bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue
Authors: Schwanstecher Mathias , Löser Stefan , Chudziak Franz , Bachmann Cornelius , Panten Uwe
Published: July 2010

Abstract: In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [3H]glibenclamide or a 125I‐labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4‐azido‐5‐iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high‐affinity binding of this novel probe to the particulate (KD = 0.13 nM) and solubilized (KD = 0.56 nM) sulfonylurea receptor from cerebral cortex. The novel probe showed >100‐fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175‐kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a KD value of 0.38 nM. Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed KD values for these sulfonylureas of 0.06 nM, 1.6 nM, and 1.2 µM, respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175‐kDa protein represents the cerebral sulfonylurea receptor.
Purification and Characterization of Two Casein Kinase Type II Isozymes from Bovine Brain Gray Matter
Authors: Mitev Vanio , Pauloin Alain , Houdebine Louis‐Marie
Published: July 2010

Abstract: Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono‐Q steps. The phosphocellulose eluate showed two BBGM‐CK II activities. The first minor component (BBGM‐CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM‐CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., α (40 kDa), α′ (38 kDa), and β (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM‐CK IIa and b showed chromatographic and biochemical differences including differing Km for ATP and GTP and Ki for heparin and 2,3‐bisphosphoglycerate. The properties of the main peak (BBGM‐CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM‐CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM‐CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin β‐light chain. The unusual chromatographic behavior and biochemical properties of these BBGM‐CK II isozymes, compared with the classical CK II, could be explained at least in part by their subunit ratios.
Brain Arachidonic Acid Incorporation and Precursor Pool Specific Activity During Intravenous Infusion of Unesterified [3H]Arachidonate in the Anesthetized Rat
Authors: Washizaki Kazushige , Smith Quentin R. , Rapoport Stanley I. , Purdon A. David
Published: July 2010

Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [3H]arachidonate was infused intravenously in pentobarbital‐anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl‐CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl‐CoA rose quickly (t1/2 < 1 min) to steady‐state values that averaged <5% of plasma specific activity. Incorporation was rapid, as >85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.
Evidence that Protein Kinase C Activities Involved in Regulating Neurite Growth Are Localized to Distal Neurites
Authors: Campenot Robert B. , Draker Dwight D. , Senger Donna L.
Published: July 2010

Abstract: Previously, we observed that long‐term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12‐myristate 13‐acetate (PMA) caused a reduction in the rate of neurite elongation by >50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca2+‐dependent phosphorylation of a PKC‐specific octapeptide substrate. We found that local application of 1 µM PMA for 24 h to distal neurites caused nearly complete down‐regulation of Ca2+‐dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC50 value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.
Adrenalectomy Attenuates Kainic Acid‐Induced Spectrin Proteolysis and Heat Shock Protein 70 Induction in Hippocampus and Cortex
Authors: Lowy Martin T. , Wittenberg Lee , Novotney Susan
Published: July 2010

Abstract: Glucocorticoids have been shown to exacerbate the damaging effects of a variety of neurotoxic insults in the hippocampus and other brain areas. Evidence suggests that the endangering effects of glucocorticoids may be due to augmenting the cascade of events, such as elevations in intracellular calcium levels, because of excitatory amino acid (EAA) receptor stimulation. A potential mechanism responsible for EAA‐induced neuronal damage is activation of calcium‐sensitive proteases, such as calpain, which then proteolytically degrade cytoskeleton structural proteins, such as spectrin. The present study was designed to determine if glucocorticoids can regulate the spectrin proteolysis produced by the EAA agonist, kainic acid. Rats were adrenalectomized (ADX) or sham operated and 7 days later injected with kainic acid (10 mg/kg). Twenty‐four hours later rats were killed and tissues obtained for western blot analyses of the intact spectrin molecule and the proteolytically derived breakdown products. Kainic acid produced an approximate sevenfold increase in the 145–155‐kDa spectrin breakdown products in the hippocampus relative to ADX or sham rats injected with vehicle. ADX attenuated the kainic acid‐induced increase in breakdown products by 43%. In a similar way, kainic acid produced a large 10‐fold increase in spectrin breakdown products in the frontal cortex, which was also significantly attenuated (−80%) by ADX. Induction of heat shock protein 70 (hsp70) by neurotoxic insults has been suggested to be a sensitive indicator of cellular stress in neurons. Kainic acid induced large amounts of hsp70 in both hippocampus and frontal cortex of sham‐operated rats that was markedly attenuated (85–95%) by ADX. There was a strong positive correlation between the amount of spectrin proteolysis and the degree of hsp70 induction in both the hippocampus and frontal cortex. In contrast, kainic acid did not significantly produce spectrin proteolysis and induced only a very modest and inconsistent increase of hsp70 in the hypothalamus. This is consistent with the observation that the hypothalamus is relatively insensitive to the neurotoxic effects of systemically administered kainic acid. The dose of kainic acid (10 mg/kg) used in this experiment produces a 10‐fold elevation in circulating corticosterone levels at both 1 and 3 h after administration. These results suggest that part of the endangering effects of glucocorticoids on hippocampal and cortical neurons may be due to augmentation of calpain‐induced spectrin proteolysis. The attenuation of kainic acid‐induced synthesis of hsp70 by ADX indicates that the cellular stress produced by EAAs is regulated in part by glucocorticoids. In addition, the elevation in endogenous corticosterone levels produced by kainic acid appears to be a significant factor contributing to the neuronal damage produced by this agent.
Plasminogen Activator Inhibitor‐1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose
Authors: Kollros Peter R. , Konkle Barbara A. , Ambarian Anne P. , Henrikson Paul
Published: July 2010

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor‐1 (PAI‐1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI‐1 mRNA levels were decreased fivefold in cells cultured in media containing 20 mM glucose compared with BBEC cultured in media with 5.5 mM glucose, whereas expression of PAI‐1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI‐1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI‐1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI‐1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI‐1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.
Corticosterone Regulates Heme Oxygenase‐2 and NO Synthase Transcription and Protein Expression In Rat Brain
Authors: Weber Colleen M. , Eke Benay C. , Maines Mahin D.
Published: July 2010

Abstract: Heme oxygenase (HO)‐1 and ‐2 produce carbon monoxide, which is suspected, as is nitric oxide (NO), to function as a neuronal messenger. We report on glucocorticoid‐mediated modulation of HO‐2 and NO synthase expression in brain and the differential response of the two proteins to corticosterone in different brain regions. Corticosterone treatment (40 mg/kg, 20 days) had opposing effects on HO‐2 and NO synthase transcript levels: increasing the 1.3‐ and 1.9‐kb HO‐2 mRNAs and decreasing that of the brain‐specific 10.5‐kb NO synthase. Corticosterone did not uniformly affect HO‐2 protein expression in all regions, but appeared to cause a universal reduction in NO synthase, e.g., HO‐2 was decreased in hippocampus (CA1 and dentate gyrus), but not in cerebellum. In contrast, NADPH diaphorase staining was reduced in hippocampus and in molecular and granule layers of cerebellum (not detected in Purkinje cells). Striking deficits in neuronal morphology and number of diaphorase‐staining neurons were observed in the lateral tegmental area, paraventricular nucleus, and frontal cortex; HO‐2 expression was only selectively affected. In cerebellum, activity of NO synthase, but not that of HO, was reduced. Consistent with the possibility that carbon monoxide can generate cyclic GMP, the change in cyclic GMP level did not mirror the decrease in NO synthase. We suggest that glucocorticoid‐mediated deficits in hippocampal functions may reflect their negative effect on messenger‐generating systems.
A Rapid Anterograde Axonal Transport of Carboxypeptidase H in Rat Sciatic Nerves
Authors: Yajima Ryuichi , Chikuma Toshiyuki , Kato Takeshi
Published: July 2010

Abstract: Using the highly sensitive HPLC‐fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative pro‐hormone processing enzyme that removes a basic amino acid from the C‐terminus of a precursor peptide, was measured 12–72 h after double ligations of rat sciatic nerves. CPH‐like activity in rat sciatic nerves was 60‐fold lower than that in the pituitary gland. CPH‐like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is ∼50 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.
Effects of 3‐Nitropropionic Acid on Synaptosomal Energy and Transmitter Metabolism: Relevance to Neurodegenerative Brain Diseases
Authors: Erecińska Maria , Nelson David
Published: July 2010

Abstract: 3‐Nitropropionic acid (3‐NPA) inhibited synaptosomal respiration in a dose‐dependent manner; the degree of inhibition by the same concentration of the compound was greater, however, when respiration was stimulated by concomitant increase in ATP usage. The most rapid event after addition of 3‐NPA was a decrease in [creatine phosphate]/[creatine] ([CrP]/[Cr]) and an increase in [lactate]/[pyruvate]. A fall in [ATP]/[ADP] and [GTP]/[GDP] was initially less pronounced but closely followed that in [CrP]/[Cr]. In the absence of glutamine, 3‐NPA caused a pronounced decrease in internal aspartate level and a small reduction in glutamate concentration, whereas [GABA] rose; the sum of these three amino acids inside synaptosomes fell, but there were no increases in their external levels. With glutamine in the medium, the reduction in intrasynaptosomal aspartate was accompanied by increases in intrasynaptosomal glutamate and GABA. The external concentration of glutamate rose substantially in the presence of the inhibitor. 3‐NPA had no effect on basal release of either glutamate (and GABA) or biogenic amines but increased efflux occurring upon addition of nonsaturating concentrations of the depolarizing agents veratridine and KCI. The results allow the following predictions with respect to the behavior of brain metabolism in neurodegenerative diseases that involve restrictions of mitochondrial function: (1) The extent of inhibition of mitochondrial ATP generation is expected to be greater in cells with high energy demand. The earliest signs of impairment of the respiratory chain function are a fall in [PCr]/[Cr] (or a rise in [Pi]/[CrP]) and an increase in [lactate]/[pyruvate]. (2) A fall in [GTP]/[GDP] can limit protein synthesis. This may be one of the factors that contributes to cell death. (3) An increase in the concentration of inorganic phosphate stimulates neuronal glutaminase activity and leads to a release of glutamate into the external environment; the latter could activate excitatory amino acid receptors. (4) A lowered energy level limits the cell's ability to restore ion gradients. Stimulated release of transmitters from neurons may, therefore, be enhanced and their reuptake delayed.
α2‐Adrenoceptor Subtypes Identified by [3H]RX821002 Binding in the Human Brain: The Agonist Guanoxabenz Does Not Discriminate Different Forms of the Predominant α2A Subtype
Authors: Sastre Magdalena , García‐Sevilla Jesús A.
Published: July 2010

Abstract: Competition [3H]RX821002 ([3H]2‐methoxyidazoxan) binding experiments with α2‐adrenoceptor subtype‐specific antagonists—BRL 44408 (α2A selective), ARC 239 (α2B selective), and others—were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of α2‐adrenoceptors appears to belong to the α2A subtype (100%; Bmax = 34–90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the α2A subtype (87%; Bmax = 53 fmol/mg of protein), although a small population of the α2B/C subtype (13%; Bmax = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of α2A (64%; Bmax = 9 fmol/mg of protein) and α2B/C (36%; Bmax = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the α2B/C‐adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high‐ and low‐affinity states of the α2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl‐5′‐imidodiphosphate together with NaCl and EDTA (to eliminate the high‐affinity α2A‐adrenoceptor) guanoxabenz only recognized the low‐affinity α2A‐adrenoceptor. The results indicate that in the human brain the predominant α2‐adrenoceptor is of the α2A subtype and that this functionally relevant receptor subtype is not heterogeneous in nature.
Alteration of Tubulin‐Gi Protein Interaction in Rat Cerebral Cortex with Aging
Authors: Hatta Shinichi , Ozawa Hiroki , Saito Toshikazu , Ohshika Hideyo
Published: July 2010

Abstract: The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2‐month‐old and 24‐month‐old rats. The hydrolysis‐resistant GTP analogue 5′‐guanylylimidodiphosphate (GppNHp)‐dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24‐month‐old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (∼28%) in 2‐month‐old rats. Tubulin‐GppNHp‐dependent inhibition of adenylyl cyclase in 24‐month‐old rats was significantly attenuated (∼15%). In 2‐month‐old rats, when tubulin, polymerized with the hydrolysis‐resistant photoaffinity GTP analogue [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Giα. Transfer of AAGTP from tubulin to Giα was reduced in 24‐month‐old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Giα in cortex membranes was significantly decreased in 24‐month‐old rats. No differences were observed in the amounts of Gsα, Giα, or Gβ subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2‐month‐old and 24‐month‐old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24‐month‐old rats.
Two‐Site Immunoradiometric Assay of Chicken Acetylcholinesterase: Active and Inactive Molecular Forms in Brain and Muscle
Authors: Chatel Jean‐Marc , Eichler Jerry , Vallette François‐Marie , Bon Suzanne , Massoulié Jean , Grassi Jacques
Published: July 2010

Abstract: Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C‐131, which is conformation dependent and preferentially recognizes active AChE. We have set up two‐site immunoradiometric assays, using an immobilized capture antibody, C‐6 or C‐131, and a radiolabeled antibody, 125I‐C‐54. The C‐6/C‐54 assay quantifies the totality of inactive and active AChE subunits: It detects 10−3 Ellman unit (∼40 pg of protein) and yields a linear response up to at least 25 10−3 Ellman units. An analysis of gradient fractions, using C‐6/C‐54 and C‐131/C‐54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1. The proportion of inactive forms was much higher in cultured myotubes, obtained from E11 myoblasts. These results show that the proportion of inactive AChE depends on the tissue and varies during development. Thus, the cells seem to control actively the acquisition of AChE activity, as well as the formation of the various oligomeric forms.
Release of N‐Acetylaspartylglutamate from Slices of Rat Cerebellum, Striatum, and Spinal Cord, and the Effect of Climbing Fiber Deprivation
Authors: Zollinger M. , Brauchli‐Theotokis J. , Gutteck‐Amsler U. , Do K. Q. , Streit P. , Cuénod M.
Published: July 2010

Abstract: The release of endogenous N‐acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 mM K+ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography‐mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization‐induced release of NAAG was found in all three regions. The release was Ca2+ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K+‐induced release of NAAG was studied: Treatment of the animals with 3‐acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.
Influence of the CNS Environment on Chromaffin Cell Survival and Catecholamine Secretion Patterns
Authors: Sagen Jacqueline , Ortega John D.
Published: July 2010

Abstract: Chromaffin cells implanted into the CNS have been used as a potential source of sustained catecholamine delivery, although their survival and continued catecholamine secretion are controversial. In addition, chromaffin cells exhibit a high degree of neurochemical plasticity in response to environmental factors. The present aims were to determine whether the CNS provides a supportive environment for sustained catecholamine production in transplanted chromaffin cells and to assess whether this novel environment alters patterns of catecholamine secretion. Catecholamine release from bovine chromaffin cells implanted into the rat midbrain was determined in brain slices. In addition, alterations in catecholamine secretion patterns, particularly adrenaline/noradrenaline ratios, were compared in vitro versus in transplants. Results indicated that brain slices containing chromaffin cell implants released high basal and nicotine‐stimulated levels of adrenaline and noradrenaline. It is surprising that although adrenaline/noradrenaline ratios steadily declined in culture, this did not occur when cells were transplanted to the CNS in the early postharvesting phases. However, if cells were transplanted following longer periods in culture, adrenaline/noradrenaline ratios remained low. Together, these results suggest that the CNS can provide a supportive environment for chromaffin cell survival and that the pattern of catecholamine secretion can be optimized by prior in vitro manipulation.
Cerebrospinal Fluid Nitrite/Nitrate Levels in Neurologic Diseases
Authors: Milstien Sheldon , Sakai Naoki , Brew Bruce J. , Krieger Charles , Vickers James H. , Saito Kuniaki , Heyes Melvyn P.
Published: July 2010

Abstract: Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.
A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum
Authors: Sivvas Emmanuel , Voukelatou Georgia , Papaioannou Dionissios , Aletras Alexios J. , Kouvelas Elias D.
Published: July 2010

Abstract: The synthesis of (2S,3S,4S)‐4‐[1‐(4‐azidobenzamidomethyl)ethenyl]‐2‐carboxy‐3‐pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [3H]CNQX and [3H]kainic acid binding on chick cerebellar membranes. [3H]ABCPA was photoaffinity cross‐linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [3H]ABCPA incorporation in both bands was completely blocked by 2 mM CNQX. When photoaffinity labeling was performed in the presence of 2 mM kainic acid, incorporation of [3H]ABCPA was blocked by ∼70% in the 45‐kDa band and by 18% in the 33.5‐kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 mM glutamate.
Regional Specificity in Alterations of Rat Brain Copper and Catecholamines Following Perinatal Copper Deficiency
Authors: Prohaska Joseph R. , Bailey William R.
Published: July 2010

Abstract: Perinatal copper deficiency was studied in 1‐month‐old female and male Sprague‐Dawley rat offspring to investigate regional changes in brain copper and catecholamine levels. Offspring of dams given the low copper treatment beginning at day 7 of gestation exhibited signs characteristic of deficiency such as impaired growth and 10‐fold lower liver copper levels compared with copper‐adequate controls. Regional analysis of brain copper by graphite furnace atomic absorption spectroscopy revealed uniform and severe reduction of copper to levels 20 ± 3% of controls in all regions, except the hypothalamus, where reductions to 56 and 28% of those in copper‐adequate females and males, respectively, were measured. HPLC analysis revealed significant reductions in norepinephrine levels in cerebrum, midbrain, corpus striatum, cerebellum, and medulla‐pons of copper‐deficient offspring ranging between 39 and 67% of control values. There were no significant differences in norepinephrine concentration in the hypothalamus. There was a significant, one‐third reduction of dopamine in the corpus striatum of copper‐deficient male rats. Consistent with altered in vivo dopamine β‐monooxygenase activity, there were five‐, three‐, and twofold elevations of dopamine in cerebellum, medulla‐pons, and hypothalamus of copper‐deficient rats. Spectrophotometric measurement of in vitro dopamine β‐monooxygenase activity of brain and adrenal homogenates was higher in copper‐deficient rats, confirming prior work. An explanation for the in vitro data is unclear. Changes in copper and catecholamine levels were influenced by diet and were regionally selective, especially in the hypothalamus.
Differential Proteolysis of the Full‐Length Form of the L‐Type Calcium Channel α1 Subunit by Calpain
Authors: De Jongh Karen S. , Colvin Anita A. , Wang Kevin K. W. , Catterall William A.
Published: July 2010

Abstract: This study examines the proteolysis of the carboxy terminal domain of the full‐length (α1212) and truncated (α1190) forms of the rabbit skeletal muscle L‐type calcium channel α1 subunit by calpain I and calpain II. Although both forms of the α1 subunit show little sensitivity to proteolysis by calpain II, α1212 is relatively more sensitive than α1190 to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37‐kDa fragment from the C‐terminus of α1212 in a time‐ and concentration‐dependent manner and proteolysis is independent of the α1212 phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP‐dependent phosphorylation from α1212 and may provide a mechanism for modifying the cyclic AMP‐dependent regulation of L‐type calcium channels in skeletal muscle.
α‐Guanidinoglutaric Acid, an Endogenous Convulsant, as a Novel Nitric Oxide Synthase Inhibitor
Authors: Yokoi Isao , Kabuto Hideaki , Habu Hitoshi , Mori Akitane
Published: July 2010

Abstract: The effects of α‐guanidinoglutaric acid (GGA), the levels of which were increased in the cobalt‐induced epileptic focus tissue in the cerebral cortex of cats, on brain nitric oxide synthase (NOS) activity were observed. GGA inhibited NOS activity in a linear mixed manner (Ki = 2.69 µM) and was as effective as NG‐monomethyl‐l‐arginine (MeArg; Ki = 3.51 µM), a well‐known NOS inhibitor. Although MeArg was synthesized by substituting the guanidino nitrogen of l‐arginine (Arg), GGA was a non‐guanidino nitrogen‐substituted guanidino compound. On the other hand, Arg, which is an endogenous NOS substrate, elevates the threshold of seizures induced by GGA. There is evidence that GGA is an endogenous, potent, and non‐guanidino nitrogen‐substituted NOS inhibitor and that suppression of nitric oxide biosynthesis may be involved in GGA‐induced convulsions. Therefore, GGA may be a useful tool in elucidating the chemical nature of NOS and the physiological function of nitric oxide.
Synapsin IIa Bundles Actin Filaments
Authors: Chilcote Tamie J. , Siow Yaw L. , Schaeffer Eric , Greengard Paul , Thiel Gerald
Published: July 2010

Abstract: Synapsins are neuron‐specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F‐actin in vitro, has been postulated to regulate neurotransmitter release by cross‐linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F‐actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose‐response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross‐link synaptic vesicles and actin filaments in the nerve terminal.
(−)‐Deprenyl Reduces PC12 Cell Apoptosis by Inducing New Protein Synthesis
Authors: Tatton W. G. , Ju W. Y. L. , Holland D. P. , Tai C. , Kwan M.
Published: July 2010

Abstract: (−)‐Deprenyl, a monoamine oxidase (MAO)‐B inhibitor, has been shown to increase neuronal survival and to alter protein synthesis and gene expression in astrocytic or PC12 cells independently of MAO‐B inhibition. We used serum and nerve growth factor withdrawal to induce apoptotic death in PC12 cells to determine whether (−)‐deprenyl increases neuronal survival by reducing apoptosis. (−)‐Deprenyl reduced both cell death and internucleosomal DNA degradation in a concentration‐dependent manner and was effective at concentrations too low to inhibit MAO (<10−9M). (+)‐Deprenyl did not increase PC12 cell survival, and, with the exception of pargyline, other MAO‐A and MAO‐B inhibitors did not alter apoptotic death. Transcriptional and translational inhibition showed that the reduction in apoptosis required the induction of new protein synthesis by (−)‐deprenyl. Increased survival was induced if transcription was maintained for 4 h and translation for 6 h after (−)‐deprenyl addition. The findings suggest that transcriptional induction may underlie the other MAO‐independent actions of (−)‐deprenyl.
A New Inhibitor of the Chymotrypsin‐Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin‐Protein Conjugates in a Neuronal Cell
Authors: Figueiredo‐Pereira Maria E. , Berg Kelly A. , Wilk Sherwin
Published: July 2010

Abstract: Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin‐like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin‐protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin‐protein conjugates is commonly detected in abnormal brain inclusions.
Transcription and Motoneuron Size
Authors: Sato Shoko , Burgess S. B. , McIlwain D. L.
Published: July 2010

Abstract: Nuclear size and total RNA synthesis were compared in single lumbar motoneurons isolated from the grass frog. Transcription was found to correlate significantly, but not exclusively, with nuclear area or volume over a wide range of nuclear size, the largest nuclei having the highest mean transcriptional activity. Flow cytometric analysis of propidium iodide‐stained nuclei excluded polyploidy or polyteny as an explanation for the increased transcription, but left open the possibility of a small increase in DNA with increasing nuclear size. Alternatively, motoneurons may increase transcription and nuclear size without increasing their DNA content, possibly by increasing the proportion of dispersed chromatin (euchromatin). These two mechanisms for size‐related changes in RNA synthesis in motoneurons present an interesting contrast to mechanisms used by many other large animal cells.
Membrane Lipids of Adult Human Brain: Lipid Composition of Frontal and Temporal Lobe in Subjects of Age 20 to 100 Years
Authors: Svennerholm L. , Boström K. , Jungbjer B. , Olsson L.
Published: July 2010

Abstract: The membrane lipid composition of human frontal and temporal cortices and white matter has been studied in 118 subjects, age 20–100 years. The brain specimens were selected from subjects who lived a normal social life and died suddenly and unexpectedly with no history of neurologic or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The sudden death eliminated all risk of changes over a long agonal stage. The data for total solids and major lipids are summarized in graphic form. Total solids, phospholipids, and cholesterol diminished linearly from 20 years of age in frontal and temporal cortices, whereas total solids phospholipids, cholesterol, cerebroside, and sulfatide showed a curvilinear diminution in frontal and temporal white matter. Gangliosides differed from the other lipids, showing an almost constant concentration between 20 and 70 years of age with a slight peak around 50 years of age. The ganglioside pattern showed continuous change with aging, with decreasing proportions of GM1 and GD1a and increasing proportions of GD1b, GM3, and GD3. Equations are given that can be used to calculate the lipid composition of normal human frontal and temporal cortices and white matter at any age between 20 and 100 years of age. These data can be used where data by direct analysis are not available for comparison with values for various pathological states.
Neuroprotective Effects of Some Monoamine Oxidase‐B Inhibitors Against DSP‐4‐Induced Noradrenaline Depletion in the Mouse Hippocampus
Authors: Yu P. H. , Davis B. A. , Fang J. , Boulton A. A.
Published: July 2010

Abstract: DSP‐4 [N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine], a selective noradrenaline (NA) uptake blocker, is capable of inducing long‐lasting depletion of NA in some noradrenergic axon terminals and of subsequently causing cell death to NA neuronal cell bodies in rodents. R(−)‐Deprenyl, a selective monoamine oxidase (MAO)‐B inhibitor, has been shown to be capable of protecting animals against this DSP‐4‐induced neuronal degeneration. Its action, however, has been claimed to be unrelated to the inhibition of MAO‐B activity but rather due to competition for the NA uptake sites. The effects of several types of MAO inhibitors against DSP‐4 toxicity, MAO‐B activity both in vivo and in vitro, and NA uptake into the hippocampus have been assessed. N‐(2‐Hexyl)‐N‐methylpropargylamine (2‐HxMP), a potent MAO‐B inhibitor, for example, exerts no appreciable effect on NA uptake but is quite potent in counteracting the NA‐depleting effect of DSP‐4. Such results rule out the possibility that the neuroprotective effect of the MAO‐B inhibitors is due mainly to their effect on NA uptake. The in vitro inhibition of MAO‐B activity seems to correlate positively with their neuroprotective effects against DSP‐4. In comparison to the MAO‐B inhibitors, NA uptake blockers, such as desipramine and S(+)‐deprenyl, exhibit relatively low efficacy in protecting the NA axon terminals from the effects of DSP‐4‐induced damage. The restoration of hippocampal NA levels is significantly enhanced with repeated treatments of R(−)‐deprenyl or 2‐HxMP even at very low doses following the DSP‐4 insult. This suggests that in addition to neuroprotection, these MAO‐B inhibitors may rescue some of the noradrenergic axon terminals damaged by DSP‐4.
Metabolic Abnormalities and Grade of Encephalopathy in Acute Hepatic Failure
Authors: Mans Anke M. , DeJoseph M. Regina , Hawkins Richard A.
Published: July 2010

Abstract: Acute hepatic failure is associated with many biochemical abnormalities in plasma and brain. Changes that correlate well with the degree of behavioral impairment may be important factors in the development of encephalopathy. We measured the concentrations of intermediary metabolites, ammonia, and amino acids in brain and plasma and the rate of whole‐brain glucose utilization in rats with an acutely devascularized liver. In all rats an estimate of the grade of encephalopathy (reflected by behavioral impairment) was made. Rats underwent portacaval shunting and hepatic artery ligation (or sham operation) and were kept normoglycemic and normothermic thereafter. We sampled blood and whole brain (by near‐instantaneous freeze‐blowing) 2, 4, or 6 h later. There were no alterations in levels of high‐energy phosphate metabolites in the brain or in metabolites associated with the glycolytic pathway and Krebs cycle, except lactate and pyruvate. Brain glucose use was decreased similarly at all times after surgery. Levels of ammonia and many amino acids were increased in brain and plasma; brain aspartate, glutamate, and arginine levels were decreased. The increases in content of plasma ammonia and brain glutamine, proline, alanine, and aromatic amino acids and the decreases in brain aspartate and glutamate were most strongly correlated with behavioral impairment.
Characterization of Lysophospholipid Metabolizing Enzymes in Human Brain
Authors: Ross Brian M. , Kish Stephen J.
Published: July 2010

Abstract: Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl‐CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl‐CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N‐ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two‐fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 µM. Acyltransferase displayed an approximately threefold preference for arachidonyl‐CoA over palmitoyl‐CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1‐palmitoyl lysophosphatidylcholine > 1‐oleoyl lysophosphatidylcholine ≫ lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane‐associated processes.
Activation of Protein Kinase C by Purified Bovine Brain 14‐3‐3: Comparison with Tyrosine Hydroxylase Activation
Authors: Tanji M. , Horwitz R. , Rosenfeld G. , Waymire J. C.
Published: July 2010

Abstract: In the course of the purification of 14‐3‐3 protein (14‐3‐3) we found that 14‐3‐3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14‐3‐3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14‐3‐3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient‐gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse‐phase HPLC. The concentration dependence of PKC activation by 14‐3‐3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14‐3‐3 activation of tyrosine hydroxylase; a maximal stimulation of two‐ to three‐fold occurs at 40–100 µg/ml. 14‐3‐3's activation of PKC is sensitive to α‐chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP‐ and calcium/calmodulin‐dependent protein kinases, are not activated. The activation of PKC by 14‐3‐3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.
Activation and Tyrosine Phosphorylation of 44‐kDa Mitogen‐Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus
Authors: Kang Ung Gu , Hong Kyung Sue , Jung Hee Yeon , Kim Yong Sik , Seong Yeon‐Sun , Yang Yun Chung , Park Joo‐Bae
Published: July 2010

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42‐kDa, but not 44‐kDa, mitogen‐activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42‐ and 44‐kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42‐kDa MAPK in rat hippocampus after ECS, but not on that of 44‐kDa MAPK. However, when we examined the immunoprecipitated 44‐kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44‐kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44‐kDa MAPK, as well as 42‐kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44‐kDa MAPK than in 42‐kDa MAPK.