bluuebunny/crossref_metadata_2025 huggingface dataset:
2‐Oxoacids Regulate Kynurenic Acid Production in the Rat Brain
Authors: Hodgkins Paul S. , Wu Hui‐Qiu , Zielke Ronald H. , Schwarcz Robert
Published: March 2003

Abstract : This study was designed to examine the role of 2‐oxoacids in the enzymatic transamination of L‐kynurenine to the excitatory amino acid receptor antagonist, kynurenate, in the rat brain. In brain tissue slices incubated in Krebs‐Ringer buffer with a physiological concentration of L‐kynurenine, pyruvate, and several other straight‐ and branched‐chain 2‐oxoacids, substantially restored basal kynurenate production in a dose‐dependent manner without increasing the intracellular concentration of L‐kynurenine. All 2‐oxoacids tested also reversed or attenuated the hypoglycemia‐induced decrease in kynurenate synthesis, but only pyruvate and oxaloacetate also substantially restored intracellular L‐kynurenine accumulation. Thus, 2‐oxoacids increase kynurenate formation in the brain primarily by functioning as co‐substrates of the transamination reaction. This was supported further by the fact that the nonspecific kynurenine aminotransferase inhibitors (aminooxy)acetic acid and dichlorovinylcysteine prevented the effect of pyruvate on kynurenate production in a dose‐dependent manner. Moreover, all 2‐oxoacids tested attenuated or prevented the effects of veratridine, quisqualate, or L‐α‐aminoadipate, which reduce the transamination of L‐kynurenine to kynurenate. Finally, dose‐dependent increases in extracellular kynurenate levels in response to an intracerebral perfusion with pyruvate or α‐ketoisocaproate were demonstrated by in vivo microdialysis. Taken together, these data show that 2‐oxoacids can directly augment the de novo production of kynurenate in several areas of the rat brain. 2‐Oxoacids may therefore provide a novel pharmacological approach for the manipulation of excitatory amino acid receptor function and dysfunction.
Quantification of Axonal Damage in Traumatic Brain Injury
Authors: Zemlan Frank , Rosenberg William S. , Luebbe Patricia A. , Campbell Thomas A. , Dean Gary E. , Weiner Ned E. , Cohen Jeffery A. , Rudick Richard A. , Woo Daniel
Published: March 2003

Abstract : Diffuse axonal injury is a primary feature of head trauma
and is one of the most frequent causes of mortality and morbidity. Diffuse
axonal injury is microscopic in nature and difficult or impossible to detect
with imaging techniques. The objective of the present study was to determine
whether axonal injury in head trauma patients could be quantified by measuring
levels of CSF tau proteins. Tau proteins are structural microtubule binding
proteins primarily localized in the axonal compartment of neurons. Monoclonal
antibodies recognizing the form of tau found in the CSF of head trauma
patients were developed by differential CSF hybridoma screening using CSF from
head trauma and control patients. Clones positive for head trauma CSF tau
proteins were used to characterize this form of tau and for ELISA development.
Using the developed ELISA, CSF tau levels were elevated >1,000‐fold in head
trauma patients (mean, 1,519 ng/ml of CSF) when compared with patients with
multiple sclerosis (mean, 0.014 ng/ml of CSF ; p < 0.001), normal
pressure hydrocephalus (nondetectable CSF tau), neurologic controls (mean,
0.031 ng/ml of CSF ; p < 0.001), or nonneurologic controls
(nondetectable CSF tau ; p < 0.001). In head trauma, a relationship between clinical improvement and decreased CSF tau levels was observed. These data suggest that CSF tau levels may prove a clinically useful assay for quantifying the axonal injury associated with head trauma and monitoring efficacy of neuroprotective agents. Affinity purification of CSF tau from head trauma patients indicated a uniform cleavage of ~ 18 kDa from all six tau isoforms, reducing their apparent molecular sizes to 30‐50 kDa. These cleaved forms of CSF tau consisted of the interior portion of the tau sequence, including the microtubule binding domain, as judged by cyanogen bromide digestion. Consistent with these data, CSF cleaved tau bound taxolpolymerized microtubules, indicating a functionally intact microtubule binding domain. Furthermore, epitope mapping studies suggested that CSF cleaved tau proteins consist of the interior portion of the tau sequence with cleavage at both N and C terminals.
Protein‐Bound Acrolein
Authors: Calingasan Noel Y. , Uchida Koji , Gibson Gary E.
Published: March 2003

Abstract : Several lines of evidence support the role of oxidative stress, including increased lipid peroxidation, in the pathogenesis of Alzheimer's disease (AD). Lipid peroxidation generates various reactive aldehydes, such as 4‐hydroxynonenal (HNE), which have been detected immunochemically in AD, particularly in neurofibrillary tangels, one of the major diagnostic lesions in AD brains. A recent study demonstrated that acrolein, the most reactive among the α, β‐unsaturated aldehyde products of lipid peroxidation, could be rapidly incorporated into proteins, generating a carbonyl derivative, a marker of oxidative stress to proteins. The current studies used an antibody raised against acrolein‐modified keyhole limpet hemocyanin (KLH) to test whether acrolein modification of proteins occurs in AD. Double immunofluorescence revealed strong acrolein‐KLH immunoreactivity in more than half of all paired helical filament (PHF)‐1‐labeled neurofibrillary tangles in AD cases. Acrolein‐KLH immunoreactivity was also evident in a few neurons lacking PHF‐1‐positive neurofibrillary tangles. Light acrolein‐KLH immunoreactivity occurred in dystrophic neurites surrounding the amyloid‐β core, which itself lacked acrolein‐KLH staining. The pattern of acrolein‐KLH immunostaining was similar to that of HNE. Control brains did not contain any acrolein‐KLH‐immunoreactive structures. The current results suggest that protein‐bound acrolein is a powerful marker of oxidative damage to protein and support the hypothesis that lipid peroxidation and oxidative damage to protein may play a crucial role in the formation of neurofibrillary tangles and to neuronal death in AD.
Induction of Two DNA Mismatch Repair Proteins, MSH2 and MSH6, in Differentiated Human Neuroblastoma SH‐SY5Y Cells
Authors: Belloni Marco , Uberti Daniela , Rizzini Claudia , Jiricny Josef , Memo Maurizio
Published: March 2003

Abstract : The MutS homologues MSH2 and MSH6 form a heterodimeric
protein complex that is involved in the recognition of base/base mismatches
and insertion/deletion loops, as well as some other types of DNA damage. We
investigated the expression of these proteins in undifferentiated and retinoic
acid‐differentiated human neuroblastoma SH‐SY5Y cells by immunocytochemistry,
western blot analysis, and RT‐PCR. Nuclei from undifferentiated SH‐SY5Y cells
were found to be immunoreactive to anti‐MSH2 and anti‐MSH6 antibodies.
Following differentiation, the cells stop dividing and change morphology to
acquire a neuron‐like phenotype. Under these conditions, both anti‐MSH2 and
anti‐MSH6 immunoreactivities were still detectable, although the signals were
somewhat less intense. When these cells were exposed for 2 h to neurotoxic
concentrations of doxorubicin (50 nM), they exhibited a marked and homogeneous increase of both anti‐MSH2 and anti‐MSH6 immunoreactivities. As revealed by western blot analysis, these effects were associated with increased protein content and were dose‐dependent. Using RT‐PCR technology, we also found that doxorubicin treatment did not change MSH2 or MSH6 mRNA levels. Our data indicate that human postmitotic, neuron‐like cells constitutively express the molecular machinery devoted to recognition of DNA mismatches and that this system is activated by specific treatment leading to cell death. These findings might help clarify the molecular mechanisms underlying various human neurological diseases that are associated with deficiencies in DNA repair and/or a high rate of DNA damage acquisition.
Differential Regulation of Adenine Nucleotide Translocators by Hypertonicity in the Brain
Authors: Yamashita Toshihide , Yamauchi Atsushi , Miyai Akiko , Taniguchi Masaaki , Yoshimine Toshiki , Tohyama Masaya
Published: March 2003

Abstract : To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up‐regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT) 2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up‐regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of Nacl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.
Phosphatidic Acid as the Biosynthetic Precursor of the Endocannabinoid 2‐Arachidonoylglycerol in Intact Mouse Neuroblastoma Cells Stimulated with Ionomycin
Authors: Bisogno Tiziana , Melck Dominique , De Petrocellis Luciano , Di Marzo Vincenzo
Published: March 2003

Abstract: In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2‐arachidonoylglycerol (2‐AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2‐AG. [3H]2‐AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N‐ethylmaleimide, two inhibitors of the Mg2+/Ca2+‐dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin‐induced [3H]2‐AG formation. These data indicate that DAG precursors for 2‐AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl‐CoA acylase inhibitor thimerosal significantly reduced [3H]2‐AG biosynthesis. The implications of these latter findings, and of the PA‐dependent pathways of 2‐AG formation described here, are discussed.
Antisilencing
Authors: Dobretsova Anna , Wight Patricia A.
Published: March 2003

Abstract: Antisilencer or antirepressor elements have been
described, thus far, for only a few eukaryotic genes and were identified by
their ability not to augment gene expression per se but to override repression
mediated via negative transcription regulatory elements. Here we report the
first case of antisilencing for a neural‐specific gene, the myelin proteolipid
protein (PLP) gene (Plp). PLP is the most abundant protein found in
CNS myelin. The protein is synthesized in oligodendrocytes, and its expression
is regulated developmentally. Previously we have shown that a
PLP‐lacZ transgene (which includes the entire sequence for
Plp intron 1) is regulated in mice, in a manner consistent with the
spatial and temporal expression of the endogenous Plp gene. In the
present report, we demonstrate by transfection analyses, using various
PLP‐lacZ deletion constructs, that Plp intron 1 DNA contains
multiple elements that collectively regulate Plp gene expression in
oligodendrocytes. One of these regulatory elements functions as an
antisilencer element, which acts to override repression mediated by at least
two negative regulatory elements located elsewhere within Plp intron 1 DNA. The mechanism for antisilencing appears to be complex as the intragenic region that mediates this function binds multiple nuclear factors specifically.
Bone Morphogenetic Protein‐2 Promotes Survival and Differentiation of Striatal GABAergic Neurons in the Absence of Glial Cell Proliferation
Authors: Hattori Akira , Katayama Masahiro , Iwasaki Shoji , Ishii Kazuhiro , Tsujimoto Masafumi , Kohno Michiaki
Published: March 2003

Abstract: We examined the potential neurotrophic effects of bone morphogenetic protein (BMP)‐2 on the survival and differentiation of neurons cultured from the rat developing striatum at embryonic day 16, a period during which the mRNAs for BMP‐2 and its receptor subunits (types IA, IB, and II) were detected. BMP‐2 exerted potent activity to promote the survival of striatal neurons and increased the number of surviving microtubule‐associated protein‐2‐positive cells by 2.4‐fold as compared with the control cultures after 4 days in vitro. Although basic fibroblast growth factor (bFGF) also showed relatively high activity to promote the survival of striatal neurons, transforming growth factor‐β1, ‐β2, and ‐β3, glial cell line‐derived neurotrophic factor, or brain‐derived neurotrophic factor promoted their survival weakly. Striatal neurons cultured in the presence of BMP‐2 or bFGF possessed extensive neurite outgrowths, the majority of which were GABA‐immunoreactive. Inhibition of glial cell proliferation by 5‐fluorodeoxyuridine did not affect the capacity of BMP‐2 to promote the survival of striatal GABAergic neurons. In contrast, the ability of bFGF to promote the survival of striatal neurons was inhibited significantly by the treatment of cells with 5‐fluorodeoxyuridine. All these results suggest that BMP‐2 exerts potent neurotrophic effects on the striatal GABAergic neurons in a glial cell‐independent manner.
Expression of Mouse Sialic Acid on Gangliosides of a Human Glioma Grown as a Xenograft in SCID Mice
Authors: Ecsedy Jeffrey A. , Holthaus Kathryn A. , Yohe Herbert C. , Seyfried Thomas N.
Published: March 2003

Abstract: Ganglioside sialic acid content was examined in the U87‐MG human glioma grown as cultured cells and as a xenograft in severe combined imunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N‐glycolylneuraminic acid (NeuGc)‐containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N‐acetylneuraminic acid (NeuAc)‐containing gangliosides, whereas mouse cells express both NeuAc‐ and NeuGc‐containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2‐3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87‐MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87‐MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87‐MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo‐GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.
Regulation of the Heparan Sulfate Proteoglycan, Perlecan, by Injury and Interleukin‐1α
Authors: De Yébenes Esther García , Ho Angela , Damani Tanuja , Fillit Howard , Blum Mariann
Published: March 2003

Abstract : Perlecan is a specific proteoglycan that binds to amyloid precursor protein and β‐amyloid peptide, is present within amyloid deposits, and has been implicated in plaque formation. Because plaque formation is associated with local inflammation, we hypothesized that the mechanisms involved in brain inflammatory responses could influence perlecan biosynthesis. To test this hypothesis, we first studied perlecan regulation in mice after inflammation induced by a brain stab wound. Perlecan mRNA and immunoreactivity were both increased 3 days after injury. Interleukin‐1α (IL‐1α) is a cytokine induced after injury and plays an important role in inflammation. As such, IL‐1α may be one of the factors participating in regulating perlecan synthesis. We thus studied perlecan regulation by IL‐1α, in vivo. Regulation of perlecan mRNA by this cytokine was area‐specific, showing up‐regulation in hippocampus, whereas in striatum, perlecan mRNA was unchanged. To support this differential regulation of perlecan mRNA by IL‐1α, basic fibroblast growth factor (bFGF), a growth factor also present in plaques, was studied in parallel. bFGF mRNA did not show any regional difference, being up‐regulated in both hippocampus and striatum in vivo. In vitro, both astrocyte and microglia were immunoreactive for perlecan. Moreover, perlecan mRNA was increased in hippocampal glial cultures after II‐1α but not in striatal glia. These results show an increase in perlecan biosynthesis after injury and suggest a specific regulation of perlecan mRNA by IL‐1α, which depends on brain area. Such regulation may have important implications in the understanding of regional brain variations in amyloid plaque formation.
The Role of the 7B2 CT Peptide in the Inhibition of Prohormone Convertase 2 in Endocrine Cell Lines
Authors: Fortenberry Yolanda , Liu June , Lindberg Iris
Published: March 2003

Abstract : Prohormone convertase (PC) 2 plays an important role in the processing of neuropeptide precursors via the regulated secretory pathway in neuronal and endocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21‐kDa domain involved in proPC2 maturation and a carboxyl‐terminal peptide (CT peptide) that represents a potent inhibitor of PC2 in vitro. A role for the CT peptide as an inhibitor in vivo has not yet been established. To study the involvement of the CT peptide in PC2‐mediated cleavages in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expression vector containing PE with its carboxyl‐terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably transfected into two PC2‐expressing cell lines, AtT‐20/PC2 and Rin cells. Although recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitro, cellular PC2‐mediated cleavages of PE were not inhibited by the PECT chimera, nor was proopiomelanocortin cleavage (as assessed by adrenocorticotropin cleavage to α‐melanocyte‐stimulating hormone) inhibited further than in control cells expressing only the competitive substrate PE. Tests of stimulated secretion showed that both the CT peptide and the PE portion of the chimera were stored in regulated secretory granules of transfected clones. In both AtT‐20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the observed lack of inhibition. Taken together, our data suggest that overexpressed CT peptide derived from PECT is unable to inhibit PC2 in mature secretory granules, most likely due to its inactivation by PC2 or by other enzyme(s).
The K‐Opioid Agonist, U‐69593, Decreases Acute Amphetamine‐Evoked Behaviors and Calcium‐Dependent Dialysate Levels of Dopamine and Glutamate in the Ventral Striatum
Authors: Gray Alex M. , Rawls Scott M. , Shippenberg Toni S. , McGinty Jacqueline F.
Published: March 2003

Abstract : The effect of a k‐opioid receptor agonist on acute amphetamine‐induced behavioral activation and dialysate levels of dopamine and glutamate in the ventral striatum were investigated. Amphetamine (2.5 mg/kg i.p.) evoked a substantial increase in rearing, sniffing, and hole‐poking behavior as well as dopamine and glutamate levels in the ventral striatum of awake rats. U‐69593 (0.32 mg/kg s.c.) significantly decreased the amphetamine‐evoked increase in behavior and dopamine and glutamate levels in the ventral striatum. Reverse dialysis of the selective k‐opioid receptor antagonist, nor‐binaltorphimine, into the ventral striatum antagonized the effects of U‐69593 on amphetamine‐induced behavior and dopamine and glutamate levels. Reverse dialysis of low calcium (0.1 mM) into the ventral striatum decreased basal dopamine, but not glutamate, dialysate levels by 91% 45 min after initiation of perfusion. Strikingly, 0.1 mM calcium perfusion significantly reduced the 2.5 mg/kg amphetamine‐evoked increase in dopamine and glutamate levels in the ventral striatum, distinguishing a calcium‐dependent and a calcium‐independent component of release. U‐69593 did not alter the calcium‐independent component of amphetamine‐evoked dopamine and glutamate levels. These data are consistent with the view that a transsynaptic mechanism augments the increase in dopamine and glutamate levels in the ventral striatum evoked by a moderately high dose of amphetamine and that stimulation of k‐opioid receptors suppresses the calcium‐dependent component of amphetamine’s effects.
Increase in External Glutamate and NMDA Receptor Activation Contribute to H2O2‐Induced Neuronal Apoptosis
Authors: Mailly Franck , Marin Philippe , Israël Maurice , Glowinski Jacques , Prémont Joël
Published: March 2003

Abstract : The present study aims to investigate the role of extracellular glutamate and NMDA receptor stimulation in the neuronal death induced by a transient exposure to H2O2 of cultured neurons originating from mouse cerebral cortex. Most of the neuronal loss following a transient exposure to H2O2 of cortical neurons results from an apoptotic process involving a secondary stimulation of NMDA receptors, which occurs after H2O2 washout. Indeed, (a) the neurotoxic effect of H2O2 was strongly reduced by antagonists of NMDA receptors, (b) the neurotoxic effect of H2O2 was enhanced in the absence of Mg2+, (c) the protective effect of MK‐801 progressively decayed when it was applied with increasing delay time after H2O2 exposure, and (d), finally, the extracellular concentration of glutamate was increased after H2O2 exposure. The major part of H2O2‐induced neurotoxicity is mediated by the formation of hydroxyl radicals, which might be involved in (a) the delayed accumulation of extracellular glutamate and NMDA receptor activation and (b) the poly(ADP‐ribose) polymerase activation and the related NAD content decrease. The combination of these two mechanisms could lead to both an increase in ATP consumption and a decrease of ATP synthesis. The resulting large decrease in ATP content might be finally responsible for the neuronal death.
Genomic Organization of Human DLG4, the Gene Encoding Postsynaptic Density 95
Authors: Stathakis Dean G. , Udar Nitin , Sandgren Ola , Andreasson Sten , Bryant Peter J. , Small Kent , Forsman‐Semb Kristina
Published: March 2003

Abstract : We have determined the exon‐intron organization and

characterized the 5′‐flanking promoter region of DLG4.

Encompassing ~30 kb, the DLG4 locus is composed of 22 exons that

range in size from 28 to 1,218 nucleotides. All splice sites conform to the

GT‐AG rule, except for the splice acceptor site of intron 5, which is TG

instead of AG. Three different exons of DLG4 were found to be

alternatively spliced in a subset of tissues. Two of these variants result in

altered postsynaptic density 95 (PSD95) isoforms that dramatically truncate

the protein. The third splicing variant represents an extension of exon 4 that

encodes an additional 33‐amino acid segment. Analysis of the core promoter

region for DLG4 suggests that the expression of this gene is

controlled by a TATA‐less promoter using a single transcriptional start site

embedded within a CpG island. DLG4 maps to a region on chromosome

17p13.1 known to contain a locus for autosomal dominant cone dystrophy 5.

Scanning for mutations in the DLG4 coding region and splice sites was

performed in 15 cone dystrophy patients, including probands from five families

showing linkage to the DLG4 region. No disease‐causing mutations were

identified in any patients, suggesting that DLG4 is not the causative gene for this genetic eye disorder.
Antisense‐Induced Reduction of Presenilin 1 Expression Selectively Increases the Production of Amyloid β42 in Transfected Cells
Authors: Refolo Lawrence M. , Eckman Chris , Prada Christian‐Mihail , Yager Debora , Sambamurti Kumar , Mehta Nitin , Hardy John , Younkin Steven G.
Published: March 2003

Abstract : Autosomal dominant mutations in the presenilin 1 (PS1)

gene are associated with familial, early‐onset Alzheimer's disease. Although

the pathogenic mechanism of these mutations is unclear, their common feature

is that they lead to an increased concentration of amyloid β‐peptide

(Aβ) 42 in the plasma of early‐onset patients, in the conditioned media

of transfected cells, and in the brains of transgenic mice that overexpress

mutant PS1. To address the mechanism(s) by which the pathogenic PS1 mutations

increase Aβ42, we constructed human cell lines expressing a doxycyclin

(dox)‐inducible antisense PS1 RNA and measured its effects on the levels of

PS1, amyloid precursor protein (APP), and Aβ. In time course experiments,

we observed a statistically significant (p = 0.0038) more than

twofold elevation in secreted Aβ42 as early as 12 days after addition of

dox. This correlated with an 80% decrease in the 46‐kDa PS1 holoprotein and a

30% decrease in the 26‐kDa N‐terminal fragment (NTF). Furthermore, there was a

significant fivefold (p = 0.002) increase in Aβ42 after 14‐day dox treatment ; this correlated with a >90% decrease in PS1 holoprotein and 60% decrease in NTF. At no time point did we observe significant changes in Aβ40, APP holoprotein, presenilin 2, or tubulin. Ten days after the removal of dox, we observed a return to constitutive levels for Aβ42, PS1 holoprotein, and NTF. These results suggest that in human cell lines, the reduction of normal PS1 activity results in the increased production of Aβ42. Furthermore, our results are consistent with a loss of function or dominant negative mechanism for the pathogenic PS1 mutations.
Stimulation of the Brain NO/Cyclic GMP Pathway by Peripheral Administration of Tetrahydrobiopterin in the hph‐1 Mouse
Authors: Canevari Laura , Land John M. , Clark John B. , Heales Simon J. R.
Published: March 2003

Abstract : Mutations in GTP‐cyclohydrolase I (GTP‐CH) have been

identified as causing a range of inborn errors of metabolism, including

dopa‐responsive dystonia. GTP‐CH catalyses the first step in the biosynthesis

of tetrahydrobiopterin (BH4), a cofactor necessary for the

synthesis of catecholamines and serotonin. Current therapy based on monoamine

neurotransmitter replacement may be only partially successful in correcting

the neurological deficits. The reason might be that BH4 is also a

cofactor for nitric oxide synthase. Using a strain of mutant GTP‐CH‐deficient

(hph‐1) mice, we demonstrate that in addition to impaired monoamine

metabolism, BH4 deficiency is also associated with diminished

nitric oxide synthesis in the brain (as evaluated by measuring the levels of

cyclic GMP), when compared with wild‐type animals. We have found a decline in

the levels of BH4 with age in all animals, but no gender‐related

differences. We found a strong association between the levels of

BH4 and cyclic GMP in hph‐1 mice but not in wild‐type

animals. We also demonstrate that acute peripheral administration of

BH4 (100 μmol/kg s.c.) in hph‐1 mice significantly

elevated the brain BH4 concentration and subsequently cyclic GMP

levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that

BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.
Cloning and Characterization of zRICH, a 2′,3′‐Cyclic‐Nucleotide 3′‐Phosphodiesterase Induced During Zebrafish Optic Nerve Regeneration
Authors: Ballestero Rafael P. , Dybowski Joseph A. , Levy Gallia , Agranoff Bernard W. , Uhler Michael D.
Published: March 2003

Abstract: We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterases (CNPases). CNPases are well‐established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration‐Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three‐fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved β‐ketoacyl synthase motif in zRICH to CNPase activity by means of site‐directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for β‐ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.
Characterization of a Novel Serotonin Receptor from Caenorhabditis elegans
Authors: Hamdan Fadi F. , Ungrin Mark D. , Abramovitz Mark , Ribeiro Paula
Published: March 2003

Abstract: Serotonin [5‐hydroxytryptamine (5‐HT)] modulates feeding activity, egg‐laying, and mating behavior in the free‐living nematode, Caenorhabditis elegans. We have cloned a novel receptor cDNA from C. elegans (5‐HT2Ce) that has high sequence homology with 5‐HT2 receptors from other species. When transiently expressed in COS‐7 cells, 5‐HT2Ce exhibited 5‐HT binding activity and activated Ca2+ ‐mediated signaling in a manner analogous to other 5‐HT2 receptors. However, 5‐HT2Ce displayed unusual pharmacological properties, which resembled both 5‐HT2 and 5‐HT1‐like receptors but did not correlate well with any of the known 5‐HT2 subtypes. Two splice variants of 5‐HT2Ce that differ by 48 N‐terminal amino acids were identified. The two isoforms were found to have virtually identical binding and signaling properties but differed in their levels of mRNA expression, with the longer variant being four times more abundant than the shorter species in all developmental stages tested. Taken together, the results describe two variants of a novel C. elegans 5‐HT receptor, which has some of the properties of the 5‐HT2 family but whose pharmacological profile does not conform to any known class of receptor.
Pregnenolone Sulfate, a Naturally Occurring Excitotoxin Involved in Delayed Retinal Cell Death
Authors: Cascio C. , Guarneri R. , Russo D. , De Leo G. , Guarneri M. , Piccoli F. , Guarneri P.
Published: March 2003

Abstract: The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30‐min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid‐reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL‐positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS‐induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4‐(3‐phosphonopropyl)‐2‐piperazinecarboxylic acid and 3α‐hydroxy‐5β‐pregnan‐20‐one sulfate but completely blocked after concomitant addition of the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17β‐estradiol) differently prevented PS‐induced delayed cell death. Cycloheximide treatment protected against DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cycloheximide‐sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.
Evidence for Expression of Heteromeric Serotonin 5‐HT3 Receptors in Rodents
Authors: Hanna Michael C. , Davies Paul A. , Hales Tim G. , Kirkness Ewen F.
Published: March 2003

Abstract : The gene and cDNAs that encode a novel subunit of rodent

serotonin 5‐HT3 receptors were isolated from mouse and rat tissues.

Each of the new rodent subunits shares 40% amino acid identity with the rat

5‐HT3A subunit and 73% identity with the human 5‐HT3B

subunit. Despite a relatively low level of structural conservation, sequence

analysis and functional studies suggest that the new rodent subunits are

orthologues of the human 5‐HT3B subunit. In common with homologous

human receptors, rat heteromeric 5‐HT3 receptors displayed a

substantially larger single‐channel conductance than homomeric

5‐HT3A receptors. In addition, the rat heteromeric receptors were

less sensitive to antagonism by tubocurarine. However, in contrast to human

heteromeric receptors, those of the rat displayed pronounced inward

rectification of both the whole‐cell and single‐channel current amplitudes.

Transcripts of the mouse 5‐HT3A and 5‐HT3B subunits are

coexpressed in several cell lines that possess endogenous 5‐HT3

receptors. In addition, treatment of rat PC12 cells with nerve growth factor

induced expression of both subunit mRNAs, with a similar time course for

accumulation of each transcript. The combination of functional data and

expression patterns is consistent with the existence of heteromeric

5‐HT3 receptors in rodent neurons.
Behavioral, Pharmacological, and Molecular Characterization of an Amphibian Cannabinoid Receptor
Authors: Soderstrom Ken , Leid Mark , Moore Frank L. , Murray Thomas F.
Published: March 2003

Abstract : Investigation of cannabinoid pharmacology in a vertebrate

with a phylogenetic history distinct from that of mammals may allow better

understanding of the physiological significance of cannabinoid neurochemistry.

Taricha granulosa, the roughskin newt, was used here to characterize

an amphibian cannabinoid receptor. Behavioral experiments demonstrated that

the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor

activity and courtship clasping behavior. Inhibition of clasping was

dose‐dependent and potent (IC50 = 1.2 μg per animal).

Radioligand binding studies using [3H]CP‐55940 allowed

identification of a specific binding site (KD = 6.5

nM, Bmax = 1,853 fmol/mg of protein) in brain

membranes. Rank order of affinity of several ligands was consistent with that

reported for mammalian species (KD, nM) :

CP‐55940 (3.8) > levonantradol (13.0) > WIN55212‐2 (25.7) ▴

anandamide (1,665)

>> anandamide + 100 μM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid‐mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose‐dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.
Mobilizing Store Ca2+ in the Presence of La3+ Evokes Exocytosis in Bovine Chromaffin Cells
Authors: Marley Philip D. , Bales Peter J. R. , Zerbes Mariann , Powis David A. , O'Farrell Melanie
Published: March 2003

Abstract: The effect on exocytosis of La3+, a known

inhibitor of plasma membrane Ca2+‐ATPases and

Na+/Ca2+ exchangers, was studied using cultured bovine

adrenal chromaffin cells. At high concentrations (0.3‐3 mM),

La3+ substantially increased histamine‐induced catecholamine

secretion. This action was mimicked by other lanthanide ions (Nd3+,

Eu3+, Gd3+, and Tb3+), but not several

divalent cations. In the presence of La3+, the secretory response

to histamine became independent of extracellular Ca2+.

La3+ enhanced secretion evoked by other agents that mobilize

intracellular Ca2+ stores (angiotensin II, bradykinin, caffeine,

and thapsigargin), but not that due to passive depolarization with 20

mM K+. La3+ still enhanced histamine‐induced

secretion in the presence of the nonselective inhibitors of

Ca2+‐permeant channels SKF96365 and Cd2+, but the

enhancement was abolished by prior depletion of intracellular Ca2+

stores with thapsigargin. La3+ inhibited

45Ca2+ efflux from preloaded chromaffin cells in the

presence or absence of Na+. It also enhanced and prolonged the rise

in cytosolic [Ca2+] measured with fura‐2 during mobilization of

intracellular Ca2+ stores with histamine in Ca2+‐free

buffer. The results suggest that the efficacy of intracellular Ca2+

stores in evoking exocytosis is enhanced dramatically by inhibiting

Ca2+ efflux from the cell.
Functional Characterization of a cis‐Acting DNA Antisilencer Region that Modulates Myelin Proteolipid Protein Gene Expression
Authors: Dobretsova Anna , Kokorina Natalia A. , Wight Patricia A.
Published: March 2003

Abstract: Regulation of myelin proteolipid protein (Plp)
gene expression is tightly controlled, both spatially and temporally.
Previously, we have shown with transgenic mice that a Plp‐lacZ fusion
gene (which includes the entire sequence for Plp intron 1 DNA) is
regulated in a similar manner to endogenous Plp gene expression.
Furthermore, by deletion‐transfection analyses using assorted
Plp‐lacZ constructs with partial deletion of Plp intron 1
sequences, we have shown that the first intron possesses an antisilencer
region that is capable of over‐coming repression mediated by two distinct
regions located elsewhere within intron 1 DNA. Here, we report the ability of
various fragments encompassing the antisilencer region to restore
β‐galactosidase activity when inserted into Plp‐lacZ constructs,
which originally exhibited low levels of β‐galactosidase activity.
Additional constructs were generated to test the effects of these
antisilencer‐containing fragments in constructs that are missing either one or
both of the negative regulatory regions that are overridden during
antisilencing. Transfection analyses, in conjunction with protein‐DNA binding
assays, suggest that several nuclear factors are necessary for derepression of
Plp gene activity in an oligodendroglial cell line. Moreover, either
the “core” or complete antisilencing region can act in an additive
or synergistic fashion when multiple copies are inserted into the
Plp‐lacZ constructs.
BIT/SHPS‐1 Enhances Brain‐Derived Neurotrophic Factor‐Promoted Neuronal Survival in Cultured Cerebral Cortical Neurons
Authors: Araki Toshiyuki , Yamada Masashi , Ohnishi Hiroshi , Sano Shin‐ichiro , Hatanaka Hiroshi
Published: March 2003

Abstract: Brain‐derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS‐1 (brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/SHP substrate 1) is a substrate of Shp‐2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS‐1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS‐1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS‐1 were replaced with phenylalanine. Overexpression of wild‐type BIT/SHPS‐1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/SHPS‐1 itself and an association of Shp‐2 with BIT/SHPS‐1 even without addition of BDNF. We found that BDNF‐promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS‐1 does not depend on its tyrosine phosphorylation. BDNF‐induced activation of mitogen‐activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF‐induced activation of Akt was enhanced in neurons expressing wild‐type or 4F mutant BIT/SHPS‐1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3‐kinase, blocked the enhancement of BDNF‐promoted neuronal survival in both neurons expressing wild‐type and 4F mutant BIT/SHPS‐1. These results indicate that BIT/SHPS‐1 contributes to BDNF‐promoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3‐kinase‐Akt pathway. Our results suggest that a novel action of BIT/SHPS‐1 does not occur through tyrosine phosphorylation of BIT/SHPS‐1 in cultured cerebral cortical neurons.
Characterization of GAR‐2, a Novel G Protein‐Linked Acetylcholine Receptor from Caenorhabditis elegans
Authors: Lee Yong‐Seok , Park Yang‐Seo , Nam Seunghee , Suh SuJeong , Lee Junho , Kaang Bong‐Kiun , Cho Nam Jeong
Published: March 2003

Abstract: We have previously identified two G protein‐linked
acetylcholine receptors (GARs), GAR‐1 and GAR‐3, in the nematode
Caenorhabditis elegans. Whereas GAR‐3 is a homologue of muscarinic
acetylcholine receptors (mAChRs), GAR‐1 is similar to but pharmacologically
distinct from mAChRs. In the current work we isolated a new type of GAR using
C. elegans genome sequence information. This receptor, named GAR‐2,
consists of 614 amino acid residues and has seven putative transmembrane
domains. Database searches indicate that GAR‐2 is most similar to GAR‐1 and
closely related to GAR‐3/mAChRs. The overall amino acid sequence identities to
GAR‐1 and GAR‐3 are ∼32 and ∼23%, respectively. When GAR‐2 was
coexpressed with the G protein‐activated inwardly rectifying K+
(GIRK1) channel in Xenopus oocytes, acetylcholine was able to evoke
the GIRK current in a dose‐dependent fashion. Oxotremorine, a classical
muscarinic agonist, had little effect on the receptor, indicating that GAR‐2
is pharmacologically different from mAChRs but rather similar to GAR‐1. GAR‐2
differs from GAR‐1, however, in that it showed virtually no response to
muscarinic antagonists such as atropine, scopolamine, and pirenzepine.
Expression studies using green fluorescent protein reporter gene fusion
revealed that GAR‐2 is expressed in a subset of C. elegans neurons,
distinct from those expressing GAR‐1. Together with our previous reports, this
study demonstrates that diverse types of GARs are present in C.
elegans.
Degradation of Glial Glutamate Transporter mRNAs Is Selectively Blockedby Inhibition of Cellular Transcription
Authors: Zelenaia Olga A. , Robinson Michael B.
Published: March 2003

Abstract: Recent studies have demonstrated that the expression ofthe glial glutamate transporters GLT‐1 (glutamate transporter 1) and GLAST(glutamate aspartate transporter) is regulated both in vivo and in vitro. Forexample, co‐culturing with neurons, treatment withN6,2′‐O‐dibutyryladenosine3′:5′‐cyclic monophosphate (dbcAMP), and treatment with epidermalgrowth factor all increase the steady‐state levels of GLT‐1 and GLAST proteinin astrocyte cultures. These changes in protein expression are correlated withincreased mRNA levels. In the present study, the degradation of GLT‐1 andGLAST mRNAs was examined in control and dbcAMP‐treated astrocyte culturesafter inhibiting transcription with actinomycin D. Although one would predictthat inhibition of transcription would cause a decrease in GLT‐1 and GLASTmRNAs and that this decrease would depend on the rate of mRNA degradation, thelevels of GLT‐1 and GLAST mRNAs did not decrease even after 24 h of treatmentwith actinomycin D. Withdrawal of dbcAMP caused the levels of GLT‐1 and GLASTmRNAs to fall to basal levels within 24 h, but this degradation was blocked ifactinomycin D was added at the time of dbcAMP withdrawal. Importantly,actinomycin D did not block the degradation of c‐fos mRNA also induced bydbcAMP in these cultures. Inhibition of translation with cycloheximide did notstabilize GLT‐1 but partially attenuated the degradation of GLAST mRNA.Although the mechanism of this effect remains to be defined, these studiessuggest that GLT‐1 and GLAST mRNAs belong to a select class of inducible mRNAsstabilized by inhibitors of transcription. The possible relevance of thesedata to astrocyte differentiation is briefly discussed.
Tau Protein Is Hyperphosphorylated in a Site‐Specific Manner in Apoptotic Neuronal PC12 Cells
Authors: Zhang Jianwen , Johnson Gail V. W.
Published: March 2003

Abstract: Alterations in the status of microtubules contribute tothe cytoskeletal rearrangements that occur during apoptosis. Themicrotubule‐associated protein tau regulates microtubule dynamics and thus islikely to play an important role in the cytoskeletal changes that occur inapoptotic cells. Previously, we demonstrated that the phosphorylation of tauat the Tau‐1 epitope was increased during neuronal PC12 cell apoptosis, andfurther that the microtubule binding of tau from apoptotic cells wassignificantly impaired because of altered phosphorylation. The fact that themicrotubule‐binding capacity of tau from apoptotic cells was reduced to∼30% of control values indicated that sites in addition to those withinthe Tau‐1 epitope were hyperphosphorylated during apoptosis. In this studyusing a combination of immunological and biochemical approaches, numeroussites were found to be hyperphosphorylated on tau isolated from apoptoticcells. Further, during apoptosis, the activities of cell division controlprotein kinase (cdc2) and cyclin‐dependent kinase 5 (cdk5) were selectivelyand significantly increased. The association of these two protein kinases withtau was also increased during apoptosis. These findings are intriguing becausemany of the sites found to be hyperphosphorylated on tau during apoptosis arealso hyperphosphorylated on tau from Alzheimer's disease brain. Likewise,there are data indicating that in Alzheimer's disease the activities of cdc2and cdk5 are also increased.