bluuebunny/crossref_metadata_2025_split huggingface dataset:
CREB is required for acquisition of ischemic tolerance in gerbil hippocampal CA1 region
Authors: Hara Tsuyoshi , Hamada Jun‐ichiro , Yano Shigetoshi , Morioka Motohiro , Kai Yutaka , Ushio Yukitaka 
Published: July 2003

SummaryIschemic tolerance is well known as a neuroprotective effect of pre‐conditioning ischemia against delayed neuronal death, however, the mechanism or mechanisms underlying this effect are not fully understood. We investigated the relationship between CREB and ischemic tolerance in gerbil hippocampal CA1 neurons using CRE decoy oligonucleotide. Sublethal ischemia led to an increase in the level of CREB phosphorylation in CA1 regions while lethal ischemia did not. Experiments with NG108‐15 cells showed that adding CRE decoy oligonucleotide to culture media significantly inhibited the cell growth rate. The administration of CRE decoy oligonucleotide into gerbil cerebral ventricle decreased CREB‐DNA binding activity to 38% of the control. Pre‐treatment with CRE decoy oligonucleotide 24 h before the induction of ischemic tolerance decreased CA1 neuronal cell survival to 21% of the control. The present findings suggest that a CREB‐mediated transcription system is necessary for the induction of ischemic tolerance.
Dynorphin A(1–17) biotransformation in striatum of freely moving rats using microdialysis and matrix‐assisted laser desorption/ionization mass spectrometry
Authors: Reed Brian , Zhang Yong , Chait Brian T. , Kreek Mary Jeanne 
Published: July 2003

AbstractThe biotransformation of the opioid peptide dynorphin A(1–17) was investigated in striatum of freely moving Fischer rats, by direct infusion of this peptide, followed by recovery of the resulting biotransformation products via microdialysis and identification using matrix‐assisted laser desorption/ionization mass spectrometry. The observed peptides are consistent with enzymatic cleavage at the Arg7‐Ile8 position of dynorphin A(1–17), followed by terminal degradation of the resulting dynorphin A(1–7) and dynorphin A(8–17) peptides. Unexpectedly, novel post‐translational modifications were found on C‐terminal fragments of dynorphin A(1–17). Using tandem mass spectrometry, a covalent modification of mass 172 Da, the nature of which is not understood, was found on the tryptophan residue of C‐terminal fragments (Trp14). Additional modifications, of mass 42 and 113 Da, were also found on the N‐terminus (Ile8 or Pro10) of these same C‐terminal fragments. The role of these modifications of C‐terminal fragments has not yet been characterized.
Distinct cleavage patterns of normal and pathologic forms of α‐synuclein by calpain I in vitro
Authors: Mishizen‐Eberz Amanda J. , Guttmann Rodney P. , Giasson Benoit I. , Day George A. , Hodara Roberto , Ischiropoulos Harry , Lee Virginia M.‐Y. , Trojanowski John Q. , Lynch David R. 
Published: July 2003

AbstractParkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha‐synuclein (α‐syn), the protein mutated in some patients with familial PD. The mechanisms of α‐syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave α‐syn in vitro. Calpain I cleaved wild‐type α‐syn predominantly after amino acid 57 and within the non‐amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized α‐syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild‐type α‐syn after amino acid 57, this did not occur in mutant A53T α‐syn. This paucity of proteolysis could increase the stability of A53T α‐syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild‐type α‐syn. However, once α‐syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of α‐syn by cleaving at aberrant sites within the C‐terminal region. Elucidating the role of calpain I in the proteolytic processing of α‐syn in normal and diseased brains may clarify mechanisms of neurodegenerative α‐synucleinopathies.
Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum
Authors: Ghoumari A. M. , Ibanez C. , El‐Etr M. , Leclerc P. , Eychenne B. , O'Malley B. W. , Baulieu E. E. , Schumacher M. 
Published: July 2003

AbstractWe have previously shown that progesterone (PROG) is synthesized by Schwann cells and promotes myelin formation in the peripheral nervous system (PNS). We now report that this neurosteroid also stimulates myelination in organotypic slice cultures of 7‐day‐old (P7) rat and mouse cerebellum. Myelination was evaluated by immunofluorescence analysis of the myelin basic protein (MBP). After 7 days in culture (7DIV), we found that adding PROG (2–5 × 10−5 M) to the culture medium caused a fourfold increase in MBP expression when compared to control slices. The effect of PROG on MBP expression involves the classical intracellular PROG receptor (PR): the selective PR agonist R5020 significantly increased MBP expression and the PR antagonist mifepristone (RU486) completely abolished the effect of PROG on this MBP expression. Moreover, treatment of P7‐cerebellar slice cultures from PR knockout (PRKO) mice with PROG had no significant effect on MBP expression. PROG was metabolized in the cerebellar slices to 5α‐dihydroprogesterone (5α‐DHP) and to the GABAA receptor‐active metabolite 3α,5α‐tetrahydroprogesterone (3α,5α‐THP, allopregnanolone). The 5α‐reductase inhibitor L685‐273 partially inhibited the effect of PROG, and 3α,5α‐THP (2–5 × 10−5 M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3α,5α‐THP involved GABAA receptors, as it could be inhibited by the selective GABAA receptor antagonist bicuculline. These findings suggest that progestins stimulate MBP expression and consequently suggest an increase in CNS myelination via two signalling systems, the intracellular PR and membrane GABAA receptors, and they confirm a new role of GABAA receptors in myelination.
Effect of middle cerebral artery occlusion on mRNA expression for the sodium‐coupled vitamin C transporter SVCT2 in rat brain
Authors: Berger Urs V. , Lu Xi‐Chun M. , Liu Weilin , Tang Zhaocheng , Slusher Barbara S. , Hediger Matthias A. 
Published: July 2003

AbstractThe sodium‐vitamin C co‐transporter SVCT2 is primarily responsible for the accumulation of the important antioxidant ascorbate into brain cells. In vitro studies have demonstrated strong expression of this transporter in cultured astrocytes, whereas in situ hybridization analysis has so far detected SVCT2 only in neurons. In the present study, we examined the response of SVCT2 mRNA expression in the brain to focal ischemia induced for 2 h by unilateral middle cerebral artery occlusion. The mRNA expression patterns of SVCT2 and the glutamate‐activated immediate early gene Arc were investigated at 2 and 22 h after ischemia. SVCT2 and Arc mRNA expression was lost in the ischemic core at both time points. In areas outside the core, Arc was strongly up‐regulated, primarily at 2 h, whereas SVCT2 showed an increase at 2 and 22 h. SVCT2 expression was increased in neurons as well as in astrocytes, providing the first evidence for SVCT2 expression in astrocytes in situ. These findings underscore the importance of ascorbate as a neuroprotective agent and may have implications for therapeutic strategies. In addition, the increase of SVCT2 in astrocytes after ischemia suggests that cultured astrocytes are exposed to chronic oxidative stress.
Cloning and characterization of a Caenorhabditis elegans D2‐like dopamine receptor
Authors: Suo Satoshi , Sasagawa Noboru , Ishiura Shoichi 
Published: July 2003

AbstractThe neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1‐like and D2‐like receptors. However, D2‐like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2‐like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2‐like dopamine receptors than to D1‐like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo‐lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2‐like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.
Phorbol ester induces CYP2E1 in astrocytes, through a protein kinase C‐ and tyrosine kinase‐dependent mechanism
Authors: Tindberg Niclas 
Published: July 2003

AbstractCytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin‐1β. In the present study, phorbol‐12,13‐dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21–24 h of PDBu treatment. CYP2E1 expression in PDBu‐treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin‐6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.
Mitochondrial apoptotic cell death and moderate superoxide generation upon selective activation of non‐desensitizing AMPA receptors in hippocampal cultures
Authors: Rego A. Cristina , Monteiro Nuno M. , Silva Ana P. , Gil Joana , Malva João O. , Oliveira Catarina R. 
Published: July 2003

AbstractIn the present work we investigated the effect of selective stimulation of non‐desensitizing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(f)quinoxaline (NBQX), an AMPA‐preferring receptor antagonist. (+)‐5‐Methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non‐desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK‐801, significantly decreased the number of Co2+‐positive neurones, used as a cytochemical marker of Ca2+‐permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA‐mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases‐1 and ‐3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn‐ superoxide dismutase (SOD) was observed on selective activation of non‐desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor‐mediated excitotoxicity in cultured rat hippocampal neurones.
Functional coupling of serotonin and noradrenaline transporters
Authors: Horschitz Sandra , Hummerich René , Schloss Patrick 
Published: July 2003

AbstractRe‐uptake of the neurotransmitters serotonin and noradrenaline out of the synaptic cleft is mediated by selective transporter proteins, the serotonin transporter and the noradrenaline transporter respectively. Both are integral membrane proteins that are have a high degree of homology and represent members of a larger neurotransmitter transporter superfamily. Several studies have indicated that the serotonin transporter has an an oligomeric structure. To determine whether monoamine transporters can also function in oligomeric structures in situ, we constructed a concatenate consisting of one molecule of serotonin transporter covalently linked to one molecule of noradrenaline transporter. Heterologous expression of this hybrid construct allowed us to analyse the function, i.e. transport activity, and the structure, i.e. the molecular weight of the total construct and of its single components, at the same time. We showed that serotonin–noradrenaline transporter fusion proteins are fully active and exhibit the pharmacological profile of both their individual components. These findings support the hypothesis that monoamine transporters are expressed and may function as oligomeric proteins composed of non‐interacting monomers.
Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma
Authors: Bungay Peter M. , Newton‐Vinson Paige , Isele Wanda , Garris Paul A. , Justice Joseph B. 
Published: July 2003

AbstractAlthough microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady‐state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no‐net‐flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast‐scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no‐net‐flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post‐implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no‐net‐flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.
Characterization of the N‐acetylaspartate biosynthetic enzyme from rat brain
Authors: Madhavarao C. N. , Chinopoulos C. , Chandrasekaran K. , Namboodiri M. A. A. 
Published: July 2003

AbstractAspartate N‐acetyltransferase (Asp‐NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp‐NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp‐NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size‐exclusion high performance liquid chromatography. Asp‐NAT activity and the optical density pattern of the eluted protein from size‐exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi‐subunit complex Asp‐NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3‐[(3‐cholamidopropyl)dimethylammonia]‐1‐propanesulfonate (CHAPS) treatment followed by size‐exclusion chromatography. Asp‐NAT showed an order of magnitude difference in Km between l‐aspartate (l‐Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp‐NAT showed high specificity towards l‐Asp with 3% or less activity towards l‐Glu, l‐Asn, l‐Gln and Asp‐Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.
Molecular modeling and mutagenesis of the ligand‐binding pocket of the mGlu3 subtype of metabotropic glutamate receptor
Authors: Yao Yi , Pattabiraman N. , Michne William F. , Huang Xi‐Ping , Hampson David R. 
Published: July 2003

AbstractA homology model of the extracellular domain of the mGlu3 subtype of metabotropic glutamate (mGlu) receptor was generated and tested using site‐directed mutagenesis, a radioligand‐binding assay using the Group II selective agonist (2S,2′R,3′R)‐2‐(2′,3′‐[3H]dicarboxycyclopropyl) glycine ([3H]DCG‐IV), and in a fluorescence‐based functional assay in live transiently transfected human embryonic kidney cells. Ten of the 12 mGlu3 mutants (R64A, R68A, Y150A, S151A, T174A, D194A, Y222A, R277A, D301A and K389) showed either no binding or a 90% or greater loss of specific [3H]DCG‐IV binding. Several analogous mutations in mGlu2 supported the results obtained with mGlu3. These results demonstrate that the binding of [3H]DCG‐IV to mGlu3 is exceptionally sensitive to mutagenesis‐induced perturbations. In silico docking of DCG‐IV into the agonist binding pocket of mGlu3 facilitated the interpretation the mutagenesis results. Tyrosines 150 and 222, and arginine 277 show close contacts with the third carboxylic acid group in DCG‐IV, which is not present in glutamate or (2S,1′S,2′S)‐2‐(carboxycyclopropyl)glycine (L‐CCG‐I). Mutation of these three amino acids to alanine resulted in a near complete loss of receptor activation by DCG‐IV and retention of near wild‐type affinity for L‐CCG‐I. It is proposed that hydrogen bonding between this carboxylate and tyrosines 150 and 222 and arginine 277 provide a partial explanation for the high affinity and Group II selectivity of DCG‐IV. These findings define the essential features of the ligand‐binding pocket of mGlu3 and, together with other recent studies on mGlu receptors, provide new opportunities for structure‐based drug design.
Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARγ) in murine primary astrocytes and neurons
Authors: Klotz Luisa , Sastre Magdalena , Kreutz Anne , Gavrilyuk Vitaliy , Klockgether Thomas , Feinstein Douglas L. , Heneka Michael T. 
Published: July 2003

AbstractCerebral inflammatory events play an important part in the pathogenesis of Alzheimer's disease (AD). Agonists of the peroxisome proliferator‐activated receptor gamma (PPARγ), a nuclear hormone receptor that mediates anti‐inflammatory actions of non‐steroidal anti‐inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) – a pathological hallmark of AD – plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARγ‐mediated suppression of inflammation we investigated the influence of NA on PPARγ expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 µm NA resulted in an increase of PPARγ mRNA as well as PPARγ protein levels in both cell types. These effects were blocked by the β‐adrenergic antagonist propranolol but not by the α‐adrenergic antagonist phentolamine, suggesting that they might be mediated by β‐adrenergic receptors. Our results indicate for the first time that PPARγ expression can be modulated by the cAMP signalling pathway, and suggest that the anti‐inflammatory effects of NA on brain cells may be partly mediated by increasing PPARγ levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARγ expression and therefore potentiate neuroinflammatory processes.
Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis
Authors: Battaglioli Gino , Liu Hongcheng , Martin David L. 
Published: July 2003

AbstractGlutamate decarboxylase (GAD) exists as two isoforms, GAD65 and GAD67. GAD activity is regulated by a cycle of activation and inactivation determined by the binding and release of its co‐factor, pyridoxal 5′‐phosphate. Holoenzyme (GAD with bound co‐factor) decarboxylates glutamate to form GABA, but it also catalyzes a slower transamination reaction that produces inactive apoGAD (without bound co‐factor). Apoenzyme can reassociate with pyridoxal phosphate to form holoGAD, thus completing the cycle. Within cells, GAD65 is largely apoenzyme (∼93%) while GAD67 is mainly holoenzyme (∼72%). We found striking kinetic differences between the GAD isoforms that appear to account for this difference in co‐factor saturation. The glutamate dependent conversion of holoGAD65 to apoGAD was about 15 times faster than that of holoGAD67 at saturating glutamate. Aspartate and GABA also converted holoGAD65 to apoGAD at higher rates than they did holoGAD67. Nucleoside triphosphates (such as ATP) are known to affect the activation reactions of the cycle. ATP slowed the activation of GAD65 and markedly reduced its steady‐state activity, but had little affect on the activation of GAD67 or its steady‐state activity. Inorganic phosphate opposed the effect of ATP; it increased the rate of apoGAD65 activation but had little effect on apoGAD67 activation. We conclude that the apo‐/holoenzyme cycle of inactivation and reactivation is more important in regulating the activity of GAD65 than of GAD67.