bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds
Authors: Kang Jiesheng , Teeter John H. , Brazier Stephen P. , Nguyen Nguyen D. , Chang Christine C. , Puchalski Ralph B.
Published: March 2003

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full‐length cDNA for Kv2‐catfish, a novel K+ channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56–59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage‐gated K+ channels. The Kv2‐catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in Xenopus oocytes were selective for K+, and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two‐electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mm. The channels showed voltage‐dependent activation, and did not inactivate within 200 ms. Functionally, Kv2‐catfish is a voltage‐gated, delayed rectifier K+ channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K+ channels, being related equally well to Kv2.1 and Kv2.2.
Fibulin‐1 binds the amino‐terminal head of β‐amyloid precursor protein and modulates its physiological function
Authors: Ohsawa Ikuroh , Takamura Chizuko , Kohsaka Shinichi
Published: March 2003

Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two‐hybrid system to identify potential interacting factors. We now report that fibulin‐1, which contains repetitive Ca2+‐binding EGF‐like elements, binds to APP at its amino‐terminal growth factor‐like domain, the region that is responsible for its neurotrophic activities. Fibulin‐1 expression in the brain is confined to neurons, and is not expressed significantly by astrocytes or microglia. Direct binding of fibulin‐1 to the secreted form of APP (sAPP) was demonstrated with a pull‐down assay using fragments of both fibulin‐1 fused with glutathione‐S transferase and sAPP, produced in bacteria and yeast, respectively. The fibulin‐1/sAPP heteromer was shown to form in the conditioned medium of transfected COS‐7 cells. Furthermore, fibulin‐1 blocks sAPP‐mediated proliferation of primary cultured rat neural stem cells. These results suggest that fibulin‐1 may play a significant role in modulating the neurotrophic activities of APP.
Visualization of oxygen‐concentration‐dependent production of nitric oxide in rat hippocampal slices during aglycemia
Authors: Kojima Hirotatsu , Hirata Mihoko , Kudo Yoshihisa , Kikuchi Kazuya , Nagano Tetsuo
Published: March 2003

Our novel fluorescent indicator, DAF‐FM, permits the bioimaging of nitric oxide (NO) in living cells with high resolution in space and time, with stable intensity above pH 5.8. A membrane‐permeable derivative, DAF‐FM DA, was applied to imaging of NO generated in rat hippocampal slices by exposure to an aglycemic medium. NO production was observed mainly in the CA1 area, and was dependent on the concentration of O2. During exposure to an anoxic‐aglycemic medium, NO was hardly produced, while marked elevation of intracellular Ca2+ was observed. Production of NO increased sharply as soon as the perfusate was changed to the normal medium. These results suggest that NO synthase is activated after reperfusion rather than during ischemia.
Increases in cortical glutamate concentrations in transgenic amyotrophic lateral sclerosis mice are attenuated by creatine supplementation
Authors: Andreassen Ole A. , Jenkins Bruce G. , Dedeoglu Alpaslan , Ferrante Kimberly L. , Bogdanov Mikhail B. , Kaddurah‐Daouk Rima , Beal M. Flint
Published: March 2003

Several lines of evidence implicate excitotoxic mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS). Transgenic mice with a superoxide dismutase mutation (G93A) have been utilized as an animal model of familial ALS (FALS). We examined the cortical concentrations of glutamate using in vivo microdialysis and in vivo nuclear magnetic resonance (NMR) spectroscopy, and the effect of long‐term creatine supplementation. NMDA‐stimulated and

ltrans‐pyrrolidine‐2,4‐dicarboxylate (LTPD)‐induced increases in glutamate were significantly higher in G93A mice compared with littermate wild‐type mice at 115 days of age. At this age, the tissue concentrations of glutamate were also significantly increased as measured with NMR spectroscopy. Creatine significantly increased longevity and motor performance of the G93A mice, and significantly attenuated the increases in glutamate measured with spectroscopy at 75 days of age, but had no effect at 115 days of age. These results are consistent with impaired glutamate transport in G93A transgenic mice. The beneficial effect of creatine may be partially mediated by improved function of the glutamate transporter, which has a high demand for energy and is susceptible to oxidative stress.
Complete inhibition of in vivo glioma growth by oncostatin M
Authors: Friedrich Matthias , Höss Norbert , Stögbauer Florian , Senner Volker , Paulus Werner , Ringelstein E. bernd , Halfter Hartmut
Published: March 2003

We describe here the oncostatin M (OSM)‐dependent inhibition of in vivo tumour formation after intracerebral inoculation of glioblastoma cells in mice. We generated human glioblastoma cells transfected with the OSM gene under the control of a tetracycline‐response promoter. Upon removal of tetracycline from the medium, cells exhibited a differentiated cell morphology, while proliferation was significantly inhibited. After implantation of these cells into nude mice brains, large tumours developed in animals lacking OSM expression, whereas no tumour formation was observed in mice with induced OSM expression. Our results suggest that OSM exerts pronounced antitumorigenic effects on glioblastoma cells in vivo and provide arguments for a therapeutic application of OSM in humans.
Probing the mechanism of rhodopsin‐catalyzed transducin activation
Authors: Natochin Michael , Moussaif Mustapha , Artemyev Nikolai O.
Published: March 2003

An agonist‐bound G protein‐coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein α‐subunit (Gα) followed by the release of GαGTP and Gβγ which, subsequently, activate their targets. The C‐terminal regions of Gα subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR‐induced conformational change is communicated from the Gα C‐terminus, via the α5 helix, to the nucleotide‐binding β6/α5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C‐terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three‐turn α‐helical segment was inserted between the 11 C‐terminal residues and the α5 helix of Gαt‐like chimeric Gα, Gαti. The mutant Gα subunits with the Gly‐loop (GαtiL) and the extended α5 helix (GαtiH) retained intact interactions with Gβγt, and displayed modestly reduced binding to R*. GαtiH was capable of efficient activation by R*. In contrast, R* failed to activate GαtiL, suggesting that the Gly‐loop absorbs a conformational change at the C‐terminus and blocks G protein activation. Our results provide evidence for the role of Gα C‐terminus/α5 helix/β6/α5 loop route as a dominant channel for transmission of the GPCR‐induced conformational change leading to G protein activation.
Chronic cocaine‐mediated changes in non‐human primate nucleus accumbens gene expression
Authors: Freeman Willard M. , Nader Michael A. , Nader Susan H. , Robertson Daniel J. , Gioia Lynda , Mitchell Samara M. , Daunais James B. , Porrino Linda J. , Friedman David P. , Vrana Kent E.
Published: March 2003

Chronic cocaine use elicits changes in the pattern of gene expression within reinforcement‐related, dopaminergic regions. cDNA hybridization arrays were used to illuminate cocaine‐regulated genes in the nucleus accumbens (NAcc) of non‐human primates (Macaca fascicularis; cynomolgus macaque), treated daily with escalating doses of cocaine over one year. Changes seen in mRNA levels by hybridization array analysis were confirmed at the level of protein (via specific immunoblots). Significantly up‐regulated genes included: protein kinase A α catalytic subunit (PKAcα); cell adhesion tyrosine kinase beta (PYK2); mitogen activated protein kinase kinase 1 (MEK1); and β‐catenin. While some of these changes exist in previously described cocaine‐responsive models, others are novel to any model of cocaine use. All of these adaptive responses coexist within a signaling scheme that could account for known inductions of genes(e.g. fos and jun proteins, and cyclic AMP response element binding protein) previously shown to be relevant to cocaine's behavioral actions. The complete data set from this experiment has been posted to the newly created Drug and Alcohol Abuse Array Data Consortium (http://www.arraydata.org) for mining by the general research community.
The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site‐directed mutagenesis
Authors: Chen Nianhang , Vaughan Roxanne A. , Reith Maarten E. A.
Published: March 2003

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na+/Cl−‐dependent transporters. We have explored the roles of these residues using non‐conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10–24‐fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [3H]DA uptake, but caused undetectable [3H]CFT binding. Upon anti‐C‐terminal‐hDAT immunoblotting, D345N appeared as broad bands of 66–97 kDa, but this band could not be photoaffinity labeled with cocaine analog [125I]‐3β‐(p‐chlorophenyl)tropane‐2β‐carboxylic acid ([125I]RTI‐82). Unexpectedly, in this mutant, cocaine‐like drugs remained potent inhibitors of [3H]DA uptake. CFT solely raised the Km of [3H]DA uptake in wild‐type hDAT, but increased Km and decreased Vmax in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.
Dual phases of functional change in norepinephrine transporter in cultured bovine adrenal medullary cells by long‐term treatment with clozapine
Authors: Yoshimura Reiji , Yanagihara Nobuyuki , Hara Koji , Nakamura Jun , Toyohira Yumiko , Ueno Susumu , Izumi Futoshi
Published: March 2003

The effects of long‐term treatment with clozapine, a prototype of atypical antipsychotic drugs, on the functional activity, synthesis and mRNA of norepinephrine (NE) transporter were examined in bovine adrenal medullary cells in culture. Treatment of cells with clozapine at 0.1–3.0 µm concentrations produced dual phases of changes in [3H]NE uptake, i.e. the first phase showed a decrease in [3H]NE uptake at 2–48 h, and the following phase showed an increase in uptake at 72–168 h. Treatment with clozapine for 6 h decreased Vmax to 40% of the control without changing the Km value for [3H]NE uptake. However, treatment with clozapine for 96 h increased Vmax by 56% over the control without a change in Km. Scatchard plot analysis of [3H]desipramine (DMI) binding to membranes isolated from cells treated with clozapine for 6 h revealed a decrease in Bmax without any change in Kd; in contrast, treatment with clozapine for 96 h caused an increase in Bmax without any change in Kd. Both actinomycin D and cycloheximide, which are inhibitors of protein synthesis, suppressed the clozapine (96 h)‐induced increase in [3H]NE uptake. Treatment of cells with clozapine for 12–96 h increased the level of NE transporter mRNA in a concentration‐dependent manner (0.3–3.0 µm). These findings suggest that treatment of cells with clozapine results in the down‐regulation and subsequent up‐regulation of NE transporter. The latter change may be caused by the synthesis of new proteins of NE transporter via an increase in its mRNA.
Cloning and characterization of the murine Imitation Switch (ISWI) genes: differential expression patterns suggest distinct developmental roles for Snf2h and Snf2l
Authors: Lazzaro Maribeth A. , Picketts David J.
Published: March 2003

Here we report the cloning of two cDNAs, Snf2h and Snf2l, encoding the murine members of the Imitation Switch (ISWI) family of chromatin remodeling proteins. To gain insight into their function we examined the spatial and temporal expression patterns of Snf2h and Snf2l during development. In the brain, Snf2h is prevalent in proliferating cell populations whereas, Snf2l is predominantly expressed in terminally differentiated neurons after birth and in adult animals, concomitant with the expression of a neural specific isoform. Moreover, a similar proliferation/differentiation relationship of expression for these two genes was observed in the ovaries and testes of adult mice. These results are consistent with a role of Snf2h complexes in replication‐associated nucleosome assembly and suggest that Snf2l complexes have distinct functions associated with cell maturation or differentiation.
Stromal cell‐derived factor‐1α induces astrocyte proliferation through the activation of extracellular signal‐regulated kinases 1/2 pathway
Authors: Bajetto Adriana , Barbero Simone , Bonavia Rudy , Piccioli Patrizia , Pirani Paolo , Florio Tullio , Schettini Gennaro
Published: March 2003

Stromal cell‐derived factor‐1 (SDF‐1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co‐receptor for HIV‐1 infection. We previously demonstrated that CXCR4 and SDF‐1α are expressed in cultured type‐I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type‐I cortical astrocytes and demonstrated that SDF‐1α stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF‐1α in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal‐regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF‐1α treatment selectively activated ERK1/2, but not p38 or stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF‐1α, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G‐protein and of phosphatidyl inositol‐3 kinase in the signalling of SDF‐1α. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF‐1α in primary cultures of rat type‐I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF‐1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.
Glutathione S‐transferase Pi is a dopamine‐inducible suppressor of dopamine‐induced apoptosis in PC12 cells
Authors: Ishisaki Akira , Hayashi Hisaki , Suzuki Seigo , Ozawa Kazuo , Mizukoshi Eiichi , Miyakawa Kazuko , Suzuki Masashi , Imamura Toru
Published: March 2003

The finding that the neurotransmitter dopamine induces apoptosis in neurons implies the existence of a cellular mechanism by which dopaminergic neurons protect themselves from dopamine‐induced apoptosis. By profiling the expression of a number of genes in differentiating PC12 cells which exhibit elevated levels of dopamine biosynthesis, we found that expression of glutathione S‐transferase class Pi (GSTp) mRNA was selectively up‐regulated. Interestingly, dopamine added to the culture medium of PC12 cells also augmented their expression of GSTp mRNA. Suppression of GSTp expression by transfection of its antisense expression vector augmented dopamine‐induced apoptosis of PC12 cells. Conversely, overexpression of GSTp made the resultant PC12 transfectants highly resistant to dopamine‐induced apoptosis. Transfection of the antisense or sense GSTp expression vectors also resulted in corresponding augmentation or suppression of dopamine‐induced activation of cell‐associated Jun‐N‐terminal kinase (JNK), which has been suggested to mediate dopamine‐induced apoptosis in neuronal cells. These results indicate that GSTp is a dopamine‐inducible suppressor of dopamine‐induced apoptosis in PC12 cells, and suggest that this activity is exerted through inhibition of JNK activity.
The olfactory G protein Gαolf possesses a lower GDP‐affinity and deactivates more rapidly than Gsαshort: consequences for receptor‐coupling and adenylyl cyclase activation
Authors: Liu Hui‐Yu , Wenzel‐Seifert Katharina , Seifert Roland
Published: March 2003

The olfactory G protein Gαolf differs from the short splice variant of Gsα (GsαS) in 80 amino acids, but little is known about biochemical differences between Gαolf and GsαS. We addressed this question by analyzing fusion proteins of the β2‐adrenoceptor (β2AR) and Gαolf and GsαS, respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High‐affinity agonist binding studies showed that Gαolf possesses a lower GDP‐affinity than GsαS As a result, the agonist‐free β2AR and the β2AR occupied by partial agonists were more efficient at promoting GDP‐dissociation from Gαolf than from GsαS a assessed by guanosine 5’‐O‐(3‐thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing β2AR‐Gαolf than in membranes expressing β2AR‐GsαS at similar levels, reflecting the lower abundance of Gαolf‐GDP relative to GsαS‐GDP. The maximum agonist‐stimulated AC activity with β2AR‐GsαS was more than twofold higher than with β2AR‐Gαolf, but the relative agonist‐stimulation of AC with β2AR‐Gαolf was much greater than with β2AR‐GsαS. The difference in maximum AC activity can be explained by more rapid deactivation of Gαolf‐GTP by GTP hydrolysis and GTP dissociation relative to GsαS‐GTP. Taken together, there are biochemical differences between Gαolf and GsαS, supporting different roles of these G proteins in vivo.
A neutrophil elastase inhibitor (ONO‐5046) reduces neurologic damage after spinal cord injury in rats
Authors: Tonai Takeharu , Shiba Kei‐ichiro , Taketani Yutaka , Ohmoto Yasukazu , Murata Kaori , Muraguchi Masahiro , Ohsaki Hiroyuki , Takeda Eiji , Nishisho Takehiko
Published: March 2003

In view of a cytoprotective effect of elastase inhibitor on chemokine‐mediated tissue injury, we examined the neuroprotective effect of ONO‐5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine‐induced neutrophil chemo‐attractant (CINC)‐1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC‐1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO‐5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC‐1 protein and mRNA expression. ONO‐5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC‐1 contributed to inflammation in rat spinal cord injury and ONO‐5046 attenuated neurologic damage partly by blocking CINC‐1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.
Rapid induction and Ca2+ influx‐mediated suppression of vitamin D3 up‐regulated protein 1 (VDUP1) mRNA in cerebellar granule neurons undergoing apoptosis
Authors: Saitoh Tatsuya , Tanaka Shuuitsu , Koike Tatsuro
Published: March 2003

Cerebellar granule neurons (CGNs) grown under depolarizing conditions with high K+ (HK; 30 mm) undergo apoptosis following replacement of HK by physiological K+ (5.4 mm). Differential display analysis identified eight genes up‐regulated in this paradigm of apoptosis. Vitamin D3 up‐regulated protein 1 (VDUP1) mRNA was markedly up‐regulated as early as 2 h following HK withdrawal. VDUP1 mRNA was up‐regulated in other paradigms of neuronal apoptosis as well both in vitro and in vivo. HK effectively suppressed the up‐regulation of VDUP1 mRNA in CGNs undergoing apoptosis via Ca2+ influx through voltage‐dependent L‐type Ca2+ channels, which did not require de novo protein synthesis. The up‐regulation occurred in parallel with that of the c‐jun transcript and c‐jun protein phosphorylation. Moreover, SB203580, p38 mitogen‐activated protein kinase inhibitor, suppressed up‐regulation of both c‐jun and VDUP1 mRNAs, and c‐jun phosphorylation in CGNs undergoing apoptosis. IGF‐1, one of the neuroprotective agents for CGNs, also inhibited VDUP1 mRNA up‐regulation through a phosphoinositide 3 kinase‐dependent pathway. These results suggest that the VDUP1 gene is a novel member of early response genes in neuronal apoptosis whose expression is directly regulated by Ca2+ influx and coordinately regulated with the transcription factor c‐jun in CGNs.
Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site
Authors: Hwang J. R. , Lindberg I.
Published: March 2003

The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone substrates. In the present study, we examined the role of the pentabasic site within 27‐kDa 7B2 in this process. We prepared two His‐tagged recombinant 7B2s by overexpression in bacteria: 7B2‐Ser‐Ser (SS), with an inactivating mutation in the CT peptide from Lys171‐Lys172 (KK) to SS, rendering the CT peptide non‐inhibitory; blockade‐SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site. These purified proteins were used in a cell‐free proPC2 activation assay. Both 7B2‐SS as well as blockade‐SS were able to facilitate the activation of proPC2 (as judged by efficient production of enzyme activity), suggesting that cleavage at the furin site is not required for 7B2s lacking inhibitory CT peptides. Plasmids encoding proPC2 and various 7B2s were transiently transfected into human embryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in each overnight conditioned medium were measured. Cells transfected with proPC2 and wild‐type 7B2 secreted CT peptide cleavage products, but cells transfected with proPC2 and the blockade mutant overwhelmingly secreted intact, 27‐kDa, blockaded 7B2. Medium obtained from HEK293 cells transfected with proPC2 and either wild‐type 7B2, 7B2‐SS, or blockade‐SS exhibited PC2 activity, but medium from cells expressing the 7B2 blockade mutant did not. We conclude that cleavage at the 7B2 furin consensus site is required to produce PC2 capable of efficient proteolytic inactivation of the CT peptide.
Survivin inhibition induces human neural tumor cell death through caspase‐independent and ‐dependent pathways
Authors: Shankar Sai Latha , Mani Sridhar , O'Guin Kathleen Norman , Kandimalla Ekambar R. , Agrawal Sudhir , Shafit‐Zagardo Bridget
Published: March 2003

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose‐dependent reduction in survivin protein. Although 74% of the SAO‐treated MSN cells were trypan blue+, PARP cleavage or activated caspase‐3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co‐administration of z‐Val‐Ala‐Asp(OMe)‐fluoromethyl ketone (zVAD‐fmk) with SAO did not inhibit cell death suggesting a caspase‐independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO‐treated TC620 cells were trypan blue+, PARP was cleaved, cells were TUNEL+ and PI‐staining revealed macronuclei and numerous apoptotic bodies. Co‐treatment of the TC620 cells with SAO and zVAD‐fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two‐fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase‐independent mechanism.
Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein
Authors: Xiong Liang‐Wen , Raymond Lynne D. , Hayes Stanley F. , Raymond Gregory J. , Caughey Byron
Published: March 2003

The conversion of protease‐sensitive prion protein (PrP‐sen) to a high β‐sheet, protease‐resistant and often fibrillar form (PrP‐res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP‐res itself in cell‐free conversion reactions. The detergent sodium N‐lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP‐res purifications and is known to stimulate the PrP‐res‐induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP‐sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001–0.1%) induced aggregation of PrP‐sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of β‐sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of β‐sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead‐like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP‐res was detected in the sarkosyl‐induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP‐sen and induce high β‐sheet aggregates that are distinct from scrapie‐associated PrP‐res in terms of protease‐resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high‐β aggregates of PrP are equivalent to the pathologic form, PrP‐res.
Two kinds of mitogen‐activated protein kinase phosphatases, MKP‐1 and MKP‐3, are differentially activated by acute and chronic methamphetamine treatment in the rat brain
Authors: Takaki Manabu , Ujike Hiroshi , Kodama Masafumi , Takehisa Yasushi , Nakata Kenji , Kuroda Shigetoshi
Published: March 2003

Two functionally different MAP kinase phosphatases (MKPs) were investigated to clarify their roles in behavioral sensitization to methamphetamine (METH). MKP‐1 mRNA levels increased substantially by about 60–300% in a range of brain regions, including several cortices, the striatum and thalamus 0.5–1 h after acute METH administration. After chronic METH administration its increase was less pronounced, but a more than 50% increase was still seen in the frontal cortex. MKP‐1 protein levels also increased 3 h after acute or chronic METH administration. MKP‐3 mRNA levels increased by about 30–50% in several cortices, the striatum and hippocampus 1 h after acute METH administration, but only in the hippocampus CA1 after chronic METH administration. Pre‐treatment with the D1 dopamine receptor antagonist, SCH23390, attenuated the METH‐induced increase of MKP‐1 and MKP‐3 mRNA in every brain region, while pre‐treatment with the NMDA receptor antagonist, MK‐801, attenuated it in some regions. These findings suggest that in METH‐induced sensitization, MKP‐1 and MKP‐3 play important roles in the neural plastic modification in widespread brain regions in the earlier induction process, but in the later maintenance process, they do so only in restricted brain regions such as MKP‐1 in the frontal cortices and MKP‐3 in the hippocampus.
In vivo effects of adenosine A2 receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo13C MR spectroscopy
Authors: Hammer Janniche , Qu Hong , Håberg Asta , Sonnewald Ursula
Published: March 2003

The effect of adenosine A2 receptor agonist 2‐[p‐(2‐carboxyethyl)phenylethylamino]‐5′‐ethylcarboxamidoadenosine (CGS 21680) and antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX) on [1‐13C]glucose and [1,2‐13C]acetate metabolism was studied in rats by 13C magnetic resonance (MR) spectroscopy and HPLC. In the cortex a significant reduction was observed in the amounts of [2‐13C]GABA and [3‐13C]aspartate from [1‐13C]glucose in CGS 21680. In the subcortex the concentration of labelled [4‐13C]glutamate was increased in both treatment groups. The amounts of [2 + 3‐13C]succinate and [3‐13C]lactate were increased in the CGS 21680 group compared to control, and the DMPX group showed an increase in the total amount of [6‐13C]N‐acetyl aspartate compared to control in the subcortex. Astrocyte metabolism was only affected in the cortex as shown by a decrease in the pyruvate carboxylase/pyruvate dehydrognase ratio in glutamate and glutamine in the treatment groups. Labelling from [1,2‐13C]acetate was not much affected by CGS 21680 or DMPX. However, the amount of [1,2‐13C]acetate in cortex and subcortex was reduced in the DMPX group. In the cortex a reduction in the labelling of [3‐13C]GABA in the DMPX group compared to control and an increase in the total amount of taurine in both treatment groups was detected. The present study shows that A2 receptor agonist and antagonist have similar effects; however, in cortex GABAergic neurones and astrocytes were affected in contrast to subcortex, where glutamatergic neurones showed the greatest changes.
Glucocorticoid inhibits growth factor‐induced differentiation of hippocampal progenitor HiB5 cells
Authors: Son Gi Hoon , Geum Dongho , Jung Hosung , Kim Kyungjin
Published: March 2003

In the present study, we investigated the effect of glucocorticoid on neuronal differentiation of hippocampal progenitor HiB5 cells. Dexamethasone (DEX), a synthetic glucocorticoid, inhibited platelet‐derived growth factor (PDGF)‐induced differentiation of HiB5 cells. The inhibitory effect of DEX was antagonized by RU486, a glucocorticoid receptor (GR) antagonist, indicating the GR‐mediated processes. Nestin mRNA level was decreased and midsize neurofilament (NF‐M) mRNA level was increased as a function of neuronal differentiation. DEX significantly blocked PDGF‐induced down‐regulation of nestin mRNA level, and up‐regulation of NF‐M mRNA level, which were similar to those of undifferentiated cells. DEX inhibited PDGF‐induced activation of cyclic AMP‐responsive element binding protein (CREB) and AP‐1, suggesting that glucocorticoid interfered with signal transduction cascades linking the PDGF receptor and downstream transcription factors. Indeed, DEX reduced PDGF‐induced phosphorylation of extracellular signal‐regulated kinases1/2 (ERK1/2). Tyrosine phosphatase inhibitor reversed the effect of DEX on ERK1/2. In accordance with this finding, blockage of ERK1/2 signaling pathway with PD098059, a potent inhibitor for Ras/ERK pathway, mimicked the inhibitory effect of DEX on differentiation processes. Taken together, these results indicate that glucocorticoid inhibits PDGF‐induced differentiation of hippocampal progenitor HiB5 cells by inhibiting the ERK1/2 signaling cascade via a tyrosine phosphatase‐dependent mechanism.
Enhanced hippocampal F2‐isoprostane formation following kainate‐induced seizures
Authors: Patel Manisha , Liang Li‐Ping , Roberts II L. Jackson
Published: March 2003

We attempted to obtain evidence for the occurrence of oxidant injury following seizure activity by measuring hippocampal F2‐isoprostanes (F2‐IsoPs), a reliable marker of free radical‐induced lipid peroxidation. Formation of F2‐IsoPs esterified in hippocampal phospholipids was correlated with hippocampal neuronal loss and mitochondrial aconitase inactivation, a marker of superoxide production in the kainate model. F2‐IsoPs were measured in microdissected hippocampal CA1, CA3 and dentate gyrus (DG) regions at various times following kainate administration. Kainate produced a large increase in F2‐IsoP levels in the highly vulnerable CA3 region 16 h post injection. The CA1 region showed small, but statistically insignificant increases in F2‐IsoP levels. Interestingly, the DG, a region resistant to kainate‐induced neuronal death also showed marked (2.5–5‐fold) increases in F2‐IsoP levels 8, 16, and 24 h post injection. The increases in F2‐Isop levels in CA3 and DG were accompanied by inactivation of mitochondrial aconitase in these regions. This marked subregion‐specific increase in F2‐Isop following kainate administration suggests that oxidative lipid damage results from seizure activity and may play an important role in seizure‐induced death of vulnerable neurons.
Theory relatingin vitroandin vivomicrodialysis with one or two probes
Authors: Chen Kevin C. , Höistad Malin , Kehr Jan , Fuxe Kjell , Nicholson Charles
Published: March 2003

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungayet al. to provide a theoretical basis forin vitroandin vivomicrodialysis. Specifically, we considered the effect of active clearance processes onin vivomicrodialysis, and thereby elaborated the theory of Benvenisteet al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on thein vitroandin vivorecoveries in dual‐probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi‐steady state is about 5.5 × 10−5s−1. This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.
5‐S‐Cysteinyl‐conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase‐3 activity in neurons
Authors: Spencer Jeremy P. E. , Whiteman Matthew , Jenner Peter , Halliwell Barry
Published: March 2003

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl‐adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5‐S‐cysteinyl conjugates of dopamine,l‐DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase‐3 activity in cells. Damage to neurons was apparent 12–48 h of post‐exposure and there were increases in caspase‐3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8‐OH‐guanine suggesting that 5‐S‐cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase‐3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.
Desensitization of pigment granule aggregation in Xenopus leavis melanophores: melatonin degradation rather than receptor down‐regulation is responsible
Authors: Teh Muy‐Teck , Sugden David
Published: March 2003

Xenopus laevis melanophores express a high density (Bmax 1224 fmol/mg protein) of high‐affinity (Kd 37 pm) cell membrane melatonin receptors. Treatment of melanophores with melatonin resulted in a loss of membrane melatonin receptors reaching a maximum (∼60%) by 6 h. In addition to receptor loss, a decline in the potency of melatonin to produce pigment aggregation was observed on prolonged treatment. However, the loss of potency (3.8‐fold in 24 h and 162‐fold in 96 h) was much slower than loss of receptors, and was completely prevented by inclusion of eserine (100 µm), an inhibitor of melatonin deacetylation in the culture medium. Incubation of melanophores with [3H]‐melatonin showed that eserine prevented metabolism of melatonin to 5‐methoxytryptamine. These results indicate that although receptor density does decline on prolonged treatment, this is not responsible for the diminishing melatonin potency, which is entirely due to degradation of melatonin by deacetylation and subsequent deamination in melanophores.
Blockade of cannabinoid CB1 receptor function protects against in vivo disseminating brain damage following NMDA‐induced excitotoxicity
Authors: Hansen Henrik H. , Azcoitia Iñigo , Pons Sebastián , Romero Julián , García‐Segura Luis Miguel , Ramos José Antonio , Hansen Harald S. , Fernández‐Ruiz Javier
Published: March 2003

The ability of cannabinoid CB1 receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB1 receptor function on NMDA‐induced excitotoxicity. Neonatal (6‐day‐old) rat pups received a systemic injection of a mixed CB1/CB2 receptor agonist (WIN55,212–2) or their respective antagonists (SR141716A for CB1 and SR144528 for CB2) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA‐induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist‐stimulated CB1 receptor function. In contrast, blockade of CB1, but not CB2, receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB1 receptor tonus on neuronal survival following NMDA receptor‐induced excitotoxicity in vivo.
CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones
Authors: Holm Ninna R. , Christophersen Palle , Hounsgaard Jørn , Gammeltoft Steen , Olesen Søren‐Peter
Published: March 2003

Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high‐threshold voltage‐activated Ca channels in cultured fetal mouse brain cortical neurones was investigated. Addition of CNTF into serum‐free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18 h. CNTF reduced the current of all pharmacological subtypes of Ca channels as shown by use of selective blockers of L, N, and P/Q type Ca channels (nifedipine, ω‐conotoxin MVIIA, ω‐agatoxin IVA). The Ca channel depression was mediated via the CNTF receptor, because enzymatic cleavage of the α‐subunit glycerophosphatidylinositol anchor of the receptor eliminated the response. The CNTF effect was not elicited through pertussis toxin‐sensitive G proteins. Other neurotrophic factors like neurotrophin‐3 and insulin‐like growth factor‐I had no effect on the Ca2+ currents. These results may have important implications for the possible functions of CNTF in the nervous system, such as altered synaptic activity, neuronal excitability and susceptibility to brain ischaemia.
In vivo monitoring of evoked noradrenaline release in the rat anteroventral thalamic nucleus by continuous amperometry
Authors: Dugast C. , Cespuglio R. , Suaud‐Chagny M. F.
Published: March 2003

Continuous amperometry coupled with untreated carbon‐fibre electrodes was used in anaesthetized rats to measure the noradrenaline release evoked in the anteroventral thalamic nucleus by electrical stimulation of the dorsal noradrenergic bundle. As expected, the variations in the oxidation current detected in the anteroventral thalamic nucleus exhibited the characteristics of the in vivo noradrenaline release. They were closely correlated with stimulation and consistent with the anatomy of the noradrenergic system involved. They were abolished by the ejection of tetrodotoxin in the vicinity of the carbon‐fibre electrode, diminished by clonidine, an alpha‐2 agonist, and restored by yohimbine, an alpha‐2 antagonist. Furthermore, the time course of these variations was dramatically increased by desipramine, a specific noradrenaline reuptake blocker. In contrast, neither dopamine nor serotonin reuptake blockers, nor the monoamine oxidase inhibitor pargyline were able to alter them. The main advantage of the present approach is its excellent time resolution. We show here for the first time that after single pulse stimulation, noradrenaline is released and eliminated in 118 milliseconds, this time lapse corresponding to the maximal period beyond which subsequent noradrenaline releases could not add up. These observations are in good agreement with the physiological relationship previously observed between impulse flow and noradrenaline overflow.
Homologous regulation of the heptahelical D1A receptor responsiveness: specific cytoplasmic tail regions mediate dopamine‐induced phosphorylation, desensitization and endocytosis
Authors: Jackson Adele , Iwasiow Rafal M. , Chaar Ziad Y. , Nantel Marie‐France , Tiberi Mario
Published: March 2003

In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)‐induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild‐type (WT) or truncation forms (Δ425, Δ379 and Δ351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA‐mediated phosphorylation‐dependent desensitization of D1A receptors. However, the longer truncation mutant Δ351 failed to undergo detectable DA‐induced phosphorylation while exhibiting DA‐induced desensitization features similar to the shorter truncation mutant Δ379. These data potentially suggest a novel role for a receptor phosphorylation‐independent process in the DA‐promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA‐mediated receptor endocytosis. Additionally, time‐course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation orkobs, a parameter linked to the receptor‐G protein coupling status. In agreement with the desensitization data, Δ425‐ and Δ379‐expressing cells exhibit an increase ofkobsin comparison with WT‐expressing cells. Nevertheless, Δ351‐expressing cells, which harbor similar desensitization features of Δ379‐expressing cells, display no change inkobswhen compared with WT‐expressing cells. Our results suggest that a defective DA‐induced endocytosis may hamper Δ351 resensitization and concomitant increase inkobs. Thus, our study showing that specific D1A receptor CT sequences regulate DA‐induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.
Low‐density lipoprotein receptor‐related protein levels and endocytic function are reduced by overexpression of the FE65 adaptor protein, FE65L1
Authors: Guénette Suzanne Y. , Chang Yang , Hyman Bradley T. , Tanzi Rudolph E. , Rebeck G. William
Published: March 2003

The FE65 adaptor protein family was identified in two‐hybrid screens as proteins that bind the cytoplasmic domain of the amyloid precursor protein (APP). Studies have shown that FE65 binding to APP modulates APP processing. Increased levels of α‐secretase derived secreted APP (APPsα) and β‐amyloid (Aβ) were recovered from conditioned media upon FE65L1 or FE65 overexpression. These effects were associated with an increase in the ratio of mature/immature APP and increased cell‐surface APP. FE65 has also been reported to bind low‐density lipoprotein receptor‐related protein (LRP). Here we show that FE65L1 overexpression results in decreased LRP steady state levels, LRPs, and LRP endocytic receptor function. These changes in LRP protein levels are not due to decreased transcription of LRP. Furthermore, pulse/chase experiments demonstrate that changes in LRP protein only occurred 12–18 h after translation. We conclude that the decreases in LRP levels likely reflect routing of LRP away from the cell surface into a degradative pathway. Previous studies suggested that LRP plays an important role for Aβ production of Kunitz protease inhibitor forms of APP in the endocytic pathway. These data show that FE65L1 can differentially affect the metabolic fate of APP and LRP. In addition, these data suggest that the LRP decrease observed in FE65L1 overexpressing cells may in part contribute to altered APP processing.