaffected at this pH. There were also no changes observed in choline, glucose, or butanol transport. The Ki of arginine transport inhibition by H+ was 2.4 ± 0.5 μM; i.e., pH 5.6 ± 0.1. No change with pH occurred in the Km of arginine transport, while a significant decrease (p < 0.01) was observed in the Vmax (10.2 ± 2.3 nmol min−1 and 5.6 ± 2.3 nmol min−1 g−1 at pH 7.5 and pH 5.5, respectively). This noncompetitive inhibition was found to be transient as arginine uptake at pH 7.5; it was measured by carotid injection 30 sec following a previous bolus which was buffered to pH 4.5, and was not significantly different from the control. This selective inhibition of the blood‐brain barrier basic amino acid carrier demonstrates the advantage of the carotid injection approach in exposing the capillary exchange site to extreme alterations in chemical composition which could not be tolerated systemically.
Hypoprolactinemic Action of Calcitonin and the Tuberoinfundibular Dopaminergic System
Authors: Clementi G. , Nicoletti F. , Patacchioli F. , Prato A. , Patti F. , Fiore C.E. , Matera M. , Scapagnini U.
Published: October 2006

Abstract: The effects of calcitonin on neurochemical parameters related to the tuberoinfundibular dopaminergic system have been investigated in an attempt to elucidate how calcitonin decreases serum prolactin levels. Intracerebroventricular human or salmon calcitonin injection decreases serum prolactin, medial basal hypothalamic dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) and hypophysial DA and increases hypophysial DOPAC. Results suggest that calcitonin may decrease prolactin secretion via the tuberoinfundibular dopaminergic system.
Effects of Preweaning Undernutrition and Continued Postweaning Protein Deficiency or Nutritional Rehabilitation on Polyphosphoinositides in Rat Brain
Authors: Uma S. , Ramakrishnan C. V.
Published: October 2006

Metabolically inert polyphosphoinositides seem to play an important role in the structural development of neurons, glia, and myelin. The metabolically active pool of Phlpp appears to be important for the functional development of glia and myelin during the post‐weaning period, whereas Phlp seems to be more important for the functional development of neurons during the preweaning period. Neonatal undernutrition reduces the concentrations of structural polyphosphoinositides and metabolic PhIp while metabolic Phlpp remains unaltered. These effects can be reversed by postweaning nutritional rehabilitation. A continued postweaning protein deficiency of neonatally undernourished rats affects structural Phlpp more than PhIp. Metabolically active Phlpp is drastically reduced.
Selective Alterations of Opiate Receptor Subtypes in Mono sodium Glutamate‐Treated Rats
Authors: Young Elizabeth , Olney John , Akil Huda
Published: October 2006

Neonatal treatment of rats with monosodium glutamate (MSG) has been demonstrated to destroy cell bodies of neurons in the arcuate nucleus including the brain beta‐endorphin (B‐END) system. The effects on opiate receptors of the loss of B‐END is unknown. Neonatal rats were treated with MSG as previously described. After reaching maturity (7‐9 months), MSG‐treated rats and litter‐matched untreated control rats were decapitated and brains dissected into brain regions. Opiate receptor assays were run with [3H]morphine (mu receptor ligand) and [3H]D‐alanine2‐D‐leucine5 (DADL) enkephalin (delta receptor ligand) for each brain region for both MSG and control rats simultaneously. Scatchard plot analyses showed a selective increase in delta receptors in the thala‐mus only. No corresponding change in mu receptors in the thalamus was found. The cross‐competition IC50 data supported this conclusion, showing a loss in the potency of morphine in displacing [3H]DADL enkephalin in the thalamus of MSG‐treated rats. This shift in delta receptors produced an IC50 displacement pattern in thalamus, ordinarily a mu‐rich area, similar to that of striatum or cortex, delta‐rich areas, again indicating an increase in delta receptors. Similar changes in delta receptors in other brain regions were not found. These results represent one of the few examples of a selective and localized shift in delta with no change in mu sites. Furthermore, the delta increase may reflect an up‐regulation of the receptors in thalamus after chronic loss of the endogenous opioid B‐END.
Norepinephrine Depletion in the Spinal Cord Gray Matter of Rats with Experimental Allergic Encephalomyelitis
Authors: White S. R. , Bhatnagar R. K. , Bardo M. T.
Published: October 2006

Norepinephrine and dopamine concentrations were determined by radioenzymatic assay in discrete gray matter regions of the spinal cords of rats with experimental allergic encephalomyelitis (EAE). Norepinephrine was depleted in most spinal cord regions of EAE rats compared with controls, whereas dopamine depletion in EAE rats was restricted to the cervical dorsal horn. There was a rostrocaudal gradient of norepinephrine reduction in the spinal cords of the EAE rats with most severe depletion in the lumbar region. The results of this experiment confirmed recent anatomical observations that suggested that catecholamine‐fluorescent axons and terminals were damaged in spinal cords of rats with EAE.
Low Concentrations of Trifluoperazine Affect Striatal Cells in Culture
Authors: Messer Anne , Jennings Rosalie , Maskin Paul
Published: October 2006

Abstract: Primary monolayer cultures of the newborn rat corpus striatum were treated with the phenothiazine trifluoperazine after various times in culture. When the drug is added to cells at least 3 weeks old, concentrations of 10−7 and 10−8M appear morphologically identical to controls but show significant changes in synthesis of acetylcholine and γ‐aminobutyric acid, particularly the former. The results with these very low concentrations suggest that the drug has a highly specific effect directly on striatal cells, and that it is not acting via calmodulin.
Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations
Authors: Siman Robert , Baudry Michel , Lynch Gary
Published: October 2006

Abstract: Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium‐stimulated proteolytic activity. Addition of calcium to SPMs caused a dose‐dependent increase in trichloroacetic acid‐soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low‐ionic‐strength buffer and the extract was fractionated on DEAE‐cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1= 35 μM; Aact2= 500 μM). The specific thiol‐protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low‐sensitivity protease was converted to a high‐sensitivity enzyme (Kact= 20 μM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550‐fold from SPMs. The high‐ and low‐sensitivity membrane‐associated calcium‐dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.
Multiple Forms of Choline‐O‐Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization
Authors: Benishin Christina G. , Carroll Paul T.
Published: October 2006

Abstract: Three forms of acetyl coenzyme A: choline‐O‐acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high‐salt solution (500 mM NaCl), and a 2% Triton DN‐65 solution, respectively. The Triton‐solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl‐solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4–(1‐naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane‐bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP‐ and Triton‐solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl‐solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.
Rat Brain Norepinephrine Metabolism: Substantial Clearance Through 3,4‐Dihydroxyphenylethyleneglycol Formation
Authors: Li Peter P. , Warsh Jerry J. , Godse Damodar D.
Published: October 2006

Abstract: To assess whether the metabolic clearance of rat brain norepinephrine (NE) through 3,4‐dihydroxyphenylethyleneglycol (DHPG) formation is quantitatively comparable or greater than through 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) production, we studied the accumulation rates of conjugated DHPG and MHPG following probenecid administration in whole brain as well as in several brain regions. Administration of increasing doses of probenecid (100–500 mg/kg, i.p.) 1.5 h before sacrifice produced a dose‐dependent increase of conjugated DHPG and MHPG levels. The maximum increment of these conjugated metabolites occurred at a dose of 300 mg/kg or higher. During the first hour following probenecid administration (300 mg/kg, i.p.), rat brain conjugated DHPG and MHPG levels accumulated linearly at a rate of 646 and 319 pmol/g/h, respectively. With the probenecid technique, the estimated appearance rates of conjugated DHPG significantly exceeded those of conjugated MHPG in hypothalamus, midbrain, brainstem, hippocampus, and cerebral cortex. These results clearly indicate that under resting conditions, formation and efflux of conjugated DHPG is the major route of metabolic clearance of rat brain NE.
Fast‐Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate
Authors: Stone George C. , Hammerschlag Richard , Bobinski Judith A.
Published: October 2006

Abstract: 35SO4‐labeled fast‐transported proteins of bullfrog dorsal root ganglion neurons were separated by two‐dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine‐labeled fast‐transported proteins. The majority of these well resolved spots appeared as “families,” previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side‐chain charge that underlies microheterogeneity. The most heavily 35SO4‐labeled species, however, corresponded to fast‐transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.
Identification of an Axolemma‐Enriched Fraction from Peripheral Nerve
Authors: Yoshino Jun E. , Griffin John W. , DeVries George H.
Published: October 2006

Abstract: A method has been devised for the fractiona‐tion of whole peripheral nerve. The procedure utilizes differential centrifugation and separation on a linear sucrose gradient (10–40%, wt/wt). A membrane fraction localized between 26% and 29% sucrose was not only enriched for the plasma membrane markers, 5′‐nucleotidase and acetylcholinesterase (AChE), but also possessed the highest binding of [3H]saxitoxin, a specific marker for sodium channels. Neurons in the lumbar dorsal roots and ventral horns of rats were injected with [3H]fucose to label glycoproteins associated with the axolemma from sciatic nerve. Fractionation of the labeled nerves demonstrated a coincidence in the distribution of [3H]fucose‐labeled material and AChE activity in the sucrose density gradient. The increase in the specific activity of marker enzymes for plasma membrane, sodium channels, and labeled membrane, previously demonstrated to be of axolemmal origin, identified the 26–29% region of the sucrose gradient as enriched for axolemma derived from peripheral nerve.
[3H]Muscimol Binding Site on Purified Benzodiazepine Receptor
Authors: Martini C. , Rigacci T. , Lucacchini A.
Published: October 2006

Abstract: The presence of a [3H]muscimol binding site on the purified benzodiazepine receptor was demonstrated. The purified protein was apparently homogeneous as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (stained with silver), with a molecular weight of 60,000 ± 3000. The benzodiazepine binding sites were characterized as being of the central type and the [3H]flunitrazepam binding was enhanced by GABA. This activation was antagonized by bicuculline. [3H]Muscimol specifically binds to the benzodiazepine receptor. The Scatchard plot indicates a Kd of 23 nM and the ratio [3H]flunitrazepam/[3H]muscimol is approximately unity.
Regulation of the Muscarinic Acetylcholine Receptor: Effects of Phosphorylating Conditions on Agonist and Antagonist Binding
Authors: Burgoyne Robert D.
Published: October 2006

Abstract: Incubation of rat brain synaptic membranes under phosphorylating conditions (i.e., in the presence of Mg2+, ATP and cyclic AMP) leads to a loss in muscarinic acetylcholine receptors, detectable as specific binding of the muscarinic antagonist L‐[3H]quinuclidinyl benzilate. A role for protein phosphorylation in this receptor loss is indicated by the finding that 5′‐adenylyl imidodiphosphate, a nonhydrolysable analogue of ATP, does not support receptor loss. Furthermore, receptor loss is inhibited by adenosine and 2‐deoxyadenosine, both of which inhibit protein kinase activity. The loss of muscarinic receptors is calmodulin dependent and it has been demonstrated here that this requirement is probably at the level of calmodulin‐dependent phosphorylation. An investigation of the effects of phosphorylation on the binding of the agonist carbachol to synaptic membranes from the cortex and cerebellum demonstrated that phosphorylation altered the relative proportions of the super‐high‐, high‐ and low‐affinity binding sites. The results were consistent with an apparent conversion of high‐ into super‐high‐affinity sites. In the presence of 5′‐guanylyl imidodiphosphate, agonist binding demonstrated the properties expected of a population of largely low‐affinity sites. This conversion of super‐high‐ and high‐affinity sites into low‐affinity sites by 5′‐guanylyl imidodiphosphate was partially inhibited by phosphorylation.
A Quantitative Regional Analysis of Amino Acids Involved in Rat Brain Protein Synthesis by High Performance Liquid Chromatography
Authors: Bodsch Wolfram , Hossmann Konstantin‐Alexander
Published: October 2006

Abstract: A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood‐brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl‐labeled amino acids and terminated 2 min after infusion of 3H‐labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C‐ or 3H‐labeled phenyl‐alanine, tyrosine, leucine, isoleucine and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl‐tRNAs and proteins. Amino acids were converted to their corresponding 5‐dimethylamino‐naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed‐phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu > Tyr > Ile > Phe > Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl‐tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane‐anesthetized rat were determined to be 30 and 67–91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.
β‐N‐γ‐Glutamyl Diaminopropionic Acid, a Convulsant and an Inhibitor of Brain Glutamate Decarboxylase
Authors: Kazi S. J. , Rao S. L. N.
Published: October 2006

Abstract: The different isomers of the dipeptide β‐N‐γ‐ glutamyl diaminopropionate inhibit L‐glutamate‐1–carbox‐ylyase (GAD, EC‐4.1.1.15) activity in mouse brain homog‐enates. The l‐d isomer is the most effective as an inhibi, while the d‐d isomer is least inhibitory. The different isomers are neurotoxic to mice and the chick, the l‐d isomer being the most toxic. The neurotoxicity of the isomers in mice was also associated with a significant lowering in GAD activity in the brains of convulsing mice.
Adrenocorticotropin: AdH1–38 Is a Major Product of Biotransformation by Brain Synaptic Membranes
Authors: Codd Ellen E. , Burbach J. Peter H. , Verhoef J. Coos , Wang Xin‐Chang , Witter Albert
Published: October 2006

Abstract: Since adrenocorticotropic hormone is found in the brain, and several of its fragments affect adaptive behavior, the formation of fragments of ACTH,1–39 by a rat brain synaptic membrane fraction was investigated. Following the incubations at physiological pH conditions, the digests were fractionated by HPLC to quantitate the amounts of ACTH,1–39 remaining and products formed. Time‐ and enzyme‐dependent disappearance of ACTH,1–39 was accompanied by the accumulation of a major peptide metabolite (product B). Amino acid analysis and NH2‐‐terminal end‐group determination revealed that product B was identical to ACTH,1–38. These results indicate the predominance of carboxypeptidase activity in the degradation of ACTH 1–39 by brain synaptic membranes.
Predicted Folding of β‐Structure in Myelin Basic Protein
Authors: Stoner Gerald L.
Published: October 2006

Abstract: Predictions of myelin basic protein secondary structure have not previously considered a major role for β‐structure in the organization of the native molecule because optical rotatory dispersion and circular dichroism studies have provided little, if any, evidence for β‐structure, and because a polycationic protein is generally considered to resist folding into a compact structure. However, the Chou‐Fasman, Lim, and Robson algorithms identify a total of five β‐strands in the amino acid sequence. Four of these hydrophobic amino acid sequences (37–45, 87–95, 110–118, and 150–158) could form a hairpin intermediate that initiates folding of a Greek‐key‐type β‐structure. A second fold on the more hydrophobic side, with the addition of a strand from the N‐terminus (residues 13–21), would complete the five‐stranded an‐tiparallel β‐sheet. A unique strand alignment can be predicted by phasing the hydrophobic residues. The unusual triproline sequence of myelin basic protein (100–102) is enclosed in the 14‐residue hairpin loop. If these prolines are in the trans conformation, models show that a reverse turn could occur at residues 102–105 (Pro‐Ser‐Gln‐Gly). Algorithms do not agree on the prediction of α‐helices, but each of the two large loops could accommodate an α‐helix. Myelin basic protein is known to be phosphor‐ylated in vivo on as many as five Ser/Thr residues. Phos‐phorylation might alter the dynamics of folding if the nascent polypeptide were phosphorylated in the cytoplasm. In particular, phosphorylation of Thr‐99 could neutralize cationic residues Lys‐106 and Arg‐108 within the hairpin loop. In addition, the methylation of Arg‐108 might stabilize the hairpin loop structure through hydrophobic interaction with the side chain of Pro‐97. The cationic side chains of arginine and lysine residues located on the faces of the β‐sheet (Arg‐43, Arg‐114, Lys‐13, Lys‐92, Lys‐153, and Lys‐156) could provide sites for interaction with phospholipids and other anionic structures on the surface of the myelin lipid bilayer.