bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of GABAA Receptor Subunit mRNAs in Rat Cerebellar Granule Neurons: Importance of Environmental Cues
Authors: Behringer K. A. , Gault L. M. , Siegel R. E.
Published: July 2010

Abstract: Levels of the GABAA receptor α1‐, α6‐, β2‐, β3‐, γ2‐, and δ‐subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase‐PCR was used to examine transcript expression in cultured granule neurons prepared at 2‐day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the α1‐ and α6‐subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the β2‐, β3‐, and γ2‐subunit mRNAs were constant in cultures prepared at postnatal days 2–6 but increased in those prepared at days 8–10. Third, the δ‐subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only δ‐subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are differentially regulated by multiple environmental cues.
Characterization and Localization of Adrenal Nicotinic Acetylcholine Receptors: Evidence that mAb35‐Nicotinic Receptors Are the Principal Receptors Mediating Adrenal Catecholamine Secretion
Authors: Gu Hyangja , Wenger Bryan W. , Lopez Isabel , McKay Susan B. , Boyd R. Thomas , McKay Dennis B.
Published: July 2010

Abstract: Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the α‐subunit of muscle nAChRs and cross‐reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR‐stimulated catecholamine release (IC50 of ∼10 nM). mAb35 had no effects on release stimulated through non‐nAChR mechanisms. Unlike agonist‐induced nAChR desensitization, the mAb35‐induced reduction in nAChR‐mediated secretion developed slowly. Although not immediately reversible, nAChR‐stimulated release recovered after mAb35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was partially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat α3 cDNA revealed two strong bands and two fainter bands: two higher‐molecular‐weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of a 2.3‐kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on α3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35‐nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells.
Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells
Authors: Kagaya Ariyuki , Uchitomi Yosuke , Kugaya Akira , Takebayashi Minoru , Nagaoka Ikuo , Muraoka Mitsutaro , Yokota Norio , Yamawaki Shigeto
Published: July 2010

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca2+ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca2+‐sensitive dye fura‐2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration‐dependent manner. This enhancement was abolished by pretreatment with 100 µM BAPTA tetraacetoxymethyl ester or in the presence of W‐7 in a concentration‐dependent manner. NG‐Monomethyl‐l‐arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF‐induced generation of cGMP. These results suggest that NaF‐induced cGMP generation occurs via a calcium/calmodulin‐ and NOS‐dependent pathway. (b) The basal intracellular Ca2+ concentration ([Ca2+]i) was transiently greater at 1 and 3 h after pretreatment with NaF. W‐7 and W‐13 antagonized the increase in [Ca2+]i, whereas NMMA had little effect. This suggests that the NaF‐induced change in basal [Ca2+]i was mediated by a calmodulin‐dependent pathway but was independent of a NOS‐sensitive pathway. (c) The serotonin (5‐HT)‐induced intracellular mobilization of Ca2+ was reduced by pretreating the cells with NaF. The reduction in Ca2+ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W‐7, W‐5, and H‐8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca2+]i, and 5‐HT2A receptor function in C6 glioma cells.
Synaptic Membrane G Proteins Are Complexed with Tubulin In Situ
Authors: Yan Kun , Greene Erin , Belga Ferdinand , Rasenick Mark M.
Published: July 2010

Abstract: The G proteins Gs and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenylyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express Gsα but not Giα1, responded to the addition of tubulin with a stable activation of adenylyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both Gs and Gi1, responded to exogenous tubulin with a stable inhibition of adenylyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and Gi1 or Gs from detergent extracts of these membranes. Nearly 90% of the Gsα from Triton X‐114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein‐cytoskeleton link that participates in the modulation of cellular signal transduction.
Cellular Mechanisms of Protection by Metabotropic Glutamate Receptors During Anoxia and Nitric Oxide Toxicity
Authors: Maiese Kenneth , Swiriduk Margaret , TenBroeke Michelle
Published: July 2010

Abstract: Metabotropic glutamate receptors, nitric oxide (NO), and the signal transduction pathways of protein kinase C (PKC) and protein kinase A (PKA) can independently alter ischemic‐induced neuronal cell death. We therefore examined whether the protective effects of metabotropic glutamate receptors during anoxia and NO toxicity were mediated through the cellular pathways of PKC or PKA in primary hippocampal neurons. Pretreatment with the metabotropic glutamate receptor agonists (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid, (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), and l(+)‐2‐amino‐4‐phosphonobutyric acid (l‐AP4) 1 h before anoxia or NO exposure increased hippocampal neuronal cell survival from ∼30 to 70%. In addition, posttreatment with 1S,3R‐ACPD or l‐AP4 up to 6 h following an insult attenuated anoxic‐ or NO‐induced neurodegeneration. In contrast, treatment with l‐(+)‐2‐amino‐3‐phosphonopropionic acid, an antagonist of the metabotropic glutamate receptor, did not significantly alter neuronal survival during anoxia or NO exposure. Protection by the ACPD‐sensitive metabotropic receptors, such as the subtypes mGluR1α, mGluR2, and mGluR5, appears to be dependent on the modulation of PKC activity. In contrast, l‐AP4‐sensitive metabotropic glutamate receptors, such as the subtype mGluR4, may increase neuronal survival through PKA rather than PKC. Thus, activation of specific metabotropic glutamate receptors is protective during anoxia and NO toxicity, but the signal transduction pathways mediating protection differ among the metabotropic glutamate receptor subtypes.
Intrasynaptosomal Free Calcium Concentration During Rat Brain Development: Effects of Hypoxia, Aglycaemia, and Ischaemia
Authors: Keelan Julie , Bates Timothy E. , Clark John B.
Published: July 2010

Abstract: The effects of hypoxia, aglycaemia, and hypoxia‐aglycaemia on intrasynaptosomal free Ca2+ concentration ([Ca2+]i) have been investigated in rat brain synaptosomes prepared from animals aged 5, 10, 15, 20, 25, and 60 days. After 60 min of hypoxia there was no significant difference, when compared with controls, in basal [Ca2+]i or [Ca2+]i following depolarisation in all of the ages studied. Following 60 min of aglycaemia there was no significant difference from controls in [Ca2+]i of synaptosomes prepared from pups of ≤20 days, although a significant rise in [Ca2+]i was seen in preparations from animals >20 days old. Sixty minutes of hypoxia‐aglycaemia led to a significant rise in [Ca2+]i only in preparations from animals 15–60 days old. With both aglycaemia and hypoxia‐aglycaemia a progressive increase in the magnitude of the rise in [Ca2+]i was seen with development. These data suggest increases in [Ca2+]i in adult nerve terminals following prolonged aglycaemia and hypoxia‐aglycaemia but no change following prolonged hypoxia. In contrast, no significant changes in [Ca2+]i values were apparent in neonatal nerve terminals under any of these conditions. In control synaptosomes with glucose and oxygen freely available, a decrease in resting and depolarised [Ca2+]i during development was seen, suggesting a change in calcium homeostasis within the nerve terminal as the brain develops. It is suggested that the mechanism underlying the relative resistance to ischaemic damage of neonatal brain as compared with adult brain may be related to the regulation of calcium at the nerve ending.
Acute and Prolonged Alterations in Brain Free Magnesium Following Fluid Percussion‐Induced Brain Trauma in Rats
Authors: Vink Robert , Heath Deanne L. , McIntosh Tracy K.
Published: July 2010

Abstract: Several studies have reported declines in brain total and free magnesium concentration after a traumatic insult to the CNS. Although the evidence suggests that this magnesium decline is associated with eventual neurologic outcome after trauma, the duration of free magnesium decline and its impact on related bioenergetic variables are relatively unknown. The present study has therefore used phosphorus magnetic resonance spectroscopy to determine the length of time that free magnesium remains suppressed after traumatic brain injury in rats. Immediately after the traumatic event, brain intracellular free magnesium declined to <60% of preinjury values and remained significantly depressed (50 ± 8%; p < 0.001) for 5 days before recovering to preinjury levels by day 8. Cytosolic phosphorylation ratio and mitochondrial oxidative capacity also significantly decreased (p = 0.008) and increased (p = 0.002), respectively, after trauma. However, unlike the time of maximum magnesium change, the maximum changes in these bioenergetic variables occurred at 16–24 h after trauma and thereafter remained stable until after the magnesium had recovered. We conclude that free magnesium decline after trauma precedes changes in bioenergetic variables. Furthermore, therapies targeted at reestablishing magnesium homeostasis after trauma may require administration over a 1‐week period.
Ca2+/Calmodulin‐Dependent Protein Kinase II Inhibitor KN‐62 Inhibits Adrenal Medullary Chromaffin Cell Functions Independent of Its Action on the Kinase
Authors: Tsutsui Masato , Yanagihara Nobuyuki , Fukunaga Kohji , Minami Kouichiro , Nakashima Yasuhide , Kuroiwa Akio , Miyamoto Eishichi , Izumi Futoshi
Published: July 2010

Abstract: KN‐62, an inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN‐62, however, showed an additional inhibitory effect on acetylcholine‐induced 45Ca2+ influx, which is essential for functional responses. Carbachol‐stimulated 22Na+ influx, veratridine‐induced 22Na+ influx, and 56 mM K+‐evoked 45Ca2+ influx were also attenuated by KN‐62. Inhibitions by KN‐62 of these ion influxes were correlated closely with those of catecholamine secretion. KN‐04, which is a structural analogue of KN‐62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant‐evoked ion influxes with an inhibitory potency similar to KN‐62. These results suggest that KN‐62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.
Flesinoxan Dose‐Dependently Reduces Extracellular 5‐Hydroxytryptamine (5‐HT) in Rat Median Raphe and Dorsal Hippocampus Through Activation of 5‐HT1A Receptors
Authors: Bosker Fokko J. , De Winter Tessa Y. C. E. , Klompmakers André A. , Westenberg Herman G. M.
Published: July 2010

Abstract: The effects of systemic administration of the serotonin (5‐hydroxytryptamine) 5‐HT1A receptor agonists flesinoxan and 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin on extracellular 5‐HT were measured using microdialysis probes in both median raphe nucleus and dorsal hippocampus. Both 5‐HT1A agonists dose‐dependently decreased dialysate 5‐HT levels from both brain regions. The effects of flesinoxan in the median raphe (0.3 mg/kg) and dorsal hippocampus (1.0 mg/kg) could be blocked by the 5‐HT1A receptor antagonist N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridyl)cyclohexane carboxamide trihydrochloride (WAY 100,635) at a dose of 0.05 mg/kg s.c. The antagonist itself had no effect at this dosage. Local perfusion of flesinoxan for 30 min through the dialysis probe into the median raphe region at concentrations of 20, 100, and 1,000 nM resulted in a significant decrease in dialysate 5‐HT content from both regions. The effect of 100 nM flesinoxan could be blocked by coperfusion of 1,000 nM WAY 100,635. The data indicate that flesinoxan is a potent 5‐HT1A receptor agonist and also support the notion that somatodendritic 5‐HT1A autoreceptors regulate both terminal and somatodendritic 5‐HT release.
Ganglioside‐Induced Adherence of Botulinum and Tetanus Neurotoxins to Adducin
Authors: Schengrund Cara‐Lynne , DasGupta Bibhuti R. , Hughes Christine A. , Ringler Nancy J.
Published: July 2010

Abstract: Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an ∼116‐kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the ∼116‐kDa protein more than that with GT1b. A purified preparation of the water‐soluble ∼116‐kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and two‐dimensional gel electrophoresis. N‐Terminal amino acid sequences were obtained for two tryptic fragments of the ∼116‐kDa protein. These sequences matched with the data bank sequences for β‐adducin, a cytoskeletal protein. The carboxy‐terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).
Altered Glycosaminoglycan Chain Structure in a Variant of the C2 Mouse Muscle Cell Line
Authors: Bowen David C. , Gordon Herman , Hall Zach W.
Published: July 2010

Abstract: Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of 35SO4 into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation.
Calcium Channel Subunits in the Mouse Cochlea
Authors: Green Glenn E. , Khan Khalid M. , Beisel Kirk W. , Drescher Marian J. , Hatfield James S. , Drescher Dennis G.
Published: July 2010

Abstract: Messages for subunits of voltage‐gated calcium channels were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 16–18‐day‐old animals. After reverse transcription, resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to 12 different calcium channel subunits. PCR products representing subunit gene expression were strongly and consistently amplified for α1C, α1D, α1E, α2δ, β1, β3, and β4 but not for α1A, α1B, α1S, β2, or γ. The chosen primers amplified cochlear cDNA to yield an overall pattern of bands different from that of any tissue studied thus far, in particular with respect to the α2δ and β1 subunits; the α2δ product was found to be significantly shorter than the corresponding brain and skeletal muscle isoforms. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. The results suggest that L‐type and presumptive R‐type calcium channels are expressed in the mammalian cochlea and that the α2δ subunits may be coded by a characteristic splice‐variant mRNA.
Molecular Cloning and Characterization of Annexin V‐Binding Proteins with Highly Hydrophilic Peptide Structure
Authors: Ohsawa Keiko , Imai Yoshinori , Ito Daisuke , Kohsaka Shinichi
Published: July 2010

Abstract: We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. Herein, we screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S‐transferase‐annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca2+ ‐ and phospholipid‐dependent manner. Although these cDNAs encoded different polypeptides, all four polypeptides shared the unique feature of containing highly hydrophilic stretches with high Lys, Glu, and Ser contents. Deduced amino acid sequences of two clones showed high homology with human X‐linked Helicase2 (XH2) and DNA (cytosine‐5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence. These results suggest that XH2 and DMTase are candidates for annexin V‐binding proteins and thus may mediate the biological activity of annexin V.
Increased Superoxide Dismutase Activity Improves Survival of Cultured Postnatal Midbrain Neurons
Authors: Przedborski Serge , Khan Uzma , Kostic Vladimir , Carlson Elaine , Epstein Charles J. , Sulzer David
Published: July 2010

Abstract: Copper/zinc superoxide dismutase (Cu/Zn‐SOD) is a major free radical scavenging enzyme. Increased Cu/Zn‐SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn‐SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn‐SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn‐SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn‐SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn‐SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn‐SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn‐SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn‐SOD activity does not seem to play a critical role in determining the mode of cell death in this culture system.
Signaling Pathway Downstream of GABAA Receptor in the Growth Cone
Authors: Fukura Haruhiko , Komiya Yoshiaki , Igarashi Michihiro
Published: July 2010

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABAA receptor appear even in early stages of CNS development. We investigated the GABAA receptor‐mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABAA binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin‐sensitive Cl− flux (not influx but efflux) and increased the intracellular Ca2+ concentration in a picrotoxin‐ and verapamil‐sensitive manner. Protein kinase C (PKC)‐dependent phosphorylation of two proteins identified as GAP‐43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP‐43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABAA receptor localized in the growth cone membrane → Cl− efflux induction through the GABAA‐associated Cl− channel → Ca2+ influx through an L‐type voltage‐sensitive Ca2+ channel → Ca2+‐dependent phosphorylation of GAP‐43 and MARCKS protein by PKC.
Ethanol Inhibits Basic Fibroblast Growth Factor‐Mediated Proliferation of C6 Astrocytoma Cells
Authors: Luo Jia , Miller Michael W.
Published: July 2010

Abstract: Early ethanol exposure alters the proliferative activity of glial and neuronal precursors in the developing CNS. The present study tests the hypothesis that ethanol‐induced alterations in cell proliferation result from interference with growth factors. An in vitro model of astroglia (C6 astrocytoma cells) was used to study the effects of ethanol on proliferation mediated by basic fibroblast growth factor (bFGF). bFGF stimulated the proliferation of C6 cells. This bFGF‐enhanced proliferation was evident by increases in total cell number, DNA synthesis (as measured by [3H]thymidine incorporation), and the number of cells that took up bromodeoxyuridine. A synthetic peptide that specifically blocked the binding of bFGF to its high‐affinity receptor completely abolished the proliferation‐promoting effect of bFGF. The action of another mitogen for C6 cells, insulin‐like growth factor‐1, was not affected by this peptide. Therefore, the bFGF‐stimulated proliferation was mediated through a specific bFGF receptor. Ethanol inhibited bFGF‐mediated proliferation in a concentration‐dependent manner. Ethanol concentrations of 100 and 200 mg/dl partially inhibited bFGF‐mediated proliferation (by 58 and 74%, respectively), whereas concentrations of ≥400 mg/dl completely abolished the growth‐stimulating effect of bFGF. Our data show that ethanol alters proliferative activity of C6 cells by disrupting the action of bFGF. The target of ethanol neurotoxicity is a receptor‐mediated activity. bFGF can affect cell proliferation by a non‐receptor‐mediated intracellular pathway, but ethanol does not have an impact on this pathway.
Formation of d‐Serine from l‐Phosphoserine in Brain Synaptosomes
Authors: Wood Paul L. , Hawkinson Jon E. , Goodnough Dayan B.
Published: July 2010

Abstract: Although glycine has been assumed to be the sole endogenous coagonist at NMDA‐associated glycine receptors, recent descriptions of endogenous d‐serine in the brain indicate that this assumption is probably not valid. d‐Serine is a stereospecific agonist of the NMDA‐associated glycine receptor, with an affinity equal to or greater than that of glycine but with no affinity for the strychnine‐sensitive glycine receptor. In the current studies, we assessed the levels and metabolic sources of d‐serine in rat neocortical synaptosomal preparations. Previous studies have demonstrated that CNS serine and glycine are synthesized de novo primarily via a phosphorylated pathway, originating with the glycolytic intermediate phosphoglycerate. The rate‐limiting step in the synthesis of serine is the hydrolysis of phosphoserine by phosphoserine phosphatase (EC 3.1.3.3). In synaptosomal preparations we have demonstrated high endogenous levels of d‐serine and the uptake of l‐phosphoserine along with its hydrolysis to both l‐serine and d‐serine, which are preferentially released into the medium. Experiments with both intact and lysed synaptosomal preparations demonstrated hydrolysis of d‐phosphoserine to only d‐serine and inhibition of hydrolysis by the phosphoserine phosphatase inhibitor 2‐amino‐3‐phosphonopropionic acid (AP3). The lack of stereospecificity for synaptosomal hydrolysis of phosphoserine and the inhibitory actions of AP3 are consistent with the presence of phosphoserine phosphatase in synaptosomes and further indicate that epimerization of serine can occur during or subsequent to the hydrolysis of l‐phosphoserine but not d‐phosphoserine. In conclusion, these studies demonstrate that phosphoserine phosphatase may be an important enzyme in regulating the steady‐state levels of d‐serine in neocortical synaptosomes.
Acidic Fibroblast Growth Factor and Catecholamines Synergistically Up‐Regulate Tyrosine Hydroxylase Activity in Developing and Damaged Dopamine Neurons in Culture
Authors: Stull Natalie D. , Iacovitti Lorraine
Published: July 2010

Abstract: Our previous studies indicate that, in certain non‐catecholamine (CA) neurons, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the obligatory interaction of acidic fibroblast growth factor (aFGF) and a CA activator. In this study, we sought to determine whether these same differentiation factors also play a role in regulating existing TH expression in CA neurons. Thus, the effects of exogenous aFGF and CAs on TH were studied in developing or toxin‐damaged dopamine (DA) neurons from the embryonic day 15 rat ventral midbrain, where it was likely to be at physiologically low levels. Cultures were incubated with various concentrations of aFGF, DA, or aFGF and DA. Some cultures were first damaged with 2.5 µM 1‐methyl‐4‐phenylpyridinium. In developing DA neurons, an 80% increase in TH activity was found only after cotreatment with aFGF (100 ng/ml) and DA (1 µM) or other monoamines. Likewise, in damaged DA neurons, aFGF and DA reversed the 50% loss in TH activity caused by toxin. This was observed within 4 h of treatment and was not associated with changes in the number or appearance of DA neurons, suggesting a biochemical rather than a trophic effect. Pretreatment with protein or RNA synthesis inhibitors eliminated the increase. In PC12 cells, where TH is highly expressed, activity was unaltered by treatment. We conclude that the aFGF and CAs may be involved in not only the initiation but also the regulation of TH.
Cerebral Energy Metabolism During Severe Ischemia of Varying Duration and Following Reperfusion
Authors: Phillis J. W. , O'Regan M. H. , Estevez A. Y. , Song D. , VanderHeide S. J.
Published: July 2010

Abstract: Changes in cerebral cortical adenine nucleotide and adenosine levels during 10‐, 20‐, or 40‐min periods of four‐vessel occlusion producing cerebral ischemia in rats and reperfusions of 10, 45, or 90 min were determined to evaluate the effects of ischemia duration on mitochondrial function. Substantial recovery was evident following 10 or 20 min of cerebral ischemia but not, however, after a 40‐min period of ischemia. A secondary decline in the cortical levels of ATP became evident following 40 min of cerebral ischemia and 90 min of reperfusion. Longer periods of ischemia may be associated with a loss of adenosine, limiting the resynthesis of ATP during reperfusion. A separate group of rats, resuscitated with 100% O2, demonstrated a more rapid recovery of mitochondrial function compared with animals that received room air during reperfusion following 20 min of cerebral ischemia. No detrimental effects of 100% O2 were observed during the 90‐min period of reperfusion, indicating that 100% O2 does not promote early mitochondrial dysfunction.
Accumulation of DNA Damage in Aging Neurons Occurs Through a Mechanism Other than Apoptosis
Authors: Mandavilli Bhaskar S. , Rao Kalluri Subba
Published: July 2010

Abstract: Two biochemical strategies using nick translation‐type of incubation and terminal transferase‐catalyzed reaction were used to assess single‐ (SSB) and double‐strand (DSB) breaks in DNA of permeabilized neurons isolated from young, adult, and old rat cerebral cortex. Both SSBs and DSBs accumulate with age. On prior treatment of neuronal cells with 1 mM glutamate or 50 µMN‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), more extensive damage was seen at all ages, with the old neurons suffering maximal damage. When neuronal DNA was subjected to agarose electrophoresis, increasingly diffused bands were seen with age in normally aging neurons. However, a typical nucleosomal ladder, characteristic of apoptosis, was seen only when the cells were exposed to either glutamate or MNNG irrespective of the age of the neurons. Furthermore, this apoptotic fragmentation of DNA was prevented by prior treatment of the cells with either cycloheximide or aurintricarboxylic acid, indicating that both glutamate and MNNG induce programmed cell death. Fluorescence microscopic observation of glutamate‐ and MNNG‐treated neurons after acridine orange staining revealed a high degree of staining and marked condensation of nuclear DNA. On the other hand, no such phenomenon was observed in normally aging neurons either histologically or in biochemical assays of damage. It is concluded that both glutamate and MNNG induce programmed cell death in neurons independent of age and that accumulation of DNA damage in naturally aging neurons occurs through a process other than that of apoptosis.
μ‐Calpain Activation and Calpain‐Mediated Cytoskeletal Proteolysis Following Traumatic Brain Injury
Authors: Kampfl A. , Posmantur R. , Nixon R. , Grynspan F. , Zhao X. , Liu S. J. , Newcomb J. K. , Clifton G. L. , Hayes R. L.
Published: July 2010

Abstract: Increasing evidence suggests that excessive activation of the calcium‐activated neutral protease μ‐calpain could play a major role in calcium‐mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ‐calpain after unilateral cortical impact injury in vivo, the ratio of the 76‐kDa activated isoform of μ‐calpain to its 80‐kDa precursor was measured by western blotting. This μ‐calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ‐calpain activation on proteolysis of the neuronal cytoskeletal protein α‐spectrin. Immunoreactivity for α‐spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α‐spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α‐spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ‐calpain autolysis, calpain‐mediated α‐spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ‐calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain‐mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ‐calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.
The Neuronal Nitric Oxide Synthase Inhibitor, 7‐Nitroindazole, Protects Against Methamphetamine‐Induced Neurotoxicity In Vivo
Authors: Itzhak Yossef , Ali Syed F.
Published: July 2010

Abstract: The present study was undertaken to investigate whether the relatively selective neuronal nitric oxide synthase (NOS) inhibitor, 7‐nitroindazole (7‐NI), protects against methamphetamine (METH)‐induced neurotoxicity. Male Swiss Webster mice received the following treatments (i.p.; q 3 h × 3): (a) vehicle/saline, (b) 7‐NI (25 mg/kg)/saline, (c) vehicle/METH (5 mg/kg), and (d) 7‐NI (25 mg/kg)/METH (5 mg/kg). On the second day, groups (a) and (b) received two vehicle injections, and groups (c) and (d) received two 7‐NI injections (25 mg/kg, each). Administration of vehicle/METH resulted in 68, 44, and 55% decreases in the concentration of dopamine, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid, respectively, and a 48% decrease in the number of [3H]mazindol binding sites in the striatum compared with control values. Treatment with 7‐NI (group d) provided full protection against the depletion of dopamine and its metabolites and the loss of dopamine transporter binding sites. Administration of 7‐NI/saline (group b) affected neither the tissue concentration of dopamine and its metabolites nor the binding parameters of [3H]mazindol compared with control values. 7‐NI had no significant effect on animals' body temperature, and it did not affect METH‐induced hyperthermia. These findings indicate a role for nitric oxide in methamphetamine‐induced neurotoxicity and also suggest that blockade of NOS may be beneficial for the management of Parkinson's disease.
Human Astrocytoma Cells (U‐87 MG) Exhibit a Specific Substance P Binding Site with the Characteristics of an NK‐1 Receptor
Authors: Ogo Hiroki , Kuroyanagi Naoyoshi , Inoue Atsuko , Nishio Hiroaki , Hirai Yuko , Akiyama Mitoshi , DiMaggio Debora A. , Krause James E. , Nakata Yoshihiro
Published: July 2010

Abstract: To investigate substance P (SP) receptors on an established human astrocytoma cell line (U‐87 MG), [3H][Sar9,Met(O2)11]‐SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti‐SP binding protein antibody. In U‐87 MG and U‐373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U‐87 MG cell membrane‐enriched preparations, the binding of [3H][Sar9,Met(O2)11]‐SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 ± 0.15 nM and aBmax of 108 ± 9.8 fmol/mg of protein. [3H][Sar9,Met(O2)11]‐SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl‐5′‐imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]‐SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U‐87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U‐87 MG have either a biantennary complex‐type or a high mannose‐type of carbohydrate chain and may be regulated by GTP‐binding protein(s).
Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l‐threo‐1‐Phenyl‐2‐Decanoylamino‐3‐Morpholino‐1‐Propanol
Authors: Usuki Seigou , Hamanoue Makoto , Kohsaka Shinichi , Inokuchi Jin‐ichi
Published: July 2010

Abstract: We reported previously that stereoisomers of 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), the d‐threo and l‐threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l‐ or d‐threo‐PDMP. These isomers exhibited opposite effects on neurite outgrowth: d‐PDMP was inhibitory at concentrations ranging from 5 to 20 µM, whereas l‐PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µM. Rat neocortical explants were doubly labeled with [14C]serine and [3H]galactose at 15 µMl‐ or d‐PDMP. l‐PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d‐PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l‐PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N‐acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l‐PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.
β‐Adrenergic Stimulation Promotes Homocysteic Acid Release from Astrocyte Cultures: Evidence for a Role of Astrocytes in the Modulation of Synaptic Transmission
Authors: Do Kim Q. , Benz Beatrix , Sorg Olivier , Pellerin Luc , Magistretti Pierre J.
Published: July 2010

Abstract: The sulfur‐containing amino acid homocysteic acid (HCA) is present in and released from nervous tissue, exerting excitatory effects on neurons by predominantly activating NMDA receptors. It is interesting that HCA appears to be exclusively localized in glial cells, not in neurons. This profile of glial localization and excitatory action on neurons has led to the hypothesis that HCA could participate in intercellular communication in the brain as a “gliotransmitter.” To test this hypothesis further, we searched for specific, receptor‐mediated stimuli that could induce release of HCA from cultured astrocytes. For this reason we tested the effect of noradrenaline and vasoactive intestinal peptide, two transmitters known to interact with specific receptors on astrocytes, on the release of HCA from these cells. Noradrenaline and the β‐adrenergic agonist isoproterenol induced an efflux of HCA from astrocyte cultures. Further stressing the β‐adrenergic mediation of this effect is the blockade by atenolol of the HCA release evoked by isoproterenol. The stimulation of HCA release from astrocytes was not observed with the α‐noradrenergic agonist methoxamine and with vasoactive intestinal peptide. These results taken together further strengthen the role of HCA as a gliotransmitter. Its efflux from glia could be controlled by noradrenaline, activating β‐adrenergic receptors on astrocytes. The present study provides the first evidence for an influence of β‐adrenergic receptor activation on the release of an excitatory amino acid from astrocytes and further supports the notion that glial‐neuronal interactions play a role in synaptic transmission.
Thyroid Hormone‐Regulated Expression of RC3/Neurogranin in the Immortalized Hypothalamic Cell Line GT1‐7
Authors: Morte Beatriz , Iñiguez Miguel Angel , Lorenzo Petra Isabel , Bernal Juan
Published: July 2010

Abstract: The calmodulin‐binding, protein kinase C substrate RC3/neurogranin is the product of a neuron‐specific gene expressed in the forebrain that is under specific regional and temporal control by thyroid hormone (3,5,3′‐triiodothyronine, T3). In vivo, some neuronal populations are sensitive and others are insensitive to T3. The goal of this study was to identify neuronal cell cultures that express RC3/neurogranin, to check whether they are sensitive to T3, and to examine the mechanism of regulation. We found that RC3 is induced by T3 in the hypothalamic cell line GT1‐7 at the transcriptional level. The half‐life of the mature mRNA was 20 h and was not affected by the hormone. Addition of T3 to the cell culture induces neurogranin mRNA after 6 h in the absence of new protein synthesis. These results suggest a direct transcriptional effect of T3 mediated through nuclear receptors. Indeed, GT1‐7 cells express functional T3 receptors, as shown by northern blotting, nuclear T3‐binding assays, and transactivation of reporter genes. The role of retinoic acid and glucocorticoids on RC3 expression was also evaluated, because we have previously noted the presence of consensus response elements for these hormones in the RC3 upstream promoter region. In contrast to T3, neither retinoic acid nor dexamethasone influences neurogranin expression despite the presence of respective functional receptors.
On the efficiency of biased sampling of the multiple state path ensemble
Authors: Rogal Jutta , Bolhuis Peter G.
Published: July 2010

Developed for complex systems undergoing rare events involving many (meta)stable states, the multiple state transition path sampling aims to sample from an extended path ensemble including all possible trajectories between any pair of (meta)stable states. The key issue for an efficient sampling of the path space in this extended ensemble is sufficient switching between different types of trajectories. When some transitions are much more likely than others the collective sampling of the different path types can become difficult. Here we introduce a Wang–Landau based biasing approach to improve the sampling. We find that the biasing of the multiple state path ensemble does not influence the switching behavior, but does improve the sampling and thus the quality of the individual path ensembles.
Conjugate degradability and the quantum capacity of cloning channels
Authors: Brádler Kamil , Dutil Nicolas , Hayden Patrick , Muhammad Abubakr
Published: July 2010

A quantum channel is conjugate degradable if the channel’s environment can be simulated up to complex conjugation using the channel’s output. For all such channels, the quantum capacity can be evaluated using a single-letter formula. In this article we introduce conjugate degradability and establish a number of its basic properties. We then use it to calculate the quantum capacity of N to N+1 and 1 to M universal quantum cloning machines as well as the quantum capacity of a channel that arises naturally when data are being transmitted to an accelerating receiver. All the channels considered turn out to have strictly positive quantum capacity, meaning they could be used as part of a communication system to send quantum states reliably.
Slow gas microflow past a sphere: Analytical solution based on moment equations
Authors: Torrilhon Manuel
Published: July 2010

The regularized 13-moment equations are solved analytically for the microflow of a gas past a sphere in the case of low Mach numbers. The result is given in fully explicit expressions and shows nontrivial behavior for all fluid fields including stress, heat flux, and temperature. Various aspects of the flow such as temperature polarization and total force are reproduced correctly for moderate Knudsen number. The analytical solution allows studying the rise of Knudsen layers and their interaction and coupling to the fluid variables in the bulk. Additionally, based on the regularized 13-moment equations system, hybrid boundary conditions are given for the standard Stokes equations in order to enable them to predict nonequilibrium effects in the flow past a sphere.
Short vertical tube furnace for the fabrication of doped glass microsphere lasers
Authors: Ward Jonathan M. , Wu Yuqiang , Khalfi Krimo , Chormaic Síle Nic
Published: July 2010

We report on the design of an electric tube furnace that can be used for the fabrication of doped glass microsphere lasers. The tube furnace has a short hot zone of length 133 mm and is based on a quartz tube design. Doped laser glass particles, specifically Er:Yb phosphate glass (IOG-2), of ∼1 μm diameter are blown into the furnace using a 60 ml syringe and microspheres ranging in size from 10 to 400 μm are collected at the output of the tube furnace in a Petri dish. The furnace operates at a wall temperature of ∼900 °C and is capable of making microspheres from glasses with glass transition temperatures of at least 375 °C. High quality (Q∼105) whispering gallery modes have been excited within the microspheres by optically pumping at 978 nm via a tapered optical fiber.