bluuebunny/crossref_metadata_2025 huggingface dataset:
Excitatory Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of Adenosine Receptor Agonists and Antagonists
Authors: Simpson R. E. , O'Regan M. H. , Perkins L. M. , Phillis J. W.
Published: October 2006

Abstract: The effects of selective adenosine receptor agonists [N6‐cyclopentyladenosine (CPA) and N‐ethylcarboxamidoadenosine (NECA)] and antagonists {8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) and 9‐chloro‐2‐(2‐furanyl)‐5,6‐dihydro‐1,2,4‐triazolo[1,5‐c]quinazoline‐5‐imine (CGS‐15943A)} on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four‐vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug‐treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10−10M) and NECA (10−9M) significantly inhibited the ischemia‐evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia‐evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10−6M) or NECA (10−5M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia‐evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS‐15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia‐evoked release of excitatory neurotransmitter amino acids via high‐affinity A1 receptors, whereas coactivation of lower‐affinity A2 receptors may block (or reverse) the A1‐mediated response.
Glucocorticoids Exacerbate Kainic Acid–Induced Extracellular Accumulation of Excitatory Amino Acids in the Rat Hippocampus
Authors: Stein‐Behrens Becky A. , Elliott Elicia M. , Miller Chris A. , Schilling James W. , Newcombe Robert , Sapolsky Robert M.
Published: October 2006

Abstract: Glucocorticoids (GCs) compromise the ability of hippocampal neurons to survive various insults, and do so, at least in part, by exacerbating steps in the glutamate/N‐methyl‐D‐aspartate (NMDA)/calcium cascade of damage. As evidence, GCs impair uptake of glutamate by hippocampal astrocytes, the GC endangerment of the hippocampus is NMDA receptor dependent, and GCs exacerbate kainic acid (KA)‐induced calcium mobilization. These observations predict that GCs should also exacerbate KA‐induced accumulation of extracellular glutamate and aspartate. To test this, adrenalectomized rats were given replacement GCs in either the low or high physiological range. Three days later, rats were anesthetized and 1 mM KA was infused through a dialysis probe placed in the dorsal hippocampus. Extracellular amino acid concentrations in the dialysate were then assessed by HPLC. After KA infusion, high‐GC rats (30 ± 3 μg/dl) had significantly elevated concentrations of glutamate and aspartate compared with low‐GC rats (all <0.95 μg/dl). The glutamate accumulation was due to GCs raising pre‐KA concentrations, whereas the aspartate accumulation was due to GCs exacerbating the KA‐induced rise. Glutamine concentrations were unaffected by KA, whereas the high‐GC regimen elevated glutamine concentrations both before and after KA. Taurine concentrations rose after infusion of KA, but were unaffected by GC regime, whereas alanine concentrations were unaffected by either manipulation. Serine concentrations were unaffected by KA, but were depressed both before and after KA in high‐GC rats. The high‐GC effects on glutamate, aspartate, and serine concentrations could be reversed by supplementing rats with the sugar mannose; this agrees with prior observations that GC endangerment of the hippocampus and GC exacerbation of other steps in the glutamate/NMDA/calcium cascade are reversible with energy supplementation. A variety of steps are suggested by which GCs, exerting their known disruptive effects on hippocampal energetics, could cause excessive accumulation of extracellular glutamate and aspartate during neurological insults and thus exacerbate the subsequent toxicity.
Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells
Authors: Kuchler Sabine , Lehmann Sylvain , Vincendon Guy , Zanetta Jean‐Pierre
Published: October 2006

Abstract: In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in nonmyelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31‐kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.
Expression of Functional Membrane‐Bound and Soluble Catechol‐O‐Methyltransferase in Escherichia coli and a Mammalian Cell Line
Authors: Malherbe Parichehr , Bertocci Barbara , Caspers Patrick , Zürcher Gerhard , Da Prada Mosé
Published: October 2006

Abstract: Human catechol‐O‐methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane‐bound catechol‐O‐methyltransferase (MB‐COMT; amino acids 1‐271) and the soluble catechol‐O‐methyltransferase COMT (S‐COMT; Δ membrane anchor hCOMT; amino acids 27‐271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high‐level synthesis of catalytically active enzymes was obtained. Insertion of the human MB‐COMT‐coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV‐hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol‐O‐methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37°C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 μM for MB‐COMT and 108 μM for S‐COMT, indicating that recombinant MB‐COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of ∼ 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue. Using the polymerase chain reaction (PCR) with primers surrounding the putative membrane anchor region, we have clearly identified a single‐size PCR product generated from hCOMT mRNA of various human tissues. Hence, the two forms of the enzyme cannot be the products of an alternative splicing of transcripts. We suggest that S‐COMT is generated by proteolytic cleavage between the NH2‐terminal membrane anchor and the catalytic domain of the membrane‐bound form. Lack of the N‐terminal fragments reduces the catalytic activity of the enzyme.
A Rabbit Autoantibody Specific for the 46‐kDa Form of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase
Authors: Möller Johanna R. , Ramaswamy Sengoda G. , Jacobowitz David M. , Quarles Richard H.
Published: October 2006

Abstract: An autoantibody occurring in the serum of an apparently normal rabbit that immunocytochemically stains myelin sheaths and oligodendrocytes in rat brain was shown to react specifically with the 46‐kDa isoform of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) (EC 3.1.4.37) in a number of species. Identification of the shorter isoform of the enzyme (CNP1) as the antigen was achieved by comparing the immunostaining of Western blots by the autoantibody with that of a well‐characterized anti‐CNP antiserum. The 46‐kDa antigen reacting with the autoantibody exhibited the same Mr and pI as the small isoform of CNP on two‐dimensional gels and showed a similar enrichment in purified CNS myelin. The autoantibody has very high affinity for CNP1 and is capable of detecting the very low amounts of this enzyme in peripheral nerve, spleen, adrenal gland, pancreas, testis, and intestine. Testing the reactivity of the autoantibody with synthetic peptides by enzyme‐linked immunosorbent assay revealed that it reacted with the N‐acetylated decapeptide corresponding to the N‐terminus of CNP1, but did not react if the peptide was not acetylated or if the acetyl group was replaced with a palmityl group. The lack of reactivity with CNP2, which differs from CNP1 by a 20‐amino acid extension at the N‐terminus of the protein as a result of alternative splicing, may be due to the absence of the N‐acetyl moiety that is part of the epitope and/or blocking of antibody binding to the decapeptide by extension of the polypeptide chain. This highly specific antibody for the shorter isoform of CNP may be useful in studies to establish possible differences in the location or function of the two isoforms of this enzyme.
Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat
Authors: Soares Holly D. , Thomas Mark , Cloherty Keri , McIntosh Tracy K.
Published: October 2006

Abstract: The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid‐percussion brain injury. Animals receiving fluid‐percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid‐percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p < 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p < 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p < 0.05) and injured hippocampus (10 min post injury, p < 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p < 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24‐h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid‐percussion brain injury and that these changes may persist for a prolonged period after brain injury.
Changes in the Turnover of Monoamines in Prefrontal Cortex of Rats Fed on Vitamin E‐Deficient Diet
Authors: Castaño Angélica , Venero José Luis , Cano Josefina , Machado Alberto
Published: October 2006

Abstract: Turnover of noradrenaline (NA), dopamine (DA), serotonin (5‐HT), and their metabolites has been measured after a 15‐day vitamin E‐deficient diet in adult rat prefrontal cortex. Turnover rates of 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxy‐3‐indoleacetic acid have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase. NA, DA, and 5‐HT turnover rates have been measured as accumulation rates of NA, DA, and 5‐HT after pargyline inhibition of monoamine oxidase. No change was found in the turnover rate of NA between control and experimental animals. In contrast, turnover rates of DA and homovanillic acid significantly increased in the animals fed on a low‐vitamin E diet. However, the most striking results were found on the serotoninergic system. Levels of 5‐HT and its main metabolite, 5‐hydroxy‐3‐indoleacetic acid, and their respective turnover rates were lower in the vitamin E‐deficient diet. These results could indicate that vitamin E is necessary for the normal functioning of the serotoninergic neurons in the rat prefrontal cortex. The involvement of vitamin E in preventing the formation of free radicals is well known. Therefore, this lack of protective effect after a 15‐day vitamin E‐deficient diet could be responsible for the neuronal damage to the serotoninergic system. The opposing results found in DA (increase) and 5‐HT (decrease) turnover could provide further evidence for an inhibitory control of the serotoninergic ascending pathways to the dopaminergic system in the prefrontal cortex.
Release of Excitatory Amino Acids from Cultured Hippocampal Astrocytes Induced by a Hypoxic‐Hypoglycemic Stimulation
Authors: Ogata Tadanori , Nakamura Yoichi , Shibata Taiho , Kataoka Kiyoshi
Published: October 2006

Abstract: An excess release of excitatory amino acids (EAA) is an important factor for postischemic brain damage. In the present communication, we demonstrate that cultured hippocampal cells release EAA after hypoxic‐hypoglycemic treatment. The amounts of EAA released from astrocytes were appreciably above those released from neurons. Furthermore, the amount of aspartate released from astrocytes was comparable to that of glutamate, although the endogenous content of aspartate was one‐fifth that of glutamate. The endogenous content of aspartate in astrocytes increased even after hypoxic‐hypoglycemic treatment. These results suggests that ischemic neuronal death is due, at least in part, to the excitotoxicity of aspartate and glutamate‐derived from surrounding astrocytes.
Evidence for Distinct Neuronal Localization of γ and δ Subunits of Ca2+/Calmodulin‐Dependent Protein Kinase II in the Rat Brain
Authors: Takaishi Tomoaki , Saito Naoaki , Tanaka Chikako
Published: October 2006

Abstract: Localization of the γ and δ types of mRNAs for Ca2+/calmodulin‐dependent protein kinase II (CaM‐kinase II) was determined in the rat brain, making use of in situ hybridization histochemistry. The γ and δ mRNAs as well as the α and β mRNAs for CaM‐kinase II were heterogeneously and distinctly distributed. In the Purkinje cell layer of the cerebellum, α, γ, and δ mRNAs but not δ mRNA were present, whereas β, γ, and δ mRNAs were present in the locus coeruleus. These findings provide evidence that CaM‐kinase II exists in a variety of forms in different cells composed of a variable number and type of subunits.
A τ‐Related Protein of 130 kDa Is Present in Alzheimer Brain
Authors: Gache Y. , Guilleminot J. , Ricolfi F. , Theiss G. , Nunez J.
Published: October 2006

Abstract: Two abnormal entities of 69 and 130 kDa, immunologically related to the microtubule‐associated τ proteins, are present in the hippocampus and the frontal cortex of the Alzheimer brain, which contain a large number of neurofibrillary tangles (NFTs), but are absent in the cerebellum, which does not contain these structures. Epitope mapping with antibodies spanning domains present in the N‐terminal, middle, and C‐terminal τ sequence demonstrated that the 69‐ and 130‐kDa entities belong to the τ family. Both the 69‐ and the 130‐kDa proteins were found in an insoluble form and were the major τ species present in purified NFTs. A procedure was devised that allowed us to prepare from Alzheimer hippocampi two NFT fractions differing in size (20 and 3 μm), both of which contained the τ entities of 130 and 69 kDa.
Secretion of Carboxypeptidase E from Cultured Astrocytes and from AtT‐20 Cells, a Neuroendocrine Cell Line: Implications for Neuropeptide Biosynthesis
Authors: Klein Robyn S. , Das Banasree , Fricker Lloyd D.
Published: October 2006

Abstract: Cultured astrocytes have recently been shown to produce certain neuropeptides, as well as neuropeptide processing enzymes. To characterize the secretory pathway in cultured astrocytes, we used the neuropeptide processing enzyme carboxypeptidase E (CPE) as a marker for neuropeptide secretion. Cultured astrocytes and AtT‐20 cells, a mouse pituitary‐derived neuroendocrine cell line, were labeled with [35S]Met for 15 min and then chased with unlabeled Met. CPE was isolated from either medium or cell extracts using a substrate affinity column. The time course of secretion of radiolabeled CPE was significantly different for cultured astrocytes as compared with AtT‐20 cells. CPE was rapidly secreted from the astrocytes after a 30‐min lag time, presumably reflecting transport through the endoplasmic reticulum and Golgi apparatus, followed by constitutive secretion. The secretion of radiolabeled CPE was essentially complete by 2 h. In contrast, only a portion of the radiolabeled CPE was secreted from AtT‐20 cells over a 2–3‐h period, indicating that the majority of newly synthesized CPE is stored, presumably in secretory granules within the AtT‐20 cells. The regulation of CPE secretion from astrocytes was also examined. CPE secretion is stimulated two‐ to threefold by prolonged treatment (3–48 h) with the phorbol ester 12‐O‐tetra‐decanoylphorbol 13‐acetate (TPA) but not by treatment with other secretagogues that stimulate CPE secretion from AtT‐20 cells (forskolin, isoproterenol, A23187, and vasoactive intestinal peptide) or short (<3 h) exposure to TPA. Taken together, these results indicate that the secretory pathway for CPE, and presumably neuropeptides, is substantially different in astrocytes than the secretory pathway for CPE in neuroendocrine cells.
Actin Depolymerizing Factor Is a Component of Slow Axonal Transport
Authors: Bray John J. , Fernyhough Paul , Bamburg James R. , Bray Dennis
Published: October 2006

Abstract: We examined the low molecular weight proteins transported with actin in the chicken sciatic nerve after injection of [35S]methionine into the lumbar spinal cord. A prominent component of slow axonal transport with apparent molecular mass 19 kDa comigrated on two‐dimensional gels with chicken actin depolymerizing factor (ADF), previously shown to be a major actin‐binding protein in brain. There was comparatively little radioactivity associated with the actin monomer sequestering proteins, profilin or cofilin, and examination of the rapid component of axonal transport failed to reveal appreciable quantities of actin, ADF, profilin, or cofilin. These results show that both actin and ADF are carried by slow axonal transport and raise the possibility that actin travels within the axon in an unpolymerized form in a complex with ADF.
Membrane Localization of Endopeptidase‐24.11 and Peptidyl Dipeptidase A (Angiotensin Converting Enzyme) in the Pig Brain: A Study Using Subcellular Fractionation and Electron Microscopic Immunocytochemistry
Authors: Barnes Kay , Turner Anthony J. , Kenny A. John
Published: October 2006

Abstract: Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand‐binding assays using (–)‐[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase‐24. 11 exhibited a biphasic distribution, consistent with its presence on both pre‐ and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity‐purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity‐purified antibody to endopeptidase‐24. 11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre‐ and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase‐24.11 is present on both pre‐ and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.
Inhibition of Tyrosine Hydroxylase by R and S Enantiomers of Salsolinol, 1‐Methyl‐6,7‐Dihydroxy‐1,2,3,4‐ Tetrahydroisoquinoline
Authors: Minami Midori , Takahashi Tsutomu , Maruyama Wakako , Takahashi Akira , Dostert Philippe , Nagatsu Toshiharu , Naoi Makoto
Published: October 2006

Abstract: Salsolinol is one of the dopamine‐derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood‐brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3‐hydroxylating); EC 1.14.16.2], the rate‐limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring co‐factor of tyrosine hydroxylase, l‐erythro‐5,6,7,8‐tetra‐hydrobioptein, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using l‐erythro‐5,6,7,8‐tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C‐1 played an important role in changing the affinity to l‐tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.
A Transcriptional Regulatory Element of the Gene Encoding the 67,000‐Mr Form of Human Glutamate Decarboxylase Is Similar to a Drosophila Regulatory Element
Authors: Erlander M. G. , Tobin A. J.
Published: October 2006

Abstract: We have isolated the 5’flanking DNA sequences of the human gene encoding the 67,000‐Mr form of glutamate decarboxylase (GAD67), the γ ‐aminobutyric acid synthetic enzyme. Transcription begins at a single promoter (P1) in adult brain but at two tandem promoters, P1 and P2, in fetal brain. P1, which is 3’to P2, resembles the promoter regions of many constitutively expressed genes, whereas P2 resembles a tissue‐specific promoter. P1 contains a 10‐base sequence (dec‐1) that closely matches the element I cis‐regulatory sequence identified in the promoter region of Drosophila 3,4‐dihydroxyphenylalanine decarboxylase. Gel shift and transient expression assays demonstrate that the dec‐1 sequence plays a role in the transcription of the human GAD67 gene.
Muscarinic Binding of Pial Vessels and Arachnoid Membrane
Authors: Lasbennes F. , Verrecchia C. , Philipson V. , Seylaz J.
Published: October 2006

Abstract: The muscarinic sites in arachnoid and pial vessels were compared by analysis of the binding of quinuclidinyl benzilate (QNB) to membrane preparations. Saturation analysis indicated that the process was saturable, high affinity, and related to protein concentration in both structures. Although the affinities in the two structures [KD= 0.039 (arachnoid) and 0.097 nM (pial vessels)] were similar, the arachnoid had ∼ 10‐fold more binding sites (Bmax= 2,100 fmol/mg of protein) than the pial vessels (Bmax= 250 fmol/ mg of protein). This difference was found in both bovine and porcine fractions. Pharmacological analysis of [3H]‐QNB displacement by muscarinic and nonmuscarinic ligands gave the typical pattern of muscarinic receptors in both structures. Inhibition of binding to pial vessels by the M, antagonist pirenzepine revealed only one low‐affinity site (Ki= 7.8 × 10−7M), whereas, the arachnoid had a small proportion (21%) of high‐affinity sites (Ki= 2.2 × 10−9M) associated with low‐affinity sites (Ki= 5.50 × 10−7M). It is concluded that muscarinic‐mediated effects that do not involve the M, subtype are induced in bovine pial vessels by a relatively low concentration of binding sites. The high content of muscarinic binding sites and their diversity in the arachnoid suggest a functional role for muscarinic cholinergic receptors in this structure.
Tirilazad Mesylate Protects Vitamins C and E in Brain Ischemia‐Reperfusion Injury
Authors: Sato Paul H. , Hall Edward D.
Published: October 2006

Abstract: Brain concentrations of the antioxidant vitamins C and E decreased following unilateral carotid occlusion and reperfusion for 2 or 24 h in gerbils. Administration of the 21‐aminosteroid inhibitor of lipid peroxidation, tirilazad mesylate (U74006F), prevented the decrease in level of both of these vitamins following 2 h of reperfusion. After 24 h of reperfusion, however, α‐tocopherol (vitamin E) continued to be protected, but ascorbic acid (vitamin C) showed a pronounced decrease in content. The changes in concentrations of these vitamins are consistent with U74006F acting to inhibit peroxidation in the CNS by scavenging of lipid peroxyl radicals and suggest that, in the presence of this agent, injury‐induced depletion of ascorbic acid may occur without irreversible tissue damage.
Blood–Cerebrospinal Fluid and Blood‐Brain Barriers Imaged by 18F‐Labeled Metabolites of 18F‐Setoperone Studied in Humans Using Positron Emission Tomography
Authors: Blin Jérôme , Crouzel Christian
Published: October 2006

Abstract: 18F‐Setoperone, a sensitive radioligand for brain serotonin 5‐HT2 receptor positron emission tomography studies, is metabolized into 18F‐labeled metabolites, which participate in blood 18F radioactivity. Its main metabolite, identified as reduced 18F‐setoperone, was synthesized and studied in humans to determine if 18F‐labeled metabolites of 18F‐setoperone (a) enter into the brain, (b) bind to the 5‐HT2 receptor, and (c) explain the increase of 18F radioactivity in the free fraction in blood measured following 18F‐setoper‐one injection. After reduced 18F‐setoperone injection, the brain‐to‐blood 18F radioactivity concentration ratio (a) was low, at the beginning, indicating that this metabolite did not cross the blood‐brain barrier; (b) was increased thereafter, with a higher radioactivity level in the choroid plexus than in brain tissue, suggesting a blood‐CSF barrier crossing due to radioligand hydrophilicity; and (c) showed similar kinetics for cerebellum and frontal cortex, indicating that radioactive metabolites of 18F‐setoperone did not bind to the 5‐HT2 receptor. Because hydrophilic 18F‐labeled metabolites of 18F‐setoperone increased 18F radioactivity in the free fraction in blood, we quantified the relation between 18F‐se‐toperone metabolism and free fraction kinetics in blood. A significant negative correlation was found between metabolism and free fraction rate constants in blood, showing it was possible to predict the 18F‐setoperone metabolism rate using free fraction kinetics in blood. This will allow us to avoid the use of radio‐TLC, a reference method that is difficult to use when multiple samples must be analyzed. A hydrophilic positron‐emitter radioligand could also be used to study the blood‐CSF barrier.
Phosphatidylinositol Kinase Is Reduced in Alzheimer's Disease
Authors: Jolles J. , Bothmer J. , Markerink M. , Ravid R.
Published: October 2006

Abstract: Phosphatidylinositol (PI) kinase and PI phosphate (PIP) kinase activities were measured in postmortem samples of brain tissue from patients with Alzheimer's disease and nondemented control subjects. A membrane‐free cytosolic fraction from four neocortical locations, with exogenous inositol lipids as the substrate, was used. Tissue from patients with Alzheimer's disease was characterized by reduced PIP formation; the reduction was 50% in prefrontal cortex, temporal cortex, and parietal cortex and 40% in precentral gyrus. In contrast, no alterations were found in PI bisphosphate formation in these four neocortical locations. The specific changes in PI kinase but not PIP kinase activity suggest that the findings may have functional relevance to the involvement of brain membrane processes in Alzheimer's disease.
Reactive Oxygen Species and the Central Nervous System
Authors: Halliwell Barry
Published: October 2006

Abstract: Radicals are species containing one or more unpaired electrons, such as nitric oxide (NO•−). The oxygen radical superoxide (O2•−) and the nonradical hydrogen peroxide (H2O2) are produced during normal metabolism and perform several useful functions. Excessive production of O2•‐ and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxyl radical (•−OH) and other oxidants in the presence of “catalytic” iron or copper ions. An important form of antioxidant defense is the storage and transport of iron and copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e.g., by ischemia or trauma, can cause increased metal ion availability and accelerate free radical reactions. This may be especially important in the brain because areas of this organ are rich in iron and CSF cannot bind released iron ions. Oxidative stress on nervous tissue can produce damage by several interacting mechanisms, including increases in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminum and in damage to the substantia nigra in patients with Parkinson's disease are reviewed. Finally, the nature of antioxidants is discussed, it being suggested that antioxidant enzymes and chelators of transition metal ions may be more generally useful protective agents than chain‐breaking antioxidants. Careful precautions must be used in the design of antioxidants for therapeutic use.
Effect of Quinine on Autoreceptor‐Regulated Dopamine Release in the Rat Striatum
Authors: Tanaka Takahiko , Vincent Steven R. , Nomikos George G. , Fibiger Hans C.
Published: October 2006

Abstract: In vivo brain microdialysis was used to examine the role of potassium channel activation in dopamine (DA) autoreceptor function in the striatum of freely moving rats. Local application of the D2 receptor agonists quinpirole or N‐0437 through the dialysis probe significantly reduced extracellular concentrations of DA. Local application of the D2 antagonist (—)‐sulpiride produced significant increases in DA. Local perfusion with quinine, a K+ channel blocker, completely blocked the (—)‐sulpiride‐induced increases in DA but did not affect the DA agonist‐induced decreases. (—)‐Sulpiride completely blocked the effect of quinpirole on DA both in control and in quinine‐treated animals. At the highest dose used, quinine caused a large transient increase in extracellular DA. Local application of tetrodotoxin or infusion of Mg2+ in the absence of Ca2+ did not prevent this quinine‐induced transient increase in extracellular DA. These results demonstrate that DA autoreceptors in the striatum regulate DA release in awake, behaving animals. Local application of (—)‐sulpiride increases DA levels by blocking the tonic activation of autoreceptors by endogenous DA. Quinine blocks the neuroleptic‐induced increase in DA, perhaps by preventing the K+ channel opening that would normally accompany endogenous autoreceptor activation. The fact that exogenously applied DA receptor agonists can decrease extracellular DA levels in the presence of quinine suggests that they may be acting at extrasynaptic autoreceptors that are not tonically active in vivo. The effect of DA agonists on this site is via a DA receptor because it is blocked by (—)‐sulpiride. However, this receptor does not appear to be coupled to a quinine‐sensitive potassium channel.
Lipid Composition in Different Regions of the Brain in Alzheimer's Disease/Senile Dementia of Alzheimer's Type
Authors: SoOderberg M. , Edlund C. , Alafuzoff I. , Kristensson K. , Dallner G.
Published: October 2006

Abstract: The lipid compositions of 10 different brain regions from patients affected by Alzheimer's disease/senile dementia of Alzheimer's type were analyzed. The total phospholipid amount decreased somewhat in nucleus caudatus and in white matter. The cortical areas that are morphologically affected by Alzheimer's disease, i.e., frontal and temporal cortex and the hippocampus, showed elevated contents of lipid solvent‐extractable phosphatidylinositol. Sphingomyelin content was decreased in regions rich in myelin. There was a 20–50% decrease in dolichol amount in all investigated parts of the brain, but no change was seen in the polyisoprenoid pattern. Levels of α‐unsaturated polyprenes were decreased in Alzheimer brains. Dolichyl‐phosphate content increased in most regions, up to 100%. In both control and Alzheimer tissue almost all of the dolichyl‐phosphate was covalently bound, apparently through glycosylation. Cholesterol amounts were highly variable but mostly unchanged, whereas ubiquinone concentrations increased by 30–100% in most regions in brains affected by Alzheimer's disease. These results demonstrate that both phospholipids and neutral lipids are modified in brains affected by Alzheimer's disease/senile dementia of Alzheimer's type.
Hypoxia Induces Synthesis of a Novel 22‐kDa Protein in Neonatal Rat Oligodendrocytes
Authors: Qi Yan , Dawson Glyn
Published: October 2006

Abstract: Neonatal (3‐day‐old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S‐label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22‐kDa protein. Reoxygenation of cultures reversed the synthesis of the 22‐kDa protein, and thiol and calpain protease inhibitors (EP‐459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22‐kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti‐actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S‐label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22‐kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton‐associated Rho or Rac low‐molecular‐mass (20–24 kDa) GTP‐binding proteins because it did not bind [α‐32P]GTP on western blots. Oligodendrocytes did not synthesize a 22‐kDa protein in response to heat shock but did synthesize the typical 70‐ and 90‐kDa heat‐shock proteins. The novel aspects of this protein were further emphasized by the fact that increased synthesis of this 22‐kDa protein was not detected in cultured embryonic chick neurons, rat astrocytes, mouse, rat, or human gliomas, and oligodendrogliomas subjected to similar periods of hypoxia. Thus the induction of the hypoxin appears specific for nontransformed oligodendrocytes subjected to hypoxic injury.
Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells
Authors: Berti‐Mattera Liliana N. , Douglas Janice G. , Mattera Rafael , Goraya Tauqir Y.
Published: October 2006

Abstract: In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39–40‐kDa pertussis toxin substrates that were resolved on separation on urea‐gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45‐kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of Goα, Gi1α, Gi2α, Gi3α, Gq/G11α, and the two isoforms of Gsα in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified Goα, Gi2α,Gi3α, and proteins from the G family, but no immunoreactivity toward anti‐Gi1 antiserum was detected. In these membranes, anti‐Gsa antibody recognized the three cholera toxin substrates mentioned above, with the 45‐kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3α and the absence of Gi1α. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.
Amygdala Kindling Potentiates Seizure‐Stimulated Immediate‐Early Gene Expression in Rat Cerebral Cortex
Authors: Duman Ronald S. , Craig Jennifer S. , Winston Sheryl M. , Deutch Ariel Y. , Hernandez Theresa D.
Published: October 2006

Abstract: Kindling induces long‐term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate‐early genes (lEGs) c‐fos, c‐jun, and NGFI‐A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c‐fos mRNA in cerebral cortex, ECS‐stimulated levels of c‐fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham‐treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS‐stimulated levels of c‐fos mRNA were not significantly increased in stage 2 kindled animals. ECS‐stimulated levels ofc‐jun and NGFI‐A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham‐treated controls. The influence of kindling on IEG expression was long‐lasting because an acute ECS stimulus significantly elevated levels of c‐fos and c‐jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS‐stimulated levels of c‐fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina‐specific changes in the cerebral cortex. In sham‐treated controls, acute ECS resulted in an increase in the number of Fos‐like immunoreactive (Fos‐li) neurons in layers II + III and V of prefrontal cortex, which approached significance; ECS did not influence the number of Fos‐li neurons in layer VI. However, in kindled animals acute ECS significantly increased the number of Fos‐li neurons in layers II + III and V of prefrontal cortex. These results suggest that kindling increases the responsiveness of cerebral cortical neurons to certain stimuli and that regulation of IEG expression could be involved in the molecular adaptations that underlie kindling.
Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine
Authors: Amejdki‐Chab N. , Benmansour S. , Costentin J. , Bonnet J.‐J.
Published: October 2006

Abstract: We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenyl‐2‐[1‐3H]propenyl)piperazine ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+= Li+ gtm Cs+ gtm Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ gtm K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.
Relationship Between Plasma and Brain Large Neutral Amino Acids in Rats Fed Diets with Different Compositions at Different Times of the Day
Authors: Voog Lennart , Eriksson Tomas
Published: October 2006

Abstract: Large neutral amino acids (LNAAs) compete with each other for carrier‐mediated transport through the blood‐brain barrier into the brain. The relative plasma concentration, expressed as the ratio of each LNAA to the sum of LNAAs, is considered the main regulator of brain LNAA concentrations. In order to investigate the consistency of this assumption throughout a 24‐h period, we have compared the relationship of plasma LNAAs to brain LNAAs among groups of rats fed diets containing various amounts of protein (in order to obtain a wide range of plasma LNAA levels) at two different phases of the light/dark cycle (0900 and 2100 hours). The relationship between plasma and brain LNAAs was found to be dependent on both diet and the time of day. Similar plasma amino acid concentrations in the morning and in the evening contrasted with different brain concentrations. Furthermore, previous findings that brain LNAA concentrations are influenced by plasma amino acid concentrations were confirmed.
Multiple Mechanisms for Inhibition of Excitatory Amino Acid Receptors Coupled to Phosphoinositide Hydrolysis
Authors: Littman Louis , Munir Muhammad , Flagg Stephanie D. , Robinson Michael B.
Published: October 2006

Abstract: Excitatory amino acid (EAA) analogues activate receptors that are coupled to the increased hydrolysis of phosphoinositides (Pis). In these studies, hippocampal slices were prepared from neonatal rats (6–11 days old) to characterize the effects of EAA analogues on these receptors. The concentrations of ibotenate and trans‐(pmn)‐1‐amino‐1,3‐cyclopentanedicarboxylate (trans‐ACPD) required to evoke half‐maximal stimulation (EC50 values) were 28 and 51 μM, respectively. Although the data for stimulation of PI hydrolysis by ibotenate and trans‐ACPD were best fit to theoretical curves that had Hill slopes of 1, data for stimulation of PI hydrolysis by quisqualate were best fit to two sites. The EC50 values were 0.43 and 44 μM. The high‐affinity sites were 70% of the total. A number of EAA analogues were tested for inhibition of PI metabolism. One of these, L‐aspartate‐β‐hydroxamate (L‐AβHA), was identified as a novel inhibitor of this response. L‐AβHA was equipotent as an inhibitor of PI metabolism stimulated by ibotenate, quisqualate, and trans‐ACPD. The data for this inhibition were best fit to two sites. Between 32 and 48% of the total sites had high affinity with IC50 values in the range of 1.2–6.3 μM. The low‐affinity sites had IC50 values between 610 and 2,700 μM. DL‐2‐Amino‐3‐phosphonopro‐pionate (DL‐AP3) was also equipotent as an inhibitor of PI hydrolysis stimulated by ibotenate, quisqualate, and trans‐ACPD (IC50 values were 480–850 μM). In contrast to the data for L‐AβHA, the data for DL‐AP3 were best fit to a single site. Both of these inhibitors reduced the maximal response caused by the agonists, consistent with noncompetitive mechanisms of action. Several experiments were designed to examine potential mechanisms for these noncompetitive effects. These studies suggest that either L‐AβHA and DL‐AP3 bind to a site on the receptor and irreversibly block activation of the receptor, or that these inhibitors act via a distinct site that specifically regulates EAA receptors coupled to PI hydrolysis.
Tyrosine Phosphorylation of Microtubule‐Associated Protein Kinase After Transient Ischemia in the Gerbil Brain
Authors: Campos‐González Roberto , Kindy Mark S.
Published: October 2006

Abstract: The tyrosine phosphorylation of microtubule‐associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine‐phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N‐methyl‐D‐aspartate (NMDA) receptor blocker (+)‐MK‐801, whereas a non‐NMDA receptor blocker, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.
Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor‐Directed Neurite Outgrowth in PC 12 Cells
Authors: Chiou Jin‐Yi , Westhead Edward W.
Published: October 2006

Abstract: The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20–25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50, of sim7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF‐primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid‐sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGF.