bluuebunny/crossref_metadata_2025 huggingface dataset:
The Neuroendocrine Proteins Secretogranin II and III Are Regionally Conserved and Coordinately Expressed with Proopiomelanocortin in Xenopus Intermediate Pituitary
Authors: Holthuis Joost C. M. , Martens Gerard J. M.
Published: July 2010

Abstract: Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretory granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans‐Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15‐ and 35‐fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30‐fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family.
Ras‐Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells
Authors: Li HongZhen , Kawasaki Hiroshi , Nishida Eisuke , Hattori Seisuke , Nakamura Shun
Published: July 2010

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin‐6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa‐Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras‐dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.
Denervation, but Not Decentralization, Reduces Nerve Growth Factor Content of the Mesenteric Artery
Authors: Liu Dennis T. , Reid Michelle T. , McL. Bridges Diana C. , Rush Robert A.
Published: July 2010

Abstract: In the present study we applied an improved nerve growth factor (NGF) extraction method to examine the effects of denervation and sympathetic decentralization on NGF levels in vascular tissue. Adult male Wistar Kyoto rats underwent mesenteric arterial denervation or splanchnic nerve transection. Four days after operation, animals were killed, and the mesenteric artery and coeliac‐superior mesenteric ganglia were removed. The arterial adventitia was stripped from the media to measure NGF levels in nerve and smooth muscle separately. A high concentration of NGF was detected in the normal artery, 90% of which was in the adventitial layer. Surgical denervation significantly reduced the NGF levels in the artery and ganglia by 78 and 71%, respectively. However, within the artery the level of NGF was reduced in the adventitia but not in the media. Thus, the large reduction of NGF content resulted from the loss of nerve plexus from the artery. In contrast, decentralization did not alter the NGF content in the artery, in either the adventitia or media. Our results are in marked contrast to previous studies reporting elevated levels of NGF following denervation. This discrepancy is explained by the ability of our new procedure to extract much greater amounts of NGF from the tissue.
Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia
Authors: LeBlanc Andréa C. , Xue Run , Gambetti Pierluigi
Published: July 2010

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C‐terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.
Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors
Authors: Pagh‐Roehl Kathryn , Lin Danny , Burnside Beth
Published: July 2010

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS‐ROS). Isolated RIS‐ROS were cultured in the presence of membrane‐permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP‐dependent protein kinase (PKA)‐selective inhibitors (H‐89 and KT5720), (2) a protein kinase C (PKC)‐selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H‐85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light‐induced elongation; these include the myosin light chain kinase inhibitors ML‐7 and ML‐9, the calcium‐calmodulin kinase II inhibitor KN‐62, and inhibitors or activators of diacylglycerol‐dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H‐89 and GF 109203X all enhanced light‐induced elongation. These observations suggest that light‐induced RIS‐ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol‐independent form of PKC.
A Transgenic Mouse Model to Study Transsynaptic Regulation of Tyrosine Hydroxylase Gene Expression
Authors: Min Nan , Joh Tong H. , Corp Eric S. , Baker Harriet , Cubells Joseph F. , Son Jin H.
Published: July 2010

Abstract: Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5′ flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type‐specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity‐dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down‐regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg, s.c.), which up‐regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X‐gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine‐treated transgenic animals. These data showed that the 9‐kb sequence also mediates cell type‐specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.
Recoverin in Cultured Human Retinoblastoma Cells: Enhanced Expression During Morphological Differentiation
Authors: Wiechmann Allan F.
Published: July 2010

Abstract: Recoverin is a calcium‐binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 mM butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite‐like cellular processes. Addition of 4 mM dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP‐treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.
Acetylcholinesterase and Nicotinic Acetylcholine Receptor Expression Diverge in Muscular Dysgenic Mice Lacking the L‐Type Calcium Channel
Authors: Luo Z. David , Pincon‐Raymond Martine , Taylor Palmer
Published: July 2010

Abstract: L‐type Ca2+ channels play critical roles in achieving stabilization of acetylcholinesterase (AChE) mRNA during myogenesis in C2–C12 skeletal muscle cells. To ascertain the importance of this signaling pathway in AChE expression during skeletal muscle development in the animal, we examined AChE mRNA levels in skeletal muscle and heart from control (+/+) and muscular dysgenic (mdg/mdg) mice that lack the skeletal, but not the cardiac, muscle L‐type Ca2+ channels. RNase protection analysis showed 40–60% reductions in content of AChE mRNA in leg muscle, but not heart, from newborn and day 18 embryonic dysgenic mice. AChE activity was also reduced uniquely in skeletal muscle. In contrast to AChE transcripts, mRNA levels of the α‐subunit of the nicotinic acetylcholine receptors (nAChRs) were increased in dysgenic skeletal muscle. Similar alterations in activity and mRNA levels of AChE were also observed from skeletal muscle cell lines derived from mdg mice. Because run‐on transcription revealed no corresponding decrease in transcription rate, the decrease in mRNA content is likely a consequence of the inability of the dysgenic muscle cells to stabilize AChE mRNA during differentiation. These findings indicate that L‐type Ca2+ channels play an important role in regulation of AChE expression during skeletal muscle development in vivo. The differential influence of muscle dysgenesis on mRNA levels of AChE and nAChRs provides additional evidence for distinct mechanisms of regulation of these two proteins.
Biochemical Evidence of Functional Interaction Between μ‐ and δ‐Opioid Receptors in SK‐N‐BE Neuroblastoma Cell Line
Authors: Palazzi Elisabetta , Ceppi Elena , Guglielmetti Filippo , Catozzi Lidia , Amoroso Daniela , Groppetti Antonio
Published: July 2010

Abstract: Radioligand binding assays and functional experiments revealed that the SK‐N‐BE neuroblastoma cell line expresses a similar ratio of μ‐ and δ‐opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin‐sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long‐term exposure to [d‐Ala2‐N‐methyl‐Phe4‐Gly‐ol5]enkephalin (DAMGO), a μ‐selective agonist, sensitized the functional response of the δ‐selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration‐effect curve of either DAMGO or [d‐Pen2,5]enkephalin (DPDPE), a δ‐selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for μ receptors, increased the inhibitory effect of [d‐Ala2,d‐Met5]enkephalinamide (DAME), a mixed μ/δ agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between μ and δ receptors in the SK‐N‐BE cell line.
Ethanol‐Induced Inhibition of [3H]Thienylcyclohexylpiperidine (TCP) Binding to NMDA Receptors in Brain Synaptic Membranes and to a Purified Protein Complex
Authors: Michaelis Elias K. , Chen Xingyu , Joseph David B. , Hurlbert Marc , Kumar Keshava N. , Michaelis Mary L.
Published: July 2010

Abstract: N‐Methyl‐d‐asparate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol‐insensitive, but dizocilpine (MK‐801)‐sensitive, binding of N‐[1‐(2‐[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration‐dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by l‐Glu when l‐Glu was present at concentrations < 100 µM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of l‐Glu (200–400 µM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has l‐Glu‐, Gly‐, and [3H]TCP‐binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either l‐Glu or Gly. Activation constants (Kact) for l‐Glu and Gly acting on the purified complex were 12 and 28 µM, respectively. Ethanol had no significant effect on the Kact of l‐Glu but caused an increase in the Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both l‐Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.
Chimeric D2/D3 Dopamine Receptors Efficiently Inhibit Adenylyl Cyclase in HEK 293 Cells
Authors: Robinson Susan W. , Caron Marc G.
Published: July 2010

Abstract: Despite a high degree of sequence homology, the dopamine D2 and D3 receptors have substantially different second messenger coupling properties. We have used chimeric D2/D3 receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D2 receptors inhibit prostaglandin E1‐stimulated cyclic AMP levels by >90%, whereas D3 receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D2 and D3 receptors, the maximal inhibition of adenylyl cyclase is 60–90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild‐type receptors. It appears that the intracellular loops of the D3 receptor are capable of interacting with G proteins, as when these loops are expressed in the D2 receptor, the chimeras inhibit adenylyl cyclase similarly to the wild‐type D2 receptor. Our data suggest that the overall conformation of the D3 receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D3 receptor structure is altered by the introduction of D2 receptor sequence, this constraint may be lifted.
Sodium and Chloride Ion‐Dependent Transport of β‐Alanine Across the Blood‐Brain Barrier
Authors: Komura Junko , Tamai Ikumi , Senmaru Mizuho , Terasaki Tetsuya , Sai Yoshimichi , Tsuji Akira
Published: July 2010

Abstract: The characteristics of β‐alanine transport at the blood‐brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β‐[3H]alanine transport indicated that the transporter for β‐alanine functions with Kt of 25.3 ± 2.5 µM and Jmax of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β‐[3H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4‐dinitrophenol and NaN3) and low temperature reduced the uptake significantly. Furthermore, the uptake of β‐[3H]alanine required Na+ and Cl− in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β‐alanine molecule. The Na+ and Cl−‐dependent uptake of β‐[3H]alanine was stimulated by a valinomycin‐induced inside‐negative K+‐diffusion potential. β‐Amino acids (β‐alanine, taurine, and hypotaurine) inhibited strongly the uptake of β‐[3H]alanine, whereas α‐ and γ‐amino acids had little or no inhibitory effect. In ATP‐depleted cells, the uptake of β‐[3H]alanine was stimulated by preloading of β‐alanine or taurine but not l‐leucine. These results show that β‐alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β‐amino acids.
K252a Modulates the Expression of Nerve Growth Factor‐Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones
Authors: Buck Helen , Winter Janet
Published: July 2010

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor‐induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor‐dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 nM) of K252a. At higher concentrations (3 µM), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor‐dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.
Agonist‐Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5‐Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones
Authors: Simpson Peter B. , Nahorski Stefan R. , Challiss R. A. John
Published: July 2010

Abstract: The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N‐methyl‐d‐aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh‐evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA‐evoked [Ca2+]i response. Both CCh (1 mM) and 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle‐treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24‐h CCh pretreatment decreased the subsequent inositol 1,4,5‐trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR‐mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24‐h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR‐mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh‐ and NMDA‐evoked responses, it is possible that the directly InsP3‐sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA‐evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.
Bidirectional Regulation of GABAA Receptor α1 and α6 Subunit Expression by a Cyclic AMP‐Mediated Signalling Mechanism in Cerebellar Granule Cells in Primary Culture
Authors: Thompson Christopher L. , Pollard Simon , Stephenson F. Anne
Published: July 2010

Abstract: Forskolin treatment of cerebellar granule cells in culture resulted in bidirectional regulation of the expression of GABAA receptor α1 and α6 subunits. Thus, forskolin applied at 2 days in vitro (DIV) increased expression of the α1 subunit but decreased the expression of the α6 subunit. Values with respect to control cultures, both assayed at 9 DIV by immunoblotting, were 310 ± 48% for α1 and 25 ± 16% for the α6 subunit. Similar effects were evoked following chronic treatment with both dibutyryl cyclic AMP and 3‐isobutyl‐1‐methylxanthine. Dideoxyforskolin had no effect on the level of expression of either the α1 or the α6 GABAA receptor subunits. The changes in subunit expression were accompanied by a 1.7‐fold increase in number of total specific [3H]Ro 15‐4513 binding sites expressed by intact cerebellar granule cells. This increase in total binding sites was accommodated by a 2.7‐fold increase in number of diazepam‐sensitive Ro 15‐4513 binding sites in accordance with the observed increase in α1 subunit expression. The number of diazepam‐insensitive subtype of binding sites were not significantly changed. These results suggest that GABAA receptor subtype expression can be differentially regulated by intracellular cyclic AMP concentration.
Human JC Virus Nuclear Factor 1 Binding Motifs and Large Tumor Antigen Region Required for Transactivation of Late Promoter
Authors: Kumar Kotlo U. , Devireddy Laxminarayana R. , Tang Shou‐Ching , Pater Alan , Pater Mary M.
Published: July 2010

Abstract: The nuclear factor 1 (NF‐1) motifs, NF‐1 II/III, in the two 98‐bp repeats of the transcription‐regulatory region of JC virus (JCV), have a critical role in brain‐specific transcription from the JCV early promoter‐enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter‐enhancer (JCVL) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T‐Ag) in the glial cells was shown by double immunofluorescence assays. By using site‐directed mutagenesis and in vivo assays, the two wild‐type NF‐1 II/III sites, but not the third site, were found to be essential for the transactivation of JCVL by JCV T‐Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T‐Ag antibody. In electrophoretic mobility shift assays, expression of JCV T‐Ag increased the binding of a factor(s) to the 98‐bp repeat. T‐Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF‐1 II/III motifs showed that the increased binding specifically required the wild‐type NF‐1 II/III sequences and confirmed the requirement of T‐Ag. To determine the region of T‐Ag necessary for transactivation of JCVL, the coding sequences were mutated. The amino‐terminal region of JCV Ag in amino acids 1–437 was essentially required for efficient transactivation. These results indicated that transactivation of JCVL and increased binding require a factor(s) found specifically in glial cells, the JCV NF‐1 II/III sites, and the T‐Ag amino‐terminal region.
Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells
Authors: Huang Chun‐Ming , Tsay Kun‐Eng , Kao Lung‐Sen
Published: July 2010

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca2+ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca2+ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP‐induced Ca2+ influx, and Ca2+ mobilization from intracellular Ca2+ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca2+ accumulation by nonmitochondrial Ca2+ pools or the thapsigargin (TG)‐sensitive Ca2+ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG‐sensitive Ca2+ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP‐induced influx of extracellular Ca2+. Therefore, NGF and dbcAMP induced different effects on Ca2+ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG‐sensitive Ca2+ pool, the dbcAMP‐ or dbcAMP plus NGF‐mediated neurite outgrowth was significantly inhibited, whereas NGF‐mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca2+ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.
Insulin‐Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor‐Differentiated PC12 Cells
Authors: Yakovchenko Eugenia , Whalin Michael , Movsesyan Vilen , Guroff Gordon
Published: July 2010

Abstract: Receptors for insulin‐like growth factor I (IGF‐I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF‐I receptors nor their affinity for IGF‐I. IGF‐I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF‐I also increased the proportion of differentiated cells found in S‐phase. But although the addition of IGF‐I to naive cells caused an increase in cell number, there was no comparable increase when IGF‐I was added to differentiated cells. Thus, although the receptor for IGF‐I continues to be present and functional, IGF‐I fails to induce cell proliferation in differentiated PC12 cells.
Changes in Arachidonic Acid Levels and Formation and in Lipid Synthesis in the Human Neuroblastoma SK‐N‐BE During Retinoic Acid‐Induced Differentiation
Authors: Petroni A. , Blasevich M. , Papini N. , La Spada P. , Galli C.
Published: July 2010

Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK‐N‐BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n‐6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n‐6 series, total conversion of linoleic acid, especially to 20:3 n‐6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20‐carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n‐3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK‐N‐BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20‐carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.
Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor
Authors: Yonezawa Mihoko , Back Stephen A. , Gan Xiaodong , Rosenberg Paul A. , Volpe Joseph J.
Published: July 2010

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1‐day‐old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC50 values for cystine for a 24‐h exposure ranged from 2 to 65 µM. After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α‐tocopherol, ascorbate, idebenone, and N‐tert‐butyl‐α‐phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3‐day‐old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine‐depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine‐depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.
Modification of Dopamine Transporter Function: Effect of Reactive Oxygen Species and Dopamine
Authors: Berman Sarah B. , Zigmond Michael J. , Hastings Teresa G.
Published: July 2010

Abstract: Dopamine can oxidize to form reactive oxygen species and quinones, and we have previously shown that dopamine quinones bind covalently to cysteinyl residues on striatal proteins. The dopamine transporter is one of the proteins at risk for this modification, because it has a high affinity for dopamine and contains several cysteinyl residues. Therefore, we tested whether dopamine transport in rat striatal synaptosomes could be affected by generators of reactive oxygen species, including dopamine. Uptake of [3H]dopamine (250 nM) was inhibited by ascorbate (0.85 mM; −44%), and this inhibition was prevented by the iron chelator diethylenetriaminepentaacetic acid (1 mM), suggesting that ascorbate was acting as a prooxidant in the presence of iron. Preincubation with xanthine (500 µM) and xanthine oxidase (50 mU/ml) also reduced [3H]dopamine uptake (−76%). Preincubation with dopamine (100 µM) caused a 60% inhibition of subsequent [3H]dopamine uptake. This dopamine‐induced inhibition was attenuated by diethylenetriaminepentaacetic acid (1 mM), which can prevent iron‐catalyzed oxidation of dopamine during the preincubation, but was unaffected by the monoamine oxidase inhibitor pargyline (10 µM). None of these incubations caused a loss of membrane integrity as indicated by lactate dehydrogenase release. These findings suggest that reactive oxygen species and possibly dopamine quinones can modify dopamine transport function.
Regulation of l‐DOPA Biosynthesis by Site‐Specific Phosphorylation of Tyrosine Hydroxylase in AtT‐20 Cells Expressing Wild‐Type and Serine 40‐Substituted Enzyme
Authors: Harada K. , Wu J. , Haycock J. W. , Goldstein M.
Published: July 2010

Abstract: De novo l‐DOPA biosynthesis was studied in stably transfected AtT‐20 cells expressing wild‐type‐ or [Leu40]‐recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser40 (S40L‐rTH) than by those expressing wild‐type rTH (WT‐rTH). Treatment of WT‐rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT‐rTH phospho‐Ser40 content, whereas DOPA production by cells expressing S40L‐rTH was entirely unaffected by forskolin. After forskolin treatment of 32Pi‐prelabeled cells, WT‐rTH was phosphorylated at Ser8, Ser19, Ser31, and Ser40, whereas 32P incorporation into S40L‐rTH was restricted to Ser8, Ser19, and Ser31. Relatively prolonged treatment of AtT‐20 cells expressing WT‐rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser40; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L‐rTH. Thus, the present studies demonstrate that Ser40 phosphorylation mediates forskolin‐induced increases in DOPA biosynthesis directly but that mechanisms other than Ser40 phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.
Ascorbate‐Stimulated Lipid Peroxidation in Human Brain Is Dependent on Iron but Not on Hydroxyl Radical
Authors: Andorn Anne C. , Britton Robert S. , Bacon Bruce R.
Published: July 2010

Abstract: Lipid peroxidation has been postulated as a cause of neuronal damage in both aging and neurodegenerative disease. In studies of neurodegenerative disease, iron, iron plus ascorbate, or ascorbate alone has been used to stimulate lipid peroxidation. The mechanism by which ascorbate stimulates lipid peroxidation in human brain is unclear. The studies reported here were performed to determine whether ascorbate‐stimulated lipid peroxidation in human brain membrane fragments was dependent on iron, superoxide radical, hydrogen peroxide, or hydroxyl radical. Our findings indicate that although ascorbate‐stimulated lipid peroxidation in brain is iron dependent, it apparently does not require hydrogen peroxide or hydroxyl radical, but rather is dependent on superoxide radical formation.
Brain Insulin‐Like Growth Factor‐II mRNA Content Is Reduced in Insulin‐Dependent and Non‐Insulin‐Dependent Diabetes Mellitus
Authors: Wuarin Laura , Namdev Ritu , Burns J. Gregory , Fei Zhi‐Jian , Ishii Douglas N.
Published: July 2010

Abstract: Diabetic encephalopathy, characterized by structural, electrophysiological, neurochemical, and cognitive abnormalities, is observed in insulin‐dependent diabetes mellitus (IDDM) and non‐IDDM (NIDDM). Identification of early biochemical lesions potentially may provide clues pointing to its pathogenesis. Insulin‐like growth factors (IGFs) are neurotrophic factors that recently have been implicated in the pathogenesis of diabetic neuropathy. Because IGF‐II is the predominant IGF in adult brain, we tested the hypothesis that IGF‐II gene expression is decreased in the CNS in both IDDM and NIDDM. Brain and spinal cord were isolated from streptozotocin‐diabetic rats, a model of IDDM with weight loss and impaired insulin production. IGF‐II mRNA content was measured by northern and slot blots. After 2 weeks of diabetes, IGF‐II mRNA content per milligram of tissue wet weight, as well as per unit of poly(A)+ RNA, declined significantly (p≤ 0.05) in brain and spinal cord. Insulin replacement therapy partially restored IGF‐II mRNA levels in brain, cortex, medulla, and spinal cord. The obese, hyperinsulinemic, and spontaneously diabetic (fa/fa) Zucker rat was used as a model of NIDDM. Brain weight (p < 0.025) and IGF‐II mRNA contents (p < 0.01) were significantly decreased in (fa/fa) versus lean nondiabetic (+/?) rats. Therefore, the decline in IGF‐II mRNA levels in diabetic brain was independent of the type of diabetes, the direction of change in body weight, and the insulinemic state. We speculate that this early biochemical lesion may contribute to the development of diabetic encephalopathy.
Chronic Lithium‐Induced Down‐Regulation of MARCKS in Immortalized Hippocampal Cells: Potentiation by Muscarinic Receptor Activation
Authors: Watson David G. , Lenox Robert H.
Published: July 2010

Abstract: Previous studies in our laboratory have demonstrated that exposure of rats to chronic lithium results in a significant reduction in the hippocampus of levels of the protein kinase C (PKC) phosphoprotein substrate MARCKS (myristoylated alanine‐rich C kinase substrate), which persists after withdrawal and is not observed following acute administration. In an immortalized hippocampal cell line (HN33), we have determined that phorbol esters rapidly down‐regulate PKC activity and lead to a subsequent PKC‐dependent reduction in content of MARCKS protein. We now report that chronic exposure of HN33 cells to LiCl (1–10 mM) produces a dose‐ and time‐dependent down‐regulation of MARCKS protein. The lithium‐induced reduction in MARCKS is dependent on the concentration of inositol present in the medium and is reversed and prevented in the presence of elevated inositol concentrations. When HN33 cells were exposed to lithium at clinically relevant concentrations (1 mM) under limiting inositol conditions, activation of muscarinic receptor‐coupled phosphoinositide signaling significantly potentiated the lithium‐induced down‐regulation of MARCKS protein. It has been suggested that a major action of lithium in the brain is linked to its inositol monophosphatase inhibitory activity in receptor‐mediated signaling through the inositol trisphosphate/diacylglycerol pathway, resulting in a relative inositol depletion. Our data provide evidence that this initial action of lithium may translate into a PKC‐dependent long‐term down‐regulation of MARCKS protein expression in the hippocampus.
A Low‐Km 5′‐Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP
Authors: Orford Michael R. , Saggerson E. David
Published: July 2010

Abstract: A readily soluble 5′‐nucleotidase was purified 1,800‐fold from rat brain 105,000‐g supernatant. The enzyme showed similarity to the 5′‐nucleotidase ectoenzyme of plasma membranes. It exhibited a low Km for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β‐methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol‐specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5′‐nucleotidase ectoenzyme or for other low‐Km“soluble” 5′‐nucleotidases. The soluble 5′‐nucleotidase showed an elution profile on AMP‐Sepharose affinity chromatography or on Mono Q ion‐exchange chromatography different from that of the brain ectoenzyme. Forty‐two percent of the soluble 5′‐nucleotidase in brain 105,000‐g supernatant did not bind to a Mono Q ion‐exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A‐Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5′‐nucleotidase toward the inhibitor ATP by 20‐fold. This factor was also able to increase the inhibition of brain 5′‐nucleotidase ectoenzyme by ATP.
Endothelin‐1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium‐Modulated Influx of Calcium
Authors: Dunica Dara J. , Griffiths Roger , Williams D. Clive , O'Neill Luke A. J.
Published: July 2010

Abstract: Endothelin‐1 (Et‐1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et‐1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et‐1 caused an early increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca2+, and the demonstration of an increase in phospholipase A2 (PLA2) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA2 in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et‐1‐induced AA release but abolished that by phorbol 12‐myristate 13‐acetate, demonstrating that the Et‐1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca2+]i triggered by Et‐1, whereas valinomycin, which causes K+ efflux, not only caused a rapid rise in [Ca2+]i but also caused AA mobilisation. The results therefore suggest that Et‐1 activation of PLA2 in this cell type requires calcium influx dependent on K+ efflux.
Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester‐Stimulated Secretory Cleavage of Amyloid Precursor Protein
Authors: Efthimiopoulos Spiros , Punj Suzy , Manolopoulos Vangelis , Pangalos Menelas , Wang Gian P. , Refolo Lawrence M. , Robakis Nikolaos K.
Published: July 2010

Abstract: α‐Secretase cleaves the full‐length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the α‐secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester‐stimulated secretion of nexin II (NXII), the secreted product of the α‐secretase cleavage of APP751. At 1 µM, forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9‐dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α‐secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.
Fate of Cerebrospinal Fluid‐Borne Amyloid β‐Peptide: Rapid Clearance into Blood and Appreciable Accumulation by Cerebral Arteries
Authors: Ghersi‐Egea J.‐F. , Gorevic P. D. , Ghiso J. , Frangione B. , Patlak C. S. , Fenstermacher J. D.
Published: July 2010

Abstract: In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid β‐peptides. In the present study, either soluble 40‐residue amyloid β‐peptide radiolabeled with 125I (I‐sAβ) or [14C]polyethylene glycol ([14C]‐PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I‐sAβ was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [14C]PEG was lost from the CSF‐brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I‐sAβ that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I‐sAβ was found in brain. The clearance of amyloid β‐peptides from the CSF‐brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.
Retinoic Acid Increases Hydroxyindole‐O‐Methyltransferase Activity and mRNA in Human Y‐79 Retinoblastoma Cells
Authors: Bernard Marianne , Klein David C.
Published: July 2010

Abstract: Hydroxyindole‐O‐methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma‐derived Y‐79 cell line. Treatment with all‐trans‐, 13‐cis‐, and 9‐cis‐RA induced a gradual 10‐fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde‐3‐phosphate dehydrogenase, actin, S‐antigen, and interphotoreceptor retinoid‐binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.