bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Morphine Administration Increases β‐Adrenergic Receptor Kinase (βARK) Levels in the Rat Locus Coeruleus
Authors: Terwilliger Rose Z. , Ortiz Jordi , Guitart Xavier , Nestler Eric J.
Published: July 2010

Abstract: Based on the established role of β‐adrenergic receptor kinase (βARK) and β‐arrestin in the desensitization of several G protein‐coupled receptors, we investigated the effect of chronic morphine administration on βARK and β‐arrestin levels in selected brain areas. Levels of βARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of βARK, i.e., βARK1 and βARK2. It was found that chronic morphine treatment produced an ∼35% increase in levels of βARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of βARK2 immunoreactivity in any of the brain regions studied. Levels of β‐arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by ∼20%) in the locus coeruleus. It is proposed that chronic morphine regulation of βARK1 and β‐arrestin levels may contribute to opioid‐receptor tolerance that is known to occur in this brain region.
n‐3 Fatty Acid Deficiency Increases Brain Protein Synthesis in the Free‐Moving Adult Rat
Authors: Giaume Murielle , Gay Nadine , Baubet Valérie , Gharib Abdallah , Durand Georges , Bobillier Pierre , Sarda Nicole
Published: July 2010

Abstract: The autoradiographic method with l‐[35S]methionine was used to determine the effects of an n‐3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n‐3 fatty acid‐deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.
Partial Structure and Mapping of the Human Myelin P2 Protein Gene
Authors: Narayanan Vinodh , Ripepi Benedetta , Jabs Ethylin Wang , Hawkins Anita , Griffin Constance , Tennekoon Gihan
Published: July 2010

Abstract: The myelin P2 protein, a 14,800‐Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin‐producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin‐producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P2 protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human‐hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P2 gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P2 gene as a candidate gene for autosomal recessive Charcot‐Marie‐Tooth disease type 4A.
Destabilization of Glial Fibrillary Acidic Protein mRNA in Astrocytes by Ammonia and Protection by Extracellular ATP
Authors: Neary J. T. , Whittemore S. R. , Zhu Q. , Norenberg M. D.
Published: July 2010

Abstract: The expression of the astrocyte‐specific intermediate filament protein, glial fibrillary acidic protein (GFAP), is decreased in hepatic encephalopathy and increased in numerous neurological conditions including brain injury. However, little is known about the molecular mechanisms that regulate GFAP expression. Here it is reported that treatment of cultured astrocytes with ammonium chloride reduces GFAP mRNA by up to 85% without inhibiting total RNA synthesis. The effect of NH4Cl was time and dose dependent. The reduction in GFAP mRNA was detected 3 h after initiation of ammonia treatment with a maximum effect observed at 24 h. Significant decreases in GFAP mRNA were observed at 2, 5, and 10 mM NH4Cl. Concurrent treatment with extracellular ATP prevented the loss of GFAP mRNA, possibly by activation of purinergic receptors. In addition, removal of ammonium chloride restored GFAP mRNA to normal levels. Nuclear runoff experiments indicated that NH4Cl did not inhibit GFAP mRNA transcription. Studies using α‐amanitin, an inhibitor of RNA polymerase II, showed that NH4Cl decreased the stability of GFAP mRNA by ∼50%. This destabilization of GFAP mRNA may be an important factor in the pathogenesis of hepatic encephalopathy. Because increased GFAP is an important component of reactive gliosis, understanding the mechanisms that destabilize GFAP mRNA may facilitate strategies to minimize the gliosis associated with brain injury.
l‐Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l‐Glutamate Without Direct Activation of mGluR1a
Authors: Thomsen Christian , Hansen Liselotte , Suzdak Peter D.
Published: July 2010

Abstract: The functional efficacies of inhibitors of l‐glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l‐Serine‐O‐sulfate was an agonist at mGluR1a (EC50 = 70 µM), mGluR2 (EC50 = 25 µM), and mGluR4 (EC50 = 324 µM). l‐Cysteine sulfinate, 1‐aminocyclobutane‐trans‐1,3‐dicarboxylate, l‐cysteine, and dl‐threo‐3‐methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC50 values of 43, 64, 463, and 488 µM, respectively, and displaced l‐[3H]glutamate binding from membranes prepared from these cells with respective IC50 values of 48, 44, 79, and 139 µM. However, d‐aspartate,l‐trans‐pyrrolidine‐2,4‐dicarboxylate, l‐threo‐3‐hydroxyaspartate, and l‐aspartate‐β‐hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC50 values of 73, 54, 57, and 430 µM) but did not displace l‐[3H]glutamate binding. These compounds inhibited Na+‐dependent l‐glutamate uptake into baby hamster kidney cells with IC50 values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l‐glutamate transporter, was significantly attenuated in the presence of l‐glutamate decarboxylase (EC 4.1.1.15) or l‐alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl‐trans‐pyrrolidine‐2,4‐dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four‐ to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l‐glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.
Plasminogen Binds Specifically to α‐Enolase on Rat Neuronal Plasma Membrane
Authors: Nakajima Kazuyuki , Hamanoue Makoto , Takemoto Nagisa , Hattori Tatsuya , Kato Kanefusa , Kohsaka Shinichi
Published: July 2010

Abstract: Plasminogen (PGn) that we identified in microglial‐conditioned medium has a neurotrophic factor‐like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor‐like molecule on the neuronal surface, this 45‐kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45‐kDa protein is identical to rat α‐enolase. In fact, PGn was found to bind to purified rat α‐enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α‐enolase. Physical properties of the 45‐kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α‐enolase. The 45‐kDa PGn‐binding protein in the plasma membrane was also recognized by anti‐rat α‐enolase antibody, and pretreatment with α‐enolase antibody markedly diminished the PGn‐binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α‐enolase is present on the cell surface of a certain population of neurons. These results suggest that α‐enolase may function as a PGn‐binding molecule on the neuronal cell surface.
Muscarinic Stimulation Promotes Cultured Purkinje Cell Survival: A Role for Acetylcholine in Cerebellar Development?
Authors: Mount Howard T. J. , Dreyfus Cheryl F. , Black Ira B.
Published: July 2010

Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µM) and pirenzepine (10 nM), but not by methoctramine (25 nM). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high‐affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine‐sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.
Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles
Authors: Wu Anfan , Colvin Robert A.
Published: July 2010

Abstract: The inhibitory effects of Na+/Ca2+ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na+/Ca2+ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na+‐dependent Ca2+ uptake in both rat brain [IC50 = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC50 = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ∼80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na+‐dependent Ca2+ release, and the inhibitory effect was enhanced by increasing the extravesicular Na+ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na+. When XIP was added at steady state (5 min after the initiation of intravesicular Na+‐dependent Ca2+ uptake), it was found that the intravesicular Ca2+ content declined with time. Analysis of unidirectional fluxes for Ca2+ at steady state showed that 50 µM XIP inhibited Ca2+ influx and efflux ∼85 and 70%, respectively. This result suggested that XIP inhibited both Na+/Ca2+ exchange and Ca2+/Ca2+ exchange but had no effect on the passive release pathway for Ca2+. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na+ or other monovalent cations, e.g., K+, is required for XIP to have its inhibitory effect on Ca2+ transport.
Cortical Cytochrome Oxidase Activity Is Reduced in Alzheimer's Disease
Authors: Mutisya Elizabeth M. , Bowling Allen C. , Beal M. Flint
Published: July 2010

Abstract: A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age‐matched controls. Complex I and complex II–III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25–30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy‐metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.
Activity of Ca2+/Calmodulin‐Dependent Protein Kinase II Following Ischemia: A Comparison Between CA1 and Dentate Gyrus in a Hippocampal Slice Model
Authors: Westgate Sara A. , Brown Jean , Aronowski Jaroslaw , Waxham M. Neal
Published: July 2010

Abstract: Both CA1 and dentate gyrus regions of the hippocampal slice exhibit an irreversible loss of synaptic transmission after exposure to in vitro ischemic conditions (buffer without oxygen and glucose). However, after shorter durations of ischemia (8–10 min) the CA1 region shows an irreversible loss of synaptic responses, whereas the dentate gyrus region completely recovers synaptic responses upon reoxygenation. To determine biochemical mechanisms underlying this differential susceptibility, we have examined changes in Ca2+/calmodulin‐dependent protein kinase II (CaM‐KII) and cyclic AMP‐dependent protein kinase activities in homogenates from CA1 and dentate gyrus regions of the hippocampal slice after increasing durations of in vitro ischemia. Time‐dependent changes in CaM‐KII activities were correlated with changes in electrophysiological responses. CA1 homogenates from slices exposed to 1 min of ischemia showed significant increases in CaM‐KII activity, whereas there was no significant change in kinase activity in dentate homogenates after 1 min of ischemia. However, after longer durations of ischemia (5, 10, and 20 min) we found a time‐dependent reduction in CaM‐KII activity in both CA1 and dentate gyrus regions, whereas no change was detected in cyclic AMP‐dependent protein kinase activity. Irreversible depression of CaM‐KII activity was seen at shorter durations of ischemia (10 min) in the CA1 region than in dentate region (20 min), which correlated with irreversible effects on synaptic responses. Immunoblot analysis showed that the decrease in CaM‐KII activity was not due to degradation of CaM‐KII protein. However, the microtubule‐associated protein MAP2, known to be a substrate for the Ca2+‐dependent proteases (calpains), showed extensive proteolysis evident after 90 min of reoxygenation after ischemia.
Selective Patterns of Expression of G Protein α Subunits During In Vitro Development of Hypothalamic Neurons
Authors: Viollet Cécile , Loudes Catherine , Kordon Claude , Faivre‐Bauman Annie
Published: July 2010

Abstract: Expression of different α subunits of G proteins was studied in hypothalamic primary cultures grown in defined medium and enriched in either neurons or glial (astrocyte) cells. In parallel, the cellular distribution of Gi, Gs, and GoA subunits was visualized by in situ hybridization. Immunoblots using specific antisera and hybridization of mRNAs with specific oligonucleotide probes allowed us to characterize Gs, Gi2, and GoA as major neuronal G proteins in the hypothalamus, whereas the glial cells expressed mostly Gs, Gi2, and GoB forms. Gi was found to be expressed very early and transiently in the culture, whereas expression of Gs and GoA increased regularly with time.
Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System
Authors: Tsutsumi Keisuke , Niwa Masami , Kitagawa Naoki , Yamaga Sei‐ich , Anda Takeo , Himeno Akihiko , Sato Takaya , Khalid Humayun , Taniyama Kohtaro , Shibata Shobu
Published: July 2010

Abstract: We identified and characterized 125I‐endothelin‐1 (125I‐ET‐1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High‐affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (KD = 4.7 ± 1.0 × 10−10 and 1.6 ± 0.3 × 10−10M, respectively; Bmax = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ‐123, a selective antagonist for the ETA receptor, potently competed for 125I‐ET‐1 binding to sections of the microvessels with IC50 values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10−6M BQ‐123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET‐3 showed two components (IC50 = 5.7 ± 2.5 × 10−10 and 1.4 ± 0.2 × 10−6M for tumor microvessels, 1.8 ± 0.6 × 10−10 and 1.1 ± 0.3 × 10−6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high‐affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ETA subtype.
Localization of Specific Epitopes on Human Microtubule‐Associated Protein 2
Authors: Kalcheva N. , Albala J. S. , Binder L. I. , Shafit‐Zagardo B.
Published: July 2010

Abstract: Microtubule‐associated protein 2 (MAP‐2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP‐2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP‐14, AP‐20, AP‐21, AP‐23, and AP‐25) share epitopes with only the high molecular weight isoforms (MAP‐2a, MAP‐2b); two of the mAbs (AP‐18 and tau 46) recognize MAP‐2a, MAP‐2b, and MAP‐2c. Although AP‐18 immunoreactivity was detected within heat‐stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP‐2 were negative, suggesting that the AP‐18 epitope is phosphorylated. Furthermore, AP‐18 immunoreactivity was lost after alkaline phosphatase treatment of heat‐stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP‐2. AP‐21 maps to a region between amino acids 553 and 645. AP‐23 maps between amino acids 645 and 993, whereas AP‐20, AP‐14, and AP‐25 map between amino acids 995 and 1332. Expression of the region of MAP‐2 between amino acids 1787 and 1824 was positive to tau 46.
Characterization and Expression of the cDNA Coding for the Human Myelin/Oligodendrocyte Glycoprotein
Authors: Pham‐Dinh Danielle , Allinquant Bernadette , Ruberg Merle , Della Gaspera Bruno , Nussbaum Jean‐Louis , Dautigny André
Published: July 2010

Abstract: We report here the characterization of a full‐length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3′ untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3′ untranslated Alu‐containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.
A Ewing's Sarcoma Cell Line Showing Some, but Not All, of the Traits of a Cholinergic Neuron
Authors: O'Regan Seana , Diebler Marie‐Françoise , Meunier François‐Marie , Vyas Sheela
Published: July 2010

Abstract: The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high‐affinity, hemicholinium‐3‐sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High‐affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15‐kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium‐dependent release but lacks an organized release mechanism.
myc‐Immortalized Microglial Cells Express a Functional Platelet‐Activating Factor Receptor
Authors: Righi Marco , Letari Ornella , Sacerdote Paola , Marangoni Franca , Miozzo Amelia , Nicosia Simonetta
Published: July 2010

Abstract: The autacoid platelet‐activating factor (PAF) takes part in a complex network of interactions regarding the cellular components of nervous tissues. Efforts aimed at characterizing the effects of PAF in the brain have been recently focalized on neurons because PAF exerts pleiotropic effects on these cells. Less attention has instead been paid to the glial component of the brain. We have used microglial cell lines immortalized from 13‐day‐old mouse embryo brains by a myc‐transducing retrovirus. When exposed to physiological doses of PAF, immortalized microglial cells showed increases in intracellular free calcium concentrations due to release of calcium from internal stores, as well as to extracellular calcium influxes. These profiles of reactivity were independent from the immortalizing process, being observable in primary microglial cultures and in immortalized clones showing different proliferative rates. PAF was also able to induce transient expression of the c‐fos protooncogene in serum‐starved cultures and induced a strong chemotactic response in microglial cells. In contrast with control macrophage cultures, PAF did not promote prostaglandin or leukotriene synthesis in immortalized cells. This was most likely due to the low amount of total arachidonic acid found in immortal microglia, with respect to that observed in freshly isolated cells. Our data suggest that several of the effects observed after PAF stimulation might be independent from PAF‐induced arachidonic acid metabolism. The availability of an in vitro microglial model might now help in studying the proinflammatory effects of PAF, both direct or microglia mediated, in the neural environment.
Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla
Authors: Petit Koen , De Block Jan , De Potter Werner
Published: July 2010

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A2 (PLA2), which is localized in the cytosol. In the present study, this PLA2 was purified 1,097‐fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µM Ca2+ and has a pH optimum near 8.0. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA2 is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X‐100 (0.01%) and deoxycholate (1 mM) but inhibited at higher concentrations (0.1% and 3 mM, respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p‐Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA2, has little effect until 100 µM, and 2–10 mM dithiothreitol totally inactivated the enzyme. The purified PLA2 displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn‐2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA2, which was isolated and characterized in this study, represents a type of PLA2 that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA2 will help to reveal whether the enzyme is important for exocytosis.
Uptake and Release of d‐Aspartate, GABA, and Glycine in Guinea Pig Brainstem Auditory Nuclei
Authors: Suneja S. K. , Benson C. G. , Gross J. , Potashner S. J.
Published: July 2010

Abstract: This study attempts to determine if the medial (MSO) and lateral superior olive (LSO), medial nucleus of the trapezoid body (MNTB), ventral nucleus of the lateral lemniscus (VNLL), and central nucleus of the inferior colliculus (ICc) contain glutamatergic synaptic endings. Micropunch and microdissection procedures provided fresh samples of these auditory nuclei for the measurement of the high‐affinity uptake and electrically evoked release of exogenous d‐[3H]ASP. The study also determined if the LSO and MSO contain glycinergic synaptic endings by measuring uptake and release of [14C]‐Gly in these nuclei, and whether the MNTB, VNLL, and ICc contain GABAergic endings by assessing the uptake and release of [14C]GABA in these structures. Several strategies optimized the evoked Ca2+‐dependent release of the labeled amino acids. These included the enhancement of high‐affinity uptake during loading of the markers into the tissues, inhibition of uptake during the subsequent measurement of release, and use of an electrical stimulus current that evoked maximal Ca2+‐dependent release. Each of these nuclei manifested the high‐affinity uptake and the evoked Ca2+‐dependent release of d‐[3H]Asp, suggesting the presence of synaptic endings that may use Glu or Asp as a transmitter. Similar findings suggest the presence of glycinergic synaptic endings in the LSO and MSO, and of GABAergic synaptic endings in the MNTB, VNLL, and ICc.
Nerve Growth Factor Amplifies Cyclic AMP Production in the HT4 Neuronal Cell Line
Authors: Berg Kelly A. , Maayani Saul , McKay Ronald , Clarke William P.
Published: July 2010

Abstract: There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml 15 min) amplified cAMP accumulation (≈75%) in response to activation of adenosine A2 receptors (5 min) with 5′‐N‐ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down‐regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF‐induced amplification of cAMP accumulation. Although NGF‐induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF‐mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.
Intracerebral Dialysis and the Blood‐Brain Barrier
Authors: Westergren Irena , Nyström Britta , Hamberger Anders , Johansson Barbro B.
Published: July 2010

Abstract: The aim of the study was to evaluate how implantation of a dialysis probe influences the blood‐brain barrier. Leakage of endogenous serum albumin was evaluated by Evans blue/albumin staining and by immunohistochemistry. The passage from blood to dialysate of two substances that normally do not pass into the brain, [3H]inulin and glutamate, was studied 3 and 24 h after insertion of a dialysis probe. Evans blue, given 20 min before rats were killed, was observed around the probe and surrounding brain tissue. Albumin immunoreactivity was seen at considerable distance from the probe with larger spread at 24 h than at 3 h after probe insertion. Glutamate and [3H]inulin were detected in the dialysate with no significant further increase of radioactivity after intracarotid infusion of protamine sulfate that enhances the permeability over the blood‐brain barrier. When protamine was followed by infusion of glutamate, the concentrations of taurine increased in the dialysate in four of eight rats. That plasma constituents have access to the brain around the dialysis probe is essential to consider, particularly in studies using substances and drugs that do not pass an intact blood‐brain barrier.
Characterization of the Substance P (NK‐1) Receptor in Tunicamycin‐Treated Transfected Cells Using a Photoaffinity Analogue of Substance P
Authors: Kage R. , Hershey A. D. , Krause J. E. , Boyd N. D. , Leeman S. E.
Published: July 2010

Abstract: Chinese hamster ovary cells expressing the N‐glycosylated substance P (NK‐1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with 125I‐Bolton‐Hunter‐p‐benzoyl‐l‐phenylalanine8‐substance P. Two radioactive proteins of Mr 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK‐1) receptor, respectively, were observed. The IC50 for the inhibition of photolabeling of both receptor forms was 0.3 ± 0.1 nM for substance P and 30 ± 5 nM for neurokinin A (substance K). Thus, glycosylation of the substance P (NK‐1) receptor has no detectable effect on the affinity of the substance P (NK‐1) receptor for substance P or neurokinin A (substance K).
Evaluation of Potential Effectors of Agonal Glycolytic Rate in Developing Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy
Authors: Corbett Ronald J. T. , Sterett Rick , Laptook Abbot R.
Published: July 2010

Abstract: Previously we have shown that hypercarbia produces a larger decrease in agonal glycolytic rate in 1‐month‐old swine than in newborns. In an effort to understand the mechanism responsible for this difference, we tested the hypothesis that hypercarbia produces age‐related changes in the concentration of one or more effectors of phosphofructokinase activity. Specifically, in vivo 31P and 1H NMR spectroscopy was used to compare changes in lactate levels, intracellular pH, free magnesium concentration, and content of phosphorylated metabolites for these two age groups at three intervals during the first 1.5 min of complete ischemia in the presence or absence of hypercarbia (Paco2 = 102–106 mm Hg). Hypercarbia produced the same drop in intracellular brain pH for both age groups, but the decrease in phosphocreatine level and increase in inorganic phosphate content were greater in 1‐month‐olds compared with newborns. During ischemia there was no difference between the magnitude of change in intracellular pH and levels of phosphocreatine and inorganic phosphate in hypercarbic 1‐month‐olds versus newborns. Under control conditions, i.e., normocarbia and normoxia, the free Mg2+ concentration was lower and the fraction of magnesium‐free ATP was higher for newborns than 1‐month‐olds. However, there was no change in these variables for either age group during hypercarbia and early during ischemia. Thus, age‐related differences in the relative decrease in agonal glycolytic rate during hypercarbia could not be explained by differences in intracellular pH, inorganic phosphate content, or free magnesium concentration. The [ADP]free at control was higher in newborns compared with 1‐month‐olds, and there was no age‐related difference in [AMP]free. These variables did not change for newborns when exposed to hypercarbia, but for 1‐month‐olds [ADP]free and [AMP]free increased during hypercarbia relative to control values. High‐energy phosphate utilization during ischemia for hypercarbic 1‐month‐olds was reduced by 74% compared with normocarbic 1‐month‐olds during ischemia, whereas the reduction in energy utilization (14%) was not significant for hypercarbic versus normocarbic newborns during ischemia. Because hypercarbia reduces the rate of ATP depletion during ischemia in 1‐month‐olds to a greater extent than in newborns, the increase in [ADP]free and [AMP]free will be slower in the former age group. It follows therefore that for 1‐month‐olds, the agonal glycolytic rate would not be accelerated by ADP and AMP to the same degree during hypercarbia plus ischemia compared with normocarbic plus ischemia, whereas for newborns hypercarbia has relatively little impact on agonal glycolytic rate.
Rat Brain Protein Phosphatase 2A: An Enzyme that May Regulate Autophosphorylated Protein Kinases
Authors: Barnes Gregory N. , Slevin John T. , Vanaman Thomas C.
Published: July 2010

Abstract: Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B′ subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves (Ki = 0.05 nM) nearly identical to that reported for skeletal muscle PP2A. The isolated 38‐kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross‐reactivity with a specific monoclonal antibody (PY‐20). Amino acid compositions and sequences of peptides isolated from the 65‐ and 38‐kDa species correspond to regions of the cDNA‐deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca2+‐dependent K+‐depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45‐s time course. The decrease was blocked by 1 nM okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.
Two Stages in Neurite Formation Distinguished by Differences in Tubulin Metabolism
Authors: Sekimoto Sumito , Tashiro Tomoko , Komiya Yoshiaki
Published: July 2010

Abstract: Changes in tubulin solubility during neurite formation were studied biochemically using rat dorsal root ganglion neurons in culture. When fractionated with Ca2+‐containing buffer at low temperature, a considerable proportion of total cellular tubulin was recovered in the insoluble fraction. We designated this cold/Ca2+‐insoluble tubulin (InsT) and distinguished it from cold/Ca2+‐soluble tubulin (SoIT). From the relative amount of InsT, neurite formation was found to proceed through two distinct stages. The first 6 days after plating (stage 1) in which the proportion of InsT increased dramatically (from 5 to 60%) coincided with neurite outgrowth. In the following period (stage 2), a constant level of InsT was maintained, whereas neurite maturation took place. Pulse‐labeling experiments further revealed that the two stages differed significantly in terms of tubulin metabolism. High rates of synthesis as well as conversion from SoIT to InsT were observed in stage 1, whereas stage 2 was characterized by a decrease in both of these rates and an increase in the rate of degradation. The results show for the first time the coordinated changes in tubulin metabolism that underlie the process of neurite formation.
Production and Characterization of Monoclonal Antibodies to Bovine Brain Succinic Semialdehyde Reductase
Authors: Choi Eui Yul , Park Sang Yeol , Jang Sang Ho , Song Min‐Sun , Cho Sung‐Woo , Choi Soo Young
Published: July 2010

Abstract: Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross‐reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass—34 kDa—in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.
Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo
Authors: Pirttilä Tiina‐R. M. , Auriola Seppo O. K. , Kauppinen Risto A.
Published: July 2010

Abstract: Metabolism of [1‐13C]glucose was monitored in superfused cerebral cortex slice preparations from 1‐, 2‐, and 5‐week‐old rats using 1H‐observed/13C‐edited (1H{13C}) NMR spectroscopy. The rate of label incorporation into glutamate C‐4 did not differ among the three age groups: 0.52–0.67% of total 1H NMR‐detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1‐, 2‐, and 5‐week‐old animals, respectively. Steady‐state glutamate C‐4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C‐4 enrichments were smaller, however, in 1‐ and 2‐week‐old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5‐week‐old rats (22.7 ± 0.7%) after 75 min of incubation with 5 mM [1‐13C]glucose. We add a new assignment to the 1H{13C} NMR spectroscopy, as acetate C‐2 was detected in slice preparations from 5‐week‐old animals. In the acid extracts of slice preparations acetate C‐2 was labeled by ∼30% in 5‐week‐old rats but by 15% in both 1‐ and 2‐week‐old animals, showing that the turnover rate was increased in 5‐week‐old animals. In the extracts 3–4% of the C‐6 of N‐acetyl‐aspartate (NAA; CH3 of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1‐13C]glucose but not from [2‐13C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.
Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve
Authors: García R. A. G. , Villegas J.
Published: July 2010

Abstract: The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea. Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm‐free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.
Dexamethasone Up‐Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture
Authors: Baltrons Maria Antonia , Agulló Luis , García Agustina
Published: July 2010

Abstract: Treatment of rat cerebellar astrocyte‐enriched primary cultures with dexamethasone enhances the nitric oxide‐dependent cyclic GMP formation induced by noradrenaline in a time‐(>6 h) and concentration‐dependent manner (half‐maximal effect at 1 nM). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [3H]arginine to [3H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up‐regulate astroglial calcium‐dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long‐term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.
NMDA Receptor Involvement in Toxicity to Dopamine Neurons In Vitro Caused by the Succinate Dehydrogenase Inhibitor 3‐Nitropropionic Acid
Authors: Zeevalk Gail D. , Derr‐Yellin Ethel , Nicklas William J.
Published: July 2010

Abstract: Exposure of mesencephalic dopamine neurons to an irreversible inhibitor of succinate dehydrogenase (SDH), 3‐nitropropionic acid (3‐NPA), for 24 h on day 12 in vitro, produced a dose‐dependent loss of high‐affinity dopamine uptake when measured 48 h following 3‐NPA removal. ATP concentrations in the cultures were reduced by 57% after 3 h of treatment with the highest concentration of 3‐NPA tested (500 µM). To determine whether glutamate receptors mediated the dopamine toxicity by 3‐NPA, cultures were examined for their sensitivity to excitatory amino acid‐induced toxicity. Mesencephalic cultures exposed to either 100 µM NMDA or kainate, on day 12 for 24 h, showed complete loss of dopamine uptake following 48 h of recovery. The NMDA and non‐NMDA antagonists, MK‐801 (1 µM) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 15 µM), completely prevented the effects of NMDA or kainate, respectively, when present at the time of toxin exposure. In cultures treated with 3‐NPA, MK‐801, but not CNQX, significantly attenuated the loss of dopamine uptake. Direct measurement of the effect of 3‐NPA on SDH activity showed that 3‐NPA dose‐dependently inhibited SDH in vitro in a manner commensurate with the loss of dopamine uptake by 3‐NPA. MK‐801 had no effect on basal SDH activity or on 3‐NPA inhibition of SDH. These data are consistent with the interpretation that metabolic inhibition in dopamine neurons can trigger a secondary excitotoxicity that is mediated by NMDA receptors.
An HSV‐1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l‐DOPA from Cultured Rat Striatal Cells
Authors: Geller Alfred I. , During Matthew J. , Oh Young J. , Freese Andrew , O'Malley Karen
Published: July 2010

Abstract: In this report we demonstrate that a defective herpes simplex virus type one (HSV‐1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l‐DOPA‐producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV‐1 vector (pHSVth) and packaged into virus particles using an HSV‐1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of l‐DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of l‐DOPA and low levels of dopamine were observed from cells in pHSVth‐infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.
Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain
Authors: Zhong Jie , Carrozza David P. , Williams Keith , Pritchett Dolan B. , Molinoff Perry B.
Published: July 2010

Abstract: Developmental changes in the levels of N‐methyl‐d‐aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low‐affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5′ insert and with or without both 3′ inserts. In cerebellum, however, the major NR1 variants were those containing the 5′ insert and lacking both 3′ inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.