bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential Active‐Site Lysine of Brain Glutamate Dehydrogenase Isoproteins
Authors: Kim Seong Who , Lee Jongweon , Song Min‐Sun , Choi Soo Young , Cho Sung‐Woo
Published: July 2010

Abstract: Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5′‐phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5′‐phosphate‐inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5′‐phosphate. A combination of 10 mM 2‐oxoglutarate with 2 mM NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.
Polyglutamine Domains Are Substrates of Tissue Transglutaminase: Does Transglutaminase Play a Role in Expanded CAG/Poly‐Q Neurodegenerative Diseases?
Authors: Cooper Arthur J. L. , Sheu Kwan‐Fu Rex , Burke James R. , Onodera Osamu , Strittmatter Warren J. , Roses Allen D. , Blass John P.
Published: July 2010

Abstract: Huntington's disease and six other neurodegenerative diseases are associated with abnormal gene products containing expanded polyglutamine (poly‐Q; Qn) domains (n ≥ 40). In the present work, we show that glutathione S‐transferase (GST) fusion proteins containing a small, physiological‐length poly‐Q domain (GSTQ10) or a large, pathological‐length poly‐Q domain (GSTQ62) are excellent substrates of guinea pig liver (tissue) transglutaminase and that both GSTQ10 and GSTQ62 are activators of tissue transglutaminase‐catalyzed hydroxaminolysis of N‐α‐carbobenzoxyglutaminylglycine. The present findings have implications for understanding the pathophysiological mechanisms of expanded CAG/poly‐Q domain diseases.
GABAA Receptors Containing the α4‐Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ
Authors: Benke Dietmar , Michel Claudia , Mohler Hanns
Published: July 2010

Abstract: Recombinant GABAA receptors, expressed from α‐, β‐, and γ2‐subunits, are diazepam‐insensitive when the α‐subunit is either α4 or α6. In situ, diazepam‐insensitive receptors containing the α6‐subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam‐insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam‐insensitive GABAA receptors in various brain areas containing the α4‐subunit. GABAA receptors immunoprecipitated with a newly developed α4‐subunit‐specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2‐recombinant receptors and diazepam‐insensitive [3H]Ro 15‐4513 binding sites in rat brain membranes. In addition, α4‐subunit containing receptors and forebrain diazepam‐insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4‐subunit containing receptors revealed that the α4‐subunit contributes to several receptor subtypes. Depending on the brain region, the α4‐subunit can be coassembled with a second type of α4‐subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4‐subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam‐insensitive [3H]Ro 15‐4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15‐4513 and bretazenil.
Oxidant Injury in PC12 Cells—A Possible Model of Calcium “Dysregulation” in Aging: II. Interactions with Membrane Lipids
Authors: Denisova Natalia A. , Strain Joy G. , Joseph James A.
Published: July 2010

Abstract: In a model recently developed to study the parameters altering vulnerability to oxidative stress, it was shown via image analysis that H2O2‐exposed PC12 cells exhibited increased levels of intracellular Ca2+ (baseline), decreases in K+‐stimulated Ca2+ levels (peak), and decreased poststimulation Ca2+ clearance (recovery). The present experiments were performed to determine if the response patterns in these parameters to oxidative stress would be altered after modification of membrane lipid composition induced by incubating the PC12 cells with 660 µM cholesterol (CHL) in the presence or absence of 500 µM sphingomyelin (SPH) before low (5 µM) or high (300 µM) H2O2 exposure. Neither CHL nor SPH had synergistic effects with high concentrations of H2O2 on baseline. However, CHL in the presence or absence of SPH reversed the effect of low concentrations of H2O2 on baseline. SPH decreased significantly the cell's ability to clear excess Ca2+ in the presence or absence of H2O2 and increased significantly the level of conjugated dienes (CDs). It is surprising that in the cells pretreated with CHL, the CD levels were not significantly different from controls. However, in the presence of SPH, the effects of CHL on CDs were altered. These results suggest that the ratios of membrane lipids could be of critical importance in determining the vulnerability to oxidative stress and Ca2+ translocation in membranes. This may be of critical importance in aging where there is increased membrane SPH and significant loss of calcium homeostasis.
Native Serotonin 5‐HT3 Receptors Expressed in Xenopus Oocytes Differ from Homopentameric 5‐HT3 Receptors
Authors: Van Hooft Johannes A. , Kreikamp Angélique P. , Vijverberg Henk P. M.
Published: July 2010

Abstract: Efficacies of the 5‐hydroxytryptamine (serotonin) 5‐HT3 receptor (5‐HT3R) agonists 2‐methyl‐5‐HT, dopamine, and m‐chlorophenylbiguanide on 5‐HT3R native to N1E‐115 cells and on homopentameric 5‐HT3R expressed in Xenopus oocytes were determined relative to that of 5‐HT. Efficacies of 2‐methyl‐5‐HT and dopamine on 5‐HT3R native to differentiated N1E‐115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5‐HT3R‐AL and 5‐HT3R‐As receptors expressed in oocytes (4–8%). m‐Chlorophenylbiguanide does not distinguish between 5‐HT3R in N1E‐115 cells and in oocytes. The distinct pharmacological profile of 5‐HT3R native to differentiated N1E‐115 cells is conserved when poly(A)+ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5‐HT3R subunits, N1E‐115 cells express additional proteins involved in 5‐HT3R function.
A Generalised Increase in Protein Carbonyls in the Brain in Parkinson's but Not Incidental Lewy Body Disease
Authors: Alam Zafar I. , Daniel Susan E. , Lees Andrew J. , Marsden David C. , Jenner Peter , Halliwell Barry
Published: July 2010

Abstract: The degeneration of neurones in Parkinson's disease (PD) may involve oxidative stress. Previously, increased lipid peroxidation and oxidative DNA damage have been reported in parkinsonian substantia nigra. In the present study the protein carbonyl assay was used to assess oxidative protein damage in postmortem brain tissue from patients with PD and age‐matched controls. In brain areas associated with PD, such as substantia nigra, caudate nucleus, and putamen, there was a significant increase in carbonyl levels. However, increased carbonyl levels were also found in areas of the brain not thought to be affected in PD. This perhaps suggests that protein carbonyl formation is related to therapy with l‐DOPA, which can exert prooxidant properties in vitro. Consistent with this possibility, brain regions from individuals with incidental Lewy body disease (putative presymptomatic PD) showed no rise in carbonyls in any brain areas. Our data show that either oxidative protein damage occurs widely but late in PD brain, and/or that l‐DOPA treatment contributes to protein oxidation.
TPO1, a Member of a Novel Protein Family, Is Developmentally Regulated in Cultured Oligodendrocytes
Authors: Krueger W. H. H. , Gonye G. E. , Madison D. L. , Murray K. E. , Kumar M. , Spoerel N. , Pfeiffer S. E.
Published: July 2010

Abstract: Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5‐kb mRNA that is strongly up‐regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5′‐termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pl of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis.
Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression
Authors: Koenigsberger C. , Chiappa S. , Brimijoin S.
Published: July 2010

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5‐fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE‐overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.
Effects of Chronic Ethanol Treatment on the Expression of Calcium Transport Carriers and NMDA/Glutamate Receptor Proteins in Brain Synaptic Membranes
Authors: Chen Xingyu , Michaelis Mary L. , Michaelis Elias K.
Published: July 2010

Abstract: Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca‐exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca2+. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair‐fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120‐kDa exchanger proteins was increased by 14% and that of a 36‐kDa exchanger‐associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36–72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca2+, Mg2+‐ATPases. The enhanced expression of receptor and exchanger‐associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.
Caffeine Stimulates Amyloid β‐Peptide Release from β‐Amyloid Precursor Protein‐Transfected HEK293 Cells
Authors: Querfurth Henry W. , Jiang Jinwei , Geiger Jonathan D. , Selkoe Dennis J.
Published: July 2010

Abstract: Extracellular amyloid β‐peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor‐based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)‐regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 mM) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine‐stimulated Aβ release. NH4Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.
Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment
Authors: Springer Joe E. , Azbill Robert D. , Kennedy Sarah E. , George Jones , Geddes James W.
Published: July 2010

Abstract: Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain‐sensitive cytoskeletal proteins microtubule‐associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24‐h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I‐specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca2+‐dependent protease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown.
Characterization of Additional Casein Kinase I Sites in the C‐Terminal “Tail” Region of Chicken and Rat Neurofilament‐M
Authors: Shaw Gerry , Miller Rehae , Wang Deng‐Shun , Tang Dali , Hollander Brian A. , Bennett Gudrun S.
Published: July 2010

Abstract: In previous studies we have identified Ser502, Ser528, and Ser534 as target sites in chicken neurofilament middle molecular mass protein (NF‐M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF‐M, Ser464 and Ser471, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF‐M molecules, and recombinant protein constructs containing the homologous rat NF‐M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE‐IIEET‐V) found once in higher vertebrate NF‐M molecules and twice in lamprey NF‐180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF‐L and NF‐H) and α‐internexin, and in mammalian NF‐L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.
Protease‐Activated Receptor‐2 (PAR‐2) Is Present in the Rat Hippocampus and Is Associated with Neurodegeneration
Authors: Smith‐Swintosky Virginia L. , Cheo‐Isaacs Catherine T. , D'Andrea Michael R. , Santulli Rosemary J. , Darrow Andrew L. , Andrade‐Gordon Patricia
Published: July 2010

Abstract: Protease‐activated receptor‐2 (PAR‐2) is a seven‐transmembrane G protein‐coupled receptor that possesses a structure and activation mechanism similar to those of the thrombin receptor. It is activated by low concentrations of trypsin (300 pM) and a synthetic hexapeptide [sequence of serine, leucine, isoleucine, glycine, arginine, leucine (SLIGRL), the rodent PAR‐2 “tethered ligand”] representing the first six amino acids following the putative PAR‐2 cleavage site. Previous studies have indicated that α‐thrombin and SFLLRN (synthetic hexapeptide sequence of serine, phenylalanine, leucine, leucine, arginine, asparagine; the human thrombin receptor “tethered ligand”) induce neurite retraction and neurotoxicity. Because of the strong similarities between thrombin receptor and PAR‐2, we have proposed that PAR‐2 may also participate in neurodegeneration. In the present study, we used reverse transcriptase polymerase chain reaction and immunocytochemistry to provide the first evidence that PAR‐2 is present in the rat hippocampus. Moreover, we found SLIGRL to be toxic to hippocampal neurons in a concentration‐dependent manner (≥100 µM). Calcium signaling studies were performed to aid in determining the mechanism by which PAR‐2 activation is neurotoxic.
Isoform‐Specific Effects of Apolipoproteins E2, E3, and E4 on Cerebral Capillary Sequestration and Blood‐Brain Barrier Transport of Circulating Alzheimer's Amyloid β
Authors: Martel Cynthia L. , Mackic Jasmina B. , Matsubara Etsuro , Governale Samuel , Miguel Calero , Miao Wesley , McComb J. Gordon , Frangione Blas , Ghiso Jorge , Zlokovic Berislav V.
Published: July 2010

Abstract: Cerebral capillary sequestration and blood‐brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ1–40, a peptide homologous to the major form of soluble Alzheimer's amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood‐to‐brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ1–40 in vitro was similar with a KD between 11.8 and 12.9 nM. Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ1–40‐apoE2 and sAβ1–40‐apoE3, but significant for sAβ1–40‐apoE4. After 10 min, 85% of sAβ1–40‐apoE4 taken up at the BBB remained as intact complex, whereas free sAβ1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood‐to‐brain transport of sAβ1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.
Regulation of Serotonin Release in the Frontal Cortex and Ventral Hippocampus of Homozygous Mice Lacking 5‐HT1B Receptors: In Vivo Microdialysis Studies
Authors: Trillat A.‐C. , Malagié I. , Scearce K. , Pons D. , Anmella M.‐C. , Jacquot C. , Hen R. , Gardier A. M.
Published: July 2010

Abstract: To assess the involvement of the serotonin receptor subtype 5‐HT1B as terminal autoreceptor regulating 5‐HT release in mice, we compared basal values and potassium‐evoked changes of extracellular 5‐HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild‐type mice with those measured in homozygous mutant mice lacking the gene encoding the 5‐HT1B receptor. In the frontal cortex and ventral hippocampus, basal and K+‐evoked 5‐HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5‐HT1B receptor agonist CP‐93,129 (500 nM) decreased significantly K+‐evoked 5‐HT release in the frontal cortex (by −44%) and ventral hippocampus (by −32%) of wild‐type mice but had no effect in mutants. In a similar manner, the mixed 5‐HT1B‐5‐HT1D receptor agonist sumatriptan (800 nM) decreased significantly K+‐evoked 5‐HT release in the frontal cortex (by −46%) of wild‐type mice but had no effect in mutants. These results demonstrated that 5‐HT1B knockout mice are not as sensitive to full (CP‐93,129) and mixed (sumatriptan) 5‐HT1B receptor agonists as are wild‐type mice. These data provide in vivo evidence that, in mice, 5‐HT1B, but not 5‐HT1D, autoreceptors inhibit 5‐HT release at nerve terminals located in the frontal cortex and ventral hippocampus.
Cooperative Interception of Neuronal Apoptosis by BCL‐2 and BAG‐1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species
Authors: Schulz Jörg B. , Bremen Dirk , Reed John C. , Lommatzsch Jürgen , Takayama Shinichi , Wüllner Ullrich , Löschmann Peter‐A. , Klockgether Thomas , Weller Michael
Published: July 2010

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D‐ and cycloheximide‐sensitive caspase (ICE‐like) activity. The peptide inhibitor of caspase activity, N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde, was more potent than acetyl‐Tyr‐Val‐Ala‐Asp‐chloromethyl ketone in preventing NGF withdrawal‐induced apoptosis, suggesting an important role for caspase‐3 (CPP32)‐like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N‐tert‐butyl‐α‐phenylnitrone, and the antioxidant, N‐acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL‐2 or the BCL‐2‐binding protein BAG‐1 blocked NGF withdrawal‐induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL‐2 and BAG‐1 was more protective than expression of either protein alone.
Glutamate Uptake Stimulates Na+,K+‐ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain
Authors: Pellerin Luc , Magistretti Pierre J.
Published: July 2010

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+‐dependent transporter. Evidence had been provided that Na+,K+‐ATPase might be involved in this process. We have now measured the activity of Na+,K+‐ATPase in cultured astrocytes, using ouabain‐sensitive 86Rb uptake as an index. l‐Glutamate increases glial Na+,K+‐ATPase activity in a concentration‐dependent manner with an EC50 = 67 µM. Both l‐ and d‐aspartate, but not d‐glutamate, produce a similar response, an observation that is consistent with an uptake‐related effect rather than a receptor‐mediated one. Under basal conditions, concentration‐dependent inhibition of Na+,K+‐ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain (K0.5 = 113 µM), compatible with the presence of an α1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain (K0.5 = 20 nM), thus revealing a high‐affinity site akin to the α2 isozyme. These results suggest that astrocytes possess a glutamate‐sensitive isoform of Na+,K+‐ATPase that can be mobilized in response to increased neuronal activity.
Modulation of GH4 Cell Cycle via A1 Adenosine Receptors
Authors: Navarro Angels , Zapata Rafael , Canela Enric I. , Mallol Josefa , Lluis Carmen , Franco Rafael
Published: July 2010

Abstract: Identification and characterization of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunocytochemistry. Subsequently, the involvement of A1Rs in the regulation of cell proliferation was studied in these cells. The agonist N6‐(R)‐phenylisopropyladenosine (R‐PIA) did not modify the number of cultured cells, but it regulated the kinetics of the cell cycle. By means of experiments of pulse and of pulse and chase with bromodeoxyuridine and further labeling with Hoechst 33258, propidium iodide, and/or fluorescein‐conjugated antibodies against bromodeoxyuridine, it was demonstrated that R‐PIA, via A1Rs, accelerated progression from G0/G1 to S phase and from S to G2/M phase of the cell cycle, whereas the initiation of a new cycle occurred at the same time in treated and untreated cells. As a consequence, R‐PIA did not change the total length of the cycle. This is the first description of cell cycle regulation without modification of cell proliferation. Although pertussis toxin blocked the R‐PIA‐induced inhibition of cyclic AMP production in these cells, it did not affect the R‐PIA action on the cell cycle. In contrast, cholera toxin mimicked the action of R‐PIA. Thus, it is likely that regulation of the cell cycle via A1Rs is mediated by heterotrimeric G proteins different from those that mediate inhibition of adenylate cyclase. Due to the fact that cells in G0/G1 phase were less susceptible to secretory signals, adenosine, in an autocrine manner and by regulating the cell cycle kinetics, may contribute to the modulation of the secretory capacity of pituitary cells.
Nicotinic Acetylcholine Receptors of Muscle and Neuronal (α7) Types Coexpressed in a Small Cell Lung Carcinoma
Authors: Sciamanna Michele A. , Griesmann Guy E. , Williams Carol L. , Lennon Vanda A.
Published: July 2010

Abstract: SCC‐37 is a small cell lung carcinoma line that aberrantly expresses muscle‐type nicotinic acetylcholine receptors (nAChRs). It was established from a patient with a paraneoplastic autoimmune neuromuscular disorder, myasthenia gravis. When grown as a xenograft tumor, SCC‐37 cells express plasma membrane receptors that bind 125I‐labeled α‐bungarotoxin (125I‐α‐BTx), cosediment with 9S nAChR pentamers, and bind to a monoclonal antibody (MAb 35) specific for muscle‐type (α1 subunit) α‐BTx receptors. The agonist carbamylcholine (carbachol) stimulates influx of 22Na+ in SCC‐37 cells; this is inhibited by α‐BTx and by d‐tubocurarine. Long‐term cultured SCC‐37 cells have functional and ligand‐binding evidence for surface coexpression of both α1 and neuronal‐type (α7 subunit) α‐BTx receptors. A subclone of SCC‐37, designated SCC‐A9, expresses only the neuronal‐type (α7 subunit) α‐BTx receptors on its surface. Carbachol does not stimulate 22Na+ influx in SCC‐A9 cells, but cytisine initiates a sustained influx of Ca2+. Activation of this response is inhibited by α‐BTx and by the α7‐selective antagonist methyllycaconitine. Addition of Co2+ abrogates the sustained elevation of intracellular free Ca2+ concentration, implying that the cytisine‐stimulated influx of Ca2+ is sustained by secondary opening of voltage‐sensitive channels in the plasma membrane. Surface receptors for 125I‐α‐BTx are blocked by methyllycaconitine and d‐tubocurarine. Solubilized α‐BTx receptors from plasma membranes of SCC‐A9 cells cosediment with 10S neuronal nAChR pentamers and bind to an α7‐specific monoclonal antibody (MAb P27) but not to the muscle nAChR‐reactive MAb 35. However, MAb P27 and MAb 35 both bind to α‐BTx receptors solubilized from the cytoplasmic compartments of SCC‐A9 and the parental SCC‐37 line. Reverse transcription‐PCR analysis revealed RNA transcripts for α7 and α1 subunits in both SCC‐A9 and SCC‐37 cells. The nAChRs that are expressed in these novel human cell lines can regulate cation fluxes directly as well as indirectly by synergizing with the activity of voltage‐sensitive Ca2+ channels. These activities may influence the secretion of autocrine growth factors and the transcription of growth regulatory genes and thus be pertinent to the growth and metastasis of malignant neuroendocrine neoplasms.
Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes
Authors: Gamberino William C. , Berkich Deborah A. , Lynch Christopher J. , Xu Baiyang , LaNoue Kathryn F.
Published: July 2010

Abstract: CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C‐labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α‐ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C‐labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α‐[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α‐ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α‐ketoglutarate to glutamate may frequently be the rate‐limiting step in this process of glutamate synthesis.
α‐Secretase‐Derived Product of β‐Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease
Authors: Ancolio Karine , Marambaud Philippe , Dauch Pascale , Checler Frédéric
Published: July 2010

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early‐onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42‐amino‐acid‐long species of β‐amyloid peptide (Aβ). Here we show that overexpression of wild‐type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD‐linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild‐type PS1 augments the α‐secretase‐derived C‐terminally truncated fragment of β‐amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild‐type PS1. This decrease was also observed when comparing double transfectants overexpressing wild‐type β‐amyloid precursor protein and either PS1 or its mutated congener M146V‐PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down‐regulate the production of APPα.
S‐Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography‐Mass Spectrometry
Authors: Kluge Ina , Gutteck‐Amsler Ursula , Zollinger Markus , Do Kim Quang
Published: July 2010

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S‐nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S‐nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [35S]cysteine‐prelabeled slices suggested that S‐nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography‐mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using 15N‐labeled GSNO ([15N]‐GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.
Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia
Authors: Gegelashvili Georgi , Danbolt Niels Christian , Schousboe Arne
Published: July 2010

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down‐regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron‐astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron‐glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.
Changes in Monoamine Turnover in the Brain of Cachectic Mice Bearing Colon‐26 Tumor Cells
Authors: Uomoto Masashi , Nishibori Masahiro , Nakaya Naoki , Takeuchi Yoshiaki , Iwagaki Hiromi , Tanaka Noriaki , Saeki Kiyomi
Published: July 2010

Abstract: Patients with cancer cachexia often suffer from psychiatric disorders. In the present study, we investigated the changes in monoaminergic activities in the brain in tumor‐bearing mice with reference to the development of cachexia. Two clones, clone‐5 (noncachectic clone) and clone‐20 (cachectic clone), derived from the murine Colon‐26 adenocarcinoma cell line (Nippon Roche Research Center), were inoculated subcutaneously at 1 × 106 cells/0.2 ml into the right lower back of BALB/c mice. In clone‐20 mice, body weight and locomotor activity decreased significantly 10–15 days after tumor inoculation. The levels of noradrenaline, dopamine, and 3,4‐dihydroxyphenylacetic acid showed no significant change among the three groups. The noradrenaline turnover rate in clone‐20 mice was increased in cerebral cortex, hypothalamus, and midbrain. The 5‐hydroxytryptamine turnover rate in clone‐20 mice was increased in hippocampus, cerebral cortex, midbrain, and pons‐medulla oblongata. In contrast, the dopamine turnover rate in clone‐20 mice was decreased markedly in hippocampus, cerebral cortex, striatum, hypothalamus, and cerebellum. There was no significant change in amine turnover between control and clone‐5 mice except for dopamine in hippocampus, cerebral cortex, and striatum and 5‐hydroxytryptamine in striatum. No significant change in the levels of amino acids in the brain was observed among the three groups of mice. It is concluded that some of the psychiatric disorders from which cancer cachectic patients suffer might be ascribable to changes in monoaminergic activities in the brain.
Inhibition of Maitotoxin‐Induced Ca2+ Influx in Rat Glioma C6 Cells by Brevetoxins and Synthetic Fragments of Maitotoxin
Authors: Konoki Keiichi , Hashimoto Masaki , Nonomura Taro , Sasaki Makoto , Murata Michio , Tachibana Kazuo
Published: July 2010

Abstract: 45Ca2+ influx in rat glioma C6 cells induced by 0.3 nM maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC50 values of 16 and 13 µM, respectively. This inhibition was observed immediately after addition of MTX when monitored with fura‐2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage‐sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel‐forming peptide, did not significantly block MTX‐induced 45Ca2+ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX‐binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF‐GH rings (200 µM) and LM‐NO rings (500 µM) of MTX significantly reduced MTX‐elicited Ca2+ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.
Ras‐Signaling Pathways: Positive and Negative Regulation of Tau Expression in PC12 Cells
Authors: Sadot Einat , Jaaro Hanna , Seger Rony , Ginzburg Irith
Published: July 2010

Abstract: Tau is a microtubule‐associated protein whose promoter is activated during the first phase of nerve growth factor‐induced PC12 cell differentiation, whereas levels of its mRNA are accumulating throughout differentiation. In this study, we have followed the signal transduction cascades regulating tau induction. Using dominant negative Ras‐expressing PC12 cells, we show that ras regulates tau expression during the first phase of PC12 cell differentiation. The ERK and JNK cascades, which are downstream of Ras; have opposing effects on tau promoter activity: ERK induces tau promoter activity, JNK inhibits it. Tau promoter activity in PC12 cells is correlated with a short‐term activation of ERK, which declines after a few hours and is followed by an activation of the inhibitory JNK cascade 76 h later. These observations suggest that the induction and inhibition of tau promoter are mediated by alternate ERK and JNK activities, which may underlie a mechanism to turn on and off genes during PC12 cell differentiation.
Effect of Acute Systemic Inflammatory Response and Cytokines on the Transcription of the Genes Encoding Cyclooxygenase Enzymes (COX‐1 and COX‐2) in the Rat Brain
Authors: Lacroix Steve , Rivest Serge
Published: July 2010

Abstract: The aim of this study was to investigate the influence of the acute‐phase response and the proinflammatory cytokines on the transcription of the genes encoding the limiting enzymes for the production of prostaglandins, cyclooxygenase (COX)‐1 and COX‐2, in the rat brain. The bacterial endotoxin lipopolysaccharide (intravenous and intraperitoneal) and turpentine (intramuscular) were used as different models of inflammation in adult male rats. Animals were also killed at various times after intravenous administration of interleukin‐1β, tumor necrosis factor‐α, and interleukin‐6, and mRNAs encoding COX‐1 and COX‐2 were assayed by in situ hybridization histochemistry. A profound transcriptional activation of the gene encoding COX‐2 was detected over blood vessels of the entire brain microvasculature, choroid plexus, and leptomeninges of lipopolysaccharide‐challenged rats. Injection of the endotoxin intravenously also increased COX‐2 gene expression within parvocellular division of the hypothalamic paraventricular nucleus. It is interesting that intramuscular turpentine injection stimulated transcription of COX‐2 along endothelium of brain capillaries, and the signal of this transcript paralleled the inflammation of the left hind limb. A robust COX‐2 mRNA signal was detected rapidly in the brain microvessels of interleukin‐1β‐injected rats, whereas tumor necrosis factor‐α administration caused a modest but significant induction of this transcript. In contrast, intravenous injection of interleukin‐6 did not alter genetic expression of COX‐2, and none of the above described models affected the synthesis of COX‐1 gene in the rat brain. These results indicate that specific cell populations, in particular vascular‐ and/or perivascular‐associated cells, are responsible for the central production of prostaglandins during systemic inflammation, and circulating interleukin‐1β is likely to be a potent mediator of this response.
Biochemical Pathways by Which Serotonin Regulates Translation in the Nervous System of Aplysia
Authors: Yanow Stephanie K. , Manseau Frédéric , Hislop Jonathan , Castellucci Vincent F. , Sossin Wayne S.
Published: July 2010

Abstract: In the marine mollusk Aplysia californica, serotonin initiates three phases of translational regulation: an initial decrease in translation, followed by a transient increase in protein synthesis, both of which are independent of transcription, followed by a later increase in protein synthesis that is dependent on transcription. These increases in protein synthesis may underlie translation‐dependent changes in synaptic plasticity. We have characterized the second messenger pathways that underlie these changes in the pleural ganglia of Aplysia. Activation of protein kinase C was both necessary and sufficient for the initial decrease in translation. Protein kinase C, cyclic AMP‐dependent protein kinase, and a tyrosine kinase were all required for the second phase, a transient increase in protein synthesis. The late increase in protein synthesis required both protein kinase A and spaced applications of serotonin. Rapamycin, a specific inhibitor of a downstream translational regulator, blocked the transient increase in protein synthesis (second phase), suggesting that this drug may be useful in determining the specific physiological consequences of this translational regulation. Indeed, we used rapamycin to demonstrate that one type of intermediate form of synaptic plasticity induced by serotonin did not require the rapamycin‐sensitive increase in translation.
Inflammatory Signals Induce Neurotrophin Expression in Human Microglial Cells
Authors: Heese Klaus , Hock Christoph , Otten Uwe
Published: July 2010

Abstract: Inflammatory processes involving reactive microglia, e.g., those associated with β‐amyloid containing neuritic and core plaques in Alzheimer's disease, appear to contribute to neuronal degeneration in the CNS. The fact that increased nerve growth factor (NGF) protein levels were found throughout brains of Alzheimer's disease patients led us to investigate neurotrophin synthesis in a human microglial cell line showing typical properties of human microglial cells, including expression of neurotrophins such as NGF, as well as the NGF receptor trkA and the low‐affinity neurotrophin receptor p75. We found that the cytokines interleukin‐1β and tumor necrosis factor‐α synergistically stimulate microglial NGF transcription and protein release. Moreover, exposure of microglial cells to complement factor C3a induces NGF expression. To assess the role of the transcription factor nuclear factor‐κB (NF‐κB) in inflammatory mediator‐induced microglial NGF expression, the effect of the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC) was analyzed. In the presence of PDTC, a dose‐dependent inhibition of cytokine‐activated NGF expression occurred. In contrast, the C3a‐dependent stimulation of NGF synthesis was not influenced by PDTC. In addition, microglial neurotoxicity‐mediating β‐amyloid peptides Aβ(1–40) and Aβ(1–42) failed to alter NGF synthesis, whereas Aβ(25–35) specifically induced NF‐κB‐dependent microglial NGF expression. In conclusion, inflammatory signals (cytokines and complement factors), as well as Aβ(25–35), are potent stimulators of human microglial NGF synthesis involving NF‐κB‐dependent and ‐independent mechanisms. Microglial secretion of neurotrophins appears to be involved in early processes of neuronal regeneration.