bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of a Lower Molecular Weight Protein to the μ‐Opioid Receptor Demonstrated by 125I‐β‐Endorphin Cross‐Linking Studies
Authors: Law P. Y. , Tine S. J. , McLeod L. A. , Loh H. H.
Published: March 2003

Abstract : Cross‐linking experiments using the

125I‐β‐endorphin revealed the presence of several

receptor‐related species in cell lines expressing endogenous opioid receptors,

including a small molecular mass protein (~22 kDa). Previous reports have

suggested that this 22‐kDa 125I‐β‐endorphin cross‐linked

protein could be the degradative product from a higher molecular mass species,

i.e., a fragment of the receptor. To determine if this protein is indeed a

degraded receptor fragment, 125I‐β‐endorphin was cross‐linked

to the (His)6 epitope‐tagged μ‐opioid receptor (His‐μ) stably

expressed in the murine neuroblastoma Neuro2A cells. Similar to

earlier reports with cell lines expressing endogenous receptors, two major

bands of 72‐ and 25‐kDa proteins were specifically cross‐linked. Initial

cross‐linking experiments indicated the absolute requirement of the

high‐affinity 125I‐β‐endorphin binding to the μ‐opioid

receptor prior to the appearance of the low molecular weight species,

suggesting that the 22‐kDa protein could be a degraded fragment of the

receptor. However, variations in the ratios of these protein bands being

cross‐linked by several homo‐ or heterobifunctional cross‐linking agents were

observed. Although neither the carboxyl terminus μ‐opioid receptor‐specific

antibodies nor the antibodies against the epitope at the amino terminus of the

receptor could recognize the 22‐kDa protein, this

125I‐β‐endorphin cross‐linked species could be

coimmunoprecipitated with the receptor antibodies or could be isolated with a

nickel resin affinity chromatography. The direct physical association of the

22‐kDa protein with the receptor was demonstrated also by the observation that

the 22‐kDa protein could not bind to the nickel resin alone, but that its

binding to the nickel resin was restored in the presence of the His‐μ.

Taken together, these results suggest that the 22‐kDa protein cross‐linked by

125I‐β‐endorphin is not a degradative product, but a protein located within the proximity of the μ‐opioid receptor, and that it is tightly associated with the receptor.
The Postsynaptic Density Protein PSD‐95 Differentially Regulates Insulin‐ and Src‐Mediated Current Modulation of Mouse NMDA Receptors Expressed in Xenopus Oocytes
Authors: Liao Guey‐Ying , Kreitzer Matthew A. , Sweetman Brian J. , Leonard John P.
Published: March 2003

Abstract : The NMDA subtype of glutamate receptor is physically

associated with the postsynaptic density protein PSD‐95 at glutamatergic

synapses. The channel activity of NMDA receptors is regulated by different

signaling molecules, including protein tyrosine kinases. Because previous

results have suggested a role for protein kinase C (PKC) in insulin

potentiation of NMDA currents in oocytes, the effects of coexpression of

PSD‐95 on insulin and PKC potentiation of NMDA currents from these receptors

were compared. Another primary objective was to determine if PSD‐95 could

enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors

expressed in Xenopus oocytes. The results show opposite effects of PSD‐95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD‐95 coexpression. In contrast, PSD‐95 coexpression eliminated insulin‐mediated potentiation of NR2A/NR1 receptor currents. PSD‐95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD‐95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.
Muscarinic Activation of Mitogen‐Activated Protein Kinase in PC12 Cells
Authors: Berkeley Jennifer L , Levey Allan I
Published: March 2003

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen‐activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose‐dependent and atropine‐sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT‐PCR and immunoprecipitation. RT‐PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype‐specific antibodies showed that ~95% of the expressed receptors were m4, whereas the remaining ~5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR‐induced MAPK activation was abolished by protein kinase C down‐regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.
Induction of Cyclooxygenase‐2 by Overexpression of the Human Catalase Gene in Cerebral Microvascular Endothelial Cells
Authors: Fang Xiang , Moore A Steven , Nwankwo O Joseph , Weintraub L Neal , Oberley W Larry , Snyder D Gary , Spector A Arthur
Published: March 2003

Prostaglandin (PG) formation by the inducible (type 2) cyclooxygenase (COX‐2) and reactive oxygen species (ROS) have been proposed to play important roles in cerebrovascular pathological processes. To explore the relationship between ROS and COX‐2 expression, adenovirus (Ad) vectors containing cDNA for human antioxidant enzymes including catalase (AdCat), copper/zinc

superoxide dismutase (AdCu/ZnSOD), and manganese superoxide dismutase

(AdMnSOD) were transferred into murine cerebral microvascular

endothelial cells. AdCat (100 multiplicity of infection) infection

increased the content and enzymatic activity of cellular Cat threefold and

decreased the intracellular peroxide level. The expression of COX‐2 mRNA and

protein in cell lysates was up‐regulated, and the amount of PGE2

formed from exogenous arachidonic acid increased following AdCat

infection in a dose‐dependent manner, paralleling the expression of COX‐2

protein. The AdCat‐induced increase in PGE2 formation was

inhibited by NS‐398, a selective inhibitor of COX‐2 enzymatic activity.

AdCat infection did not change the expression of the constitutive

(type 1) COX protein. Although AdCu/ZnSOD and AdMnSOD

infection increased the expression of superoxide dismutase proteins, COX‐2

expression was not induced. An in vitro nuclear transcription assay indicated

that overexpression of the Cat gene increases the transcription of the COX‐2

gene. Furthermore, the stability of COX‐2 mRNA induced by lipopolysaccharide

was increased after AdCat gene transfer. These results indicate that

AdCat gene transfer induces the transcriptional activation of the COX‐2 gene and increases COX‐2 mRNA stability. Therefore, peroxide may have regulatory effect on COX‐2 function in the cerebral microcirculation.
The Neuronal Microtubule‐Associated Protein Tau Is a Substrate for Caspase‐3 and an Effector of Apoptosis
Authors: Fasulo Luisa , Ugolini Gabriele , Visintin Michela , Bradbury Andrew , Brancolini Claudio , Verzillo Vittorio , Novak Michal , Cattaneo Antonino
Published: March 2003

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma‐derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase‐3 cleavage sites. This is the only site that can be effectively used by caspase‐3 in vitro, releasing the C‐terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self‐propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.
No Evidence for Increased Oxidative Damage to Lipids, Proteins, or DNA in Huntington's Disease
Authors: Alam Zafar I , Halliwell Barry , Jenner Peter
Published: March 2003

It has been proposed that mitochondrial dysfunction and excitotoxic mechanisms lead to oxidative damage in the brain of Huntington;s disease patients. We sought evidence that increased oxidative damage occurs by examining postmortem brain material from patients who had died with clinically and pathologically diagnosed Huntington's disease. Oxidative damage was measured using methods that have already demonstrated the presence of increased oxidative damage in Parkinson's disease, Alzheimer's disease, and senile dementia of the Lewy body type. No alterations in the levels of lipid peroxidation (as measured by lipid peroxides and thiobarbituric acid‐malondialdehyde adducts) were found in the caudate nucleus, putamen, or frontal cortex of patients with Huntington's disease compared with normal controls. Similarly, there were no elevations in the levels of 8‐hydroxyguanine or of a wide range of other markers of oxidative DNA damage. Levels of protein carbonyls in these tissues were also unaltered. Our data suggest that oxidative stress is not a major component of the degenerative processes occurring in Huntington's disease, or at least not to the extent that occurs in other neurodegenerative disorders.
Structure of a Major Oligosaccharide of PASII/PMP22 Glycoprotein in Bovine Peripheral Nerve Myelin
Authors: Kitamura Kunio , Uyemura Keiichi , Shibuya Kyoko , Sakamoto Yasushi , Yoshimura Kazunori , Nomura Masahiko
Published: March 2003

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln‐Asn‐Cys‐Ser‐Thr, where the asparagine was glycosylated. To eliminate all the contaminated Po glycopeptides from the PASII/PMP22 glycopeptide

preparation, we used a fluorescent probe,

N‐[2‐(2‐pyridylamino)ethyl]maleimide, which reacts with the cysteine

of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were

isolated by HPLC and were digested further with glycopeptidase A. The

resultant oligosaccharides were conjugated with 2‐aminopyridine (PA) as a

fluorescent tag. One major PA‐oligosaccharide, OPPE1, was purified by HPLC.

The structure of OPPE1 was elucidated by fast atom bombardment mass

spectrometry and 1H‐NMR studies and comparing the derivatives of

PA‐OPPE1 and PA‐oligosaccharides of γ‐globulin on HPLC. The structure,

SO4‐3GlcAβ1‐3Galβ1‐4GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐4)

(GlcNAcβ1‐2Manα1‐3)Manβ1‐4GlcNAcβ1‐4(Fucα1‐6)GlcNAc‐PA,

was identical to the pyridylaminated form of the major oligosaccharide D8 of

bovine Po previously reported.
Glutamate Receptor Subunit δ2 Is Highly Expressed in a Novel Population of Glial‐Like Cells in Rat Pineal Glands in Culture
Authors: Yatsushiro Shouki , Hayashi Mitsuko , Morita Mitsuhiro , Yamamoto Akitsugu , Moriyama Yoshinori
Published: March 2003

Abstract: The mammalian pineal gland uses L‐glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, δ, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT‐PCR analyses with specific probes indicated the expression of mRNA of δ2 but not that of δ1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl‐terminal region of the δ2 receptor recognized a single 110‐kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The δ2 antibodies recognized a 110‐kDa polypeptide of pineal membranes and specifically immunostained huge glial‐like cells with the occasional presence of several long, branching processes in a pineal cell culture. δ2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin‐positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The δ2‐positive cells constitute a very minor population among total pineal cells (∼0.03%). Double immunolabeling with δ2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes δ2‐positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein‐ or vimentin‐positive glial‐like cells. These results indicated that the δ2 glutamate receptor is expressed in a novel subpopulation of pineal glial‐like cells in culture and suggest the presence of a glutamate‐mediated intercellular signal transduction mechanism between pinealocytes and δ2‐expressing cells. The pineal cells may provide a good experimental system for studies on the function of glutamate receptor δ2.
Rundown of Secretion After Depletion of Intracellular Calcium Stores in Bovine Adrenal Chromaffin Cells
Authors: Pan Chien‐Yuan , Fox Aaron P.
Published: March 2003

Abstract: In this study, the relationship between intracellular

calcium stores and depolarization‐evoked stimulation was examined in bovine

chromaffin cells, using changes in membrane capacitance to monitor both

exocytosis and endocytosis. Cells were voltage‐clamped using the perforated

whole‐cell patch configuration to minimize alterations in intracellular

constituents. Control cells exhibited reproducible secretory responses each

time the cell was stimulated. However, the same stimulation protocol elicited

progressively smaller secretory responses in cells where their intracellular

calcium store was emptied by thapsigargin. Transient elevation of the

intracellular calcium concentration with a brief histamine treatment enhanced

subsequent secretory responses in control but not in thapsigargin‐treated

cells. A series of depolarizations to ‐20 mV, which allowed small amounts of

Ca2+ influx but which by itself did not trigger catecholamine

secretion, enhanced subsequent exocytosis in both control and

thapsigargin‐treated cells. Caffeine‐pretreated cells exhibited a rundown in

the secretory response that was similar to that produced by thapsigargin.

These results suggest that brief elevations of [Ca2+]i could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.
Relative Contribution of Different Receptor Subtypes in the Response of Neuroblastoma Cells to Tumor Necrosis Factor‐α
Authors: Condorelli Fabrizio , Sortino Maria Angela , Stella Anna Maria Giuffrida , Canonico Pier Luigi
Published: March 2003

Abstract: The effect of tumor necrosis factor‐α (TNF‐α) on neuronal viability has been investigated in the SK‐N‐BE neuroblastoma cell line. These cells undergo differentiation upon chronic treatment with retinoic acid. Exposure of SK‐N‐BE cells to TNF‐α produced a proliferative response in undifferentiated cells, whereas a reduced cell number was observed in retinoic acid (RA)‐differentiated cultures. This biphasic response may be related to the different expression of TNF‐α receptors (TNFRs); a significant increase in the density of TNFR1 was in fact observed following RA‐induced differentiation. Under these conditions, a pronounced increase in the formation of ceramide‐1‐phosphate (which was prevented by the selective inhibitor of phosphatidylcholine‐specific phospholipase C, D609) and an activation of caspase‐3 upon TNF‐α challenge were evident. Selective blockade of each TNFR subtype allowed a more detailed analysis of the effect observed. Preincubation with an anti‐TNFR1 antibody prevented the cytotoxic effect of TNF‐α in RA‐differentiated SK‐N‐BE cells, whereas the anti‐TNFR2 antibody blocked the proliferative activity of the cytokine in undifferentiated cultures.
Dynamic Changes in Glucose and Lactate in the Cortex of the Freely Moving Rat Monitored Using Microdialysis
Authors: Jones D. A. , Ros J. , Landolt H. , Fillenz M. , Boutelle M. G.
Published: March 2003

Abstract: These experiments for the first time examine simultaneous changes in glucose and lactate in unanaesthetised animals during moderate hypoxia. Unanaesthetised rats were exposed to moderate hypoxia for a period of 15 min by reducing inspired oxygen to 8%. Changes in glucose and lactate were monitored in rat cortex using microdialysis and a novel dual enzyme‐based assay. Samples of dialysate collected at 3‐min intervals were assayed for both glucose and lactate. There was an early rapid rise of lactate that reached a peak at the end of the period of hypoxia followed by a steep decline. Glucose showed a very much smaller delayed increase that started during the period of hypoxia and continued beyond it. The origin of the rise in glucose is discussed, using the temporal relationship between the lactate and glucose changes.
Regulation of NMDA Receptor Subunits and Nitric Oxide Synthase Expression During Cocaine Withdrawal
Authors: Loftis Jennifer M. , Janowsky Aaron
Published: March 2003

Abstract: The present study characterized the effects of withdrawal from cocaine on the expression of NMDA receptor subunits (NR1, NR2B) and neuronal nitric oxide synthase. FosB induction was measured to confirm that repeated cocaine exposure influenced protein expression, as previously reported. Administration of cocaine followed by 24 h, 72 h, or 14 days of withdrawal resulted in alterations of NR1 and NR2B subunits and neuronal nitric oxide synthase expression as measured by immunohistochemical labeling of rat brain sections. Optical density analyses revealed significant up‐regulation of NR1 in the ventral tegmental area at 72 h and 14 days of withdrawal. Structure‐specific and withdrawal time‐dependent alterations in NR2B expression were also found. After 24 h of withdrawal, cocaine‐induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine caused an ∼50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. In contrast, cocaine‐induced up‐regulation of neuronal nitric oxide synthase was transient and evident in cortical areas only at 24 h after the last drug injection. The results suggest that region‐specific changes in interactions among proteins associated with the NMDA receptor complex may underlie neuronal adaptations following repeated cocaine administration.
The Role of Reactive Nitrogen Species in Secondary Spinal Cord Injury
Authors: Liu Danxia , Ling Xiang , Wen Jing , Liu Jing
Published: March 2003

Abstract: To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide‐selective electrode dramatically increased immediately following injury and then quickly declined. Nitro‐L‐arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro‐L‐arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham‐control sections. Mn(III) tetrakis(4‐benzoic acid)‐porphyrin (a novel cell‐permeable superoxide dismutase mimetic) and nitro‐L‐arginine significantly reduced the numbers of nitrotyrosine‐positive cells. Protein‐bound nitrotyrosine was significantly higher in the injured tissue than in the sham‐operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein‐bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.
Competitive Potentiation of Acetylcholine Effects on Neuronal Nicotinic Receptors by Acetylcholinesterase‐Inhibiting Drugs
Authors: Zwart Ruud , Kleef Regina G. D. M. van , Gotti Cecilia , Smulders Chantal J. G. M. , Vijverberg Henk P. M.
Published: March 2003

Abstract: The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on α4β2 and α4β4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage‐clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh‐induced ion currents. These dual effects result in bell‐shaped concentration—effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 μM physostigmine on α4β4 nicotinic receptors and amounts to 70% at 1 μM ACh. The mechanism underlying the effects of physostigmine on α4β4 ACh receptors has been investigated in detail. Potentiation of ACh‐induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh‐induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage‐dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist 125I‐epibatidine from its recognition sites on α4β4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two‐site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh‐induced ion currents. It is concluded that these acetylcholinesterase‐inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on α4‐containing nicotinic ACh receptors.
Differential gene expression of β‐1,4‐galactosyltransferases I, II and V during mouse brain development
Authors: Nakamura Naohito , Yamakawa Naomi , Sato Takeshi , Tojo Hideaki , Tachi Chikashi , Furukawa Kiyoshi
Published: March 2003

Since most brain glycoproteins from β‐1,4‐galactosyltransferase (β‐1,4‐GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel β‐1,4‐GalTs II and V which are involved in N‐linked oligosaccharide biosynthesis in addition to β‐1,4‐GalT I was studied during mouse brain development. Isolation and characterization of β‐1,4‐GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the β‐1,4‐GalT I gene is expressed mainly in mid‐embryonic stages, while the expression level of β‐1,4‐GalT II transcript remains constant and of β‐1,4‐GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that β‐1,4‐GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of β‐1,4‐GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.
Cholesterol‐dependent modulation of tau phosphorylation in cultured neurons
Authors: Fan Qi‐Wen , Yu Wei , Senda Takao , Yanagisawa Katsuhiko , Michikawa Makoto
Published: March 2003

One of the hallmarks of Alzheimer's disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann–Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with β‐migrating very low‐density lipoprotein (β‐VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability.
Piroxicam and NS‐398 rescue neurones from hypoxia/reoxygenation damage by a mechanism independent of cyclo‐oxygenase inhibition
Authors: Vartiainen Nina , Huang Chiung‐Ying , Salminen Antero , Goldsteins Gundars , Chan Pak H. , Koistinaho Jari
Published: March 2003

We studied whether NS‐398, a selective cyclo‐oxygenase‐2 (COX‐2) enzyme inhibitor, and piroxicam, an inhibitor of COX‐2 and the constitutively expressed COX‐1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180–270 µm) and NS‐398 (30 µm). Cell counts confirmed the neuroprotection. Western blotting revealed no COX‐1 or COX‐2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS‐398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor‐kappa B (NF‐κB) binding activity, which was inhibited by piroxicam but not by NS‐398. AP‐1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS‐398. However, both COX inhibitors induced activation of extracellular signal‐regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS‐398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF‐κB and AP‐1 binding activity. Instead, piroxicam and NS‐398‐induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.
Identification of a glucocorticoid‐responsive element in the promoter region of the mouse tyrosine hydroxylase gene
Authors: Hagerty Timothy , Morgan William W. , Elango N. , Strong Randy
Published: March 2003

It has been known for nearly 30 years that glucocorticoid receptor stimulation induces increased tyrosine hydroxylase (TH) gene expression. However, the mechanism mediating this effect has remained elusive. Sequences with homology to known glucocorticoid‐responsive elements (GRE) have been identified in the 5′ flanking region of the TH gene of several vertebrate species, but none has been shown to be functional. To identify the GRE element(s) in the TH promoter, we generated chimeric constructs in which different lengths of the 5′ flanking sequences of the mouse TH gene (3.6, 1.1 and 0.8 kb) were ligated to a luciferase reporter gene. Dexamethasone treatment increased luciferase expression only in cells transiently transfected with the construct containing 3.6 kb of the TH 5′ flanking DNA. Co‐administration of mifepristone (RU486), a glucocorticoid receptor antagonist, blocked this effect. We identified a TH‐GRE sequence (5′‐GGCACAGTGTGGTCT) in the mouse 5′ flanking DNA between −2435 and −2421 from the transcription start. Responsiveness to dexamethasone was lost following deletion of this sequence. To determine the ability of this element to function in a heterologous promoter, we prepared a chimeric construct in which the TH‐GRE sequence was cloned just upstream of a minimal thymidine kinase (TK) promoter. Promoter activity was increased 2‐fold in dexamethasone‐treated PC12 cells transfected with the TH‐GRE‐TK construct. These results provide strong evidence that the 15 base‐pair sequence in the 5′ flanking DNA of the mouse TH gene functions as a glucocorticoid response element. This is the first report identifying a functional glucocorticoid response element in the promoter region of the TH gene of any species.
Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors
Authors: Yan Jun , Studer Lorenz , McKay Ronald D. G.
Published: March 2003

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10‐fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium‐dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre‐treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large‐scale generation of dopaminergic neurons for clinical transplantation.
Nerve growth factor activates the RARβ2 promoter by a Ras‐dependent mechanism
Authors: Cosgaya Jose Miguel , Aranda Ana
Published: March 2003

Nerve growth factor (NGF) and retinoic acid (RA) exert important actions on PC12 cells. We have previously shown that incubation with NGF induces retinoic acid receptor β (RARβ) binding to a hormone response element in PC12 cells. In this study we show that NGF increases RARβ protein levels by enhancing basal RARβ2 promoter activity, and potentiates stimulation by RA in transient transfection assays. The effect of RA is mediated by a RA response element (RARE) located at −37/−53 and mutation of this element abolishes activation by the retinoid, as well as cooperation with NGF. However, the action of NGF is independent of the RARE and is mediated by sequences overlapping the TATA box and the INR comprising nucleotides −59 to +14. NGF produces a strong decrease in some of the complexes that bind to the INR. These results suggest that the RARβ2 gene could be in a basal repressed state and NGF could increase RARβ2 transcription by inducing the release of some inhibitory factors from the INR. Functional Ras is required for RARβ2 promoter activation by NGF because expression of oncogenic Ras increases promoter activity and a dominant inhibitory Ras mutant blocks the effect of NGF. Oncogenic Raf also mimics the effect of NGF on the promoter. Other ligands of tyrosine kinase receptors that stimulate Ras also cause RARβ2 promoter activation and act cooperatively with RA. These results indicate the existence of cross‐coupling of the Ras–Raf signal transduction pathway with retinoid receptor pathways which could increase sensitivity to RA and be important for PC12 cell function.
Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath
Authors: Bizzozero Oscar A. , Bixler Heather A. , Davis Judi D. , Espinosa Araceli , Messier Amber M.
Published: March 2003

Myelin proteolipid protein (PLP) contains thioester‐bound, long‐chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post‐translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mm hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein‐bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mm dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP‐less myelin‐deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side‐effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle‐clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.
PC12 cells utilize the homophilic binding site of L1 for cell−cell adhesion but L1–αvβ3 interaction for neurite outgrowth
Authors: Yip Paul M. , Siu Chi‐Hung
Published: March 2003

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron‐like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1‐stimulated neurite outgrowth in PC12 cells, substrate‐immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1‐Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1‐Ig2, indicating that L1–L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1‐stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1‐Ig6 highlighted the importance of the Arg‐Gly‐Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg‐Gly‐Asp‐containing peptide and anti‐integrin antibodies suggested the involvement of αvβ3 integrin. Furthermore, neurite outgrowth stimulated by L1‐Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen‐activated protein kinase cascade.
Changes in the expression of G protein‐coupled receptor kinases and β‐arrestin 2 in rat brain during opioid tolerance and supersensitivity
Authors: Hurlé María A.
Published: March 2003

We previously demonstrated that chronic treatment of rats with the µ‐opioid receptor agonist sufentanil induced pharmacological tolerance associated with µ‐opioid receptor desensitization and down‐regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented µ‐opioid receptor down‐regulation, induced down‐stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein‐coupled receptor kinases (GRKs) and β‐arrestin 2 in agonist‐induced µ‐opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7‐day chronic infusion of sufentanil (2 µg/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 µg/h) and nimodipine (1 µg/h) for 7 days. Antinociception was evaluated by the tail‐flick test. GRK2, GRK3, GRK6 and β‐arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up‐regulation of GRK2, GRK6, and β‐arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and β‐arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and β‐arrestin 2 are involved in the short‐ and long‐term adaptive changes in µ‐opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and β‐arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.
Identification of amino acid residues responsible for the α5  subunit binding selectivity of L‐655,708, a benzodiazepine binding site ligand at the GABAA receptor
Authors: Casula M. Anna , Bromidge Frances A. , Pillai Gopalan V. , Wingrove Peter B. , Martin Karine , Maubach Karen , Seabrook Guy R. , Whiting Paul J. , Hadingham Karen L.
Published: March 2003

L‐655,708 is a ligand for the benzodiazepine site of the γ‐aminobutyric acid type A (GABAA) receptor that exhibits a 100‐fold higher affinity for α5‐containing receptors compared with α1‐containing receptors. Molecular biology approaches have been used to determine which residues in the α5 subunit are responsible for this selectivity. Two amino acids have been identified, α5Thr208 and α5Ile215, each of which individually confer approximately 10‐fold binding selectivity for the ligand and which together account for the 100‐fold higher affinity of this ligand at α5‐containing receptors. L‐655,708 is a partial inverse agonist at the GABAA receptor which exhibited no functional selectivity between α1‐ and α5‐containing receptors and showed no change in efficacy at receptors containing α1 subunits where amino acids at both of the sites had been altered to their α5 counterparts (α1ΔSer205‐Thr,Val212‐Ile). In addition to determining the binding selectivity of L‐655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABAA receptor, and further delineate a region just N‐terminal to the first transmembrane domain of the receptor α subunit that contributes to this binding site.
Protein kinase C regulation of dopamine transporter initiated by nicotinic receptor activation in slices of rat prefrontal cortex
Authors: Drew Allison E. , Werling Linda L.
Published: March 2003

We previously reported that activation of nicotinic receptors causes an enhancement in amphetamine‐stimulated release of dopamine via its transporter from slices of prefrontal cortex, but no such enhancement of release from slices of nucleus accumbens or striatum. The nicotinic receptors mediating the enhancement most likely contain α4 and β2 subunits based upon pharmacological characterization. In this study, we sought to characterize the second messenger systems associated with the nicotine‐mediated response. Sodium channel involvement was confirmed by the observation that tetrodotoxin blocked nicotine‐mediated enhancement, whereas veratridine or elevated K+mimicked the enhancement seen with nicotine. Inclusion of EGTA blocked nicotine‐mediated enhancement, suggesting that, even though no exogenous Ca2+was added, endogenous stores were required for the enhancement. The enhancement by nicotine was also abolished by the L‐type voltage‐dependent calcium channel (VDCC) antagonist nitrendipine, but not by the N‐type VDCC antagonist ω‐conotoxin GVIA. Finally, inhibition of protein kinase C also abolished the nicotine‐mediated enhancement of amphetamine‐stimulated dopamine release, whereas inhibitors of Ca2+/calmodulin kinase II did not. These findings establish that nicotine can exert selective effects on dopamine transporter activity in prefrontal cortex, an area involved in cognition and learning.
Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures
Authors: Okabe Akihito , Tawara Yoshie , Masa Takahiro , Oka Takuya , Machida Atsunori , Tanaka Tatsuya , Matsuhashi Hitomi , Shiosaka Sadao , Kato Keiko
Published: March 2003

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl‐oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I−IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT‐PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STs. Furthermore, to assay activity‐related changes in ST expression, we used amygdaloid‐kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up‐regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down‐regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.
Functional expression of M1, M3 and M5 muscarinic acetylcholine receptors in yeast
Authors: Erlenbach Isolde , Kostenis Evi , Schmidt Clarice , Hamdan Fadi F. , Pausch Mark H. , Wess Jürgen
Published: March 2003

The goal of this study was to functionally express the three Gq‐coupled muscarinic receptor subtypes, M1, M3 and M5, in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full‐length receptors resulted in very low numbers of detectable muscarinic binding sites (Bmax < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M1, M3 and M5 muscarinic receptors resulted in dramatic increases in Bmax values (53–214 fmol/mg). To monitor productive receptor/G‐protein coupling, we used specifically engineered yeast strains that required agonist‐stimulated receptor/G‐protein coupling for cell growth. These studies showed that the shortened versions of the M1, M3 and M5 receptors were unable to productively interact with the endogenous yeast G protein α‐subunit, Gpa1p, or a Gpa1 mutant subunit that contained C‐terminal mammalian Gαs sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Gαq hybrid subunit containing C‐terminal mammalian Gαq sequence, indicating that the M1, M3 and M5 muscarinic receptors retained proper G‐protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling‐competent form in yeast. The strategy described here, which involves structural modification of both receptors and co‐expressed G proteins, should facilitate the functional expression of other classes of G protein‐coupled receptors in yeast.
Nicotine‐evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage‐gated calcium channels
Authors: Kulak Jennifer M. , McIntosh J. Michael , Yoshikami Doju , Olivera Baldomero M.
Published: March 2003

It has previously been shown that nicotine‐evoked dopamine release from rat striatal synaptosomes and nicotine‐evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage‐gated calcium channels was examined. Cd2+ (200 µm), as well as ω‐conotoxin MVIIC (5 µm), blocks ∼85% of nicotine‐evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin‐susceptibility suggests that these calcium channels contain α1A and/or α1B subunits. Inhibition of nicotine‐evoked dopamine release by conotoxins α‐MII and ω‐GVIA is additive and indicates that presynaptic α3β2 nAChRs are functionally coupled to α1A, but not α1B, calcium channel subtypes. Conversely, insensitivity to α‐AuIB and sensitivity to ω‐MVIIC indicate that non‐α3β2/α3β4‐containing nAChRs are functionally coupled to α1B‐containing calcium channels. In contrast, Cd2+ blocks only 65% of nicotine‐evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd2+‐insensitive component of release is blocked by α‐AuIB and therefore appears to be triggered by Ca2+ flowing directly through the channels of presynaptic α3β4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage‐gated calcium channels.
The relationship between the aggregational state of the amyloid‐β peptides and free radical generation by the peptides
Authors: Monji Akira , Utsumi Hideo , Ueda Tadashi , Imoto Taiji , Yoshida Ichiro , Hashioka Sadayuki , Tashiro Ken‐ichiro , Tashiro Nobutada
Published: March 2003

In the present study, we investigated whether or not the amyloid‐β protein (Aβ) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Aβ and Aβ‐induced cytotoxicity. The present results demonstrated a four‐line spectrum in the presence of both Aβ40 and Aβ42 with Ntert‐butyl‐α‐phenylnitrone (PBN), but not in the presence of PBN alone in phosphate‐buffered saline (PBS). The fact that the four‐line spectrum obtained for the Aβ/PBN in PBS was completely abolished in the presence of the iron‐chelating agent Desferal demonstrated the observed four‐line spectrum to be iron‐dependent. The present study also revealed that either Aβ40 or Aβ42 with PBN in phosphate buffer (PB) did not produce any definite four‐line spectrum. Both a thioflavine‐T (Th‐T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Aβ in PBS to be much higher than that of Aβ in PB. Moreover, Aβ‐induced cytotoxicity assays showed Aβ incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Aβ‐associated free radical generation is strongly influenced by the aggregational state of the peptides.