bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Zinc Finger Protein, Zic, Is Involved in Neurogenesis, Especially in the Cell Lineage of Cerebellar Granule Cells
Authors: Aruga Jun , Yokota Naoki , Hashimoto Mitsuhiro , Furuichi Teiichi , Fukuda Mitsunori , Mikoshiba Katsuhiko
Published: July 2010

Abstract: To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI‐binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.
Appearance of Depolarization‐ and Maitotoxin‐Induced [Ca2+]i Elevation in Single LAN‐1 Human Neuroblastoma Cells on Exposure to Retinoic Acid
Authors: Fatatis Alessandro , Bassi Antonella , Iannotti Elodia , Caso Nino , Mita Gustavo D. , Di Renzo Gianfranco , Annunziato Lucio
Published: July 2010

Abstract: LAN‐1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura‐2 single‐cell microfluorimetry. The exposure of LAN‐1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage‐sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN‐1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid‐treated cells. Both high K+‐ and maitotoxin‐induced [Ca2+]i elevation in retinoic acid‐differentiated LAN‐1 cells was reversed by the lanthanide Gd3+, an inorganic Ca2+‐entry blocker, and by the snail toxin ω‐conotoxin GVIA, which interacts with the N sub‐type of voltage‐sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L‐channel sub‐type, were completely ineffective. The tumor promoter phorbol 12‐myristate 13‐acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K+‐induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K+‐induced increase of [Ca2+]i was still present. In conclusion, the results of the present study demonstrated that retinoic acid‐induced differentiation of LAN‐1 cells, which lack a high K+‐evoked [Ca2+]i increase in the undifferentiated state, induces the functional expression of an ω‐conotoxin GVIA‐sensitive, dihydropyridine‐insensitive N‐type voltage‐sensitive Ca2+ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.
Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells
Authors: Lin Lih Fang , Kao Lung‐Sen , Westhead Edward W.
Published: July 2010

Abstract: The Na+/Ca2+ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na+/Ca2+ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na+/Ca2+ exchanger of these cells. Cells treated with 1 mM dibutyryl cyclic AMP (dbcAMP), 1 µM phorbol 12,13‐dibutyrate, 1 µM okadaic acid, or 100 nM calyculin A showed lowered Na+/Ca2+ exchange activity and prolonged cytosolic Ca2+ transients caused by depolarization. A combination of 10 nM okadaic acid and 1 µM dbcAMP synergistically inhibited Na+/Ca2+ exchange activity. Conversely, 50 µM 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine, a protein kinase inhibitor, enhanced Na+/Ca2+ exchange activity. Moreover, we used cyclic AMP‐dependent protein kinase and calcium phospholipid‐dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na+/Ca2+ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na+/Ca2+ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.
Complex I Inhibitors Induce Dose‐Dependent Apoptosis in PC12 Cells: Relevance to Parkinson's Disease
Authors: Hartley A. , Stone J. M. , Heron C. , Cooper J. M. , Schapira A. H. V.
Published: July 2010

Abstract: The mode of cell death in Parkinson's disease (PD) substantia nigra is uncertain. However, evidence is accumulating that certain of the biochemical abnormalities present in PD nigra at the time of death may precipitate apoptosis. We have investigated the mode of death induced by complex I inhibition of dopaminergic cell cultures, and our results suggest that both 1‐methyl‐4‐phenylpyridinium and rotenone cause apoptosis at low concentrations and necrosis at high concentrations. This dose‐dependent shift in the mode of cell death induced by these mitochondrial toxins may have important implications for the mechanism of neuronal cell death in PD.
Differential Effects of Locally Administered Clozapine and Haloperidol on Dopamine Efflux in the Rat Prefrontal Cortex and Caudate‐Putamen
Authors: Pehek Elizabeth A. , Yamamoto Bryan K.
Published: July 2010

Abstract: Previous research has shown that systemically administered antipsychotic drugs enhance dopamine release from the nigrostriatal and mesocortical dopamine pathways. However, the degree of enhancement differs as a function of the drug used (atypical versus typical antipsychotic) and the dopamine pathway examined. The present studies examined whether these differences result from differential actions of these drugs on dopamine terminal regions. Clozapine or haloperidol was infused locally into the caudate‐putamen or prefrontal cortex through reverse microdialysis. Although both drugs increased extracellular dopamine levels, clozapine produced greater effects than haloperidol in the prefrontal cortex, whereas haloperidol produced greater effects in the caudate‐putamen. These results suggest that neurochemical differences within dopamine terminal regions may explain the differential actions of antipsychotic drugs on striatal and cortical dopamine release.
Desensitization of Nicotine‐Stimulated 86Rb+ Efflux from Mouse Brain Synaptosomes
Authors: Marks Michael J. , Grady Sharon R. , Yang Jyan‐Ming , Lippiello Patrick M. , Collins Allan C.
Published: July 2010

Abstract: The desensitization of nicotine‐stimulated 86Rb+ efflux from synaptosomes prepared from C57BL/6 mouse brain was investigated. Nicotine stimulated a saturable, concentration‐dependent efflux of 86Rb+ from synaptosomes (EC50 = 0.60 µM), but the response decreased with time of exposure to nicotine. The rate of decrease of the response (desensitization) increased as the nicotine concentration was increased (EC50 = 0.35 µM; maximal rate of desensitization = 1.1 min−1). Desensitization of nicotine‐stimulated 86Rb+ efflux was also observed when synaptosomes were exposed to low (1–200 nM) concentrations of nicotine that caused little or no stimulation of efflux (EC50 = 13 nM). The rate of desensitization observed with low nicotine concentrations (0.30 min−1) was less than that measured at stimulating concentrations. Desensitization was not fully reversible for synaptosomes exposed to nicotine concentrations between 10 nM and 10 µM: Only 60‐40% of the control response was regained after a 10‐min washout period. The kinetics of functional desensitization were compared with the kinetics of [3H]nicotine binding. [3H]Nicotine binding to midbrain particulate fractions displayed both a fast and a slow phase. The EC50 values for these two phases were 2.6 and 14 nM, respectively. Data obtained from functional desensitization and ligand binding experiments were analyzed using a two‐state model. The kinetic constants obtained from the analyses of these two processes were very similar. Overall, the results suggest that nicotinic receptor function measured with ion flux desensitizes when exposed to either stimulating or nonstimulating concentrations of nicotine. In addition, the kinetic properties calculated for the functional desensitization are comparable to those for [3H]nicotine binding.
Effect of Isonicotinic Acid Hydrazide on Extracellular Amino Acids and Convulsions in the Rat: Reversal of Neurochemical and Behavioural Deficits by Sodium Valproate
Authors: Biggs Christopher S. , Pearce Brian R. , Fowler Leslie J. , Whitton Peter S.
Published: July 2010

Abstract: We have studied the effect of isonicotinic acid hydrazide (INH), a convulsant agent, on the extracellular levels of amino acids in the hippocampus, and the effect of sodium valproate (VPA) administration in INH‐treated rats. INH (250 mg/kg) caused a rapid and sustained decrease in basal levels of GABA, and during this period convulsions of increasing severity were observed. Basal levels of glutamine, taurine, aspartate, and glutamate were unchanged by INH. When VPA was coadministered with INH, basal GABA levels were increased and no convulsions were observed. When transmitter release was evoked using 100 mMK+, the increase in dialysate GABA observed in INH‐treated animals was less than that seen in controls and convulsions increased in frequency. K+‐evoked release of glutamate and aspartate tended to be higher following INH treatment, and in the case of aspartate, this increase was significant. VPA reversed the changes in evoked release of glutamate and aspartate, and release of GABA was considerably greater than that seen in control or INH‐treated rats. No drug effect on evoked changes in taurine or glutamine level was seen. These are the first data to show decreased extracellular GABA in conjunction with convulsions in freely moving animals in vivo.
Dopamine Transporter‐Dependent and ‐Independent Endogenous Dopamine Release from Weaver Mouse Striatum In Vitro
Authors: Richter J. A. , Bare D. J. , Yu H. , Ghetti B. , Simon J. R.
Published: July 2010

Abstract: The weaver mutant mouse (wv/wv) has an ∼70% loss of nigrostriatal dopamine (DA) neurons, but the fractional DA release evoked by amphetamine (but not a high potassium level) has been shown to be greater from striatal slices of the weaver compared with +/+ mice. In the present work we tested the hypothesis that fractional DA release from weaver striatum would be greater when release was mediated by the DA transporter. Serotonin (5‐HT)‐stimulated fractional DA release was greater from weaver than from +/+ striatum. The release evoked by 5‐HT in the presence of 10 µM nomifensine (an antagonist of the DA transporter) was less than in its absence, but the difference between weaver and +/+ striatum remained. In the presence of nomifensine, 1‐(m‐chlorophenyl)biguanide, classified as a 5‐HT3 agonist, also induced a greater fractional release from weaver compared with +/+ striatum. When veratridine was used at a low concentration (1 µM), the fractional evoked release of DA was higher from the weaver in the presence and absence of nomifensine. These findings suggest that the reason for the difference in the responsiveness of the two genotypes to these release‐inducing agents is not related to DA transporter function.
Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts
Authors: Ray Jasodhara , Hogg Joanna , Beutler Andreas S. , Takayama Hideichi , Baird Andrew , Gage Fred H.
Published: July 2010

Abstract: Basic fibroblast growth factor (FGF‐2) is normally expressed as a cell‐associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF‐2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF‐2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18‐kDa form of FGF‐2 in primary fibroblasts as a cell‐associated (FGF‐2‐B) or as a secreted (FGF‐2‐S) protein. FGF‐2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF‐2‐S cells. No FGF‐2 is detected in control (untransfected) cells. FGF‐2‐S cells also grow faster than the control or FGF‐2‐B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF‐2 is active when engineered to be expressed as a cell‐associated form or secreted from cells.
Regulation of the Expression of NMDA Receptor Subunits in Rat Cerebellar Granule Cells: Effect of Chronic K+‐Induced Depolarization and NMDA Exposure
Authors: Resink A. , Villa M. , Benke D. , Möhler H. , Balázs R.
Published: July 2010

Abstract: The influence of K+‐induced membrane depolarization and NMDA treatment on the regulation of NMDA receptor subunit (NR) expression was investigated during the development of granule cells in culture, as a follow‐up of previous work on NMDA receptor activity. In spite of the increase in NMDA receptor activity elicited by these treatments (K25 or K10 + NMDA cultures), the main developmental changes in receptor mRNA levels were similar to those in untreated cells (K10) (a threefold increase in total NMDA receptor mRNA, quantitative dominance of NR1 mRNA, late expression of NR2C, and virtual absence of NR2D). However, high K+ and NMDA treatment resulted in a greater increase of NR2A mRNA levels and a retardation in the developmental changes in the relative amounts of NR2B and NR2C mRNAs. The correspondence between NMDA receptor activity and the amount of NR1 and NR2A subunit proteins was excellent, the rank order being K25 > K10 + NMDA > K10 at 9 days in vitro. Because the increase in subunit mRNA was not always paralleled by an increase in subunit protein, the control of NMDA receptor expression involves critically, in addition to gene transcription, regulation of translational and/or posttranslational events.
Cyclic AMP‐Dependent Modulation of Vesicular Monoamine Transport in Pheochromocytoma Cells
Authors: Nakanishi Nobuo , Onozawa Satoshi , Matsumoto Reiko , Hasegawa Hiroyuki , Yamada Shozo
Published: July 2010

Abstract: Cyclic AMP (cAMP) is well known to enhance tyrosine hydroxylase activity in PC12 cells. We were able to demonstrate, however, that the cellular dopamine level in PC12 was lowered by dibutyryl cAMP. Furthermore, the decrease in the cellular level of dopamine was accompanied by about a 10‐fold increase in the medium. The aim of this work was to elucidate the effect of cAMP on catecholamine transport. Dibutyryl cAMP did not induce exocytotic release of norepinephrine but rather inhibited its uptake. As with forskolin and cholera toxin, physiological signaling molecules such as vasoactive intestinal polypeptide (VIP) and AMP, for which PC12 cells are known to have receptors linked to activation of adenylate cyclase, also inhibited norepinephrine uptake. The inhibitory effects of dibutyryl cAMP, VIP, and AMP were dose dependent, and EC50 values were estimated to be 100 µM, 10 nM, and 1.0 µM, respectively. The inhibition profile of dibutyryl cAMP over the time course of norepinephrine uptake was biphasic: Inhibition became clearly detectable after the cytosolic pool of norepinephrine had been saturated. This profile is similar to that of reserpine. Nomifensine, however, inhibited uptake at a rather constant rate throughout the entire time course. The ATP‐dependent serotonin uptake by digitonin‐permeabilized cells was lowered to ∼50% that of the control by dibutyryl cAMP treatment before permeabilization, indicating inhibition of vesicular monoamine transport. This effect was also dependent on a dibutyryl cAMP concentration with an EC50 of ≤100 µM. These results suggest that cAMP may be capable of elevating extracellular dopamine levels in the nervous system by inhibiting its translocation into storage vesicles while enhancing its synthesis in the cytosol. Moreover, endogenous neurotransmitters such as VIP, AMP, and adenosine may act as intrinsic antidepressants via the cAMP pathway.
Effects of Internal pH on the Acetylcholine Transporter of Synaptic Vesicles
Authors: Nguyen Marie L. , Parsons Stanley M.
Published: July 2010

Abstract: Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [3H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [14C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [3H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [3H]ACh. It exhibited Km and Vmax values of ∼200–500 µM and 7–30 nmol [3H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP‐dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 ± 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH ∼2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear. In vivo, the steep dependence of transport on the transmembrane pH gradient might serve to minimize leakage of ACh from the cytoplasm due to ACh transporter in the plasma membrane.
Regulation of c‐fos Expression by Convulsants and Hexachlorocyclohexane Isomers in Primary Cultures of Cortical Neurons
Authors: Barrón S. , Serratosa J. , Tusell J. M.
Published: July 2010

Abstract: Primary cortical cultures were used to study the effects of four convulsants on c‐fos expression. Approximately 30% of the neurons in these cultures displayed c‐fos nuclear immunostaining under basal conditions. The addition of tetrodotoxin, nifedipine, or δ‐hexachlorocyclohexane produced a significant decrease in c‐fos basal values. Lindane (γ‐hexachlorocyclohexane), Bay K 8644, pentylenetetrazole, and picrotoxinin produced a significant increase in c‐fos immunoreactivity and in c‐fos mRNA expression. Treatment of cells with tetrodotoxin before administration of the convulsant agents lowered c‐fos staining below basal levels. In contrast, δ‐hexachlorocyclohexane or nifedipine failed to block only the picrotoxinin‐induced increase. The differential pattern of expression shown by c‐fos after these treatments suggests various mechanisms of action for the compounds studied. The results obtained with δ‐hexachlorocyclohexane and nifedipine suggest that picrotoxinin activates c‐fos expression by calcium‐requiring intracellular signaling pathways that are different from those activated by Bay K 8644, pentylenetetrazole, or γ‐hexachlorocyclohexane, which, at least in part, act via L‐type calcium channels.
Production of Monoclonal Antibodies Against Neurofilament‐Associated Proteins: Demonstration of Association with Neurofilaments by a Coimmunoprecipitation Method
Authors: Starr Robyn , Xiao Jinsong , Monteiro Mervyn J.
Published: July 2010

Abstract: A panel of monoclonal antibodies (MAbs) was produced against mouse brain proteins that bind to the tail domain of the neurofilament (NF) heavy (200‐kDa) subunit (NF‐H) in vitro. An in vivo association of the MAb ligands with NFs was confirmed by examining reactivity of the MAbs with immunoprecipitated NF‐H complexes. Using this method we demonstrated association of the ligands of three of the MAbs with NFs. In contrast, glial fibrillary acidic protein and an unknown 97‐kDa brain protein were not associated with NFs by this criterion. An 80‐kDa doublet that coimmunoprecipitated with NF‐H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib. Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF‐H and NF medium (160‐kDa) subunits. MAbs 63 and 105 recognized a more complex set of proteins that had predominantly synaptic localizations. These data suggest that NFs may provide important support for attachment and/or transport of synaptic proteins in brain.
A Cellular Stress Model for the Sequestration of Redox‐Active Glial Iron in the Aging and Degenerating Nervous System
Authors: Wang Xudong , Manganaro Fortunato , Schipper Hyman M.
Published: July 2010

Abstract: The mechanisms responsible for the accumulation of redox‐active brain iron in normal senescence and in Parkinson's disease remain poorly understood. The aminothiol compound cysteamine (CSH) induces the appearance of autofluorescent, iron‐rich cytoplasmic granules in cultured astroglia that are identical to glial inclusions that progressively accumulate in the aging periventricular brain. Both in situ and in culture, these glial inclusions appear to arise in the context of a generalized cellular stress (heat shock) response. Several laboratories have previously concluded that porphyrins and heme ferrous iron are responsible, respectively, for red‐orange autofluorescence and nonenzymatic peroxidase activity in the glial inclusions. In the present study we found that, contrary to hypothesis, CSH suppresses the incorporation of the heme precursors δ‐amino[14C]levulinic acid and [14C]glycine into astroglial porphyrin and heme in primary culture. Similar results were obtained when the cells were preloaded with radiolabeled heme precursors for 24 h before CSH treatment, suggesting that the latter directly inhibits porphyrin‐heme biosynthesis rather than limiting precursor uptake by these cells. We also demonstrated that CSH exposure results in the sequestration of iron‐59 by astroglial mitochondria (granule precursors). The results of this study suggest that stress‐related trapping of nonheme iron by astroglial mitochondria may be an important mechanism underlying the pathological accumulation of redox‐active iron in the basal ganglia of subjects with Parkinson's disease. CSH‐treated astrocytes provide a useful model to investigate the role of stress‐related dysregulation of neuroglial iron metabolism in the aging and degenerating nervous system.
Preproenkephalin mRNA and Methionine‐Enkephalin Content Are Increased in Mouse Striatum After Treatment with Nicotine
Authors: Dhatt Ravinder K. , Gudehithlu Krishnamurthy P. , Wemlinger Trina A. , Tejwani Gopi A. , Neff Norton H. , Hadjiconstantinou Maria
Published: July 2010

Abstract: A single dose of nicotine increased methionine‐enkephalin (Met‐Enk) immunoreactivity in the striatum of mice in a time‐dependent manner. Met‐Enk content reached a maximum by ∼1 h after nicotine and returned to control values by 6 h. The response to nicotine was blocked by pretreating animals with the nicotinic receptor antagonist mecamylamine. In contrast, pretreating mice with the muscarinic receptor antagonist atropine or the dopamine receptor antagonist haloperidol did not block the response. A single dose of nicotine also increased mRNA for the precursor peptide preproenkephalin (PPE). The increase of PPE mRNA preceded that of Met‐Enk and reached a maximum by ∼30 min after nicotine. PPE mRNA levels returned to near normal by ∼3 h and increased again by 6 h after nicotine. Daily administration of nicotine for 14 days increased Met‐Enk content and PPE mRNA in the striatum of mice as well. Taken together, our results suggest that nicotinic receptors modulate Met‐Enk content and PPE mRNA in the mouse striatum.
Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon
Authors: Amaratunga Anil , Leeman Susan E. , Kosik Kenneth S. , Fine Richard E.
Published: July 2010

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin‐like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide‐containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.
Protein and Molecular Characterization of Hippocampal Protein Kinase C in C57BL/6 and DBA/2 Mice
Authors: Bowers Barbara J. , Christensen Steven C. , Pauley James R. , Paylor Richard , Yuva Lisa , Dunbar Scott E. , Wehner Jeanne M.
Published: July 2010

Abstract: Measures of protein kinase C (PKC) in C57BL/6 and DBA/2 mice using [3H]phorbol 12,13‐dibutyrate binding to tissue homogenates and brain slices demonstrated that levels of activated, membrane‐bound PKC were greater in C57BL hippocampus than in DBA hippocampus. Western analysis of α‐, βI‐, βII‐, γ‐, δ‐, and ɛ‐PKC using isozyme‐specific antibodies indicated that the increase observed in C57BL hippocampus was due primarily to the γ‐PKC protein, whose immunoreactivity was greater in the membrane‐bound fraction in C57BL mice. Characterization of α‐, βI,II‐, and γ‐PKC hippocampal mRNA using northern analysis and isozyme‐specific nucleic acid probes did not reveal differences between the strains in levels of gene expression. Restriction fragment length polymorphisms (RFLP) were found in the α‐ and γ‐, but not β‐PKC genomic DNA. The RFLPs appeared to be located in noncoding, nonregulatory regions of the gene. These findings suggest that the γ‐PKC isozyme is largely responsible for the PKC activity difference in C57BL and DBA hippocampus that has been reported previously and may be closely associated with differences in learning ability observed in these strains.
Dissociation of Striatal GTPase and Dopamine Release Responses to Muscarinic Cholinergic Agonists in F344 Rats: Influence of Age and Dietary Manipulation
Authors: Kelly Jeremiah F. , Joseph James A. , Denisova Natalia A. , Erat Steven , Mason R. Preston , Roth George S.
Published: July 2010

Abstract: There is evidence that dietary lipids and age both influence neuronal membrane composition and receptor G protein‐linked signal transduction, but very little information is available on the interaction between these two factors. To investigate this, we obtained striata from 2, 12, and 22‐month‐old male F344 rats who were fed either a high‐cholesterol, high‐saturated fat or low‐fat diet for 1 month. The striata were assayed for muscarinic agonist‐stimulated low‐Km GTPase activity using 10−3M carbachol and 10−5M oxotremorine and for KCl‐evoked dopamine release enhancement by 10−5M oxotremorine. Membrane cholesterol and phospholipid content and phospholipid class composition were also determined. Mature animals showed significant but divergent changes in GTPase activity and dopamine release for high‐cholesterol and low‐fat diets: GTPase activity decreased, whereas dopamine release increased in these groups. Alterations in GTPase activity but not in dopamine release were inversely correlated with the cholesterol/phospholipid molar ratio. Old control animals showed reductions in both GTPase activity and oxotremorine‐enhanced dopamine release compared with young animals. Whereas none of the experimental diets affected GTPase activity in old animals, the low‐fat diet produced a marked decrease in dopamine release. In contrast to mature and old groups, young rats showed no significant change in either GTPase or dopamine release, suggesting a relative “resistance” to such dietary lipid modulation. The observed dissociation in GTPase and dopamine release responses to diet may reflect differing effects of these diets on discrete membrane lipid domains that preferentially influence different signal transduction components. The substantial age‐related differences in striatal membrane response to dietary lipid modulation may represent the effects of underlying age differences in membrane lipid metabolism, structure, and/or dynamics. Our findings support the work of other groups that have shown that brain membranes are susceptible to modification by exogenous lipids. They also suggest the need for a more systematic examination of the influence of age on the response to other types of dietary lipid changes.
Uptake of l‐Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter
Authors: Roseth Svein , Fykse Else Marie , Fonnum Frode
Published: July 2010

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron‐donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl−. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A1, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.
The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System
Authors: Poduslo Joseph F. , Walikonis Randall S. , Domec Marie‐Christine , Berg Carole T. , Holtz‐Heppelmann Carrie J.
Published: July 2010

Abstract: The adenylyl cyclase‐cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin‐specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P0 mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P0 gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3‐isobutyl‐1‐methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short‐ or long‐term treatment with IBMX and forskolin in the transected nerve. A three‐fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger‐dependent stimuli when Schwann cells are non‐myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.
Repeated Administration of γ‐VinylGABA Reduces Rat Brain Glutamine Synthetase Activity
Authors: Waniewski Robert A. , Martin David L.
Published: July 2010

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ‐vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ‐vinylGABA administration. Amino acid analysis of the cortex from γ‐vinylGABA‐treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ‐vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.
Alteration of Catecholamine Phenotype in Transgenic Mice Influences Expression of Adrenergic Receptor Subtypes
Authors: Kobayashi Kazuto , Ota Akira , Togari Akifumi , Morita Shinji , Mizuguchi Tomoko , Sawada Hirohide , Yamada Keiki , Nagatsu Ikuko , Matsumoto Shosei , Fujita Keisuke , Nagatsu Toshiharu
Published: July 2010

Abstract: Agonist‐induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N‐methyltransferase (PNMT) under the control of a human dopamine β‐hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high‐level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)‐[125I]iodocyanopindolol to determine changes in binding sites of β‐AR subtypes. In transgenic mice, the number of β2‐AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1‐AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down‐regulation of β2 binding sites was accompanied by a significant decrease in steady‐state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β‐AR subtypes in their target tissues.
A Quantitative Analysis of Isoferritins in Select Regions of Aged, Parkinsonian, and Alzheimer's Diseased Brains
Authors: Connor James R. , Snyder Brian S. , Arosio Paolo , Loeffler David A. , LeWitt Peter
Published: July 2010

Abstract: The brain requires a ready supply of iron for normal neurological function, but free iron is toxic. Consequently, iron bioavailability must be stringently regulated. Recent evidence has suggested that the brain iron regulatory system is dysfunctional in neurological disorders such as Alzheimer's and Parkinson's diseases (AD and PD, respectively). A key component of the iron regulatory system in the brain is ferritin. Ferritin consists of 24 subunits, which are distinguished as either a heavy‐chain (H) or light‐chain (L) isoform. These peptide subunits are genetically and functionally distinct. Thus, the ability to investigate separately the types of ferritin in brain should provide insight into iron management at both the cellular and the molecular level. In this study, the ratio of isoferritins was determined in select regions of adult elderly AD and PD human brains. The H‐rich ferritin was more abundant in the young brain, except in the globus pallidus where the ratio of H/L ferritin was 1:1. The balance of H/L isoferritins was influenced by age, brain region, and disease state. With normal aging, both H and L ferritin increased; however, the age‐associated increase in isoferritins generally failed to occur in AD and PD brain tissue. The imbalance in H/L isoferritins was disease and region specific. For example, in frontal cortex, there was a dramatic (fivefold) increase in the ratio of H/L ferritin in AD brains but not in PD brains. In PD, caudate and putamen H/L ratios were higher than in AD and the elderly control group. The analysis of isoferritin expression in brain provides insight into regional iron regulation under normal conditions and suggests a loss of ability to maintain iron homeostasis in the two disease states. This latter observation provides further evidence of dysfunction of iron homeostatic mechanisms in AD and PD and may contribute significantly to understanding the underlying pathogenesis of each, particularly in relation to iron‐induced oxidative damage.
Imidazoleacetic Acid, a γ‐Aminobutyric Acid Receptor Agonist, Can Be Formed in Rat Brain by Oxidation of Histamine
Authors: Thomas Boban , Prell George D.
Published: July 2010

Abstract: It is generally accepted that in mammalian brain histamine is metabolized solely by histamine methyltransferase (HMT), to form tele‐methylhistamine, then oxidized to tele‐methylimidazoleacetic acid. However, histamine's oxidative metabolite in the periphery, imidazoleacetic acid (IAA), is also present in brain and CSF, and its levels in brain increase after inhibition of HMT. To reinvestigate if brain has the capacity to oxidize histamine and form IAA, conscious rats were injected with [3H]histamine (10 ng), either into the lateral ventricles or cisterna magna, and decapitated 30 min later. In brains of saline‐treated rats, most radioactivity recovered was due to tele‐methylhistamine and tele‐methylimidazoleacetic acid. However, significant amounts of tritiated IAA and its metabolites, IAA‐ribotide and IAA‐riboside, were consistently recovered. In rats pretreated with metoprine, an inhibitor of HMT, labeled IAA and its metabolites usually comprised the majority of histamine's tritiated metabolites. [3H]Histamine given intracisternally produced only trace amounts of oxidative metabolites. Formation of IAA, a potent GABA‐A agonist with numerous neurochemical and behavioral effects, from minute quantities of histamine in brain indicates a need for reevaluation of histamine's metabolic pathway or pathways in brain and suggests a novel mechanism for interactions between histamine and the GABAergic system.
Evidence that 5′‐Cytidinediphosphocholine Can Affect Brain Phospholipid Composition by Increasing Choline and Cytidine Plasma Levels
Authors: López‐Coviella Ignacio , Agut Julian , Savci Vahide , Ortiz J. Alfonso , Wurtman Richard J.
Published: July 2010

Abstract: We examined the effects of orally administered 5′‐cytidinediphosphocholine (CDP‐choline) on arterial plasma choline and cytidine levels and on brain phospholipid composition in rats. Animals receiving a single oral dose of 100, 250, or 500 mg/kg showed peak plasma choline levels 6–8 h after drug administration (from 12 ± 1 to 17 ± 2, 19 ± 2, and 24 ± 2 µM, respectively). The area under the plasma choline curve at >14 µM, i.e., at a concentration that induces a net influx of choline into the brain, was significantly correlated with CDP‐choline dose. In rats receiving 500 mg/kg this area was 2.3 times that of animals consuming 250 mg/kg, which in turn was 1.8 times that of rats receiving 100 mg/kg. Plasma cytidine concentrations increased 5.4, 6.5, and 15.1 times baseline levels, respectively, 8 h after each of the three doses. When the oral CDP‐choline treatment was prolonged for 42 and 90 days, brain phosphatidylcholine concentrations increased significantly (by 22–25%; p < 0.05) in rats consuming 500 mg/kg/day. Brain phosphatidylethanolamine and phosphatidylserine concentrations also increased significantly under some experimental conditions; levels of other phospholipids were unchanged.
Potentiation of Agonist‐Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes
Authors: Ho Anthony K. , Wiest Reid , Ogiwara Takayuki , Murdoch Gordon , Chik Constance L.
Published: July 2010

Abstract: To study cross‐talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic‐stimulated cyclic AMP production was investigated. Both norepinephrine‐ and isoproterenol‐stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration‐dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic‐stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin‐or forskolin‐stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic‐stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.
Toxic NMDA‐Receptor Activation Occurs During Recovery in a Tissue Culture Model of Ischemia
Authors: Vornov James J.
Published: July 2010

Abstract: In some animal models of reversible ischemia, there is a therapeutic window during early recovery when glutamate receptor antagonists can rescue neurons from injury. We have previously reported that organotypic cultures of the hippocampus can be protected by NMDA‐receptor antagonists during recovery from a brief period of simulated ischemia. To model ischemia, we have used potassium cyanide to inhibit oxidative metabolism and 2‐deoxyglucose to inhibit glycolysis. To study the time course and mechanisms of delayed NMDA‐receptor toxicity in more detail, we have extended these studies to dissociated cortical cultures. Injury was assessed by release of lactate dehydrogenase into the culture medium. Metabolic inhibition for 15 min caused dose‐dependent injury. Morphologic signs of neuronal toxicity were delayed until the recovery period. MK‐801 reduced injury significantly when present throughout the experiment. Surprisingly, MK‐801 provided the same protection when administration was delayed until after the end of the metabolic inhibition, blocking NMDA receptors only during recovery. To examine NMDA toxicity during metabolic inhibition, the competitive NMDA‐receptor antagonist 3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid was added during exposure. The protective effect of NMDA‐receptor blockade was completely lost if the antagonist was removed during 1 min of continuing selective inhibition of oxidative metabolism. The toxic potency and effectiveness of glutamate were enhanced during metabolic inhibition, showing that receptors were not inactivated by simulated ischemia. These results are consistent with the specific hypothesis that return of oxidative metabolism triggers a critical period of toxic NMDA‐receptor activation.
Nyctohemeral Rhythm in the Levels of S‐Adenosylmethionine in the Rat Pineal Gland and Its Relationship to Melatonin Biosynthesis
Authors: Sitaram Balvant R. , Sitaram Manjula , Traut Martin , Chapman Colin B.
Published: July 2010

Abstract: Liquid chromatographic techniques that permit the simultaneous analysis of S‐adenosylmethionine, melatonin, and its intermediary metabolites N‐acetyl‐5‐hydroxytryptamine and 5‐hydroxytryptamine within individual pineal glands have been developed. S‐Adenosylmethionine has been shown to undergo a marked nyctohemeral rhythm in the pineal gland of the rat, with maximal levels occurring during the light period and minimal levels during the dark period. Detailed studies of the temporal relationships between the levels of S‐adenosylmethionine and those of melatonin and its intermediary metabolites suggest that an association exists between the levels of S‐adenosylmethionine and the status of the biosynthesis of melatonin. Exposure of animals to continuous light and the administration of the β‐adrenoreceptor antagonist propranolol were both found to inhibit the induction of melatonin synthesis and prevent the reduction in the levels of S‐adenosylmethionine during the dark period. As a corollary the induction of melatonin biosynthesis following the administration of the β‐adrenoreceptor agonist isoproterenol during the light period was accompanied by a marked decrease in the levels of S‐adenosylmethionine in the pineal gland. The significance of the link between the nyctohemeral rhythms in the levels of S‐adenosylmethionine and the biosynthesis of melatonin in the pineal gland is discussed in the context of the therapeutic efficacy of S‐adenosylmethionine as an antidepressant.