bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning of a Peptidase Against N‐Acetylaspartylglutamate from a Rat Hippocampal cDNA Library
Authors: Bzdega Tomasz , Turi Thomas , Wroblewska Barbara , She Dewei , Chung Hye Sun , Kim Hyun , Neale Joseph H.
Published: July 2010

Abstract: N‐Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate‐specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752‐amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by β‐NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5′ end, the central region, and the 3′ end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5′ end of the hippocampal cDNA.
Noradrenergic Activity Differentially Regulates the Expression of Rolipram‐Sensitive, High‐Affinity Cyclic AMP Phosphodiesterase (PDE4) in Rat Brain
Authors: Ye Ying , Conti Marco , Houslay Miles D. , Farooqui Shakeel M. , Chen Ming , O'Donnell James M.
Published: July 2010

Abstract: In a previous study, it was observed that the activity of rolipram‐sensitive, low‐Km, cyclic AMP phosphodiesterase (PDE4) was decreased in vivo with diminished noradrenergic stimulation. The results of the present experiments indicated that the reduction in the activity may be associated with down‐regulation of PDE4 protein. Immunoblot analysis using PDE4‐specific, subfamily‐nonspecific antibody (K116) revealed four major bands of PDE4 in rat cerebral cortex; those with apparent molecular masses of 109 and 102 kDa are variants of PDE4A. Diminished noradrenergic activity, produced by intracerebroventricular infusion of 6‐hydroxydopamine (6‐OHDA) or chronic subcutaneous infusion of propranolol, decreased the intensities of the protein bands for the 109‐ and 102‐kDa PDE4A variants in rat cerebral cortex but not of the 98‐ or 91‐kDa PDE4 forms. 6‐OHDA‐induced noradrenergic lesioning also decreased the content of 102‐kDa PDE4A in hippocampus as labeled by PDE4A‐specific antibody (C‐PDE4A). Enhanced noradrenergic stimulation up‐regulated PDE4 in cerebral cortex. This was indicated by the finding that repeated treatment with desipramine increased the intensity of the protein band for the 102‐kDa PDE4 but not for the other variants of PDE4. These results suggest that PDE4 subtypes are differentially regulated at the level of expression, as evidenced by an apparent change in the amount of PDE4 protein, following changes in noradrenergic activity. These observations are consistent with the notion that PDE4s, especially the PDE4A variants with molecular masses of 109 and 102 kDa, play an important role in maintaining the homeostasis of the noradrenergic signal transduction system in the brain and may be involved in the mediation of antidepressant activity.
Nicotine Enhances the Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors
Authors: Hsu Yea‐Nan , Edwards Samuel C. , Wecker Lynn
Published: July 2010

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP‐dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for α4β2 and 500 nM for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ‐32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA‐mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.
Enhanced Hemicholinium Binding and Attenuated Dendrite Branching in Cognitively Impaired Acetylcholinesterase‐Transgenic Mice
Authors: Beeri R. , Le Novère N. , Mervis R. , Huberman T. , Grauer E. , Changeux J. P. , Soreq H.
Published: July 2010

Abstract: In a search for behavioral, neuroanatomical, and metabolic characteristics of Alzheimer's disease that may result from cholinergic malfunction, we used transgenic mice overexpressing acetylcholinesterase (AChE) mRNA and active enzyme in brain neurons. Mapping by in situ hybridization revealed that transgenic and host AChE mRNAs were distributed similarly. In a Morris water maze working memory paradigm, adult transgenic mice did not display the characteristic improvement found in control mice either between or within test days and spent less time than control mice in the platform zone. In 5‐week‐old transgenic mice, the basilar dendritic trees of layer 5 pyramidal neurons from the frontoparietal cortex were essentially as developed as in age‐matched controls. However, branching totally ceased after this age, whereas in control adults it continued up to at least 7 months. Therefore, dendritic arbors became smaller in adult transgenic mice than those of controls. Furthermore, the average number of spines was significantly lower on dendritic branches of 7‐month‐old but not 5‐week‐old transgenics as compared with controls. Binding of tritiated hemicholinium‐3, a blocker of the high‐affinity choline uptake characteristic of active cholinergic terminals, was over twofold enhanced in the brain of transgenic mice. In contrast, no differences were observed in the mRNA and ligand binding levels of several different subtypes of nicotinic and muscarinic acetylcholine receptors. These findings suggest that three different hallmarks associated with Alzheimer's disease—namely, progressive cognitive failure, cessation of dendrite branching and spine formation, and enhanced high‐affinity choline uptake—are outcomes of cholinergic malfunction.
Regional Decreases in α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid ([3H]AMPA) and 6‐[3H]Cyano‐7‐Nitroquinoxaline‐2,3‐dione ([3H]CNQX) Binding in Response to Chronic Low‐Level Lead Exposure: Reversal Versus Potentiation by Chronic Dopamine Agonist Treatment
Authors: McCoy L. , Richfield E. K. , Cory‐Slechta D. A.
Published: July 2010

Abstract: This study evaluated the hypotheses that in vivo lead (Pb) exposure would alter α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptor binding and, based on known glutamate‐dopamine interactions and Pb‐induced changes in dopamine (DA) systems, that AMPA binding might be differentially influenced by DA agonist treatment under conditions of Pb exposure. Alterations in high‐affinity ([3H]AMPA) versus total AMPA [6‐[3H]cyano‐7‐nitroquinoxaline‐2,3‐dione ([3H]CNQX)] receptor binding were determined in medial frontal cortex, dorsal striatum, and nucleus accumbens of rats exposed to 0, 50, or 150 ppm of Pb acetate for 2 weeks or 8 months. Additional 8‐month groups received chronic intermittent treatment with saline, the D1 agonist SKF82958, or the general DA agonist apomorphine. Two‐week exposures increased AMPA receptor densities, whereas robust decreases occurred after 8 months of Pb; at the latter time point changes were more pronounced for high‐affinity than total AMPA receptor binding, with high‐affinity effects expressed preferentially in dorsal striatum and nucleus accumbens. DA agonist treatments almost fully reversed Pb‐related declines in [3H]AMPA binding but either had no effect (apomorphine) or even further potentiated (SKF82958) the decreases in [3H]CNQX binding. One possible basis for the long‐term (8‐month) decrease in AMPA binding is a postsynaptic glutamatergic stimulation of non‐NMDA receptors.
Constitutive and Protein Kinase C‐Regulated Secretory Cleavage of Alzheimer's β‐Amyloid Precursor Protein: Different Control of Early and Late Events by the Proteasome
Authors: Marambaud Philippe , Lopez‐Perez Elvira , Wilk Sherwin , Checler Frédéric
Published: July 2010

Abstract: The physiological processing of the β‐amyloid precursor protein (βAPP) by a protease called α‐secretase gives rise to APPα, a C‐terminally truncated fragment of βAPP with known neurotrophic and cytoprotective properties. Several lines of evidence indicate that protein kinase C (PKC)‐mediated events regulate this physiological pathway. We show here that the proteasome multicatalytic complex modulates the phorbol 12,13‐dibutyrate‐stimulated APPα secretion at several levels in human kidney 293 (HK293) cells. Two blocking agents of the proteasome, namely, Z‐IE(Ot‐Bu)A‐leucinal and lactacystin, elicit a dual effect on PKC‐regulated APPα secretion by metabolically labeled HK293 cells. Thus, short periods of preincubation (2–5 h) of the cells with the inhibitors trigger a drastic potentiation of APPα recovery, whereas long‐term treatment of the cells (15–20 h) with the blocking agents leads to an overall decrease in the secretion of APPα. Such a dual effect was not observed on constitutive APPα secretion and intracellular formation generated by HK293 cells, which both only increase upon inhibitor treatments. Similar effects on the constitutive and PKC‐regulated APPα secretion were observed with PC12 cells. Altogether, these data suggest distinct mechanisms underlying basal and PKC‐regulated APPα production, indicating that this multicatalytic complex appears as a key contributor of the α‐secretase pathway.
Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity
Authors: Okada Motohiro , Kawata Yuko , Kiryu Kazuhiro , Mizuno Kazuhisa , Wada Kazumaru , Tasaki Hiroichi , Kaneko Sunao
Published: July 2010

Abstract: To clarify the effects of adenosine receptor subtypes (A1, A2, and A3) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5‐HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A1 receptor agonist, 2‐chloro‐N6‐cyclopentyladenosine, decreased extracellular 5‐HT levels, whereas an adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT), and caffeine increased these levels. A selective A2A receptor agonist (CGS‐21680), an adenosine A2 receptor agonist (PD‐125944), an adenosine A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX), and an adenosine A3 receptor agonist, N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5‐HT levels. When the adenosine A1 receptor was blocked by CPT, the hippocampal extracellular 5‐HT level was increased by adenosine, CGS‐21680, and PD‐125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A1 and A2 receptors were blocked by CPT and DMPX, the extracellular 5‐HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5‐HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A1 receptor was blocked, irrespective of whether the adenosine A2 receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5‐HT levels, during both adenosine A1 and A2 receptor blockade, were inhibited by selective 5‐HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A2 receptor and the inhibitory effects of the A3 receptor on hippocampal extracellular 5‐HT levels are masked by the inhibitory effects of the adenosine A1 receptor.
α2‐Adrenergic Receptor Blockade Markedly Potentiates Duloxetine‐ and Fluoxetine‐Induced Increases in Noradrenaline, Dopamine, and Serotonin Levels in the Frontal Cortex of Freely Moving Rats
Authors: Gobert A. , Rivet J.‐M. , Cistarelli L. , Melon C. , Millan M. J.
Published: July 2010

Abstract: Evidence exists that a reinforcement in monoaminergic transmission in the frontal cortex (FCX) is associated with antidepressant (AD) properties. Herein, we examined whether blockade of α2‐adrenergic receptors modified the influence of monoamine reuptake inhibitors on FCX levels of serotonin (5‐HT), noradrenaline (NAD), and dopamine (DA). The selective α2‐adrenergic receptor agonist S 18616 (0.16 mg/kg, s.c.) suppressed extracellular levels of NAD, DA, and 5‐HT (by 100, 51, and 63%, respectively) in single dialysates of FCX of freely moving rats. In contrast, the selective α2‐adrenergic receptor antagonists atipamezole (0.16 mg/kg, s.c.) and 1‐(2‐pyrimidinyl)piperazine (1‐PP; 2.5 mg/kg, s.c.) increased levels of NAD (by 180 and 185%, respectively) and DA (by 130 and 90%, respectively), without affecting 5‐HT levels. Duloxetine (5.0 mg/kg, s.c.), a mixed inhibitor of 5‐HT and NAD reuptake, and fluoxetine (10.0 mg/kg, s.c.), a selective 5‐HT reuptake inhibitor, both increased levels of 5‐HT (by 150 and 120%, respectively), NAD (by 400 and 100%, respectively), and DA (by 115 and 55%, respectively). Atipamezole (0.16 mg/kg, s.c.) markedly potentiated the influence of duloxetine and fluoxetine on levels of 5‐HT (by 250 and 330%, respectively), NAD (by 1,030 and 215%, respectively), and DA (by 370 and 170%, respectively). 1‐PP similarly potentiated the influence of duloxetine on 5‐HT, NAD, and DA levels (by 290, 1,320, and 600%, respectively). These data demonstrate that α2‐adrenergic receptors tonically inhibit NAD and DA and phasically inhibit 5‐HT release in the FCX and that blockade of α2‐adrenergic receptors strikingly potentiates the increase in FCX levels of 5‐HT, NAD, and DA elicited by reuptake inhibitors. Concomitant α2‐adrenergic receptor antagonism and inhibition of monoamine uptake may thus provide a mechanism allowing for a marked increase in the efficacy of AD agents.
Retinoic Acid Induces Apoptosis‐Associated Neural Differentiation of a Murine Teratocarcinoma Cell Line
Authors: Herget Thomas , Specht Heike , Esdar Christina , Oehrlein Silke A. , Maelicke Alfred
Published: July 2010

Abstract: Incubation with all‐trans retinoic acid (RA) induces PCC7‐Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue‐like pattern of neuronal, astroglial, and fibroblast‐like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA‐induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan‐caspase inhibitor zVAD‐fmk (benzyloxycarbonyl‐valinyl‐alaninyl‐aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10−7M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron‐specific protein kinase C substrate GAP‐43. RA‐induced apoptosis increased with the plating density of PCC7‐Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA‐damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle‐arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis‐inhibiting factors by the cultured cells is indicated. Neither Apol/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl‐2 proto‐oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13‐dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long‐term treatment with phorbol 12,13‐dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apoptosis of PCC7‐Mz1 cells. We conclude that the apoptotic activity following RA treatment of cultured PCC7‐Mz1 cells probably is controlled by the same cascade of gene regulatory events that govern the early cell lineage determinations in this in vitro model system of neural development.
Conditioning Heat Stress Reduces Excitotoxic and Apoptotic Components of Oxygen‐Glucose Deprivation‐Induced Neuronal Death In Vitro
Authors: Snider B. Joy , Lobner Doug , Yamada Kelvin A. , Choi Dennis W.
Published: July 2010

Abstract: We investigated the effects of sublethal heat stress in murine cortical cell cultures exposed to combined oxygen and glucose deprivation. Pretreatment with sublethal heat stress mildly attenuated the widespread neuronal death induced a day later by 30–60 min of oxygen‐glucose deprivation. Heat stress also blunted the increase in extracellular glutamate concentrations induced by the oxygen‐glucose deprivation, as well as the neuronal death and 45Ca2+ uptake induced by exogenous addition of NMDA, although no reduction was seen in neuronal death caused by exogenous kainate or in NMDA‐induced whole‐cell currents. However, arguing against the idea that the neuroprotective effect of heat stress against neuronal death was exclusively due to reduction of excitotoxicity was the finding that heat stress also reduced the neuronal apoptosis induced by oxygen‐glucose deprivation in the presence of glutamate antagonists. This antiapoptotic effect was specific in that heat stress did not reduce neuronal vulnerability to staurosporine‐induced apoptosis. Whereas heat stress transiently suppressed protein synthesis, achieving comparable protein synthesis inhibition with cycloheximide did not reproduce the neuroprotective effects of heat stress. These studies suggest that a conditioning heat stress is able to attenuate both the excitotoxic and the apoptotic components of oxygen‐glucose deprivation‐induced neuronal death in vitro, by mechanisms independent of protein synthesis reduction.
Arginine and Lysine Aminopeptidase Activities in Chromaffin Granules of Bovine Adrenal Medulla: Relevance to Prohormone Processing
Authors: Yasothornsrikul Sukkid , Toneff Thomas , Hwang Shin‐Rong , Hook Vivian Y. H.
Published: July 2010

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH2 and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg‐methylcoumarinamide (Arg‐MCA) and Lys‐MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co‐localized with [Met]‐enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β‐mercaptoethanol (β‐ME) and inhibited by p‐hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β‐ME/NaCl‐stimulated Arg‐MCA cleaving activity and β‐ME‐stimulated Lys‐MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. Km(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg‐MCA and Lys‐MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co2+ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni2+ and Zn2+ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.
Nigrostriatal Dopamine Neurons Express Low Levels of GTP Cyclohydrolase I Protein
Authors: Hirayama Kei , Kapatos Gregory
Published: July 2010

Abstract: A previous study using the in situ hybridization technique showed that serotonin neurons contain substantially more GTP cyclohydrolase I mRNA than do either dopamine or norepinephrine neurons. The objective of the current study was to determine whether these differences in mRNA abundance are predictive of the amount of GTP cyclohydrolase I protein available for tetrahydrobiopterin biosynthesis. The double‐label immunofluorescence technique was used to localize GTP cyclohydrolase I protein to the tyrosine hydroxylase‐positive A9 dopamine neurons of the substantia nigra and the A6 norepinephrine neurons of the locus ceruleus or the tryptophan hydroxylase‐positive B6/B7 serotonin neurons of the dorsal raphe nucleus. Although GTP cyclohydrolase I immunofluorescence within serotonin and norepinephrine neurons was relatively intense, the fluorescence signal within dopamine neurons was faint to nondetectable. An immunoautoradiographic technique was developed to quantify these apparent differences in GTP cyclohydrolase I protein expression at the cellular level. Significant differences between all three neurochemical subdivisions were found and comparisons showed that on average serotonin neurons contain between 2.3‐ and 7.3‐fold more GTP cyclohydrolase I protein than do either norepinephrine or dopamine neurons, respectively. Nigrostriatal dopamine neurons thus appear to synthesize and maintain tetrahydrobiopterin at low levels. Because dopamine and norepinephrine neurons express essentially equal amounts of GTP cyclohydrolase I mRNA, posttranscriptional events may serve to maintain low levels of GTP cyclohydrolase I protein within dopamine neurons. Phenotypic differences in GTP cyclohydrolase I protein expression across populations of monoamine neurons may be an important control point in neurotransmitter biosynthesis.
In Vivo Electrochemical Studies of Dopamine Clearance in the Rat Substantia Nigra: Effects of Locally Applied Uptake Inhibitors and Unilateral 6‐Hydroxydopamine Lesions
Authors: Hoffman Alexander F. , Gerhardt Greg A.
Published: July 2010

Abstract: High‐speed chronoamperometric recordings were used to measure the uptake and clearance of locally applied dopamine (DA) within the substantia nigra (SN) of anesthetized rats. To establish that DA clearance within the SN was mediated primarily by the DA transporter (DAT) rather than the norepinephrine transporter (NET) or the serotonin transporter (SERT), we locally applied uptake inhibitors with different selectivity profiles for the various amine transporters. Nomifensine, a DAT/NET inhibitor, significantly potentiated both the amplitude and the time course of the DA signals. In contrast, neither the selective NET inhibitor desipramine, nor the selective SERT inhibitor citalopram affected the DA signal, suggesting that NET and SERT do not contribute to DA uptake and clearance within the regions of the SN studied over the concentration ranges (1–5 µM) used. In unilaterally 6‐hydroxydopamine‐lesioned rats, the time course of the DA signal was increased in both the lesioned SN and striatum, relative to the unlesioned hemisphere, indicating loss of DAT and decreased DA uptake and clearance. In addition, when identical amounts of DA were injected in the striatum and SN, peak signal amplitudes were larger in the SN, suggesting that the amplitudes are related to the number of DAT sites in a given region of brain tissue. For signals of equivalent amplitudes, clearance rates were lower in the SN than in the striatum, consistent with a lower capacity for DAT‐mediated DA uptake within the SN. These results suggest that the DAT is the major transporter responsible for DA clearance within the rat SN.
Chronic Ethanol Administration Decreases Phosphorylation of Cyclic AMP Response Element‐Binding Protein in Granule Cells of Rat Cerebellum
Authors: Yang Xiaoju , Horn Kristin , Baraban Jay M. , Wand Gary S.
Published: July 2010

Abstract: To help define the molecular basis of ethanol's actions on the nervous system, we have in previous studies demonstrated that ethanol administration triggers a robust increase in cyclic AMP‐response element‐binding protein (CREB) phosphorylation in the cerebellum. The purpose of the present study was to compare the effects of acute and chronic ethanol exposure on the phosphorylation of CREB in rat cerebellum and to determine which cell types in the cerebellum display this response to ethanol. An acute ethanol challenge (3.0 g/kg of body weight) induced a rapid increase in content of the phosphorylated form of CREB, peaking at 30 min and declining to basal levels within 2 h. Immunocytochemical studies revealed prominent ethanol‐induced changes in phosphoCREB in the granule cell layer, with little phosphoCREB apparent in Purkinje cells. Following chronic ethanol exposure (5 weeks), induction of CREB phosphorylation by a subsequent acute ethanol challenge was markedly attenuated. The attenuation in CREB phosphorylation was associated with a significant reduction in the levels of the catalytic unit of protein kinase A and calcium/calmodulin‐dependent protein kinase IV. In summary, induction of CREB phosphorylation in cerebellum is most prominent in the granule cell layer. Neuroadaptation to chronic ethanol exposure includes a reduction in nuclear protein kinase A and calcium/calmodulin‐dependent protein kinase IV levels, an event associated with impaired CREB phosphorylation.
The Pyruvate Dehydrogenase Complex Is Partially Inactivated During Early Recirculation Following Short‐Term Forebrain Ischemia in Rats
Authors: Zaidan Emad , Sheu Kwan‐Fu Rex , Sims Neil R.
Published: July 2010

Abstract: The mechanisms of selective neuronal loss after short‐term global ischemia remain undefined, but processes including increased proteolytic activity, impaired protein synthesis, and oxidative damage have been proposed to contribute. A decrease in activity of the pyruvate dehydrogenase complex in the dorsolateral striatum, an ischemia‐susceptible region, is one change apparently differentiating this region from ischemia‐resistant areas during early recirculation. To provide an insight into processes contributing to postischemic cell damage, the changes in the pyruvate dehydrogenase complex during early recirculation have been further characterized. These studies provide clear confirmation that the activity of the pyruvate dehydrogenase complex is reduced in mitochondria from the dorsolateral striatum by 3 h of recirculation. The decrease in activity was not accompanied by a loss of antigenic sites or by changes in electrophoretic mobility of the components of the complex. A reduction in activity of the E1 component of the complex (39–42% decrease), but not the E2 and E3 components, was observed that was apparently sufficient to explain the decrease in activity of the whole complex. These results indicate that the changes in activity of the pyruvate dehydrogenase complex in the dorsolateral striatum are not due to loss or gross disruption of the constituent proteins but rather most likely reflect a selective inactivation of a specific component of the complex.
Ligand Binding and Modulation of Cyclic AMP Levels Depend on the Chemical Nature of Residue 192 of the Human Cannabinoid Receptor 1
Authors: Chin Chen‐ni , Lucas‐Lenard Jean , Abadji Vasiliki , Kendall Debra A.
Published: July 2010

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein‐coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild‐type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP‐55,940 and the aminoalkylindole WIN 55,212‐2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP‐55,940, whereas WIN 55,212‐2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand‐induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212‐2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP‐55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212‐2 binding, the data suggest a role for Lys192 in WIN 55,212‐2‐induced receptor activation.
Proteolytic Processing of Chromogranin B and Secretogranin II by Prohormone Convertases
Authors: Laslop Andrea , Weiss Christian , Savaria Diane , Eiter Christine , Tooze Sharon A. , Seidah Nabil G. , Winkler Hans
Published: July 2010

Abstract: Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE‐11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE‐11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5‐A and PC5‐B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE‐11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine‐tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.
Steady‐State Cerebral Glucose Concentrations and Transport in the Human Brain
Authors: Gruetter Rolf , Ugurbil Kâmil , Seaquist Elizabeth R.
Published: July 2010

Abstract: Understanding the mechanism of brain glucose transport across the blood‐brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis‐Menten kinetics. These models predict that the steady‐state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis‐Menten constant for half‐maximal transport, Kt. In experiments where steady‐state plasma glucose content was varied from 4 to 30 mM, the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 mM, the brain glucose level approached 9 mM, which was significantly higher than predicted from the previously reported Kt of ∼4 mM (p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis‐Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower Kt of 0.6 ± 2.0 mM, which was consistent with the previously reported millimolar Km of GLUT‐1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis‐Menten kinetics.
Pretreatment of Astrocytes with Interferon‐α/β Prevents Neuronal Mitochondrial Respiratory Chain Damage
Authors: Stewart Victoria C. , Land John M. , Clark John B. , Heales Simon J. R.
Published: July 2010

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Interferon‐β is used for the treatment of multiple sclerosis, but currently little is known regarding its mode of action. Exposure of astrocytes in culture to interferon‐γ plus lipopolysaccharide results in stimulation of nitric oxide release. Using a coculture system, we have been able to use astrocytes as a source of nitric oxide/peroxynitrite in an attempt to “model” the effects of raised cytokine levels observed in multiple sclerosis and to monitor the effect on neurones. Our results indicate that stimulation of astrocytic nitric oxide synthase activity causes significant damage to the mitochondrial activities of complexes II/III and IV of neighbouring neurones. This damage was prevented by a nitric oxide synthase inhibitor, suggesting that the damage was nitric oxide‐mediated. Furthermore, interferon‐α/β also prevented this damage. In view of these results, we suggest that a possible mechanism of action of interferon‐β in the treatment of multiple sclerosis is that it prevents astrocytic nitric oxide production, thereby limiting damage to neighbouring cells, such as neurones.
Exon‐Intron Structure, Analysis of Promoter Region, and Chromosomal Localization of the Human Type 1 σ Receptor Gene
Authors: Prasad Puttur D. , Li Hui W. , Fei You‐Jun , Ganapathy Malliga E. , Fujita Takuya , Plumley Lisa H. , Yang‐Feng Teresa L. , Leibach Frederick H. , Ganapathy Vadivel
Published: July 2010

Abstract: σ receptor is a protein that interacts with a variety of psychotomimetic drugs including cocaine and amphetamines and is believed to play an important role in the cellular functions of various tissues associated with the endocrine, immune, and nervous systems. Here we report on the structure and organization of the human gene coding for this receptor. The gene is ∼7 kbp long and contains four exons, interrupted by three introns. Exon 3 is the shortest (93 bp), and exon 4 is the longest (1,132 bp). Among the introns, intron 3 is the longest (∼1,250 bp). Exon 2 codes for the single transmembrane domain present in the receptor. 5′ rapid amplification of cDNA end reactions with mRNA from the JAR human trophoblast cell line have identified 56 bp upstream of the translation start codon as the initiation site for transcription. This transcription start site has been confirmed by RNase protection analysis. Structural analysis of the 5′ flanking region has revealed that the gene is TATA‐less. This region, however, contains a CCAATC box in the reverse complement and several GC boxes that are recognition sites for SP1. There are also consensus sequences for the liver‐specific transcription factor nuclear factor‐1/L, for a variety of cytokine responsive factors, and for the xenobiotic responsive factor called the arylhydrocarbon receptor. Southern blot analysis of the genomic DNA from Chinese hamster‐human and mouse‐human hybrid cell lines and fluorescent in situ hybridization with human metaphase chromosome spreads have shown that the gene is located on human chromosome 9, band p13, a region known to be associated with different psychiatric disorders.
Synthesis, Shedding, and Intercellular Transfer of Human Medulloblastoma Gangliosides: Abrogation by a New Inhibitor of Glucosylceramide Synthase
Authors: Olshefski Randal , Ladisch Stephan
Published: July 2010

Abstract: The biological activity of human medulloblastoma tumor gangliosides very likely involves the interaction of these molecules with host cells in the tumor microenvironment. To trace the hypothesized intercellular transfer of shed medulloblastoma gangliosides, we used an in vitro dual‐chamber culture system in which the tumor cells, the shed gangliosides, and the target cells to which they might bind would not be perturbed during the transfer process. We observed that under these unmanipulated conditions, gangliosides were shed by the Daoy medulloblastoma cell line (∼300 pmol/108 cells/h), traversed the chamber membrane, and stably bound to the target fibroblasts at the very high density of 107 molecules per cell within 48 h. To determine if this substantial intercellular transfer of shed gangliosides, with its potential of modifying target cell function, could be blocked, we evaluated a new inhibitor of glucosylceramide synthase, dl‐threo‐1‐phenyl‐2‐hexadecanoylamino‐3‐pyrrolidino‐1‐propanol (PPPP). PPPP (1.0 µM) reduced (90%) Daoy cell ganglioside content strikingly, without causing toxicity or inhibiting cell proliferation. Subsequently, ganglioside shedding by the medulloblastoma cells was diminished significantly (to ∼50 pmol/108/h), and binding of radiolabeled shed medulloblastoma gangliosides to target fibroblasts was consequently almost completely abrogated. We conclude that the shedding and transfer of potentially biologically active human medulloblastoma gangliosides can be diminished effectively by PPPP.
A Conditionally Immortalized Glial Cell Line that Expresses Mature Myelin Proteins and Functional GABAA Receptors
Authors: Bronstein J. M. , Hales T. G. , Tyndale R. F. , Charles A. C.
Published: July 2010

Abstract: We have isolated and characterized a conditionally immortalized glial cell line that expresses mature myelin proteins, as well as functional GABAA receptors. Glial cells were isolated from postnatal day 1 H‐2Kb‐tsA58 transgenic mice that contain the temperature‐sensitive SV40 large T antigen oncogene under the control of an interferon‐γ‐inducible promoter. A clonal line was isolated that grew rapidly under permissive conditions (33°C in the presence of interferon‐γ), but not under nonpermissive conditions (37°C in the absence of interferon‐γ). Cells expressed mRNAs of mature myelin proteins (myelin basic proteins and proteolipid protein) when grown under either permissive or nonpermissive conditions, but myelin basic proteins were detected only when cells were maintained at 37°C. GABA induced an increase in intracellular calcium concentration, indicating the presence of functional receptors for this transmitter. Characterization of the GABA‐evoked calcium response and whole‐cell currents indicated that these responses were mediated by GABAA receptors. Reverse transcription‐polymerase chain reaction revealed differential expression of mRNAs for specific GABAA receptor subunits. Messages for the α2, α4, β1, and δ subunits were readily detected by reverse transcription‐polymerase chain reaction, whereas messages for the α1, α3, α5, α6, β2, β3, and γ1–3 subunits were not. As this cell line can be easily propagated and differentiated, it should provide a valuable tool in studying not only the development of oligodendroglia, but also the function of their transmitter receptors and myelin proteins.
Neuropathological Abnormalities in Transgenic Mice Harbouring a Phosphorylation Mutant Neurofilament Transgene
Authors: Gibb Barry J. M. , Brion Jean‐Pierre , Brownlees Janet , Anderton Brian H. , Miller Christopher C. J.
Published: July 2010

Abstract: Ser55 within the head domain of neurofilament light chain (NF‐L) is a target for phosphorylation by protein kinase A. To understand further the physiological role(s) of NF‐L Ser55 phosphorylation, we generated transgenic mice with a mutant NF‐L transgene in which Ser55 was mutated to Asp so as to mimic permanent phosphorylation. Two lines of NF‐L(Asp) mice were created and these animals express the transgene in many neurones of the central and peripheral nervous systems. Both transgenic lines display identical, early onset, and robust pathological changes in the brain. These involve the formation of NF‐L(Asp)‐containing perikaryal neurofilament inclusion bodies and the development of swollen Purkinje cell axons. Development of these pathologies was rapid and fully established in mice as young as 4 weeks of age. The two transgenic lines show no elevation of NF‐L, neurofilament middle chain (NF‐M), or neurofilament heavy chain (NF‐H), and transgenic NF‐L(Asp) represents only a minor proportion of total NF‐L protein. Because other published transgenic lines expressing higher levels of wild‐type NF‐L do not exhibit phenotypic changes that in any way resemble those in the NF‐L(Asp) mice and because the two different NF‐L(Asp) transgenic lines display identical neuropathological changes, it is likely that the pathological alterations observed in the NF‐L(Asp) mice are the result of properties of the mutant NF‐L. These results support the notion that phosphorylation of Ser55 is a mechanism for regulating neurofilament organisation in vivo.
Induction of 72‐kDa Inducible Heat Shock Protein (HSP72) in Cultured Rat Astrocytes After Energy Depletion
Authors: Imuta Naohiko , Ogawa Satoshi , Maeda Yusuke , Kuwabara Keisuke , Hori Osamu , Ueda Hirokazu , Yanagihara Takehiko , Tohyama Masaya
Published: July 2010

Abstract: Protein synthesis is important in the readaptive processes for cultured astrocytes after hypoxia and subsequent reoxygenation. We have identified 72‐kDa inducible heat shock protein (HSP72) as a major stress protein in reoxygenated astrocytes. To assess the mechanism for reoxygenation‐mediated induction of HSP72, a reporter gene that consists of a human HSP promoter fused to the luciferase gene was transfected into cultured astrocytes. Analysis of cellular energy nucleotides showed an increase of the ADP/ATP ratio after reoxygenation, which synchronized with activation of the HSP promoter. Activation of the HSP promoter was also observed after an addition of iodoacetic acid to hypoxic astrocytes, which reached the maximum when the ADP/ATP ratio reached 50%, but further decline in the energy profile caused inactivation of this promoter. Inhibition of protein synthesis after reoxygenation resulted in temporary restoration of the energy profile and suppression of the DNA binding activity of the heat shock factor. Addition of quercetin greatly decreased the [3H]leucine incorporation in the polysome fraction without any effect on the mature mRNA formation. These data suggest that the energy depletion in reoxygenation triggers induction of HSP72 after reoxygenation, which may act as a pivotal mediator in the stress response of reoxygenated astrocytes by facilitating protein synthesis.
Region‐Specific Targets of p42/p44MAPK Signaling in Rat Brain
Authors: Bhat Ratan V. , Engber Thomas M. , Finn James P. , Koury Elizabeth J. , Contreras Patricia C. , Miller Matthew S. , Dionne Craig A. , Walton Kevin M.
Published: July 2010

Abstract: In vitro studies indicate that p42/p44MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT‐induced p42/p44MAPK activation by the extracellular signal‐regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule‐associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c‐Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos‐like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44MAPK in vivo. However, the functional targets of p42/p44MAPK signaling depend on the precise location of p42/p44MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44MAPK‐mediated signal transduction within different brain regions in vivo.