Science.gov

Sample records for active site glutamate

  1. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

    PubMed

    Barinka, Cyril; Sácha, Pavel; Sklenár, Jan; Man, Petr; Bezouska, Karel; Slusher, Barbara S; Konvalinka, Jan

    2004-06-01

    Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question. PMID:15152093

  2. Structural basis for an atypical active site of an L-aspartate/glutamate-specific racemase from Escherichia coli.

    PubMed

    Ahn, Jae-Woo; Chang, Jeong Ho; Kim, Kyung-Jin

    2015-12-21

    We determined the crystal structure of EcL-DER to elucidate protein function and substrate specificity. Unlike other asp/glu racemases, EcL-DER has an unbalanced pair of catalytic residues, Thr83/Cys197, at the active site that is crucial for L- to D-unidirectional racemase activity. EcL-DER exhibited racemase activity for both L-glutamate and L-aspartate, but had threefold higher activity for L-glutamate. Based on the structure of the EcL-DER(C197S) mutant in complex with L-glutamate, we determined the binding mode of the L-glutamate substrate in EcL-DER and provide a structural basis for how the protein utilizes L-glutamate as a main substrate. The unidirectionality, despite an equilibrium constant of unity, can be understood in terms of the Haldane relationship. PMID:26555188

  3. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity. PMID:27443004

  4. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    SciTech Connect

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J.

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  5. Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents.

    PubMed

    Kozikowski, Alan P; Zhang, Jiazhong; Nan, Fajun; Petukhov, Pavel A; Grajkowska, Ewa; Wroblewski, Jarda T; Yamamoto, Tatsuo; Bzdega, Tomasz; Wroblewska, Barbara; Neale, Joseph H

    2004-03-25

    The neuropeptidase glutamate carboxypeptidase II (GCPII) hydrolyzes N-acetyl-L-aspartyl-L-glutamate (NAAG) to liberate N-acetylaspartate and glutamate. GCPII was originally cloned as PSMA, an M(r) 100,000 type II transmembrane glycoprotein highly expressed in prostate tissues. PSMA/GCPII is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. Inhibition of brain GCPII may have therapeutic potential in the treatment of certain disease states arising from pathologically overactivated glutamate receptors. Recently, we reported that certain urea-based structures act as potent inhibitors of GCPII (J. Med. Chem. 2001, 44, 298). However, many of the potent GCPII inhibitors prepared to date are highly polar compounds and therefore do not readily penetrate the blood-brain barrier. Herein, we elaborate on the synthesis of a series of potent, urea-based GCPII inhibitors from the lead compound 3 and provide assay data for these ligands against human GCPII. Moreover, we provide data revealing the ability of one of these compounds, namely, 8d, to reduce the perception of inflammatory pain. Within the present series, the gamma-tetrazole bearing glutamate isostere 7d is the most potent inhibitor with a K(i) of 0.9 nM. The biological evaluation of these compounds revealed that the active site of GCPII likely comprises two regions, namely, the pharmacophore subpocket and the nonpharmacophore subpocket. The pharmacophore subpocket is very sensitive to structural changes, and thus, it appears important to keep one of the glutamic acid moieties intact to maintain the potency of the GCPII inhibitors. The site encompassing the nonpharmacophore subpocket that binds to glutamate's alpha-carboxyl group is sensitive to structural change, as shown by compounds 6b and 7b. However, the other region of the nonpharmacophore subpocket can accommodate both hydrophobic and hydrophilic groups. Thus, an aromatic ring can be introduced to the

  6. Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate.

    PubMed

    Lummis, Sarah C R; Fletcher, Elizabeth J; Green, Tim

    2002-03-01

    Activation of N-methyl-d-aspartate (NMDA) receptors requires the binding of both glutamate and glycine to independent sites on the receptor. These ligands bind to NR2 and NR1 subunits respectively. Ligand binding residues are located in two non-contiguous domains, S1 and S2, which have been implicated in glutamate binding in other ionotropic glutamate receptor subunits. To further define the amino acids through which glutamate activates the receptor, we generated single-site mutations to the NR2A subunit, and expressed them with wild type NR1 in HEK 293 cells. Using calcium imaging and whole cell patch clamp we determined glutamate and glycine potencies. Of the eight residues mutated we identified five (E413, K484, A508, G685 and G688), whose mutation leads to a large reduction (from 4- to 1000-fold) in glutamate potency, consistent with a role for these residues in receptor activation by glutamate. The potency of glycine was largely unchanged by these mutations. Thus our results extend the knowledge base of residues involved in NMDA receptor function and identifies a new site in S1, in the region of A508, that has a role in receptor activation by glutamate. PMID:11955515

  7. Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2003-05-20

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase. PMID:12741843

  8. The structure of putative N-acetyl glutamate kinase from Thermus thermophilus reveals an intermediate active site conformation of the enzyme.

    PubMed

    Sundaresan, Ramya; Ragunathan, Preethi; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2012-04-13

    The de novo biosynthesis of arginine in microorganisms and plants is accomplished via several enzymatic steps. The enzyme N-acetyl glutamate kinase (NAGK) catalyzes the phosphorylation of the γ-COO(-) group of N-acetyl-L-glutamate (NAG) by adenosine triphosphate (ATP) which is the second rate limiting step in arginine biosynthesis pathway. Here we report the crystal structure of putative N-acetyl glutamate kinase (NAGK) from Thermus thermophilus HB8 (TtNAGK) determined at 1.92Å resolution. The structural analysis of TtNAGK suggests that the dimeric quaternary state of the enzyme and arginine insensitive nature are similar to mesophilic Escherichia coli NAGK. These features are significantly different from its thermophilic homolog Thermatoga maritima NAGK which is hexameric and arginine-sensitive. TtNAGK is devoid of its substrates but contains two sulfates at the active site. Very interestingly the active site of the enzyme adopts a conformation which is not completely open or closed and likely represents an intermediate stage in the catalytic cycle unlike its structural homologs, which all exist either in the open or closed conformation. Engineering arginine biosynthesis pathway enzymes for the production of l-arginine is an important industrial application. The structural comparison of TtNAGK with EcNAGK revealed the structural basis of thermostability of TtNAGK and this information could be very useful to generate mutants of NAGK with increased overall stability. PMID:22452987

  9. Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase.

    PubMed

    Pekkala, Satu; Martínez, Ana I; Barcelona, Belén; Gallego, José; Bendala, Elena; Yefimenko, Igor; Rubio, Vicente; Cervera, Javier

    2009-12-01

    NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central beta-sheet and two alpha-helices, opens at the beta-sheet C-edge and is roofed by a three-residue lid. It can tightly accommodate one extended NAG molecule having the delta-COO- at the pocket entry, the alpha-COO- and acetamido groups tightly hydrogen bonded to the pocket, and the terminal methyl of the acetamido substituent surrounded by hydrophobic residues. This binding mode is supported by the observation of reduced NAG affinity upon mutation of NAG-interacting residues of CPSI (recombinantly expressed using baculovirus/insect cells); by the fine-mapping of the N-chloroacetyl-L-glutamate photoaffinity labelling site of CPSI; and by previously established structure-activity relationships for NAG analogues. The location of the NAG site is identical to that of the weak bacterial CPS activator IMP (inosine monophosphate) in Escherichia coli CPS, indicating a common origin for these sites and excluding any relatedness to the binding site of the other bacterial CPS activator, ornithine. Our findings open the way to the identification of CPSI deficiency patients carrying NAG site mutations, and to the possibility of tailoring the activator to fit a given NAG site mutation, as exemplified here with N-acetyl-L(+/-)-beta-phenylglutamate for the W1410K CPSI mutation. PMID:19754428

  10. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  11. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    SciTech Connect

    Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi; Hiratake, Jun; Wada, Kei

    2014-02-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.

  12. NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    PubMed Central

    Benediktsson, A.M.; Marrs, G.S.; Tu, J.C.; Worley, P.F.; Rothstein, J.D.; Bergles, D.E.; Dailey, M.E.

    2011-01-01

    Glutamate transporters maintain a low ambient level of glutamate in the CNS and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here we examined the subcellular distribution and dynamic remodeling of the predominant glutamate transporter GLT-1 (EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. GFP-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse 3D confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of glutamate transporters in developing astrocyte membranes and their position relative to synapses. PMID:22052455

  13. Glutamate Mediated Astrocytic Filtering of Neuronal Activity

    PubMed Central

    Herzog, Nitzan; De Pittà, Maurizio; Jacob, Eshel Ben; Berry, Hugues; Hanein, Yael

    2014-01-01

    Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity. PMID:25521344

  14. Nicotine recruits glutamate receptors to postsynaptic sites.

    PubMed

    Duan, Jing-Jing; Lozada, Adrian F; Gou, Chen-Yu; Xu, Jing; Chen, Yuan; Berg, Darwin K

    2015-09-01

    Cholinergic neurons project throughout the nervous system and activate nicotinic receptors to modulate synaptic function in ways that shape higher order brain function. The acute effects of nicotinic signaling on long-term synaptic plasticity have been well-characterized. Less well understood is how chronic exposure to low levels of nicotine, such as those encountered by habitual smokers, can alter neural connections to promote addiction and other lasting behavioral effects. We show here that chronic exposure of hippocampal neurons in culture to low levels of nicotine recruits AMPA and NMDA receptors to the cell surface and sequesters them at postsynaptic sites. The receptors include GluA2-containing AMPA receptors, which are responsible for most of the excitatory postsynaptic current mediated by AMPA receptors on the neurons, and include NMDA receptors containing GluN1 and GluN2B subunits. Moreover, we find that the nicotine treatment also increases expression of the presynaptic component synapsin 1 and arranges it in puncta juxtaposed to the additional AMPA and NMDA receptor puncta, suggestive of increases in synaptic contacts. Consistent with increased synaptic input, we find that the nicotine treatment leads to an increase in the excitatory postsynaptic currents mediated by AMPA and NMDA receptors. Further, the increases skew the ratio of excitatory-to-inhibitory input that the cell receives, and this holds both for pyramidal neurons and inhibitory neurons in the hippocampal CA1 region. The GluN2B-containing NMDA receptor redistribution at synapses is associated with a significant increase in GluN2B phosphorylation at Tyr1472, a site known to prevent GluN2B endocytosis. These results suggest that chronic exposure to low levels of nicotine not only alters functional connections but also is likely to change excitability levels across networks. Further, it may increase the propensity for synaptic plasticity, given the increase in synaptic NMDA receptors. PMID

  15. Activation Requirements for Metabotropic Glutamate Receptors

    PubMed Central

    Viaene, Angela N.; Petrof, Iraklis; Sherman, S. Murray

    2013-01-01

    It has been common experimentally to use high frequency, tetanic, stimulation to activate metabotropic glutamate receptors (mGluRs) in cortex and thalamus. To determine what type of stimulation is actually necessary to activate mGluRs we examined the effects of varying stimulation duration and intensity on activating mGluR responses. We used a thalamocortical and an intracortical slice preparation from mice and performed whole cell recordings from neurons in the ventral posterior medial nucleus or in layer 4 of primary somatosensory cortex (S1) while electrically stimulating in layer 6 of S1. Extracellular ionotropic glutamate receptor antagonists and GABAA receptor antagonists were used to isolate Group I or Group II mGluR responses. We observed that high frequency stimulation is not necessary for the activation of either Group I or Group II mGluRs. Either could be activated with as few as 2-3 pulses at stimulation frequencies around 15-20Hz. Additionally, increasing the number of pulses, intensity of stimulation, or stimulation frequency increased amplitude and duration of the mGluR response. PMID:23416319

  16. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  17. Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS.

    PubMed

    Terzyan, Simon S; Burgett, Anthony W G; Heroux, Annie; Smith, Clyde A; Mooers, Blaine H M; Hanigan, Marie H

    2015-07-10

    γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use. PMID:26013825

  18. On the ionization state of the substrate in the active site of glutamate racemase. A QM/MM study about the importance of being zwitterionic.

    PubMed

    Puig, Eduard; Garcia-Viloca, Mireia; González-Lafont, Angels; Lluch, José M

    2006-01-19

    Computer simulations on a QM/MM potential energy surface have been carried out to gain insights into the catalytic mechanism of glutamate racemase (MurI). Understanding such a mechanism is a challenging task from the chemical point of view because it involves the deprotonation of a low acidic proton by a relatively weak base to give a carbanionic intermediate. First, we have examined the dependency of the kinetics and thermodynamics of the racemization process catalyzed by MurI on the ionization state of the substrate (glutamate) main chain. Second, we have employed an energy decomposition procedure to study the medium effect on the enzyme-substrate electrostatic and polarization interactions along the reaction. Importantly, the present theoretical results quantitatively support the mechanistic proposal by Rios et al. [J. Am. Chem. Soc. 2000, 122, 9373-9385] for the PLP-independent amino acid racemases. PMID:16405345

  19. Modeling of slow glutamate diffusion and AMPA receptor activation in the cerebellar glomerulus.

    PubMed

    Saftenku, E E

    2005-06-01

    Synaptic conductances are influenced markedly by the geometry of the space surrounding the synapse since the transient glutamate concentration in the synaptic cleft is determined by this geometry. Our paper is an attempt to understand the reasons for slow glutamate diffusion in the cerebellar glomerulus, a structure situated around the enlarged mossy fiber terminal in the cerebellum and surrounded by a glial sheath. For this purpose, analytical expressions for glutamate diffusion in the glomerulus were considered in models with two-, three-, and fractional two-three-dimensional (2D-3D) geometry with an absorbing boundary. The time course of average glutamate concentration in the synaptic cleft of the mossy fiber-granule cell connection was calculated for both direct release of glutamate from the same synaptic unit, and for cumulative spillover of glutamate from neighboring release sites. Several kinetic schemes were examined, and the parameters of the diffusion models were estimated by identifying theoretical activation of AMPA receptors with direct release and spillover components of published experimental AMPA receptor-mediated EPSCs. For model selection, the correspondence of simulated paired-pulse ratio and EPSC increase after prevention of desensitization to experimental values were also taken into consideration. Our results suggest at least a 7- to 10-fold lower apparent diffusion coefficient of glutamate in the porous medium of the glomerulus than in water. The modeling of glutamate diffusion in the 2D-3D geometry gives the best fit of experimental EPSCs. We show that it could be only partly explained by normal diffusion of glutamate in the complex geometry of the glomerulus. We assume that anomalous diffusion of glutamate occurs in the glomerulus. A good match of experimental estimations and theoretical parameters, obtained in the simulations that use an approximation of anomalous diffusion by a solution for fractional Brownian motion, confirms our

  20. Extrasynaptic Glutamate Receptor Activation as Cellular Bases for Dynamic Range Compression in Pyramidal Neurons

    PubMed Central

    Oikonomou, Katerina D.; Short, Shaina M.; Rich, Matthew T.; Antic, Srdjan D.

    2012-01-01

    Repetitive synaptic stimulation overcomes the ability of astrocytic processes to clear glutamate from the extracellular space, allowing some dendritic segments to become submerged in a pool of glutamate, for a brief period of time. This dynamic arrangement activates extrasynaptic NMDA receptors located on dendritic shafts. We used voltage-sensitive and calcium-sensitive dyes to probe dendritic function in this glutamate-rich location. An excess of glutamate in the extrasynaptic space was achieved either by repetitive synaptic stimulation or by glutamate iontophoresis onto the dendrites of pyramidal neurons. Two successive activations of synaptic inputs produced a typical NMDA spike, whereas five successive synaptic inputs produced characteristic plateau potentials, reminiscent of cortical UP states. While NMDA spikes were coupled with brief calcium transients highly restricted to the glutamate input site, the dendritic plateau potentials were accompanied by calcium influx along the entire dendritic branch. Once initiated, the glutamate-mediated dendritic plateau potentials could not be interrupted by negative voltage pulses. Activation of extrasynaptic NMDA receptors in cellular compartments void of spines is sufficient to initiate and support plateau potentials. The only requirement for sustained depolarizing events is a surplus of free glutamate near a group of extrasynaptic receptors. Highly non-linear dendritic spikes (plateau potentials) are summed in a highly sublinear fashion at the soma, revealing the cellular bases of signal compression in cortical circuits. Extrasynaptic NMDA receptors provide pyramidal neurons with a function analogous to a dynamic range compression in audio engineering. They limit or reduce the volume of “loud sounds” (i.e., strong glutamatergic inputs) and amplify “quiet sounds” (i.e., glutamatergic inputs that barely cross the dendritic threshold for local spike initiation). Our data also explain why consecutive cortical UP

  1. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  2. Quantitative autoradiographic distribution of L-(3H)glutamate-binding sites in rat central nervous system

    SciTech Connect

    Greenamyre, J.T.; Young, A.B.; Penney, J.B.

    1984-08-01

    Quantitative autoradiography was used to determine the distribution of L-(3H)glutamate-binding sites in the rat central nervous system. Autoradiography was carried out in the presence of Cl- and Ca2+ ions. Scatchard plots and Hill coefficients of glutamate binding suggested that glutamate was interacting with a single population of sites having a K-D of about 300 nM and a capacity of 14.5 pmol/mg of protein. In displacement studies, ibotenate also appeared to bind to a single class of non-interacting sites with a KI of 28 microM. However, quisqualate displacement of (3H)glutamate binding revealed two well-resolved sites with KIS of 12 nM and 114 microM in striatum. These sites were unevenly distributed, representing different proportions of specific glutamate binding in different brain regions. The distribution of glutamate-binding sites correlated very well with the projection areas of putative glutamatergic pathways. This technique provides an extremely sensitive assay which can be used to gather detailed pharmacological and anatomical information about L-(3H)glutamate binding in the central nervous system.

  3. Structure-activity relationships of glutamate carboxypeptidase II (GCPII) inhibitors.

    PubMed

    Ferraris, D V; Shukla, K; Tsukamoto, T

    2012-01-01

    Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc metallopeptidase that hydrolyzes N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate in the nervous system. Inhibition of GCPII has the potential to reduce extracellular glutamate and represents an opportune target for treating neurological disorders in which excess glutamate is considered pathogenic. Furthermore, GCPII was found to be identical to a tumor marker, prostate-specific membrane antigen (PSMA), and has drawn significant interest as a diagnostic and/or therapeutic target in oncology. Over the past 15 years, tremendous efforts have been made in the discovery of potent GCPII inhibitors, particularly those with phosphorus-, urea- and thiol-based zinc binding groups. In addition, significant progress has been made in understanding the three-dimensional structural characteristics of GCPII in complex with various ligands. The purpose of this review article is to analyze the structure-activity relationships (SAR) of GCPII inhibitors reported to date, which are classified on the basis of their zinc-binding group. SAR and crystallographic data are evaluated in detail for each of these series to highlight the future challenges and opportunities to identify clinically viable GCPII inhibitors. PMID:22304717

  4. S1 pocket of glutamate carboxypeptidase II: a new binding site for amyloid-β degradation.

    PubMed

    Lee, Suk Kyung; Kim, Hyunyoung; Cheong, You-Hoon; Kim, Min-Ju; Jo, Sangmee Ahn; Youn, Hyung-Seop; Park, Sang Ick

    2013-09-01

    We recently reported that glutamate carboxypeptidase II (GCPII) has a new physiological function degrading amyloid-β (Aβ), distinct from its own hydrolysis activity in N-acetyl-L-aspartyl-L-glutamate (NAAG); however, its underlying mechanism remains undiscovered. Using site-directed mutagenesis and S1 pocket-specific chemical inhibitor (compound 2), which was developed for the present study based on in sillico computational modeling, we discovered that the Aβ degradation occurs through S1 pocket but not through S1' pocket responsible for NAAG hydrolysis. Treatment with compound 2 prevented GCPII from Aβ degradation without any impairment in NAAG hydrolysis. Likewise, 2-PMPA (specific GCPII inhibitor developed targeting S1' pocket) completely blocked the NAAG hydrolysis without any effect on Aβ degradation. Pre-incubation with NAAG and Aβ did not affect Aβ degradation and NAAG hydrolysis, respectively. These data suggest that GCPII has two distinctive binding sites for two different substrates and that Aβ degradation occurs through binding to S1 pocket of GCPII. PMID:23891752

  5. Binding sites for L-(/sup 3/H)glutamate in hippocampus

    SciTech Connect

    Werling, L.L.

    1983-01-01

    Three binding sites for L-(/sup 3/H)glutamate on freshly-prepared hippocampal synaptic membranes were identified on the basis of their differing affinities for L-glutamate or quisqualate. The high affinity site yielded K/sub D/ and B/sub max/ values of 12 nM and 2.5 pmol/mg protein, respectively. Binding sites of lower affinity had K/sub D/ values of 200 nM (GLU A) and 1 ..mu..M (GLU B) and B/sub max/ values of about 30 and 60 pmol/mg protein, respectively. GLU A sites bound quisqualate with about 70 times the affinity fo GLU B sites, and thus quisoqualate could be used as a tool to discriminate them. Hill slopes indicated that each site represented a single population of non-interacting binding sites. Freezing drastically decreased GLU A binding, but nearly tripled GLU B binding. Both sites bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-asparate and discriminated poorly between L- and D-asparate. In contrast, the GLU B site bound L-aspartate with similar affinity to L-gluamate, and with much higher affinity than it bound D-aspartate. Both lesions of perforant path and destruction of the granule cells with colchicine markedly reduced radioligand binding to the GLU A site in the fascia dentata, but only the perforant path lesion significantly reduced binding to the GLU B site. The structural specificity of the GLU A site is consistent with its identification as a type of quisqualate receptor.

  6. Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

    PubMed

    Navrátil, Michal; Ptáček, Jakub; Šácha, Pavel; Starková, Jana; Lubkowski, Jacek; Bařinka, Cyril; Konvalinka, Jan

    2014-07-01

    In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical. PMID:24863754

  7. Tolerance to LSD and DOB induced shaking behaviour: differential adaptations of frontocortical 5-HT(2A) and glutamate receptor binding sites.

    PubMed

    Buchborn, Tobias; Schröder, Helmut; Dieterich, Daniela C; Grecksch, Gisela; Höllt, Volker

    2015-03-15

    Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25 mg/kg, i.p.) induce a ketanserin-sensitive (0.5 mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4 ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive [(3)H]spiroperidol and DOB induced [(35)S]GTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive [(3)H]glutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet [(3)H]glutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged [(3)H]glutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in [(3)H]glutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex. PMID:25513973

  8. Activity-Dependent Plasticity of Astroglial Potassium and Glutamate Clearance

    PubMed Central

    Cheung, Giselle; Sibille, Jérémie; Zapata, Jonathan; Rouach, Nathalie

    2015-01-01

    Recent evidence has shown that astrocytes play essential roles in synaptic transmission and plasticity. Nevertheless, how neuronal activity alters astroglial functional properties and whether such properties also display specific forms of plasticity still remain elusive. Here, we review research findings supporting this aspect of astrocytes, focusing on their roles in the clearance of extracellular potassium and glutamate, two neuroactive substances promptly released during excitatory synaptic transmission. Their subsequent removal, which is primarily carried out by glial potassium channels and glutamate transporters, is essential for proper functioning of the brain. Similar to neurons, different forms of short- and long-term plasticity in astroglial uptake have been reported. In addition, we also present novel findings showing robust potentiation of astrocytic inward currents in response to repetitive stimulations at mild frequencies, as low as 0.75 Hz, in acute hippocampal slices. Interestingly, neurotransmission was hardly affected at this frequency range, suggesting that astrocytes may be more sensitive to low frequency stimulation and may exhibit stronger plasticity than neurons to prevent hyperexcitability. Taken together, these important findings strongly indicate that astrocytes display both short- and long-term plasticity in their clearance of excess neuroactive substances from the extracellular space, thereby regulating neuronal activity and brain homeostasis. PMID:26346563

  9. Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors.

    PubMed

    Rovira, Xavier; Malhaire, Fanny; Scholler, Pauline; Rodrigo, Jordi; Gonzalez-Bulnes, Patricia; Llebaria, Amadeu; Pin, Jean-Philippe; Giraldo, Jesús; Goudet, Cyril

    2015-01-01

    Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes. PMID:25342125

  10. Structural mechanism of glutamate receptor activation and desensitization.

    PubMed

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  11. Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is mediated by protein phosphatase-2A.

    PubMed Central

    Gaussin, V; Hue, L; Stalmans, W; Bollen, M

    1996-01-01

    The activation of hepatic acetyl-CoA carboxylase by Na(+)-cotransported amino acids such as glutamine has been attributed mainly to the stimulation of its dephosphorylation by accumulating dicarboxylic acids, e.g. glutamate. We report here on a hepatic species of protein phosphatase-2A that activates acetyl-CoA carboxylase in the presence of physiological concentrations of glutamate or Mg2+ and, under these conditions, accounts for virtually all the hepatic acetyl-CoA carboxylase phosphatase activity. Glutamate also stimulated the dephosphorylation of a synthetic pentadecapeptide encompassing the Ser-79 phosphorylation site of rat acetyl-CoA carboxylase, but did not affect the dephosphorylation of other substrates such as phosphorylase. Conversely, protamine, which stimulated the dephosphorylation of phosphorylase, inhibited the activation of acetyl-CoA carboxylase. A comparison with various species of muscle protein phosphatase-2A showed that the stimulatory effects of glutamate and Mg2+ on the acetyl-CoA carboxylase phosphatase activity are largely mediated by the regulatory A subunit. Glutamate and Mg2+ emerge from our study as novel regulators of protein phosphatase-2A when acting on acetyl-CoA carboxylase. PMID:8645208

  12. Serotonin impairs copulation and attenuates ejaculation-induced glutamate activity in the medial preoptic area.

    PubMed

    Dominguez, Juan M; Hull, Elaine M

    2010-08-01

    The medial preoptic area (MPOA) is critical for male sexual behavior. Glutamate is released in the MPOA of male rats during copulation, and increasing glutamate levels by reverse dialysis of glutamate uptake inhibitors facilitates mating. Conversely, increased release of serotonin (5-HT) inhibits sexual behavior. In both rats and men, selective serotonin reuptake inhibitors (SSRIs) impair erection, ejaculation, and libido. Here we reverse-dialyzed 5-HT through concentric microdialysis probes in the MPOA of male rats; concurrently we collected 2-min samples for analysis of glutamate and measured sexual behavior. Sexual activity, and especially ejaculation, increased levels of glutamate in the MPOA. However, reverse dialysis of 5-HT into the MPOA impaired ejaculatory ability and attenuated glutamate release. Implications of these results for impairment of sexual behavior that results from administration of SSRIs are discussed. PMID:20695654

  13. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors

    PubMed Central

    Gautier, Hélène O. B.; Evans, Kimberley A.; Volbracht, Katrin; James, Rachel; Sitnikov, Sergey; Lundgaard, Iben; James, Fiona; Lao-Peregrin, Cristina; Reynolds, Richard; Franklin, Robin J. M.; Káradóttir, Ragnhildur T

    2015-01-01

    Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination. PMID:26439639

  14. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors.

    PubMed

    Gautier, Hélène O B; Evans, Kimberley A; Volbracht, Katrin; James, Rachel; Sitnikov, Sergey; Lundgaard, Iben; James, Fiona; Lao-Peregrin, Cristina; Reynolds, Richard; Franklin, Robin J M; Káradóttir, Ragnhildur T

    2015-01-01

    Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination. PMID:26439639

  15. Autoradiographic characterization of L-(/sup 3/H)glutamate binding sites in the central nervous system

    SciTech Connect

    Greenamyre, J.T.

    1986-01-01

    A quantitative autoradiographic technique was developed to study L-(/sup 3/H(glutamate binding in sections of central nervous system tissue. This technique circumvented some problems associated with conventional receptor binding methodologies and allowed direct assessment of regional distribution, numbers and affinities of glutamate binding sites. The sensitivity and high degree of anatomical resolution attainable by autoradiography obviated the need for pooled samples of microdissected specimens. Under assay conditions, (/sup 4/H)glutamate bound rapidly and reversibly to sections of rat brain and was not metabolized appreciably. The distribution of glutamate binding sites corresponded to the projection areas of putative glutamatergic pathways. Thus, there was heavy glutamate binding in regions where there is evidence for glutamatergic innervation and little binding in nuclei which apparently do not receive glutamatergic input. Scatchard and Hill plots suggested that glutamate was interacting with a single population of sites; however, competition studies revealed binding site heterogeneity. Anatomical and pharmacological evidence suggested that the NMDA-, high affinity quisqualate-, and kainate-sensitive glutamate binding sites may correspond to physiologically-defined NMDA, quisqualate and kainate receptors.

  16. Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor.

    PubMed

    Gorostiza, Pau; Volgraf, Matthew; Numano, Rika; Szobota, Stephanie; Trauner, Dirk; Isacoff, Ehud Y

    2007-06-26

    The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry. PMID:17578923

  17. Acute Modulation of Cortical Glutamate and GABA Content by Physical Activity.

    PubMed

    Maddock, Richard J; Casazza, Gretchen A; Fernandez, Dione H; Maddock, Michael I

    2016-02-24

    Converging evidence demonstrates that physical activity evokes a brain state characterized by distinctive changes in brain metabolism and cortical function. Human studies have shown that physical activity leads to a generalized increase in electroencephalography power across regions and frequencies, and a global increase in brain nonoxidative metabolism of carbohydrate substrates. This nonoxidative consumption of carbohydrate has been hypothesized to include increased de novo synthesis of amino acid neurotransmitters, especially glutamate and GABA. Here, we conducted a series of proton magnetic resonance spectroscopy studies in human volunteers before and after vigorous exercise (≥80% of predicted maximal heart rate). Results showed that the resonance signals of both glutamate and GABA increased significantly in the visual cortex following exercise. We further demonstrated a similar increase in glutamate following exercise in an executive region, the anterior cingulate cortex. The increase in glutamate was similar when using echo times of 30 and 144 ms, indicating that exercise-related T2 relaxation effects across this range of relaxation times did not account for the findings. In addition, we found preliminary evidence that more physical activity during the preceding week predicts higher resting glutamate levels. Overall, the results are consistent with an exercise-induced expansion of the cortical pools of glutamate and GABA, and add to a growing understanding of the distinctive brain state associated with physical activity. A more complete understanding of this brain state may reveal important insights into mechanisms underlying the beneficial effects of physical exercise in neuropsychiatric disorders, neurorehabilitation, aging, and cognition. PMID:26911692

  18. Maternal inflammation leads to impaired glutamate homeostasis and up-regulation of glutamate carboxypeptidase II in activated microglia in the fetal/newborn rabbit brain.

    PubMed

    Zhang, Zhi; Bassam, Bassam; Thomas, Ajit G; Williams, Monica; Liu, Jinhuan; Nance, Elizabeth; Rojas, Camilo; Slusher, Barbara S; Kannan, Sujatha

    2016-10-01

    Astrocyte dysfunction and excessive activation of glutamatergic systems have been implicated in a number of neurologic disorders, including periventricular leukomalacia (PVL) and cerebral palsy (CP). However, the role of chorioamnionitis on glutamate homeostasis in the fetal and neonatal brains is not clearly understood. We have previously shown that intrauterine endotoxin administration results in intense microglial 'activation' and increased pro-inflammatory cytokines in the periventricular region (PVR) of the neonatal rabbit brain. In this study, we assessed the effect of maternal inflammation on key components of the glutamate pathway and its relationship to astrocyte and microglial activation in the fetal and neonatal New Zealand white rabbit brain. We found that intrauterine endotoxin exposure at gestational day 28 (G28) induced acute and prolonged glutamate elevation in the PVR of fetal (G29, 1day post-injury) and postnatal day 1 (PND1, 3days post-injury) brains along with prominent morphological changes in the astrocytes (soma hypertrophy and retracted processes) in the white matter tracts. There was a significant increase in glutaminase and N-Methyl-d-Aspartate receptor (NMDAR) NR2 subunit expression along with decreased glial L-glutamate transporter 1 (GLT-1) in the PVR at G29, that would promote acute dysregulation of glutamate homeostasis. This was accompanied with significantly decreased TGF-β1 at PND1 in CP kits indicating ongoing neuroinflammation. We also show for the first time that glutamate carboxypeptidase II (GCPII) was significantly increased in the activated microglia at the periventricular white matter area in both G29 and PND1 CP kits. This was confirmed by in vitro studies demonstrating that LPS activated primary microglia markedly upregulate GCPII enzymatic activity. These results suggest that maternal intrauterine endotoxin exposure results in early onset and long-lasting dysregulation of glutamate homeostasis, which may be mediated by

  19. Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

    PubMed Central

    Rinehart, C A; Hubbard, J S

    1976-01-01

    When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate. PMID:956126

  20. Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

    PubMed Central

    McKenna, Mary C.

    2011-01-01

    Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism. PMID:21771624

  1. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    PubMed Central

    Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

  2. Traumatic Brain Injury Increases Cortical Glutamate Network Activity by Compromising GABAergic Control

    PubMed Central

    Cantu, David; Walker, Kendall; Andresen, Lauren; Taylor-Weiner, Amaro; Hampton, David; Tesco, Giuseppina; Dulla, Chris G.

    2015-01-01

    Traumatic brain injury (TBI) is a major risk factor for developing pharmaco-resistant epilepsy. Although disruptions in brain circuitry are associated with TBI, the precise mechanisms by which brain injury leads to epileptiform network activity is unknown. Using controlled cortical impact (CCI) as a model of TBI, we examined how cortical excitability and glutamatergic signaling was altered following injury. We optically mapped cortical glutamate signaling using FRET-based glutamate biosensors, while simultaneously recording cortical field potentials in acute brain slices 2–4 weeks following CCI. Cortical electrical stimulation evoked polyphasic, epileptiform field potentials and disrupted the input–output relationship in deep layers of CCI-injured cortex. High-speed glutamate biosensor imaging showed that glutamate signaling was significantly increased in the injured cortex. Elevated glutamate responses correlated with epileptiform activity, were highest directly adjacent to the injury, and spread via deep cortical layers. Immunoreactivity for markers of GABAergic interneurons were significantly decreased throughout CCI cortex. Lastly, spontaneous inhibitory postsynaptic current frequency decreased and spontaneous excitatory postsynaptic current increased after CCI injury. Our results suggest that specific cortical neuronal microcircuits may initiate and facilitate the spread of epileptiform activity following TBI. Increased glutamatergic signaling due to loss of GABAergic control may provide a mechanism by which TBI can give rise to post-traumatic epilepsy. PMID:24610117

  3. Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes

    SciTech Connect

    Ly, A.M.; Michaelis, E.K. )

    1991-04-30

    L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. ({sup 14}C)Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of ({sup 14}C)methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA{sup +} led to a transient increase in the influx of the lipid-permeable anion probe S{sup 14}CN{sup {minus}}. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the {approximately}69-kDa protein in the function of these ion channels.

  4. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy. PMID:26677078

  5. Enhancement of CA3 hippocampal network activity by activation of group II metabotropic glutamate receptors

    PubMed Central

    Ster, Jeanne; Mateos, José María; Grewe, Benjamin Friedrich; Coiret, Guyllaume; Corti, Corrado; Corsi, Mauro; Helmchen, Fritjof; Gerber, Urs

    2011-01-01

    Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3−/− but not in mGluR2−/− mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity. PMID:21628565

  6. EVALUATING THE NMDA-GLUTAMATE RECEPTOR AS A SITE OF ACTION FOR TOLUENE, IN VIVO

    EPA Science Inventory

    In vitro, toluene disrupts the function of NMDA-glutamate receptors, indicating that effects on NMDA receptor function may contribute to toluene neurotoxicity. NMDA-glutamate receptors are widely present in the visual system and contribute to pattern-elicited visual evoked potent...

  7. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  8. Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum.

    PubMed

    Xu, Meijuan; Rao, Zhiming; Dou, Wenfang; Jin, Jian; Xu, Zhenghong

    2012-02-01

    Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-L-glutamate (NAG) to N-acety-L-glutamy-L-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the L-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the L-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I₀.₅ (R) 50-60 fold, and the G287D and R209A mutants could increase the I₀.₅ (R) 30-40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition. PMID:22101454

  9. Activation of metabotropic glutamate receptor 5 in the amygdala modulates pain-like behavior

    PubMed Central

    Kolber, Benedict J.; Montana, Michael C.; Carrasquillo, Yarimar; Xu, Jian; Heinemann, Stephen F.; Muglia, Louis J.; Gereau, Robert W.

    2010-01-01

    The central nucleus of the amygdala (CeA) has been identified as a site of nociceptive processing important for sensitization induced by peripheral injury. However, the cellular signaling components underlying this function remain unknown. Here, we identify metabotropic glutamate receptor 5 (mGluR5) as an integral component of nociceptive processing in the CeA. Pharmacological activation of mGluRs with R,S-3,5-dihydroxyphenylglycine (DHPG) in the CeA of mice is sufficient to induce peripheral hypersensitivity in the absence of injury. DHPG-induced peripheral hypersensitivity is reduced via pharmacological blockade of mGluR5 or genetic disruption of mGluR5. Further, pharmacological blockade or conditional deletion of mGluR5 in the CeA abrogates inflammation-induced hypersensitivity, demonstrating the necessity of mGluR5 in CeA-mediated pain modulation. Moreover, we demonstrate that phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) is downstream of mGluR5 activation in the CeA and is necessary for the full expression of peripheral inflammation-induced behavioral sensitization. Finally, we present evidence of right hemispheric lateralization of mGluR5 modulation of amygdalar nociceptive processing. We demonstrate that unilateral pharmacological activation of mGluR5 in the CeA produces distinct behavioral responses depending on whether the right or left amygdala is injected. We also demonstrate significantly higher levels of mGluR5 expression in the right amygdala compared to the left under baseline conditions, suggesting a potential mechanism for right hemispheric lateralization of amygdala function in pain processing. Taken together, these results establish an integral role for mGluR5 and ERK1/2 in nociceptive processing in the CeA. PMID:20554871

  10. Activation of metabotropic glutamate receptor 5 in the amygdala modulates pain-like behavior.

    PubMed

    Kolber, Benedict J; Montana, Michael C; Carrasquillo, Yarimar; Xu, Jian; Heinemann, Stephen F; Muglia, Louis J; Gereau, Robert W

    2010-06-16

    The central nucleus of the amygdala (CeA) has been identified as a site of nociceptive processing important for sensitization induced by peripheral injury. However, the cellular signaling components underlying this function remain unknown. Here, we identify metabotropic glutamate receptor 5 (mGluR5) as an integral component of nociceptive processing in the CeA. Pharmacological activation of mGluRs with (R,S)-3,5-dihydroxyphenylglycine (DHPG) in the CeA of mice is sufficient to induce peripheral hypersensitivity in the absence of injury. DHPG-induced peripheral hypersensitivity is reduced via pharmacological blockade of mGluR5 or genetic disruption of mGluR5. Furthermore, pharmacological blockade or conditional deletion of mGluR5 in the CeA abrogates inflammation-induced hypersensitivity, demonstrating the necessity of mGluR5 in CeA-mediated pain modulation. Moreover, we demonstrate that phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) is downstream of mGluR5 activation in the CeA and is necessary for the full expression of peripheral inflammation-induced behavioral sensitization. Finally, we present evidence of right hemispheric lateralization of mGluR5 modulation of amygdalar nociceptive processing. We demonstrate that unilateral pharmacological activation of mGluR5 in the CeA produces distinct behavioral responses depending on whether the right or left amygdala is injected. We also demonstrate significantly higher levels of mGluR5 expression in the right amygdala compared with the left under baseline conditions, suggesting a potential mechanism for right hemispheric lateralization of amygdala function in pain processing. Together, these results establish an integral role for mGluR5 and ERK1/2 in nociceptive processing in the CeA. PMID:20554871

  11. Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

    PubMed

    Rueda, Carlos B; Llorente-Folch, Irene; Traba, Javier; Amigo, Ignacio; Gonzalez-Sanchez, Paloma; Contreras, Laura; Juaristi, Inés; Martinez-Valero, Paula; Pardo, Beatriz; Del Arco, Araceli; Satrustegui, Jorgina

    2016-08-01

    Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar

  12. Synthesis and antiproliferative activity of glutamic acid-based dipeptides.

    PubMed

    Silveira-Dorta, Gastón; Martín, Víctor S; Padrón, José M

    2015-08-01

    A small and focused library of 22 dipeptides derived from N,N-dibenzylglutamic acid α- and γ-benzyl esters was prepared in a straightforward manner. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon) provided γ-glutamyl methionine (GI50 = 6.0-41 μM) and α-glutamyl proline (GI50 = 7.5-18 μM) as lead compounds. In particular, glutamyl serine and glutamyl proline dipeptides were more active in the resistant cancer cell line WiDr than the conventional anticancer drugs cisplatin and etoposide. Glutamyl tryptophan dipeptides did not affect cell growth of HBL-100, while in T-47D cells, proliferation was inhibited. This result might be attributed to the inhibition of the ATB(0,+) transporter. PMID:25900811

  13. Brainstem metabotropic glutamate receptors reduce food intake and activate dorsal pontine and medullar structures after peripheral bacterial lipopolysaccharide administration.

    PubMed

    Chaskiel, Léa; Paul, Flora; Gerstberger, Rüdiger; Hübschle, Thomas; Konsman, Jan Pieter

    2016-08-01

    During infection-induced inflammation food intake is reduced. Vagal and brainstem pathways are important both in feeding regulation and immune-to-brain communication. Glutamate is released by vagal afferent terminals in the nucleus of the solitary tract and by its neurons projecting to the parabrachial nuclei. We therefore studied the role of brainstem glutamate receptors in spontaneous food intake of healthy animals and during sickness-associated hypophagia after peripheral administration of bacterial lipopolysaccharides or interleukin-1beta. Brainstem group I and II metabotropic, but not ionotropic, glutamate receptor antagonism increased food intake both in saline- and lipopolysaccharide-treated rats. In these animals, expression of the cellular activation marker c-Fos in the lateral parabrachial nuclei and lipopolysaccharide-induced activation of the nucleus of the solitary tract rostral to the area postrema were suppressed. Group I metabotropic glutamate receptors did not colocalize with c-Fos or neurons regulating gastric function in these structures. Group I metabotropic glutamate receptors were, however, found on raphé magnus neurons that were part of the brainstem circuit innervating the stomach and on trigeminal and hypoglossal motor neurons. In conclusion, our findings show that brainstem metabotropic glutamate receptors reduce food intake and activate the lateral parabrachial nuclei as well as the rostral nucleus of the solitary tract after peripheral bacterial lipopolysaccharide administration. They also provide insight into potential group I metabotropic glutamate receptor-dependent brainstem circuits mediating these effects. PMID:27016016

  14. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    PubMed

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  15. Determination of Glutamate Dehydrogenase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)+ to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD+ or NADP+ on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD+-dependent GDH Vmax was 2.5-fold higher than NADP+-dependent Vmax, whereas the Km was similar, 1.92 mM versus 1.66 mM, when NAD+ or NADP+ was used, respectively. With either coenzyme, Vmax was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a Ki of 12.2 and 3.95 for NAD+ and NADP+ used as coenzyme, respectively. NAD+- and NADP+-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD+ was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD+ as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. PMID:25124006

  16. Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

    PubMed

    Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

    2012-01-01

    Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas. PMID:23077590

  17. Carbon monoxide and Ca2+-activated K+ channels in cerebral arteriolar responses to glutamate and hypoxia in newborn pigs

    PubMed Central

    Kanu, Alie; Leffler, Charles W.

    2008-01-01

    Large conductance calcium activated (KCa) channels regulate the physiological functions of many tissues, including cerebrovascular smooth muscle. L-glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system and oxygen tension is a dominant local regulator of vascular tone. In vivo, glutamate and hypoxia dilate newborn pig cerebral arterioles and both dilations are blocked by inhibition of CO production. CO dilates cerebral arterioles by activating KCa channels. Therefore, the present study was designed to investigate effects of glutamate and hypoxia on cerebral CO production and the role of KCa channels in the cerebral arteriolar dilations to glutamate and hypoxia. In the presence of iberiotoxin or paxilline that block dilation to the KCa channel opener, NS1619, neither CO nor glutamate dilated pial arterioles. Conversely, neither paxilline nor iberiotoxin inhibited dilation to acute severe or moderate prolonged hypoxia. Both glutamate and hypoxia increased CSF CO concentration. Iberiotoxin that blocked dilation to glutamate did not attenuate the increase in CSF CO. The guanylyl cyclase inhibitor, 1H–(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (ODQ), that blocked dilation to sodium nitroprusside did not inhibit dilation to hypoxia. These data suggest that dilation of newborn pig pial arterioles to glutamate is mediated by activation of KCa channels consistent with the intermediary signal being CO. Surprisingly, although heme oxygenase (HO) inhibition attenuates dilation to hypoxia, hypoxia increases CSF CO concentration, and KCa channel antagonists block dilation to CO, neither KCa channel blockers nor ODQ altered dilation to hypoxia suggesting the contribution of the HO/CO system to hypoxia-induced dilation is not by stimulating vascular smooth muscle KCa channels or guanylyl cyclase. PMID:17766483

  18. Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

    PubMed

    Haas, Laura T; Strittmatter, Stephen M

    2016-08-12

    The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling. PMID:27325698

  19. Striatal Synaptosomes from Hdh140Q/140Q Knock-in Mice have Altered Protein Levels, Novel Sites of Methionine Oxidation, and Excess Glutamate Release after Stimulation

    PubMed Central

    Valencia, Antonio; Sapp, Ellen; Kimm, Jeffrey S.; McClory, Hollis; Ansong, Kwadwo A.; Yohrling, George; Kwak, Seung; Kegel, Kimberly B.; Green, Karin M.; Shaffer, Scott A.; Aronin, Neil; DiFiglia, Marian

    2014-01-01

    Background: Synaptic connections are disrupted in patients with Huntington’s disease (HD). Synaptosomes from postmortem brain are ideal for synaptic function studies because they are enriched in pre- and post-synaptic proteins important in vesicle fusion, vesicle release, and neurotransmitter receptor activation. Objective: To examine striatal synaptosomes from 3, 6 and 12 month old WT and Hdh140Q/140Q knock-in mice for levels of synaptic proteins, methionine oxidation, and glutamate release. Methods: We used Western blot analysis, glutamate release assays, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Striatal synaptosomes of 6 month old Hdh140Q/140Q mice had less DARPP32, syntaxin 1 and calmodulin compared to WT. Striatal synaptosomes of 12 month old Hdh140Q/140Q mice had lower levels of DARPP32, alpha actinin, HAP40, Na+/K+-ATPase, PSD95, SNAP-25, TrkA and VAMP1, VGlut1 and VGlut2, increased levels of VAMP2, and modifications in actin and calmodulin compared to WT. More glutamate released from vesicles of depolarized striatal synaptosomes of 6 month old Hdh140Q/140Q than from age matched WT mice but there was no difference in glutamate release in synaptosomes of 3 and 12 month old WT and Hdh140Q/140Q mice. LC-MS/MS of 6 month old Hdh140Q/140Q mice striatal synaptosomes revealed that about 4% of total proteins detected (>600 detected) had novel sites of methionine oxidation including proteins involved with vesicle fusion, trafficking, and neurotransmitter function (synaptophysin, synapsin 2, syntaxin 1, calmodulin, cytoplasmic actin 2, neurofilament, and tubulin). Altered protein levels and novel methionine oxidations were also seen in cortical synaptosomes of 12 month old Hdh140Q/140Q mice. Conclusions: Findings provide support for early synaptic dysfunction in Hdh140Q/140Q knock-in mice arising from altered protein levels, oxidative damage, and impaired glutamate neurotransmission and suggest that study of synaptosomes could be of

  20. Glutamate and GABA contributions to medial prefrontal cortical activity to emotion: implications for mood disorders.

    PubMed

    Stan, Ana D; Schirda, Claudiu V; Bertocci, Michele A; Bebko, Genna M; Kronhaus, Dina M; Aslam, Haris A; LaBarbara, Eduard J; Tanase, Costin; Lockovich, Jeanette C; Pollock, Myrna H; Stiffler, Richelle S; Phillips, Mary L

    2014-09-30

    The dorsomedial prefrontal cortex (MdPFC) and anterior cingulate cortices (ACC) play a critical role in implicit emotion regulation; however the understanding of the specific neurotransmitters that mediate such role is lacking. In this study, we examined relationships between MdPFC concentrations of two neurotransmitters, glutamate and γ-amino butyric acid (GABA), and BOLD activity in ACC during performance of an implicit facial emotion-processing task. Twenty healthy volunteers, aged 20-35 years, were scanned while performing an implicit facial emotion-processing task, whereby presented facial expressions changed from neutral to one of the four emotions: happy, anger, fear, or sad. Glutamate concentrations were measured before and after the emotion-processing task in right MdPFC using magnetic resonance spectroscopy (MRS). GABA concentrations were measured in bilateral MdPFC after the emotion-processing task. Multiple regression models were run to determine the relative contribution of glutamate and GABA concentration, age, and gender to BOLD signal in ACC to each of the four emotions. Multiple regression analyses revealed a significant negative correlation between MdPFC GABA concentration and BOLD signal in subgenual ACC (p<0.05, corrected) to sad versus shape contrast. For the anger versus shape contrast, there was a significant negative correlation between age and BOLD signal in pregenual ACC (p<0.05, corrected) and a positive correlation between MdPFC glutamate concentration (pre-task) and BOLD signal in pregenual ACC (p<0.05, corrected). Our findings are the first to provide insight into relationships between MdPFC neurotransmitter concentrations and ACC BOLD signal, and could further understanding of molecular mechanisms underlying emotion processing in healthy and mood-disordered individuals. PMID:24973815

  1. Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.

    PubMed

    Tanui, Rose; Tao, Zhen; Silverstein, Nechama; Kanner, Baruch; Grewer, Christof

    2016-05-27

    Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse. PMID:27044739

  2. Metabotropic glutamate receptor 5, but not 1, modulates NMDA receptor-mediated activation of neuronal nitric oxide synthase.

    PubMed

    Llansola, Marta; Felipo, Vicente

    2010-03-01

    In cerebellar neurons in culture, activation of group I metabotropic glutamate receptors (mGluRs) prevents glutamate and NMDA-induced neuronal death, indicating that it interferes with the excitotoxic mechanisms leading to death. However, it is not known which step of these mechanisms is affected by mGluRs. The aims of this work were to assess: (a) whether activation of group I mGluRs (mGluR1 or mGluR5) impairs NMDA-induced activation of the glutamate-nitric oxide-cGMP pathway; (b) which mGluR (1 or 5) is responsible for this impairment and (c) whether impairment of the pathway occurs at the level of activation of soluble guanylate cyclase by nitric oxide or of activation of neuronal nitric oxide synthase (nNOS) by NMDA. It is shown that activation of mGluR1 enhances the function of the glutamate-nitric oxide-cGMP pathway by increasing activation of soluble guanylate cyclase by nitric oxide. In contrast, mGluR5 activation inhibits the glutamate-nitric oxide-cGMP pathway by reducing NMDA-induced activation of nNOS. This is due to reduced NMDA-induced increase in cAMP, reduced activation of Akt by cAMP and of nNOS by Akt. The impairment of activation of the glutamate-NO-cGMP pathway by activation of mGluR5 would contribute to its neuroprotective effect against excitotoxicity in cerebellar neurons in culture. PMID:20043967

  3. Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

    PubMed

    Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2014-01-01

    We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition. PMID:25126984

  4. Potentiation of acid-sensing ion channel activity by peripheral group I metabotropic glutamate receptor signaling.

    PubMed

    Gan, Xiong; Wu, Jing; Ren, Cuixia; Qiu, Chun-Yu; Li, Yan-Kun; Hu, Wang-Ping

    2016-05-01

    Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration-response curve upwards, with a 47.3±7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia. PMID:26946972

  5. Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination.

    PubMed

    Ibáñez, Ignacio; Díez-Guerra, F Javier; Giménez, Cecilio; Zafra, Francisco

    2016-08-01

    GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated. PMID:27044663

  6. Inhibition of mammillary body neurons by direct activation of Group II metabotropic glutamate receptors

    PubMed Central

    Lee, Charles C.

    2016-01-01

    The mammillary body is an important neural component of limbic circuitry implicated in learning and memory. Excitatory and inhibitory inputs, primarily mediated by glutamate and gamma-amino butyric acid (GABA), respectively, converge and integrate in this region, before sending information to the thalamus. One potentially overlooked mechanism for inhibition of mammillary body neurons is through direct activation of Group II metabotropic glutamate receptors (mGluRs). Here, whole-cell patch clamp recordings of in vitro slice preparations containing the mammillary body nuclei of the mouse were employed to record responses to bath application of pharmacological agents to isolate the direct effect of activating Group II mGluRs. Application of the Group II mGluR specific agonist, APDC, resulted in a hyperpolarization of the membrane potential in mammillary body neurons, likely resulting from the opening of a potassium conductance. These data suggest that glutamatergic inputs to the mammillary body may be attenuated via Group II mGluRs and implicates a functional role for these receptors in memory-related circuits and broadly throughout the central nervous system. PMID:27390777

  7. Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.

    PubMed

    Al Awabdh, Sana; Gupta-Agarwal, Swati; Sheehan, David F; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E; Griffin, Lewis D; Kittler, Josef T

    2016-07-01

    The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252-1264. PMID:27189737

  8. Pivotal Enzyme in Glutamate Metabolism of Poly-γ-Glutamate-Producing Microbes

    PubMed Central

    Ashiuchi, Makoto; Yamamoto, Takashi; Kamei, Tohru

    2013-01-01

    The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-γ-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess such intracellular sources of D-glutamate. The results of our present study indicate that the D-glutamate-anabolic enzyme “glutamate racemase” is pivotal in the biosynthesis of PGA. PMID:25371338

  9. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

    SciTech Connect

    Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng; Greig, Nigel H.; Mattson, Mark P.; Camandola, Simonetta

    2013-04-19

    Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

  10. Renal sympathetic nerve activity is increased in monosodium glutamate induced hyperadipose rats.

    PubMed

    da Silva Mattos, Alexandro Márcio; Xavier, Carlos Henrique; Karlen-Amarante, Marlusa; da Cunha, Natália Veronez; Fontes, Marco Antonio Peliky; Martins-Pinge, Marli Cardoso

    2012-08-01

    The literature suggests that both obesity and hypertension are associated with increased sympathetic nerve activity. In the present study we evaluated the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) in hyperadipose rats induced by neonatal administration of monosodium glutamate (MSG). Neonatal Wistar male rats were injected with MSG (4 mg/g body weight ID) or equimolar saline (control) for 5 days. At 90th day, all rats were anesthetized (urethane 1.4 g/kg) and prepared for MAP, HR and renal sympathetic nerve activity recordings. The anesthetized MSG rats presented baseline hypertension and increased baseline RSNA compared with control. Our results suggest the involvement of the renal sympathetic nervous system in the physiopathology of the MSG obesity. PMID:22705582

  11. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  12. Alleviating pain hypersensitivity through activation of type 4 metabotropic glutamate receptor.

    PubMed

    Vilar, Bruno; Busserolles, Jérôme; Ling, Bing; Laffray, Sophie; Ulmann, Lauriane; Malhaire, Fanny; Chapuy, Eric; Aissouni, Youssef; Etienne, Monique; Bourinet, Emmanuel; Acher, Francine; Pin, Jean-Philippe; Eschalier, Alain; Goudet, Cyril

    2013-11-27

    Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain. PMID:24285900

  13. Activation of Metabotropic Glutamate Receptors Regulates Ribosomes of Cochlear Nucleus Neurons

    PubMed Central

    Carzoli, Kathryn L.; Hyson, Richard L.

    2014-01-01

    The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity. PMID:25334004

  14. TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

    SciTech Connect

    Piao, L.-H.; Fujita, Tsugumi; Jiang, C.-Y.; Liu Tao; Yue, H.-Y.; Nakatsuka, Terumasa; Kumamoto, Eiichi

    2009-02-20

    We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na{sup +}-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

  15. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    PubMed

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity. PMID:26851652

  16. A Polarographic Study of Glutamate Synthase Activity in Isolated Chloroplasts 1

    PubMed Central

    Anderson, John W.; Done, James

    1977-01-01

    Illuminated pea (Pisum sativum) chloroplasts actively catalyzed (glutamine plus α-ketoglutarate)-dependent O2 evolution (average of 12 preparations 10.6 μmole mg chlorophyll per hour). The reaction was specific for glutamine and α-ketoglutarate; concentrations of 0.2 mm α-ketoglutarate and 0.6 mm glutamine, respectively, effected half-maximum rates of O2 evolution. The reaction was inhibited by 3-(3,4-dichlorophenyl)-1-1-dimethylurea and did not occur in the dark. After osmotic shock chloroplasts did not catalyze O2 evolution. The reaction was inhibited by azaserine and glutamate but not by 10 mm ammonia, 2.5 mm methionine sulfoximine, or 5 mm amino-oxyacetate; addition of amino-oxyacetate together with aspartate inhibited O2 evolution. Arsenate (3 mm) enhanced O2 evolution. The highest molar ratio for O2 evolved per mole of α-ketoglutarate supplied was 0.40; the corresponding values for glutamine in the absence and presence of 3 mm arsenate were 0.20 and 0.24, respectively. The (glutamine plus α-ketoglutarate)-dependent O2 evolution is attributed to photosynthetically coupled glutamate synthase activity and the activity is sufficient to account for the assimilation of inorganic nitrogen. The low molar ratio for glutamine is discussed. Chloroplasts also catalyzed (aspartate plus α-ketoglutarate)-dependent O2 evolution but this reaction was inhibited by 5 mm amino-oxyacetate and it was insensitive to azaserine and methionine sulfoximine. This reaction was attributed to transaminase and photosynthetically coupled malate dehydrogenase activities. PMID:16660092

  17. Overexpression of α-synuclein simultaneously increases glutamate NMDA receptor phosphorylation and reduces glucocerebrosidase activity.

    PubMed

    Yang, Junfeng; Hertz, Ellen; Zhang, Xiaoqun; Leinartaité, Lina; Lundius, Ebba Gregorsson; Li, Jie; Svenningsson, Per

    2016-01-12

    Progressive accumulation of α-synuclein (α-syn)-containing protein aggregates throughout the nervous system is a pathological hallmark of Parkinson's disease (PD). The mechanisms whereby α-syn exerts neurodegeneration remain to be fully understood. Here we show that overexpression of α-syn in transgenic mice leads to increased phosphorylation of glutamate NMDA receptor (NMDAR) subunits NR1 and NR2B in substantia nigra and striatum as well as reduced glucocerebrosidase (GCase) levels. Similarly, molecular studies performed in mouse N2A cells stably overexpressing human α-syn ((α-syn)N2A) showed that phosphorylation states of the same NMDAR subunits were increased, whereas GCase levels and lysosomal GCase activity were reduced. (α-syn)N2A cells showed an increased sensitivity to neurotoxicity towards 6-hydroxydopamine and NMDA. However, wildtype N2A, but not (α-syn)N2A cells, showed a further reduction in viability when co-incubated with 6-hydroxydopamine and the lysosomal inhibitors NH4Cl and leupeptin, suggesting that α-syn per se perturbs lysosomal functions. NMDA treatment reduced lysosomal GCase activity to the same extent in (α-syn)N2A cells as in wildtype N2A cells, indicating that the α-syn-dependent difference in NMDA neurotoxicity is unrelated to an altered GCase activity. Nevertheless, these data provide molecular evidence that overexpression of α-syn simultaneously induces two potential neurotoxic hits by increasing glutamate NMDA receptor phosphorylation, consistent with increased NMDA receptors functionality, and reducing GCase activity. PMID:26610904

  18. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

    PubMed Central

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-01-01

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

  19. Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme–Ligand Promiscuity Revealed at the Atomistic Level

    PubMed Central

    2015-01-01

    Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of l- to d-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered molecular dynamics (MD), docking, and experimental studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-associated changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-associated phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly associated with each of the GRY53/7HC–ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC–ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-associated GR changes are consistent with the experimentally observed quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and

  20. SLC1 Glutamate Transporters

    PubMed Central

    Grewer, Christof; Gameiro, Armanda; Rauen, Thomas

    2014-01-01

    The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 (SLC1) family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na+ ions and the countertransport of one K+ in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions. PMID:24240778

  1. Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function

    PubMed Central

    Nagayach, Aarti; Patro, Nisha; Patro, Ishan

    2014-01-01

    Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes. PMID:25400546

  2. A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  3. Induction of an Olfactory Memory by the Activation of a Metabotropic Glutamate Receptor

    NASA Astrophysics Data System (ADS)

    Kaba, Hideto; Hayashi, Yasunori; Higuchi, Takashi; Nakanishi, Shigetada

    1994-07-01

    Female mice form an olfactory memory of male pheromones at mating; exposure to the pheromones of a strange male after that mating will block pregnancy. The formation of this memory is mediated by the accessory olfactory system, in which an increase in norepinephrine after mating reduces inhibitory transmission of γ-aminobutyric acid from the granule cells to the mitral cells. This study shows that the activation of mGluR2, a metabotropic glutamate receptor that suppresses the γ-aminobutyric acid inhibition of the mitral cells, permits the formation of a specific olfactory memory without the occurrence of mating by infusion of mGluR2 agonists into the female's accessory olfactory bulb. This memory faithfully reflects the memory formed at mating.

  4. Helicobacter pylori γ-Glutamyltranspeptidase Induces Tolerogenic Human Dendritic Cells by Activation of Glutamate Receptors.

    PubMed

    Käbisch, Romy; Semper, Raphaela P; Wüstner, Stefanie; Gerhard, Markus; Mejías-Luque, Raquel

    2016-05-15

    Helicobacter pylori infection is characterized by chronic persistence of the bacterium. Different virulence factors, including H. pylori γ-glutamyltranspeptidase (gGT), have been reported to induce tolerogenicity by reprogramming dendritic cells (DCs). gGT is present in all bacterial isolates, indicating an important role for gGT in the course of infection. In the current study, we have analyzed the effect of H. pylori gGT on human DCs and the subsequent adaptive immune response. We show that glutamate produced due to H. pylori gGT enzymatic activity tolerizes DCs by inhibiting cAMP signaling and dampening IL-6 secretion in response to the infection. Together, our results provide a novel molecular mechanism by which H. pylori manipulates the host's immune response to persist within its host. PMID:27183641

  5. Activation of Group II Metabotropic Glutamate Receptors Induces Depotentiation in Amygdala Slices and Reduces Fear-Potentiated Startle in Rats

    ERIC Educational Resources Information Center

    Lin, Chia-Ho; Lee, Chia-Ching; Huang, Ya-Chun; Wang, Su-Jane; Gean, Po-Wu

    2005-01-01

    There is a close correlation between long-term potentiation (LTP) in the synapses of lateral amygdala (LA) and fear conditioning in animals. We predict that reversal of LTP (depotentiation) in this area of the brain may ameliorate conditioned fear. Activation of group II metabotropic glutamate receptors (mGluR II) with DCG-IV induces…

  6. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

    SciTech Connect

    Zhang Qi; Shen Mi; Ding Mei; Shen Dingding; Ding Fei

    2011-04-01

    Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax. - Research Highlights: >PQQ attenuated glutamate-induced cell apoptosis of cultured hippocampal neurons. >PQQ inhibited glutamate-induced Ca{sup 2+} influx and caspase-3 activity. >PQQ reduced glutamate-induced increase in ROS production. >PQQ affected phosphorylation of Akt and JNK signalings after glutamate injury. >PI3K/Akt was required for neuroprotection of PQQ by modulating Bcl-2/Bax ratio.

  7. Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors.

    PubMed

    Mundell, Stuart J; Pula, Giordano; McIlhinney, R A Jeffrey; Roberts, Peter J; Kelly, Eamonn

    2004-06-15

    In this study we characterized the heterologous desensitization and internalization of the metabotropic glutamate receptor 1 (mGluR1) splice variants mGluR1a and mGluR1b following activation of endogenous G(q/11)-coupled receptors in HEK293 cells. Agonist activation of M1 muscarinic acetylcholine or P2Y1 purinergic receptors triggered the PKC- and CaMKII-dependent internalization of mGluR1a. In co-immunoprecipitation studies, both glutamate and carbachol increased the association of GRK2 with mGluR1a. Co-addition of the protein kinase C (PKC) inhibitor GF109203X and the Ca(2+) calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 blocked the ability of glutamate and carbachol to increase the association of GRK2 with mGluR1a. Glutamate also increased the association of GRK2 with mGluR1b, whereas carbachol did not. However, unlike mGluR1a, glutamate-stimulated association of GRK2 with mGluR1b was not reduced by PKC/CaMKII inhibition. Pretreatment of cells expressing mGluR1a or mGluR1b with carbachol rapidly desensitized subsequent glutamate-stimulated inositol phosphate accumulation. The carbachol-induced heterologous desensitization and internalization of mGluR1a was blocked by LY367385, an mGluR1a antagonist with inverse agonist activity. Furthermore, LY367385 blocked the ability of carbachol to increase the association of GRK2 with mGluR1a. On the other hand, LY367385 had no effect on the carbachol-induced desensitization and internalization of the nonconstitutively active mGluR1b splice variant. These results demonstrate that the internalization of mGluR1a, triggered homologously by glutamate or heterologously by carbachol, is PKC/CaMKII-, GRK2-, arrestin-, and clathrin-dependent and that PKC/CaMKII activation appears to be necessary for GRK2 to associate with mGluR1a. Furthermore, the heterologous desensitization of mGluR1a is dependent upon the splice variant being in an active conformation. PMID:15182196

  8. Metabotropic glutamate receptors inhibit microglial glutamate release

    PubMed Central

    McMullan, Stephen M; Phanavanh, Bounleut; Guo Li, Gary; Barger, Steven W

    2012-01-01

    Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action. PMID:22770428

  9. Orally active glutamate carboxypeptidase II inhibitor 2-MPPA attenuates dizocilpine-induced prepulse inhibition deficits in mice.

    PubMed

    Takatsu, Yuto; Fujita, Yuko; Tsukamoto, Takashi; Slusher, Barbara S; Hashimoto, Kenji

    2011-01-31

    Glutamate carboxypeptidase II (GCP II) is a glial enzyme responsible for the hydrolysis of N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate (NAA). Abnormalities in glutamate neurotransmission are implicated in the pathophysiology of schizophrenia. In this study, we examined the effects of a novel, orally active GCP II inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), on the prepulse inhibition (PPI) deficits after administration of the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine. Oral administration of 2-MPPA (10, 30 or 100mg/kg) significantly attenuated dizocilpine (0.1mg/kg)-induced PPI deficits in mice, in a dose dependent manner. Furthermore, the efficacy of 2-MPPA on dizocilpine-induced PPI deficits was significantly antagonized by pretreatment with the selective group II metabotropic glutamate receptor (mGluR) antagonist LY341495 (1.0mg/kg). In the same model, however, the selective group II mGluR agonist LY354740 (3, 10 or 30 mg/kg) significantly attenuated dizocilpine-induced PPI deficits at only one dose and prepulse intensity. Our findings suggest that GCP II inhibition may be useful therapeutic strategy for schizophrenia. From a mechanistic perspective, while increased NAAG and activation of group II mGluRs may contribute to the therapeutic efficacy of 2-MPPA, it is likely that additional pharmacological activities are also involved. PMID:21093418

  10. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  11. Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase*

    PubMed Central

    Mandal, Papita; Verma, Naveen; Chauhan, Sakshi; Tomar, Raghuvir S.

    2013-01-01

    Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro. PMID:23673664

  12. Activation of group III metabotropic glutamate receptors is neuroprotective in cortical cultures.

    PubMed

    Bruno, V; Copani, A; Bonanno, L; Knoepfel, T; Kuhn, R; Roberts, P J; Nicoletti, F

    1996-08-22

    (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG) and (S)-alpha-methyl-3-carboxyphenylalanine (M3CPA), two novel preferential antagonists of group III metabotropic glutamate (mGlu) receptors, antagonized the neuroprotective activity of L-2-amino-4-phosphono-butanoate (L-AP4) or L-serine-O-phosphate in mice cultured cortical cells exposed to a toxic pulse of N-methyl-D-aspartate. In contrast, MPPG did not influence the neuroprotective activity of the selective group II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxy-cyclopropyl) glycine (DCG-IV). These results indicate that activation of group III mGu receptors exerts neuroprotective activity against excitotoxic neuronal death. At least one of the two major group III mGlu receptor subtypes, i.e. mGlu4 receptor, is expressed by cultured cortical neurons, as shown by immunocytochemical analysis with specific polyclonal antibodies. PMID:8880068

  13. Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

    PubMed Central

    Volpi, Claudia; Mondanelli, Giada; Pallotta, Maria T.; Vacca, Carmine; Iacono, Alberta; Gargaro, Marco; Albini, Elisa; Bianchi, Roberta; Belladonna, Maria L.; Celanire, Sylvain; Mordant, Céline; Heroux, Madeleine; Royer-Urios, Isabelle; Schneider, Manfred; Vitte, Pierre-Alain; Cacquevel, Mathias; Galibert, Laurent; Poli, Sonia-Maria; Solari, Aldo; Bicciato, Silvio; Calvitti, Mario; Antognelli, Cinzia; Puccetti, Paolo; Orabona, Ciriana; Fallarino, Francesca; Grohmann, Ursula

    2016-01-01

    Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild—yet chronic—neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1—but not pertussis toxin, which affects Gi protein-dependent responses—abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis. PMID:26522434

  14. Calmodulin activity regulates group I metabotropic glutamate receptor-mediated signal transduction and synaptic depression.

    PubMed

    Sethna, Ferzin; Zhang, Ming; Kaphzan, Hanoch; Klann, Eric; Autio, Dawn; Cox, Charles L; Wang, Hongbing

    2016-05-01

    Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity-dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation-dependent long-term depression (LTD). Although it has been established that intracellular Ca(2+) and the Gq-regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca(2+) regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca(2+) sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N-[4-aminobutyl]-5-chloro-2-naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5-stimulated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p70-S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist-induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca(2+) /CaM-dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM-CaMK-ERK1/2 signaling cascade suppressed the mGluR1/5-stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca(2+) and protein upregulation and regulating mGluR1/5-mediated synaptic modification. PMID:26864654

  15. Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation.

    PubMed

    Paul, Mila M; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J; Heckmann, Manfred

    2015-01-01

    The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp(69) ). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp(69) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt(KD) ) in wildtype (wt), brp(69) and rab3 null mutants (rab3(rup) ), where Brp is concentrated at a small number of AZs. At wt and rab3(rup) synapses, syt(KD) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt(KD) did not alter EPSC amplitude at brp(69) synapses, but shortened delay and rise time. In fact, following syt(KD) , these kinetic properties were strikingly similar in wt and brp(69) , which supports the notion that Syt protracts release at brp(69) synapses. To gain insight into this surprising role of Syt at brp(69) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At 'tonic' type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt(KD) . Notably, syt(KD) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt(KD) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires

  16. Blockade of gap junction hemichannel protects secondary spinal cord injury from activated microglia-mediated glutamate exitoneurotoxicity.

    PubMed

    Umebayashi, Daisuke; Natsume, Atsushi; Takeuchi, Hideyuki; Hara, Masahito; Nishimura, Yusuke; Fukuyama, Ryuichi; Sumiyoshi, Naoyuki; Wakabayashi, Toshihiko

    2014-12-15

    We previously demonstrated that activated microglia release excessive glutamate through gap junction hemichannels and identified a novel gap junction hemichannel blocker, INI-0602, that was proven to penetrate the blood-brain barrier and be an effective treatment in mouse models of amyotrophic lateral sclerosis and Alzheimer disease. Spinal cord injury causes tissue damage in two successive waves. The initial injury is mechanical and directly causes primary tissue damage, which induces subsequent ischemia, inflammation, and neurotoxic factor release resulting in the secondary tissue damage. These lead to activation of glial cells. Activated glial cells such as microglia and astrocytes are common pathological observations in the damaged lesion. Activated microglia release glutamate, the major neurotoxic factor released into the extracellular space after neural injury, which causes neuronal death at high concentration. In the present study, we demonstrate that reduction of glutamate-mediated exitotoxicity via intraperitoneal administration of INI-0602 in the microenvironment of the injured spinal cord elicited neurobehavioral recovery and extensive suppression of glial scar formation by reducing secondary tissue damage. Further, this intervention stimulated anti-inflammatory cytokines, and subsequently elevated brain-derived neurotrophic factor. Thus, preventing microglial activation by a gap junction hemichannel blocker, INI-0602, may be a promising therapeutic strategy in spinal cord injury. PMID:24588281

  17. Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

    PubMed Central

    Silver, R A; Cull-Candy, S G; Takahashi, T

    1996-01-01

    that practically all of the non-NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/- 0.03, n = 5) at these 'saturated' multi-site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non-stationary fluctuation analysis of EPSCs from 'saturating' multi-site synapses indicated that 170 +/- 40 postsynaptic non-NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/- 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single-site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non-NMDA receptors at cerebellar mossy fibre-granule cell synapses. However, at multi-site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi-site synapses. Images Figure 3 Figure 6 Figure 7 Figure 10 PMID:8814618

  18. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  19. Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

    PubMed

    Kuettner, E Bartholomeus; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

    2008-03-21

    Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. PMID:18258259

  20. Synthetic cathinone MDPV downregulates glutamate transporter subtype I (GLT-1) and produces rewarding and locomotor-activating effects that are reduced by a GLT-1 activator.

    PubMed

    Gregg, Ryan A; Hicks, Callum; Nayak, Sunil U; Tallarida, Christopher S; Nucero, Paul; Smith, Garry R; Reitz, Allen B; Rawls, Scott M

    2016-09-01

    Synthetic cathinones produce dysregulation of monoamine systems, but their effects on the glutamate system and the influence of glutamate on behavioral effects related to cathinone abuse are unknown. A principal regulator of glutamate homeostasis is glutamate transporter subtype 1 (GLT-1), an astrocytic protein that clears glutamate from the extracellular space and influences behavioral effects of established psychostimulants. We hypothesized that repeated administration of the synthetic cathinone, MDPV (3,4-methylenedioxypyrovalerone), would affect GLT-1 expression in the corticolimbic circuit, and that a GLT-1 activator (ceftriaxone, CTX) would reduce rewarding and locomotor-stimulant effects of MDPV in rats. GLT-1 protein expression in the nucleus accumbens (NAcc), but not prefrontal cortex (PFC), was decreased following withdrawal (2, 5 and 10 days) from repeated MDPV treatment, but not immediately after the last MDPV injection. CTX (200 mg/kg) pretreatment did not affect acute locomotor activation produced by MDPV (0.5, 1, 3 mg/kg). However, CTX (200 mg/kg) administered during a 7-day MDPV treatment paradigm attenuated the development of MDPV-induced sensitization of repetitive movements in rats challenged with MDPV following 11 days of drug abstinence. Pretreatment with CTX (200 mg/kg) during a 4-day MDPV (2 mg/kg) conditioned place preference (CPP) paradigm reduced the development of place preference produced by MDPV. The present data demonstrate dysregulation of corticolimbic glutamate transport systems during withdrawal from chronic MDPV exposure, and show that a GLT-1 transporter activator disrupts behavioral effects of MDPV that are related to synthetic cathinone abuse. PMID:27085607

  1. Glutamic acid as anticancer agent: An overview

    PubMed Central

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K.

    2013-01-01

    The objective of the article is to highlight various roles of glutamic acid like endogenic anticancer agent, conjugates to anticancer agents, and derivatives of glutamic acid as possible anticancer agents. Besides these emphases are given especially for two endogenous derivatives of glutamic acid such as glutamine and glutamate. Glutamine is a derivative of glutamic acid and is formed in the body from glutamic acid and ammonia in an energy requiring reaction catalyzed by glutamine synthase. It also possesses anticancer activity. So the transportation and metabolism of glutamine are also discussed for better understanding the role of glutamic acid. Glutamates are the carboxylate anions and salts of glutamic acid. Here the roles of various enzymes required for the metabolism of glutamates are also discussed. PMID:24227952

  2. Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

    PubMed

    Gorter, Jan A; Van Vliet, Erwin A; Proper, Evelien A; De Graan, Pierre N E; Ghijsen, Wim E J M; Lopes Da Silva, Fernando H; Aronica, Eleonora

    2002-01-21

    The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course. PMID:11793340

  3. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    PubMed

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  4. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  5. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    PubMed

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated. PMID:22886112

  6. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  7. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  8. Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Glutamate (Glu) is the major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system, including the gastrointestinal tract (GIT) of cattle. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu (Km = 1 to 4 mM) into synaptic vesicles (S...

  9. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  10. Activation of synaptic group II metabotropic glutamate receptors induces long-term depression at GABAergic synapses in CNS neurons.

    PubMed

    Tang, Zheng-Quan; Liu, Yu-Wei; Shi, Wei; Dinh, Emilie Hoang; Hamlet, William R; Curry, Rebecca J; Lu, Yong

    2013-10-01

    Metabotropic glutamate receptor (mGluR)-dependent homosynaptic long-term depression (LTD) has been studied extensively at glutamatergic synapses in the CNS. However, much less is known about heterosynaptic long-term plasticity induced by mGluRs at inhibitory synapses. Here we report that pharmacological or synaptic activation of group II mGluRs (mGluR II) induces LTD at GABAergic synapses without affecting the excitatory glutamatergic transmission in neurons of the chicken cochlear nucleus. Coefficient of variation and failure rate analysis suggested that the LTD was expressed presynaptically. The LTD requires presynaptic spike activity, but does not require the activation of NMDA receptors. The classic cAMP-dependent protein kinase A signaling is involved in the transduction pathway. Remarkably, blocking mGluR II increased spontaneous GABA release, indicating the presence of tonic activation of mGluR II by ambient glutamate. Furthermore, synaptically released glutamate induced by electrical stimulations that concurrently activated both the glutamatergic and GABAergic pathways resulted in significant and constant suppression of GABA release at various stimulus frequencies (3.3, 100, and 300 Hz). Strikingly, low-frequency stimulation (1 Hz, 15 min) of the glutamatergic synapses induced heterosynaptic LTD of GABAergic transmission, and the LTD was blocked by mGluR II antagonist, indicating that synaptic activation of mGluR II induced the LTD. This novel form of long-term plasticity in the avian auditory brainstem may play a role in the development as well as in temporal processing in the sound localization circuit. PMID:24089501

  11. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity.

    PubMed

    Bal-Price, A; Brown, G C

    2001-09-01

    Glia undergo inflammatory activation in most CNS pathologies and are capable of killing cocultured neurons. We investigated the mechanisms of this inflammatory neurodegeneration using a mixed culture of neurons, microglia, and astrocytes, either when the astrocytes were activated directly with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or LPS/IFN-gamma-activated microglia were added to mixed neuronal cultures. In either case, activated glia caused 75-100% necrotic cell death within 48 hr, which was completely prevented by inhibitors of inducible nitric oxide synthase (iNOS) (aminoguanidine or 1400W). Activated astrocytes or microglia produced nitric oxide (NO) (steady-state level approximately 0.5 microm), which immediately inhibited the cellular respiration of cocultured neurons, as did authentic NO. NO donors also decreased ATP levels and stimulated lactate production by neurons, consistent with NO-induced respiratory inhibition. NO donors or a specific respiratory inhibitor caused rapid (<1 min) release of glutamate from neuronal and neuronal-astrocytic cultures and subsequent neuronal death that was blocked by an antagonist of NMDA receptor (MK-801). MK-801 also blocked neuronal death induced by activated glia. High oxygen also prevented NO-induced neuronal death, consistent with death being induced by NO inhibition of cytochrome c oxidation in competition with oxygen. Thus activated glia kill neurons via NO from iNOS, which inhibits neuronal respiration resulting in glutamate release and subsequent excitotoxicity. This may contribute to neuronal cell death in inflammatory, infectious, ischemic, and neurodegenerative diseases. PMID:11517237

  12. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  13. Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum.

    PubMed

    Valjent, Emmanuel; Pascoli, Vincent; Svenningsson, Per; Paul, Surojit; Enslen, Hervé; Corvol, Jean-Christophe; Stipanovich, Alexandre; Caboche, Jocelyne; Lombroso, Paul J; Nairn, Angus C; Greengard, Paul; Hervé, Denis; Girault, Jean-Antoine

    2005-01-11

    Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d-amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d-amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Delta(9)-tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32). The effects of d-amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse. PMID:15608059

  14. EVALUATING THE NMDA-GLUTAMATE RECEPTOR AS A SITE OF ACTION FOR TOLUENE USING PATTERN ELICITED VISUAL EVOKED POTENTIALS.

    EPA Science Inventory

    In vitro studies have demonstrated that toluene disrupts the function of NMDA-glutamate receptors, as well as other channels. This has led to the hypothesis that effects on NMDA receptor function may contribute to toluene neurotoxicity, CNS depression, and altered visual evoked ...

  15. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production.

    PubMed

    Xu, Meijuan; Rao, Zhiming; Dou, Wenfang; Yang, Juan; Jin, Jian; Xu, Zhenghong

    2012-07-01

    N-acetyl-L-glutamate kinase (EC 2.7.2.8) is first committed in the specific L-arginine pathway of Corynebacterium sp. A limited increase of L-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that L-arginine feedback inhibition plays an influence on the L-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I0.5R efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I0.5R of L-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust L-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The L-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway. PMID:21901472

  16. Differential effects of arginine, glutamate and phosphoarginine on Ca(2+)-activation properties of muscle fibres from crayfish and rat.

    PubMed

    Jame, David W; West, Jan M; Dooley, Philip C; Stephenson, D George

    2004-01-01

    The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca2+-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log10[Ca2+]) that either contained the test solute ("test") or not ("control"). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l(-1)) significantly shifted the force-pCa curve by 0.08-0.13 pCa units in the direction of increased sensitivity to Ca2+-activated contraction in all fibre types; (ii) phosphoarginine (9-10 mmol l(-1)) induced a significant shift of the force-pCa curve by 0.18-0.24 pCa units in the direction of increased sensitivity to Ca2+ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force-pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36-40 mmol l(-1)), like arginine affected the force-pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca2+-activated contraction by 0.08-0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca2+-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca2+ and the regulatory and other myofibrillar proteins. PMID:15711880

  17. Anti-epileptogenic and anticonvulsant activity of L-2-amino-4-phosphonobutyrate, a presynaptic glutamate receptor agonist.

    PubMed

    Abdul-Ghani, A S; Attwell, P J; Singh Kent, N; Bradford, H F; Croucher, M J; Jane, D E

    1997-05-01

    The protective effect of amygdaloid (focally administered) doses of the presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate (L-AP4) was tested on the development of electrical kindling and in fully kindled animals. L-AP4 inhibited epileptogenesis at 10 nmol in 0.5 microl buffer, by preventing the increase in both seizure score and afterdischarge duration. The effects were reversible after withdrawal of the drug, with all treated animals subsequently progressing to the fully kindled state at the same rate as control animals. The same concentration of the drug was also effective when injected into fully kindled animals. It significantly decreased the mean seizure score by 88% (P < 0.005) and increased the mean generalized seizure threshold (GST) by 85% (P < 0.005). The increase in GST was accompanied by a significant delay before the onset of generalized seizure and by a 37% reduction in generalized seizure duration. MPPG ((RS)-alpha-methyl-4-phosphonophenyl glycine) a selective antagonist of L-AP4 at glutamate pre-synaptic receptors inhibited the depressant effect of L-AP4 in a dose-dependent manner. MPPG (10 nmol) inhibited the antiseizure activity of L-AP4, whilst MPPG (40 nmol) reduced both the anti-epileptogenic and antiseizure activities of L-AP4. MPPG (40 nmol) by itself had no effect on generalized seizure activity, and it had no detectable influence on the normal rate of kindled epileptogenesis. During in vitro studies using a microsuperfusion method, L-AP4 inhibited depolarization-induced release of [3H]D-aspartate from rat cortical synaptosomes (IC50 125.1 microM) and decreased the depolarization-evoked uptake of 45Ca2+ in a dose-dependent manner. Both actions of L-AP4 were reduced by the selective antagonist MPPG. When applied alone MPPG (200 microM) had no detectable action on veratridine-evoked 45Ca2+ uptake by the synaptosomes. These results suggest the mechanisms by which presynaptically active glutamate receptor

  18. Active site of the replication protein of the rolling circle plasmid pC194.

    PubMed Central

    Noirot-Gros, M F; Bidnenko, V; Ehrlich, S D

    1994-01-01

    Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step. Images PMID:7925284

  19. NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network

    PubMed Central

    Platel, Jean-Claude; Dave, Kathleen A.; Gordon, Valerie; Lacar, Benjamin; Rubio, Maria E.; Bordey, Angélique

    2010-01-01

    SUMMARY Even before integrating into existing circuitry, adult-born neurons express receptors for neurotransmitters, but the intercellular mechanisms and their impact on neurogenesis remain largely unexplored. Here, we show that neuroblasts born in the postnatal subventricular zone (SVZ) acquire NMDA receptors (NMDARs) during their migration to the olfactory bulb. Along their route, neuroblasts are ensheathed by astrocyte-like cells expressing vesicular glutamate release machinery. Increasing calcium in these specialized astrocytes induced NMDAR-activity in neuroblasts and blocking astrocytic vesicular release eliminated spontaneous NMDAR-activity. Single-cell knockout of NMDARs using neonatal electroporation resulted in neuroblast apoptosis at the time of NMDAR acquisition. This cumulated in a 40% loss of neuroblasts along their migratory route demonstrating that NMDAR acquisition is critical for neuroblast survival, prior to entering a synaptic network. In addition, our findings suggest an unexpected mechanism where SVZ astrocytes use glutamate signaling through NMDARs to control the number of adult-born neurons reaching their final destination. PMID:20346761

  20. Twenty-four hour quantitative-EEG and in-vivo glutamate biosensor detects activity and circadian rhythm dependent biomarkers of pathogenesis in Mecp2 null mice

    PubMed Central

    Johnston, Michael V.; Ammanuel, Simon; O'Driscoll, Cliona; Wozniak, Amy; Naidu, Sakkubai; Kadam, Shilpa D.

    2014-01-01

    Mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (Mecp2) cause most cases of Rett syndrome (RTT). Currently there is no cure for RTT. Abnormal EEGs are found in 100% of RTT cases and are associated with severe sleep dysfunction, the cause of which is not well understood. Mice deficient in MeCP2 protein have been studied and characterized for their neuropathological and behavioral deficits to better understand RTT. With the goal to study the non-ictal EEG correlates in symptomatic Mecp2 KO mice (Mecp2tm1.1Bird/y), and determine novel EEG biomarkers of their reported progressive neurodegeneration, we used 24 h video-EEG/EMG with synchronous in-vivo cortical glutamate biosensor in the frontal cortex. We scored the EEG for activity states and spectral analysis was performed to evaluate correlations to the synchronous extracellular glutamate fluctuations underlying Mecp2 inactivation as compared to WT. Significant alterations in sleep structure due to dark cycle-specific long wake states and poor quality of slow-wave sleep were associated with a significant increase in glutamate loads per activity cycle. The dynamics of the activity-state-dependent physiological rise and fall of glutamate indicative of glutamate homeostasis were significantly altered in the KO mice. Colorimetric quantitation of absolute glutamate levels in frontal cortex also indicated the presence of significantly higher levels in KO. This study for the first time found evidence of uncompensated sleep deprivation-like EEG biomarkers that were associated with glutamate homeostatic dysfunction in the Mecp2 KO mice. PMID:25018705

  1. Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

    PubMed

    Dal-Cim, Tharine; Martins, Wagner C; Thomaz, Daniel T; Coelho, Victor; Poluceno, Gabriela Godoy; Lanznaster, Débora; Vandresen-Filho, Samuel; Tasca, Carla I

    2016-05-01

    Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity. PMID:26858177

  2. Activation of type 5 metabotropic glutamate receptors attenuates deficits in cognitive flexibility induced by NMDA receptor blockade

    PubMed Central

    Stefani, Mark R.; Moghaddam, Bita

    2010-01-01

    Metabotropic glutamate (mGlu) receptors provide a mechanism by which the function of NMDA glutamate receptors can be modulated. As NMDA receptor hypofunction is implicated in the etiology of psychiatric disorders, including schizophrenia, the pharmacological regulation of mGlu receptor activity represents a promising therapeutic approach. We examined the effects of the positive allosteric mGlu5 receptor modulator 3- cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), alone and in combination with the NMDA receptor antagonist MK-801, on a task measuring cognitive set-shifting ability. This task measures NMDA receptor-dependent cognitive abilities analogous to those impaired in schizophrenia. Systemic administration of CDPPB (10 & 30 mg/kg i.p) blocked MK-801 (0.1 mg/kg, i.p.)-induced impairments in set-shifting ability. The effect on learning was dose-dependent, with the 30 mg/kg dose having a greater effect than the 10 mg/kg dose across all trials. This ameliorative effect of CDPPB reflected a reduction in MK-801-induced perseverative responding. These results add to the evidence that mGlu5 receptors interact functionally with NMDA receptors to regulate behavior, and suggest that positive modulators of mGlu5 receptors may have therapeutic potential in the treatment of disorders, like schizophrenia, characterized by impairments in cognitive flexibility and memory. PMID:20371234

  3. Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia

    PubMed Central

    Fazio, Francesco; Lionetto, Luana; Curto, Martina; Iacovelli, Luisa; Cavallari, Michele; Zappulla, Cristina; Ulivieri, Martina; Napoletano, Flavia; Capi, Matilde; Corigliano, Valentina; Scaccianoce, Sergio; Caruso, Alessandra; Miele, Jessica; De Fusco, Antonio; Di Menna, Luisa; Comparelli, Anna; De Carolis, Antonella; Gradini, Roberto; Nisticò, Robert; De Blasi, Antonio; Girardi, Paolo; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando; Simmaco, Maurizio

    2015-01-01

    The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia. PMID:26643205

  4. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK

    SciTech Connect

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. - Highlights: • Curcumin attenuates glutamate neurotoxicity in the hippocampus. • Curcumin suppresses ER stress in glutamate-induced hippocampus slices. • Curcumin inhibits TXNIP/NLRP3 inflammasome activation. • Regulation of AMPK by curcumin contributes to suppressing ER stress.

  5. Neuroprotective Effects of the Glutamate Transporter Activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) following Traumatic Brain Injury in the Adult Rat.

    PubMed

    Karklin Fontana, Andréia Cristina; Fox, Douglas P; Zoubroulis, Argie; Valente Mortensen, Ole; Raghupathi, Ramesh

    2016-06-01

    Traumatic brain injury (TBI) in humans and in animals leads to an acute and sustained increase in tissue glutamate concentrations within the brain, triggering glutamate-mediated excitotoxicity. Excitatory amino acid transporters (EAATs) are responsible for maintaining extracellular central nervous system glutamate concentrations below neurotoxic levels. Our results demonstrate that as early as 5 min and up to 2 h following brain trauma in brain-injured rats, the activity (Vmax) of EAAT2 in the cortex and the hippocampus was significantly decreased, compared with sham-injured animals. The affinity for glutamate (KM) and the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) were not altered by the injury. Administration of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a GLT-1 activator, beginning immediately after injury and continuing for 24 h, significantly decreased neurodegeneration, loss of microtubule-associated protein 2 and NeuN (+) immunoreactivities, and attenuated calpain activation in both the cortex and the hippocampus at 24 h after the injury; the reduction in neurodegeneration remained evident up to 14 days post-injury. In synaptosomal uptake assays, MS-153 up-regulated GLT-1 activity in the naïve rat brain but did not reverse the reduced activity of GLT-1 in traumatically-injured brains. This study demonstrates that administration of MS-153 in the acute post-traumatic period provides acute and long-term neuroprotection for TBI and suggests that the neuroprotective effects of MS-153 are related to mechanisms other than GLT-1 activation, such as the inhibition of voltage-gated calcium channels. PMID:26200170

  6. Inhibition of glutamate carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis.

    PubMed

    Rahn, Kristen A; Watkins, Crystal C; Alt, Jesse; Rais, Rana; Stathis, Marigo; Grishkan, Inna; Crainiceau, Ciprian M; Pomper, Martin G; Rojas, Camilo; Pletnikov, Mikhail V; Calabresi, Peter A; Brandt, Jason; Barker, Peter B; Slusher, Barbara S; Kaplin, Adam I

    2012-12-01

    Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function. PMID:23169655

  7. Enhanced degradation of benzene by percarbonate activated with Fe(II)-glutamate complex.

    PubMed

    Fu, Xiaori; Gu, Xiaogang; Lu, Shuguang; Miao, Zhouwei; Xu, Minhui; Zhang, Xiang; Danish, Muhammad; Cui, Hang; Farooq, Usman; Qiu, Zhaofu; Sui, Qian

    2016-04-01

    Effective degradation of benzene was achieved in sodium percarbonate (SPC)/Fe(II)-Glu system. The presence of glutamate (Glu) could enhance the regeneration of Fe(III) to Fe(II), which ensures the benzene degradation efficiency at wider pH range and eliminate the influence of HCO3 (-) in low concentration. Meanwhile, the significant scavenging effects of high HCO3 (-) concentration could also be overcome by increasing the Glu/SPC/Fe(II)/benzene molar ratio. Free radical probe compound tests, free radical scavenger tests, and electron paramagnetic resonance (EPR) analysis were conducted to explore the reaction mechanism for benzene degradation, in which hydroxyl radical (HO•) and superoxide anion radical (O2 (•-)) were confirmed as the predominant species responsible for benzene degradation. In addition, the results obtained in actual groundwater test strongly indicated that SPC/Fe(II)-Glu system is applicable for the remediation of benzene-contaminated groundwater in practice. PMID:26662563

  8. Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia

    PubMed Central

    Masuoka, Takayoshi; Kudo, Makiko; Yoshida, Junko; Ishibashi, Takaharu; Muramatsu, Ikunobu; Kato, Nobuo; Imaizumi, Noriko; Nishio, Matomo

    2016-01-01

    Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia. PMID:27064319

  9. Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia.

    PubMed

    Masuoka, Takayoshi; Kudo, Makiko; Yoshida, Junko; Ishibashi, Takaharu; Muramatsu, Ikunobu; Kato, Nobuo; Imaizumi, Noriko; Nishio, Matomo

    2016-01-01

    Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia. PMID:27064319

  10. Metabotropic glutamate receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors.

    PubMed

    Boix, Jordi; Llansola, Marta; Cabrera-Pastor, Andrea; Felipo, Vicente

    2011-04-01

    Metabotropic glutamate receptors (mGluRs) modulate important processes in cerebellum including long-term depression, which also requires formation of nitric oxide (NO) and cGMP. Some reports suggest that mGluRs could modulate the NO-cGMP pathway in cerebellum. However this modulation has not been studied in detail. The aim of this work was to assess by microdialysis in freely moving rats whether activation of mGluR5 modulates the NO-cGMP pathway in cerebellum in vivo and to analyze the underlying mechanisms. We show that mGluR5 activation increases extracellular glutamate, citrulline and cGMP in cerebellum. Blocking NMDA receptors with MK-801 does not prevent any of these effects, indicating that NMDA receptors activation is not required. However in the presence of MK-801 the effects are more transient, returning faster to basal levels. Blocking AMPA receptors prevents the increase in citrulline and cGMP induced by mGluR5 activation, but not the increase in glutamate. The release of glutamate is prevented by tetrodotoxin but not by fluoroacetate, indicating that glutamate is released from neurons and not from astrocytes. Activation of AMPA receptors increases citrulline and cGMP. These data indicate that activation of mGluR5 induces an increase of extracellular glutamate which activates AMPA receptors, leading to activation of nitric oxide synthase and increased NO, which activates guanylate cyclase, increasing cGMP. The response mediated by AMPA receptors desensitize rapidly. Activation of AMPA receptors also induces a mild depolarization, allowing activation of NMDA receptors which prolongs the duration of the effect initiated by activation of AMPA receptors. These data support that the three types of glutamate receptors: mGluR5, AMPA and NMDA cooperate in the modulation of the grade and duration of activation of the NO-cGMP pathway in cerebellum in vivo. This pathway would modulate cerebellar processes such as long-term depression. PMID:21300123

  11. Decreasing nicotinic receptor activity and the spatial learning impairment caused by the NMDA glutamate antagonist dizocilpine in rats

    PubMed Central

    Burke, Dennis A.; Heshmati, Pooneh; Kholdebarin, Ehsan; Levin, Edward D.

    2014-01-01

    Nicotinic systems have been shown by a variety of studies to be involved in cognitive function. Nicotinic receptors have an inherent property to become desensitized after activation. The relative role of nicotinic receptor activation vs. net receptor inactivation by desensitization in the cognitive effects of nicotinic drugs remains to be fully understood. In these studies, we tested the effects of the α7 nicotinic receptor antagonist methyllycaconitine (MLA), the α4β2 nicotinic receptor antagonist dihydro-β-erythroidine (DHβE), the nonspecific nicotinic channel blocker mecamylamine and the α4β2 nicotinic receptor desensitizing agent sazetidine-A on learning in a repeated acquisition test. Adult female Sprague-Dawley rats were trained on a repeated acquisition learning procedure in an 8-arm radial maze. MLA (1–4 mg/kg), DHβE (1–4 mg/kg), mecamylamine (0.125–0.5 mg/kg) or sazetidine-A (1 and 3 mg/kg) were administered in four different studies either alone or together with the NMDA glutamate antagonist dizocilpine (0.05 and 0.10 mg/kg). MLA significantly counteracted the learning impairment caused by dizocilpine. The overall choice accuracy impairment caused by dizocilpine was significantly attenuated by co-administration of DHβE. Low doses of the non-specific nicotinic antagonist mecamylamine also reduced dizocilpine-induced repeated acquisition impairment. Sazetidine-A reversed the accuracy impairment caused by dizocilpine. These studies provide evidence that a net decrease in nicotinic receptor activity can improve learning by attenuating learning impairment induced by NMDA glutamate blockade. This adds to evidence in cognitive tests that nicotinic antagonists can improve cognitive function. Further research characterizing the efficacy and mechanisms underlying nicotinic antagonist and desensitization induced cognitive improvement is warranted. PMID:25064338

  12. Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

    2015-05-28

    We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells. PMID:25923815

  13. Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction

    PubMed Central

    Walker, Adam G.; Wenthur, Cody J.; Xiang, Zixiu; Rook, Jerri M.; Emmitte, Kyle A.; Niswender, Colleen M.; Lindsley, Craig W.; Conn, P. Jeffrey

    2015-01-01

    Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu3) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu3 influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu3 can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu3 negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu3 as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu3 may be useful in alleviating prefrontal impairments associated with several CNS disorders. PMID:25583490

  14. Aspartoacylase deficiency does not affect N-acetylaspartylglutamate level or glutamate carboxypeptidase II activity in the knockout mouse brain.

    PubMed

    Surendran, Sankar; Ezell, Edward L; Quast, Michael J; Wei, Jingna; Tyring, Stephen K; Michals-Matalon, Kimberlee; Matalon, Reuben

    2004-08-01

    Aspartoacylase (ASPA)-deficient patients [Canavan disease (CD)] reportedly have increased urinary excretion of N-acetylaspartylglutamate (NAAG), a neuropeptide abundant in the brain. Whether elevated excretion of urinary NAAG is due to ASPA deficiency, resulting in an abnormal level of brain NAAG, is examined using ASPA-deficient mouse brain. The level of NAAG in the knockout mouse brain was similar to that in the wild type. The NAAG hydrolyzing enzyme, glutamate carboxypeptidase II (GCP II), activity was normal in the knockout mouse brain. These data suggest that ASPA deficiency does not affect the NAAG or GCP II level in the knockout mouse brain, if documented also in patients with CD. PMID:15246864

  15. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK.

    PubMed

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. PMID:25791922

  16. Rhinacanthus nasutus Extracts Prevent Glutamate and Amyloid-β Neurotoxicity in HT-22 Mouse Hippocampal Cells: Possible Active Compounds Include Lupeol, Stigmasterol and β-Sitosterol

    PubMed Central

    Brimson, James M.; Brimson, Sirikalaya J.; Brimson, Christopher A.; Rakkhitawatthana, Varaporn; Tencomnao, Tewin

    2012-01-01

    The Herb Rhinacanthus nasutus (L.) Kurz, which is native to Thailand and Southeast Asia, has become known for its antioxidant properties. Neuronal loss in a number of diseases including Alzheimer’s disease is thought to result, in part, from oxidative stress. Glutamate causes cell death in the mouse hippocampal cell line, HT-22, by unbalancing redox homeostasis, brought about by a reduction in glutathione levels, and amyloid-β has been shown to induce reactive oxygen species (ROS) production. Here in, we show that ethanol extracts of R. nasutus leaf and root are capable of dose dependently attenuating the neuron cell death caused by both glutamate and amyloid-β treatment. We used free radical scavenging assays to measure the extracts antioxidant activities and as well as quantifying phenolic, flavonoid and sterol content. Molecules found in R. nasutus, lupeol, stigmasterol and β-sitosterol are protective against glutamate toxicity. PMID:22606031

  17. Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

    PubMed Central

    Kamachi, Kazunari; Yonezawa, Hideo; Fukutomi, Toshiyuki; Kawakami, Hayato; Kamiya, Shigeru

    2015-01-01

    ABSTRACT Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg+ mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCE Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis

  18. Functional magnetic resonance spectroscopy of glutamate in schizophrenia and major depressive disorder: anterior cingulate activity during a color-word Stroop task

    PubMed Central

    Taylor, Reggie; Neufeld, Richard W J; Schaefer, Betsy; Densmore, Maria; Rajakumar, Nagalingam; Osuch, Elizabeth A; Williamson, Peter C; Théberge, Jean

    2015-01-01

    Background: Glutamate abnormalities have been suggested to be associated with symptoms of schizophrenia. Using functional magnetic resonance spectroscopy (1H-fMRS), it is possible to monitor glutamate dynamically in the activated brain areas, which has yet to be reported in schizophrenia. It was hypothesized that subjects with schizophrenia would have weaker glutamatergic responses in the anterior cingulate to a color-word Stroop Task. AIMS: The aim of this study was to gain insight into the health of GLU neurotransmission and the GLU-GLN cycle in SZ using a 1H-fMRS protocol. Methods: Spectra were acquired from the anterior cingulate of 16 participants with schizophrenia, 16 healthy controls and 16 participants with major depressive disorder (MDD) while performing the Stroop task in a 7T magnetic resonance imaging scanner. 1H-fMRS spectra were acquired for 20 min in which there were three 4-min blocks of cross fixation interleaved with two 4-min blocks of the Stroop paradigm. Results: A repeated-measures analysis of variance revealed a main effect of time for glutamate concentrations of all groups (P<0.001). The healthy control group increased glutamate concentrations in the first run of the Stroop task (P=0.006) followed by a decrease in the recovery period (P=0.007). Neither the schizophrenia (P=0.107) nor MDD (P=0.081) groups had significant glutamate changes in the first run of the task, while the schizophrenia group had a significant increase in glutamine (P=0.005). The MDD group decreased glutamate concentrations in the second run of the task (P=0.003), as did all the groups combined (P=0.003). Conclusions: 1H-fMRS data were successfully acquired from psychiatric subjects with schizophrenia and mood disorder using a cognitive paradigm for the first time. Future study designs should further elucidate the glutamatergic response to functional activation in schizophrenia. PMID:27336037

  19. Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

    PubMed

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-10

    Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and

  20. Role of Glutamate 64 in the Activation of the Prodrug 5-fluorocytosine by Yeast Cytosine Deaminase†

    PubMed Central

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-01

    Yeast cytosine deaminase catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. 1H and 15N NMR analysis revealed trans-hydrogen-bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has higher activation energy as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction

  1. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    PubMed

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  2. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Lobet, Y; Cluff, C W; Cieplak, W

    1991-01-01

    Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins. Images PMID:1908825

  3. Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity.

    PubMed

    Zhang, Fei; Zheng, Qing-Zhong; Jiao, Qing-Cai; Liu, Jun-Zhong; Zhao, Gen-Hai

    2010-11-01

    Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability. PMID:20238131

  4. Reactive oxygen species induced by presynaptic glutamate receptor activation is involved in [(3)H]GABA release from rat brain cortical nerve terminals.

    PubMed

    Tarasenko, A; Krupko, O; Himmelreich, N

    2012-12-01

    We investigated the production of reactive oxygen species (ROS) as a response to presynaptic glutamate receptor activation, and the role of ROS in neurotransmitter (GABA) release. Experiments were performed with rat brain cortical synaptosomes using glutamate, NMDA and kainate as agonists of glutamate receptors. ROS production was evaluated with the fluorogenic compound dichlorodihydrofluorescein diacetate (H(2)DCF-DA), and GABA release was studied using synaptosomes loaded with [(3)H]GABA. All agonists were found to stimulate ROS production, and specific antagonists of NMDA and kainate/AMPA receptors, dizocilpine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-done (CNQX), significantly inhibited the ROS increase. Spontaneous as well as agonist-evoked ROS production was effectively attenuated by diphenyleneiodonium (DPI), a commonly used potent inhibitor of NADPH oxidase activity, that suggests a high contribution of NADPH-oxidase to this process. The replacement of glucose with pyruvate or the simultaneous presence of both substrates in the medium led to the decrease in spontaneous and NMDA-evoked ROS production, but to the increase in ROS production induced by kainate. Scavenging of agonist-evoked ROS production by a potent antioxidant N-acetylcysteine was tightly correlated with the inhibition of agonist-evoked GABA release. Together, these findings show that the activation of presynaptic glutamate receptors induces an increase in ROS production, and there is a tight correlation between ROS production and GABA secretion. The pivotal role of kainate/AMPA receptors in ROS production is under discussion. PMID:22864357

  5. Adenosine Monophosphate-activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

    PubMed Central

    Maixner, Dylan W.; Yan, Xisheng; Gao, Mei; Yadav, Ruchi; Weng, Han-Rong

    2015-01-01

    Background Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn (SDH) are implicated in the genesis of neuropathic pain. We determined if adenosine monophosphate-activated protein kinase (AMPK) in the SDH regulates these processes in rodents with neuropathic pain. Methods Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. Results AMPK activities were reduced in the SDH of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1beta and activities of glycogen synthase kinase 3β, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10), and induced by inhibiting spinal AMPK in naïve rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1β (n = 4). Conclusion These findings suggest enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain. PMID:25710409

  6. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus.

    PubMed

    de Mendonça, A; Ribeiro, J A

    1997-08-01

    1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12-14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe. p.s.p.) recorded extracellularly from the stratum radiatum in the CAI area. 2. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2-50 nM), on the fe.p.s.p. slope (EC50 = 12.5 (9.2-17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 microM) (EC50 = 27.2 (21.4-34.5) nM, n = 4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 microM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). DHPG (10 microM) itself had an inhibitory effect of 20.1 +/- 1.9% (n = 4) on the fe.p.s.p. slope. 3. The concentration-response curves for the inhibitory effects of CPA (2-20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I; 1 microM), or in the presence of

  7. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

    PubMed Central

    de Mendonça, Alexandre; Ribeiro, J A

    1997-01-01

    Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC50=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC50=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non

  8. Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

    PubMed

    Wang, Hongyu; Tamba, Michiko; Kimata, Mayumi; Sakamoto, Kazuichi; Bannai, Shiro; Sato, Hideyo

    2003-06-01

    The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells. PMID:12763038

  9. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  10. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  11. Methylglyoxal and carboxyethyllysine reduce glutamate uptake and S100B secretion in the hippocampus independently of RAGE activation.

    PubMed

    Hansen, Fernanda; Battú, Cíntia Eickhoff; Dutra, Márcio Ferreira; Galland, Fabiana; Lirio, Franciane; Broetto, Núbia; Nardin, Patrícia; Gonçalves, Carlos-Alberto

    2016-02-01

    Diabetes is a metabolic disease characterized by high fasting-glucose levels. Diabetic complications have been associated with hyperglycemia and high levels of reactive compounds, such as methylglyoxal (MG) and advanced glycation endproducts (AGEs) formation derived from glucose. Diabetic patients have a higher risk of developing neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. Herein, we examined the effect of high glucose, MG and carboxyethyllysine (CEL), a MG-derived AGE of lysine, on oxidative, metabolic and astrocyte-specific parameters in acute hippocampal slices, and investigated some of the mechanisms that could mediate these effects. Glucose, MG and CEL did not alter reactive oxygen species (ROS) formation, glucose uptake or glutamine synthetase activity. However, glutamate uptake and S100B secretion were decreased after MG and CEL exposure. RAGE activation and glycation reactions, examined by aminoguanidine and L-lysine co-incubation, did not mediate these changes. Acute MG and CEL exposure, but not glucose, were able to induce similar effects on hippocampal slices, suggesting that conditions of high glucose concentrations are primarily toxic by elevating the rates of these glycation compounds, such as MG, and by generation of protein cross-links. Alterations in the secretion of S100B and the glutamatergic activity mediated by MG and AGEs can contribute to the brain dysfunction observed in diabetic patients. PMID:26347375

  12. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  13. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    PubMed Central

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.

    2009-01-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

  14. Phosphorylation and Assembly of Glutamate Receptors after Brain Ischemia

    PubMed Central

    Zhang, Fan; Guo, Ailan; Liu, Chunli; Comb, Micheal; Hu, Bingren

    2012-01-01

    Background and Purpose Over-assembly of synaptic glutamate receptors leads to excitotoxicity. The goal of this study is to investigate phosphorylation and assembly of AMPA and NMDA receptors after brain ischemia with reperfusion (I/R). Methods Rats were subjected to 15 min of global ischemia followed by 0.5, 4, and 24 h of reperfusion. Phosphotyrosine (Ptyr) peptides of glutamate receptors in synaptosomal fraction after I/R were identified and quantified by state-of-the-art immuno-affinity purification of Ptyr peptides followed by LC-MS/MS analysis (IAP-LC/MS/MS). Glutamate receptor phosphorylation and synaptic assembly after I/R were studied by biochemical methods. Results Numerous Ptyr sites of AMPA and NMDA were upregulated by about 2- to 37-fold after I/R. A core glutamate receptor kinase, Src kinase, was significantly activated. GluR2/3 and NR2A/B were rapidly clustered from extrasynaptic to synaptic membrane fractions after I/R. GluR2/3 was then translocated into the intracellular pool, whereas NR2A/B remained in the synaptic fraction for as long as 24 h. Consistently, trafficking-related phosphorylation of GluR2/3-S880 was significantly but transiently upregulated, whereas NR2A/B-Y1246 and -Y1472 were significantly and persistently upregulated after I/R. Conclusions Phosphorylation of glutamate receptors at synapses may lead to over-assembly of glutamate receptors, probably via activation of Src family kinases, after I/R. This study provides “global” proteomic information about glutamate receptor tyrosine phosphorylation after brain ischemia. PMID:23212166

  15. Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

    PubMed

    Mundell, Stuart J; Pula, Giordano; More, Julia C A; Jane, David E; Roberts, Peter J; Kelly, Eamonn

    2004-06-01

    In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins. PMID:15155843

  16. Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

    PubMed

    Romei, Cristina; Raiteri, Maurizio; Raiteri, Luca

    2013-03-01

    The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22659408

  17. Phenotypic Characterization of Mice Heterozygous for a Null Mutation of Glutamate Carboxypeptidase II

    PubMed Central

    Han, Liqun; Picker, Jonathan D.; Schaevitz, Laura R.; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C.; Basu, Alo C.; Berger-Sweeney, Joanne; Coyle, Joseph T.

    2009-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of NMDA receptors has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-alpha L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate (NAA). NAAG is a neuropeptide that is an NMDA receptor antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDA receptor activation. To manipulate the expression of GCP II, loxP sites were inserted flanking exon 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N >200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory. PMID:19347959

  18. Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase II.

    PubMed

    Han, Liqun; Picker, Jonathan D; Schaevitz, Laura R; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C; Basu, Alo C; Berger-Sweeney, Joanne; Coyle, Joseph T

    2009-08-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of N-methyl-D-aspartate receptors (NMDAR) has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-alpha L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate. NAAG is a neuropeptide that is an NMDAR antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDAR activation. To manipulate the expression of GCP II, LoxP sites were inserted flanking exons 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N > 200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory. PMID:19347959

  19. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  20. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  1. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  2. Coordinated activation of distinct Ca(2+) sources and metabotropic glutamate receptors encodes Hebbian synaptic plasticity.

    PubMed

    Tigaret, Cezar M; Olivo, Valeria; Sadowski, Josef H L P; Ashby, Michael C; Mellor, Jack R

    2016-01-01

    At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated presynaptic and postsynaptic spikes to activate postsynaptic NMDA receptors (NMDARs). The magnitudes of the ensuing Ca2+ transients within dendritic spines are thought to determine the amplitude and direction of synaptic change. In contrast, we show that at mature hippocampal Schaffer collateral synapses the magnitudes of Ca2+ transients during plasticity induction do not match this rule. Indeed, LTP induced by time-correlated pre- and postsynaptic spikes instead requires the sequential activation of NMDARs followed by voltage-sensitive Ca2+ channels within dendritic spines. Furthermore, LTP requires inhibition of SK channels by mGluR1, which removes a negative feedback loop that constitutively regulates NMDARs. Therefore, rather than being controlled simply by the magnitude of the postsynaptic calcium rise, LTP induction requires the coordinated activation of distinct sources of Ca2+ and mGluR1-dependent facilitation of NMDAR function. PMID:26758963

  3. Coordinated activation of distinct Ca2+ sources and metabotropic glutamate receptors encodes Hebbian synaptic plasticity

    PubMed Central

    Tigaret, Cezar M.; Olivo, Valeria; Sadowski, Josef H.L.P.; Ashby, Michael C.; Mellor, Jack R.

    2016-01-01

    At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated presynaptic and postsynaptic spikes to activate postsynaptic NMDA receptors (NMDARs). The magnitudes of the ensuing Ca2+ transients within dendritic spines are thought to determine the amplitude and direction of synaptic change. In contrast, we show that at mature hippocampal Schaffer collateral synapses the magnitudes of Ca2+ transients during plasticity induction do not match this rule. Indeed, LTP induced by time-correlated pre- and postsynaptic spikes instead requires the sequential activation of NMDARs followed by voltage-sensitive Ca2+ channels within dendritic spines. Furthermore, LTP requires inhibition of SK channels by mGluR1, which removes a negative feedback loop that constitutively regulates NMDARs. Therefore, rather than being controlled simply by the magnitude of the postsynaptic calcium rise, LTP induction requires the coordinated activation of distinct sources of Ca2+ and mGluR1-dependent facilitation of NMDAR function. PMID:26758963

  4. Synthesis of theanine from glutamic acid gamma-methyl ester and ethylamine catalyzed by Escherichia coli having gamma-glutamyltranspeptidase activity.

    PubMed

    Zhang, Fei; Zheng, Qing-Zhong; Jiao, Qing-Cai; Liu, Jun-Zhong; Zhao, Gen-Hai

    2010-08-01

    Glutamic acid gamma-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing gamma-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45 degrees C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h. PMID:20383735

  5. Impact of fumonisin B1 on glutamate toxicity and low magnesium-induced seizure activity in neuronal primary culture.

    PubMed

    Domijan, A-M; Kovac, S; Abramov, A Y

    2012-01-27

    Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium spp. mould that contaminates maize world-wide. Although its neurodegenerative potential is well established, mechanisms and acute effects of FB(1) on neurons are still not completely understood. Our previous study on astrocytes and neuroblastoma cells demonstrated that acute FB(1) exposure inhibits mitochondrial complex I and leads to mitochondrial membrane potential depolarization and calcium deregulation. To further explore the mechanisms of FB(1) neurotoxicity, we here investigated the effects of acute FB(1) co-exposure with glutamate and in the low magnesium model of epilepsy on neuronal calcium level, mitochondrial membrane potential, and cell death in glio-neuronal cultures. FB(1) increased the glutamate-induced calcium signal in neurons and changed neuronal calcium signals to more sustained intracellular calcium rises in the low magnesium model of epilepsy that coincided with mitochondrial membrane potential depolarization. FB(1) co-exposure increased the percentage of dead neurons in low magnesium conditions dose dependently when compared with low magnesium exposure only, whereas in FB(1) and glutamate co-exposure neuronal death remained unchanged when compared with glutamate treatment only. Our results show that FB(1) makes neurons more vulnerable to glutamate-induced toxicity and epileptiform conditions, indicating that FB(1) can enhance the detrimental effect of these conditions on neurons. PMID:22178271

  6. Disorders of glutamate metabolism.

    PubMed

    Kelly, A; Stanley, C A

    2001-01-01

    The significant role the amino acid glutamate assumes in a number of fundamental metabolic pathways is becoming better understood. As a central junction for interchange of amino nitrogen, glutamate facilitates both amino acid synthesis and degradation. In the liver, glutamate is the terminus for release of ammonia from amino acids, and the intrahepatic concentration of glutamate modulates the rate of ammonia detoxification into urea. In pancreatic beta-cells, oxidation of glutamate mediates amino acid-stimulated insulin secretion. In the central nervous system, glutamate serves as an excitatory neurotransmittor. Glutamate is also the precursor of the inhibitory neurotransmittor GABA, as well as glutamine, a potential mediator of hyperammonemic neurotoxicity. The recent identification of a novel form of congenital hyperinsulinism associated with asymptomatic hyperammonemia assigns glutamate oxidation by glutamate dehydrogenase a more important role than previously recognized in beta-cell insulin secretion and hepatic and CNS ammonia detoxification. Disruptions of glutamate metabolism have been implicated in other clinical disorders, such as pyridoxine-dependent seizures, confirming the importance of intact glutamate metabolism. This article will review glutamate metabolism and clinical disorders associated with disrupted glutamate metabolism. PMID:11754524

  7. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus

    PubMed Central

    Yang, Yang

    2015-01-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied “endbulb of Held” synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-d-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg2+) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  8. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus.

    PubMed

    Yang, Yang; Xu-Friedman, Matthew A

    2015-06-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied "endbulb of Held" synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg(2+)) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  9. Glutamate Transporter-Mediated Glutamate Secretion in the Mammalian Pineal Gland

    PubMed Central

    Kim, Mean-Hwan; Uehara, Shunsuke; Muroyama, Akiko; Hille, Bertil; Moriyama, Yoshinori; Koh, Duk-Su

    2008-01-01

    Glutamate transporters are expressed throughout the central nervous system where their major role is to clear released glutamate from presynaptic terminals. Here we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by high-performance liquid chromatography. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative ‘glutamate-induced glutamate release’. Therefore we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently down-regulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland. PMID:18945893

  10. Glutamine Assimilation and Feedback Regulation of L-acetyl-N-glutamate Kinase Activity in Chlorella variabilis NC64A Results in Changes in Arginine Pools.

    PubMed

    Minaeva, Ekaterina; Forchhammer, Karl; Ermilova, Elena

    2015-11-01

    Glutamine is a metabolite of central importance in nitrogen metabolism of microorganisms and plants. The Chlorella PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase (NAGK) that leads to arginine formation. We provide evidence that glutamine promotes effective growth of C. variabilis strain NC64A. The present study shows that externally supplied glutamine directly influences the internal pool of arginine in NC64A. Glutamine synthetase (GS) catalyzes the ATP-dependent conversion of glutamate and ammonium to glutamine. The results of this study demonstrate that glutamine acts as a negative effector of GS activity. These data emphasize the importance of glutamine-dependent coupling of metabolism and signaling as components of an efficient pathway allowing the maintenance of metabolic homeostasis and sustaining growth of Chlorella. PMID:26356535

  11. Metabotropic Glutamate Receptors

    PubMed Central

    Dillon, James; Franks, Christopher J.; Murray, Caitriona; Edwards, Richard J.; Calahorro, Fernando; Ishihara, Takeshi; Katsura, Isao; Holden-Dye, Lindy; O'Connor, Vincent

    2015-01-01

    Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior. PMID:25869139

  12. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  13. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  14. Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling.

    PubMed

    Said, H M; Chenoweth, M; Dunn, A

    1989-08-01

    Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 mumol/kg for alanine, 578 mumol/kg for glutamate and 296 mumol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline. PMID:2569659

  15. Simulation of Postsynaptic Glutamate Receptors Reveals Critical Features of Glutamatergic Transmission

    PubMed Central

    Greget, Renaud; Pernot, Fabien; Bouteiller, Jean-Marie C.; Ghaderi, Viviane; Allam, Sushmita; Keller, Anne Florence; Ambert, Nicolas; Legendre, Arnaud; Sarmis, Merdan; Haeberle, Olivier; Faupel, Michel; Bischoff, Serge; Berger, Theodore W.; Baudry, Michel

    2011-01-01

    Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following

  16. Inducible Glutamate Oxaloacetate Transaminase as a Therapeutic Target Against Ischemic Stroke

    PubMed Central

    Khanna, Savita; Briggs, Zachary

    2015-01-01

    Abstract Significance: Glutamate serves multi-faceted (patho)physiological functions in the central nervous system as the most abundant excitatory neurotransmitter and under pathological conditions as a potent neurotoxin. Regarding the latter, elevated extracellular glutamate is known to play a central role in ischemic stroke brain injury. Recent Advances: Glutamate oxaloacetate transaminase (GOT) has emerged as a new therapeutic target in protecting against ischemic stroke injury. Oxygen-sensitive induction of GOT expression and activity during ischemic stroke lowers glutamate levels at the stroke site while sustaining adenosine triphosphate levels in brain. The energy demands of the brain are among the highest of all organs underscoring the need to quickly mobilize alternative carbon skeletons for metabolism in the absence of glucose during ischemic stroke. Recent work builds on the important observation of Hans Krebs that GOT-mediated metabolism of glutamate generates tri-carboxylic acid (TCA) cycle intermediates in brain tissue. Taken together, outcomes suggest GOT may enable the transformative switch of otherwise excitotoxic glutamate into life-sustaining TCA cycle intermediates during ischemic stroke. Critical Issues: Neuroprotective strategies that focus solely on blocking mechanisms of glutamate-mediated excitotoxicity have historically failed in clinical trials. That GOT can enable glutamate to assume the role of a survival factor represents a paradigm shift necessary to develop the overall significance of glutamate in stroke biology. Future Directions: Ongoing efforts are focused to develop the therapeutic significance of GOT in stroke-affected brain. Small molecules that target induction of GOT expression and activity in the ischemic penumbra are the focus of ongoing studies. Antioxid. Redox Signal. 22, 175–186. PMID:25343301

  17. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  18. Molecular physiology of vesicular glutamate transporters in the digestive system

    PubMed Central

    Li, Tao; Ghishan, Fayez K.; Bai, Liqun

    2005-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Packaging and storage of glutamate into glutamatergic neuronal vesicles require ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. Three vesicular glutamate transporters (VGLUT1-3) have been recently identified from neuronal tissue where they play a key role to maintain the vesicular glutamate level. Recently, it has been demonstrated that glutamate signaling is also functional in peripheral neuronal and non-neuronal tissues, and occurs in sites of pituitary, adrenal, pineal glands, bone, GI tract, pancreas, skin, and testis. The glutamate receptors and VGLUTs in digestive system have been found in both neuronal and endocrinal cells. The glutamate signaling in the digestive system may have significant relevance to diabetes and GI tract motility disorders. This review will focus on the most recent update of molecular physiology of digestive VGLUTs. PMID:15793854

  19. Role of the -PEWY- Glutamate in Catalysis at the Qo-site of the Cyt bc1 Complex

    PubMed Central

    Victoria, Doreen; Burton, Rodney; Crofts, Antony R.

    2012-01-01

    We re-examine the pH dependence of partial processes of QH2 turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6–8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to wildtype. Occupancy of the Qo-site by semiquinone (SQ) was similar in wildtype and the Glu→Trp mutant. Since heme bL is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <103 s−1 for reduction of heme bL by SQ from the domain distal from heme bL, a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q.- as electron donor to heme bL, then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H+ transfer from QH., and delivery of the H+ to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme bL by allowing Q.- to move closer to the heme, accounts well for the observations. PMID:23123515

  20. REPEATED ANABOLIC/ANDROGENIC STEROID EXPOSURE DURING ADOLESCENCE ALTERS PHOSPHATE-ACTIVATED GLUTAMINASE AND GLUTAMATE RECEPTOR 1 SUBUNIT IMMUNOREACTIVITY IN HAMSTER BRAIN: CORRELATION WITH OFFENSIVE AGGRESSION

    PubMed Central

    Fischer, Shannon G.; Ricci, Lesley A.; Melloni, Richard H.

    2007-01-01

    Male Syrian hamsters (Mesocricetus auratus) treated with moderately high doses (5.0mg/kg/day) of anabolic/androgenic steroids (AAS) during adolescence (P27–P56) display highly escalated offensive aggression. The current study examined whether adolescent AAS-exposure influenced the immunohistochemical localization of phosphate-activated glutaminase (PAG), the rate-limiting enzyme in the synthesis of glutamate, a fast-acting neurotransmitter implicated in the modulation of aggression in various species and models of aggression, as well as glutamate receptor 1 subunit (GluR1). Hamsters were administered AAS during adolescence, scored for offensive aggression using the resident-intruder paradigm, and then examined for changes in PAG and GluR1 immunoreactivity in areas of the brain implicated in aggression control. When compared with sesame oil-treated control animals, aggressive AAS-treated hamsters displayed a significant increase in the number of PAG- and area density of GluR1- containing neurons in several notable aggression regions, although the differential pattern of expression did not appear to overlap across brain regions. Together, these results suggest that altered glutamate synthesis and GluR1 receptor expression in specific aggression areas may be involved in adolescent AAS-induced offensive aggression. PMID:17418431

  1. Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

    PubMed

    Lebbink, J H; Knapp, S; van der Oost, J; Rice, D; Ladenstein, R; de Vos, W M

    1999-06-01

    The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms. PMID:10366510

  2. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  3. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  4. STEREOLOGICAL ESTIMATES OF THE BASAL FOREBRAIN CELL POPULATION IN THE RAT, INCLUDING NEURONS CONTAINING CHOLINE ACETYLTRANSFERASE (ChAT), GLUTAMIC ACID DECARBOXYLASE (GAD) OR PHOSPHATE-ACTIVATED GLUTAMINASE (PAG) AND COLOCALIZING VESICULAR GLUTAMATE TRANSPORTERS (VGluTs)

    PubMed Central

    GRITTI, I.; HENNY, P.; GALLONI, F.; MAINVILLE, L.; MARIOTTI, M.; JONES, B. E.

    2006-01-01

    The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and are comprised by GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ~355,000 and the numbers of ChAT-immuno-positive (+) as ~22,000, GAD+ ~119,000 and PAG+ ~316,000, corresponding to ~5%, ~35% and ~90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also have the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, VGluT3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or

  5. Glutamate-induced activation of nitric oxide synthase is impaired in cerebral cortex in vivo in rats with chronic liver failure.

    PubMed

    Rodrigo, Regina; Erceg, Slaven; Rodriguez-Diaz, Jesus; Saez-Valero, Javier; Piedrafita, Blanca; Suarez, Isabel; Felipo, Vicente

    2007-07-01

    It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy. PMID:17286583

  6. Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

    PubMed Central

    Abbott, Stephen B. G.; Holloway, Benjamin B.; Viar, Kenneth E.; Guyenet, Patrice G.

    2016-01-01

    Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM-CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM-CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine-β-hydroxylase-expressing neurons in mice [DβHCre/0;VGLUT2flox/flox mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (DβHCre/0), using channelrhodopsin-2 (ChR2– mCherry) optogenetics. ChR2–mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90–99% colocalisation with tyrosine hydroxy-lase). RVLM-CA neurons had similar morphology and axonal projections in DβHCre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in DβHCre/0 and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in DβHCre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβHCre/0 mice only. Vagal responses were unaffected by α1-adrenoreceptor blockade. In conclusion, two responses evoked by RVLM-CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM-CA neurons are mediated by glutamate. PMID:24236954

  7. Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity.

    PubMed

    David, Hélène N; Abraini, Jacques H

    2002-03-01

    Evidence for functional interactions between metabotropic glutamate (mGlu) receptors and dopamine (DA) neurotransmission is now clearly established. In the present study, we investigated interactions between group III mGlu receptors and D1- and D2-like receptors in the nucleus accumbens (NAcc). Administration, into the NAcc, of the selective group III mGlu receptor agonist, AP4, resulted in an increase in locomotor activity, which was blocked by pretreatment with the group III mGlu receptor antagonist, MPPG. In addition, pretreatment with AP4 further blocked the increase in motor activity induced by the D1-like receptor agonist, SKF 38393, but potentiated the locomotor responses induced by either the D2-like receptor agonist, quinpirole, or coinfusion of SKF 38393 and quinpirole. MPPG reversed the effects of AP4 on the motor responses induced by D1-like and/or D2-like receptor activation. These results confirm that glutamate transmission may control DA-dependent locomotor function through mGlu receptors and further indicate that group III mGlu receptors oppose the behavioural response produced by D1-like receptor activation and favour those produced by D2-like receptor activation. PMID:11906529

  8. Spontaneous L-glutamate release enhancement in rat substantia gelatinosa neurons by (-)-carvone and (+)-carvone which activate different types of TRP channel.

    PubMed

    Kang, Qin; Jiang, Chang-Yu; Fujita, Tsugumi; Kumamoto, Eiichi

    2015-04-10

    Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of L-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (-)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (-)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (-)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (-)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (-)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (-)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous L-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG. PMID:25747716

  9. Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells.

    PubMed

    López-Colomé, Ana María; López, Edith; Mendez-Flores, Orquidia G; Ortega, Arturo

    2016-07-01

    Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function. PMID:27017513

  10. Vesicular Glutamate Transport Promotes Dopamine Storage and Glutamate Corelease In Vivo

    PubMed Central

    Hnasko, Thomas S.; Chuhma, Nao; Zhang, Hui; Goh, Germaine Y.; Sulzer, David; Palmiter, Richard D.; Rayport, Stephen; Edwards, Robert H.

    2010-01-01

    SUMMARY Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl−, indicating a distinct, presynaptic mechanism to regulate quantal size. PMID:20223200

  11. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  12. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells.

    PubMed

    Ribeiro, Mariana P C; Nunes-Correia, Isabel; Santos, Armanda E; Custódio, José B A

    2014-02-15

    Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. PMID:24240127

  13. Synergistic activities of a silver(I) glutamic acid complex and reactive oxygen species (ROS): a novel antimicrobial and chemotherapeutic agent.

    PubMed

    Batarseh, K I; Smith, M A

    2012-01-01

    The antimicrobial and chemotherapeutic activities of a silver(I) glutamic acid complex with the synergistic concomitant generation of reactive oxygen species (ROS) were investigated here. The ROS generation system employed was via Fenton chemistry. The antimicrobial and chemotherapeutic activities were investigated on Staphylococcus aureus ATCC 43300 and Escherichia coli bacteria, and Vero and MCF-7 tumor cell lines, respectively. Antimicrobial activities were conducted by determining minimum inhibitory concentration (MIC), while chemotherapeutic efficacies were done by serial dilution using standard techniques to determine the half maximal inhibitory concentration (IC50). The antimicrobial and chemotherapeutic results obtained were compared with positive control drugs gentamicin, oxacillin, penicillin, streptomycin and cisplatin, a ubiquitously used platinum-based antitumor drug, and with the silver(I) glutamic acid complex and hydrogen peroxide separately. Based on MIC and IC50 values, it was determined that this synergistic approach was very effective at extremely low concentrations, especially when compared with the other drugs evaluated here. This finding might be of great significance regarding metronomic dosing when this synergistic approach is clinically implemented. Since silver at low concentrations exhibits no toxic, mutagenic and carcinogenic activities, this might offer an alternative approach for the development of safer silver-based antimicrobial and chemotherapeutic drugs, thereby reducing or even eliminating the toxicity associated with current drugs. Accordingly, the present approach might be integrated into the systemic clinical treatment of infectious diseases and cancer. PMID:22680634

  14. Modes of glutamate receptor gating

    PubMed Central

    Popescu, Gabriela K

    2012-01-01

    Abstract The time course of excitatory synaptic currents, the major means of fast communication between neurons of the central nervous system, is encoded in the dynamic behaviour of post-synaptic glutamate-activated channels. First-pass attempts to explain the glutamate-elicited currents with mathematical models produced reaction mechanisms that included only the most basic functionally defined states: resting vs. liganded, closed vs. open, responsive vs. desensitized. In contrast, single-molecule observations afforded by the patch-clamp technique revealed an unanticipated kinetic multiplicity of transitions: from microseconds-lasting flickers to minutes-long modes. How these kinetically defined events impact the shape of the synaptic response, how they relate to rearrangements in receptor structure, and whether and how they are physiologically controlled represent currently active research directions. Modal gating, which refers to the slowest, least frequently observed ion-channel transitions, has been demonstrated for representatives of all ion channel families. However, reaction schemes have been largely confined to the short- and medium-range time scales. For glutamate receptors as well, modal gating has only recently come under rigorous scrutiny. This article reviews the evidence for modal gating of glutamate receptors and the still developing hypotheses about the mechanism(s) by which modal shifts occur and the ways in which they may impact the time course of synaptic transmission. PMID:22106181

  15. Effects of a metabotropic glutamate receptor subtype 7 negative allosteric modulator in the periaqueductal grey on pain responses and rostral ventromedial medulla cell activity in rat

    PubMed Central

    2013-01-01

    The metabotropic glutamate receptor 7 (mGluR7) negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), was locally microinjected into the ventrolateral periaqueductal gray (VL PAG) and the effect on pain responses in formalin and spare nerve injury (SNI) -induced neuropathic pain models was monitored in the rat. The activity of rostral ventromedial medulla (RVM) “pronociceptive” ON and “antinociceptive” OFF cells was also evaluated. Intra–VL PAG MMPIP blocked the first and second phase of nocifensive behaviour in the formalin pain model. MMPIP increased the tail flick latency and simultaneously increased the activity of the OFF cells while inhibiting that of ON cells in rats with SNI of the sciatic nerve. MMPIP failed to modify nociceptive responses and associated RVM ON and OFF cell activity in sham rats. An increase in mGluR7 gene, protein and staining, the latter being associated with vesicular glutamate transporter-positive profiles, has been found in the VL PAG in SNI rats. Blockade of mGluR7 within the VL PAG has an antinociceptive effect in formalin and neuropathic pain models. VL PAG mGluR7 blockade offers a target for dis-inhibiting the VL PAG-RVM pathway and silencing pain in inflammatory and neuropathic pain models. PMID:24004843

  16. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  17. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  18. Orally Active Metabotropic Glutamate Subtype 2 Receptor Positive Allosteric Modulators: Structure-Activity Relationships and Assessment in a Rat Model of Nicotine Dependence

    PubMed Central

    Sidique, Shyama; Dhanya, Raveendra-Panickar; Sheffler, Douglas J.; Nickols, Hilary Highfield; Yang, Li; Dahl, Russell; Mangravita-Novo, Arianna; Smith, Layton H.; D’Souza, Manoranjan S.; Semenova, Svetlana; Conn, P. Jeffrey; Markou, Athina; Cosford, Nicholas D. P.

    2012-01-01

    Compounds that modulate metabotropic glutamate subtype 2 (mGlu2) receptors have the potential to treat several disorders of the central nervous system (CNS) including drug dependence. Herein we describe the synthesis and structure-activity relationship (SAR) studies around a series of mGlu2 receptor positive allosteric modulators (PAMs). The effects of N-substitution (R1) and substitutions on the aryl ring (R2) were identified as key areas for SAR exploration (Figure 3). Investigation of the effects of varying substituents in both the isoindolinone (2) and benzisothiazolone (3) series led to compounds with improved in vitro potency and/or efficacy. In addition, several analogues exhibited promising pharmacokinetic (PK) properties. Furthermore, compound 2 was shown to dose-dependently decrease nicotine self-administration in rats following oral administration. Our data, showing for the first time efficacy of an mGlu2 receptor PAM in this in vivo model, suggest potential utility for the treatment of nicotine dependence in humans. PMID:23009245

  19. A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

    PubMed Central

    Uzun, Ozcan; Gabriel, Abram

    2001-01-01

    Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. PMID:11413300

  20. Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway.

    PubMed

    Mao, Xiao-Yuan; Zhou, Hong-Hao; Li, Xi; Liu, Zhao-Qian

    2016-08-01

    Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway. PMID:26440805

  1. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  2. Astrocyte origin of activity-dependent release of ATP and glutamate in hippocampal slices: real-time measurement utilizing microelectrode biosensors

    PubMed Central

    Sershen, Henry

    2012-01-01

    It is well known that astrocytic and neuronal transmitter release processes are important for signalling, and that activity-dependent release of adenosine nucleotides and transmitters occurs after stimulation. Neurons and astrocytes can account for the source of ATP efflux. In this issue of the BJP, Heinrich et al. characterized K+ depolarization-evoked release of ATP, adenosine and glutamate in hippocampal slices, utilizing microelectrode biosensors for simultaneous real-time recordings of multiple transmitter effluxes. They demonstrated efflux of ATP, adenosine and glutamate from hippocampus slices, in response to K+-depolarization, with distinct kinetics and mechanisms, suggesting a coordinated pattern of transmitter release. Surprisingly, it turned out that a considerable amount of the transmitter efflux measured under these conditions had a glial origin. For a long time, it was believed that the glial cell did not play a major role in neurotransmission, but the latter results somewhat change this view. The release of ATP and glutamate from glial cells under these conditions involved P2X7 receptors, and a source of adenosine accumulation independent of the metabolism of extracellular ATP was identified. This study also highlighted a novel use of multi-enzymatic microelectrode biosensors, which enabled a better characterization of transmitter release processes with higher temporal and spatial resolution than obtained previously. This technique was originally developed and used for the detection of purine release. In the present study, it was modified to identify the interplay between different transmitters, measured simultaneously in hippocampal slices. LINKED ARTICLE This article is a commentary on Heinrich et al., pp. 1003–1020 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.01932.x PMID:22703189

  3. Diphenyl diselenide elicits antidepressant-like activity in rats exposed to monosodium glutamate: A contribution of serotonin uptake and Na(+), K(+)-ATPase activity.

    PubMed

    Quines, Caroline B; Rosa, Suzan G; Velasquez, Daniela; Da Rocha, Juliana T; Neto, José S S; Nogueira, Cristina W

    2016-03-15

    Depression is a disorder with symptoms manifested at the psychological, behavioral and physiological levels. Monosodium glutamate (MSG) is the most widely used additive in the food industry; however, some adverse effects induced by this additive have been demonstrated in experimental animals and humans, including functional and behavioral alterations. The aim of this study was to investigate the possible antidepressant-like effect of diphenyl diselenide (PhSe)2, an organoselenium compound with pharmacological properties already documented, in the depressive-like behavior induced by MSG in rats. Male and female newborn Wistar rats were divided in control and MSG groups, which received, respectively, a daily subcutaneous injection of saline (0.9%) or MSG (4g/kg/day) from the 1st to 5th postnatal day. At 60th day of life, animals received (PhSe)2 (10mg/kg, intragastrically) 25min before spontaneous locomotor and forced swimming tests (FST). The cerebral cortices of rats were removed to determine [(3)H] serotonin (5-HT) uptake and Na(+), K(+)-ATPase activity. A single administration of (PhSe)2 was effective against locomotor hyperactivity caused by MSG in rats. (PhSe)2 treatment protected against the increase in the immobility time and a decrease in the latency for the first episode of immobility in the FST induced by MSG. Furthermore, (PhSe)2 reduced the [(3)H] 5-HT uptake and restored Na(+), K(+)-ATPase activity altered by MSG. In the present study a single administration of (PhSe)2 elicited an antidepressant-like effect and decrease the synaptosomal [(3)H] 5-HT uptake and an increase in the Na(+), K(+)-ATPase activity in MSG-treated rats. PMID:26738966

  4. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

    SciTech Connect

    Ribeiro, Mariana P.C.; Nunes-Correia, Isabel; Santos, Armanda E.; Custódio, José B.A.

    2014-02-15

    Recent reports suggest that N-methyl-D-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. - Highlights: • MK-801 and memantine decrease melanoma cell proliferation. • The combination of MK-801 with antiestrogens inhibits melanoma cell proliferation. • These combinations greatly enhance the effects of the compounds individually. • MK-801 combined with tamoxifen active metabolites induces cell cycle arrest in G1. • The combination of MK-801 and antiestrogens is an innovative strategy for melanoma.

  5. Glutamate and Neurodegenerative Disease

    NASA Astrophysics Data System (ADS)

    Schaeffer, Eric; Duplantier, Allen

    As the main excitatory neurotransmitter in the mammalian central nervous system, glutamate is critically involved in most aspects of CNS function. Given this critical role, it is not surprising that glutamatergic dysfunction is associated with many CNS disorders. In this chapter, we review the literature that links aberrant glutamate neurotransmission with CNS pathology, with a focus on neurodegenerative diseases. The biology and pharmacology of the various glutamate receptor families are discussed, along with data which links these receptors with neurodegenerative conditions. In addition, we review progress that has been made in developing small molecule modulators of glutamate receptors and transporters, and describe how these compounds have helped us understand the complex pharmacology of glutamate in normal CNS function, as well as their potential for the treatment of neurodegenerative diseases.

  6. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  7. Activation of type 5 metabotropic glutamate receptor promotes the proliferation of rat retinal progenitor cell via activation of the PI-3-K and MAPK signaling pathways.

    PubMed

    Zhang, Z; Hu, F; Liu, Y; Ma, B; Chen, X; Zhu, K; Shi, Y; Wei, T; Xing, Y; Gao, Y; Lu, H; Liu, Y; Kang, Q

    2016-05-13

    The metabotropic glutamate receptor 5 (mGluR5) regulates neurogenesis in the brain, but the effect of mGluR5 on retinal progenitor cells (RPCs) remains unknown. In this study, we found that mGluR5 promoted the proliferation of rat RPCs with activation of the phosphatidylinositol-3-kinase (PI-3-K) and mitogen-activated protein kinase (MAPK) signaling pathways in vitro. The mGluR5 agonist (S)-3,5-dihydroxyphenylglycine hydrate (DHPG) increased the cellular viability in a concentration- and time-dependent manner, whereas the mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine hydrochloride (MTEP) had the opposite effect, as shown by 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTT) assay. Treatment with DHPG (100μM) also promoted the proliferation of RPCs, as indicated by 5-Bromo-2-deoxyUridine (BrdU) staining and flow cytometry, and likewise, MTEP (100μM) and mGluR5 knockdown abolished the action of mGluR5 activity. Western blot demonstrated that the activation of mGluR5 enhanced the expression of Cyclin D1 and the phosphorylation level of PKC however, MTEP or mGluR5 knockdown also abrogated the effect of DHPG on RPCs. Furthermore, we found that activation of the extracellular signal-regulated protein kinase (ERK) and protein kinase B (AKT) signaling pathways was involved in the proliferation of RPC. After DHPG treatment, the levels of both p-ERK1/2 and p-AKT increased in a time-dependent manner. Then we used MTEP, mGluR5 knockdown, the ERK1/2 inhibitor U0126 and the AKT inhibitor LY294002 to pretreat the cells, and all of them clearly eliminated the influence of DHPG. These results demonstrated that mGluR5 regulates neurogenesis in RPCs through the MAPK and PI-3-K signaling pathways, and these findings may motivate a pharmacological study investigating a potential mechanism for the treatment of retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). PMID:26902516

  8. Activation of Group I and Group II Metabotropic Glutamate Receptors Causes LTD and LTP of Electrical Synapses in the Rat Thalamic Reticular Nucleus.

    PubMed

    Wang, Zemin; Neely, Ryan; Landisman, Carole E

    2015-05-13

    Compared with the extensive characterization of chemical synaptic plasticity, electrical synaptic plasticity remains poorly understood. Electrical synapses are strong and prevalent among the GABAergic neurons of the rodent thalamic reticular nucleus. Using paired whole-cell recordings, we show that activation of Group I metabotropic glutamate receptors (mGluRs) induces long-term depression of electrical synapses. Conversely, activation of the Group II mGluR, mGluR3, induces long-term potentiation of electrical synapses. By testing downstream targets, we show that modifications induced by both mGluR groups converge on the same signaling cascade--adenylyl cyclase to cAMP to protein kinase A--but with opposing effects. Furthermore, the magnitude of modification is inversely correlated to baseline coupling strength. Thus, electrical synapses, like their chemical counterparts, undergo both strengthening and weakening forms of plasticity, which should play a significant role in thalamocortical function. PMID:25972185

  9. Functional Insights from Glutamate Receptor Ion Channel Structures

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2014-01-01

    X-ray crystal structures for the soluble amino terminal and ligand binding domains of glutamate receptor ion channels, combined with a 3.6 Å resolution structure of the full length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of iGluR assembly and function. Increasingly sophisticated biochemical, computational and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists, and yield new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for development of subtype selective modulators. Many issues remain unsolved, including the role of the ATD in AMPA receptor signaling, and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how binding of glutamate opens the ion channel pore. PMID:22974439

  10. Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation

    PubMed Central

    Raju, Karthik; Doulias, Paschalis-Thomas; Evans, Perry; Krizman, Elizabeth N.; Jackson, Joshua G.; Horyn, Oksana; Daikhin, Yevgeny; Nissim, Ilana; Yudkoff, Marc; Nissim, Itzhak; Sharp, Kim A.; Robinson, Michael B.; Ischiropoulos, Harry

    2016-01-01

    Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS−/−) or neuronal nitric oxide synthase (nNOS−/−). In particular, nNOS−/− animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. 15N-glutamine–based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS−/− mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys373 and Cys561 in wild-type and eNOS−/− mice, but not in nNOS−/− mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism. PMID:26152695

  11. A novel GSK-3β inhibitor YQ138 prevents neuronal injury induced by glutamate and brain ischemia through activation of the Nrf2 signaling pathway

    PubMed Central

    Pang, Tao; Wang, Yun-jie; Gao, Yuan-xue; Xu, Yuan; Li, Qiu; Zhou, Yu-bo; Xu, Lei; Huang, Zhang-jian; Liao, Hong; Zhang, Lu-yong; Gao, Jian-rong; Ye, Qing; Li, Jia

    2016-01-01

    Aim: To discover neuroprotective compounds and to characterize the discovered active compound YQ138 as a novel GSK-3β inhibitor. Methods: Primary rat cerebellar granule cells (CGCs) were treated with glutamate, and cell viability was analyzed with MTT assay, which was used as in vitro model for screening neuroprotective compounds. Active compound was further tested in OGD- or serum deprivation-induced neuronal injury models. The expression levels of GSK-3β downstream proteins (Nrf2, HO-1, NQO1, Tau and β-catenin) were detected with Western blotting. For evaluating the neuroprotective effects in vivo, adult male rats were subjected to transient middle cerebral artery occlusion (tMCAO), then treated with YQ138 (10 mg/kg, iv) at 2, 4 and 6 h after ischemia onset. Results: From a compound library consisting of about 2000 potential kinase inhibitors, YQ138 was found to exert neuroprotective effects: pretreatment with YQ138 (0.1–40 μmol/L) dose-dependently inhibited glutamate-induced neuronal death. Furthermore, pretreatment with YQ138 (10 μmol/L) significantly inhibited OGD- or serum deprivation-induced neuronal death. Among a panel of seven kinases tested, YQ138 selectively inhibited the activity of GSK-3β (IC50=0.52 nmol/L). Furthermore, YQ138 dose-dependently increased the expression of β-catenin, and decreased the phosphorylation of Tau in CGCs. Moreover, YQ138 significantly increased the expression of GSK-3β downstream antioxidative proteins Nrf2, HO-1, NQO1, GSH and SOD in CGCs. In rats with tMCAO, administration of YQ138 significantly decreased infarct volume, improved the neurological deficit, and increased the expression of Nrf2 and HO-1 and the activities of SOD and GSH in the cerebral cortex. Conclusion: A novel GSK-3β inhibitor YQ138 effectively suppresses brain ischemic injury in vitro and in vivo. PMID:27108601

  12. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  13. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  14. Activation of Metabotropic Glutamate Receptor 7 Is Required for Induction of Long-Term Potentiation at SC-CA1 Synapses in the Hippocampus

    PubMed Central

    Klar, Rebecca; Walker, Adam G.; Ghose, Dipanwita; Grueter, Brad A.; Engers, Darren W.; Hopkins, Corey R.; Lindsley, Craig W.; Xiang, Zixiu

    2015-01-01

    Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders. PMID:25972184

  15. Ginkgo biloba Extract (EGb 761®) Inhibits Glutamate-induced Up-regulation of Tissue Plasminogen Activator Through Inhibition of c-Fos Translocation in Rat Primary Cortical Neurons.

    PubMed

    Cho, Kyu Suk; Lee, Ian Myungwon; Sim, Seobo; Lee, Eun Joo; Gonzales, Edson Luck; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Kwon, Kyoung Ja; Han, Seol-Heui

    2016-01-01

    EGb 761(®) , a standardized extract of Ginkgo biloba leaves, has antioxidant and antiinflammatory properties in experimental models of neurodegenerative disorders such as stroke and Alzheimer's disease. Tissue plasminogen activator (tPA) acts a neuromodulator and plays a crucial role in the manifestation of neurotoxicity leading to exaggerated neuronal cell death in neurological insult conditions. In this study, we investigated the effects of EGb 761 on the basal and glutamate-induced activity and expression of tPA in rat primary cortical neurons. Under basal condition, EGb 761 inhibited both secreted and cellular tPA activities, without altering tPA mRNA level, as modulated by the activation of p38. Compared with basal condition, EGb 761 inhibited the glutamate-induced up-regulation of tPA mRNA resulting in the normalization of overt tPA activity and expression. c-Fos is a component of AP-1, which plays a critical role in the modulation of tPA expression. Interestingly, EGb 761 inhibited c-Fos nuclear translocation without affecting c-Fos expression in glutamate-induced rat primary cortical neurons. These results demonstrated that EGb 761 can modulate tPA activity under basal and glutamate-stimulated conditions by both translational and transcriptional mechanisms. Thus, EGb 761 could be a potential and effective therapeutic strategy in tPA-excessive neurotoxic conditions. PMID:26478151

  16. Glutamate uptake block triggers deadly rhythmic bursting of neonatal rat hypoglossal motoneurons

    PubMed Central

    Sharifullina, Elina; Nistri, Andrea

    2006-01-01

    In the brain the extracellular concentration of glutamate is controlled by glial transporters that restrict the neurotransmitter action to synaptic sites and avoid excitotoxicity. Impaired transport of glutamate occurs in many cases of amyotrophic lateral sclerosis, a devastating motoneuron disease. Motoneurons of the brainstem nucleus hypoglossus are among the most vulnerable, giving early symptoms like slurred speech and dysphagia. However, the direct consequences of extracellular glutamate build-up, due to uptake block, on synaptic transmission and survival of hypoglossal motoneurons remain unclear and have been studied using the neonatal rat brainstem slice preparation as a model. Patch clamp recording from hypoglossal motoneurons showed that, in about one-third of these cells, inhibition of glutamate transport with the selective blocker dl-threo-β-benzyloxyaspartate (TBOA; 50 μ m) unexpectedly led to the emergence of rhythmic bursting consisting of inward currents of long duration with superimposed fast oscillations and synaptic events. Synaptic inhibition block facilitated bursting. Bursts had a reversal potential near 0 mV, and were blocked by tetrodotoxin, the gap junction blocker carbenoxolone, or antagonists of AMPA, NMDA or mGluR1 glutamate receptors. Intracellular Ca2+ imaging showed bursts as synchronous discharges among motoneurons. Synergy of activation of distinct classes of glutamate receptor plus gap junctions were therefore essential for bursting. Ablating the lateral reticular formation preserved bursting, suggesting independence from propagated network activity within the brainstem. TBOA significantly increased the number of dead motoneurons, an effect prevented by the same agents that suppressed bursting. Bursting thus represents a novel hallmark of motoneuron dysfunction triggered by glutamate uptake block. PMID:16455692

  17. Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

    PubMed

    Kasatkina, Ludmila A; Borisova, Tatiana A

    2013-11-01

    Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is

  18. Glutamic acid decarboxylase activity is stimulated in quail retina neuronal cells transformed by Rous sarcoma virus and is regulated by pp60v-src.

    PubMed Central

    Crisanti, P; Lorinet, A M; Calothy, G; Pessac, B

    1985-01-01

    Rous sarcoma virus (RSV) stimulates in quail embryo neuro-retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY-68, a thermodependent transformation-defective mutant of RSV, stimulation of GAD activity is regulated by pp60v-src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY-68-transformed established NR cell lines, have a high GAD activity which is regulated by pp60v-src, while other clones have a low GAD activity apparently not regulated by pp60v-src. These data indicate that pp60v-src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v-mil. PMID:2992933

  19. Glutamate Receptor Dynamics in Dendritic Microdomains

    PubMed Central

    Newpher, Thomas M.; Ehlers, Michael D.

    2008-01-01

    Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity. PMID:18498731

  20. [Glutamate transporter dysfunction and major mental illnesses].

    PubMed

    Tanaka, Kohichi

    2016-01-01

    Glutamate is the main excitatory neurotransmitter in the central nervous system and plays an important role in most aspects of normal brain function. In spite of its importance as a neurotransmitter, excess glutamate is toxic to neurons. Clearance of extracellular glutamate is critical for maintenance of low extracellular glutamate concentration, and occurs in large part through the activity of GLT1 (EAAT2) and GLAST (EAAT1), which are primarily expressed by astrocytes. Rare variants and down-regulation of GLT1 and GLAST, in psychiatric disorders have been reported. In this review, we demonstrate that various kinds of GLT1 and/or GLAST knockout mice replicate many aspects of the behavioral abnormalities seen in major mental illnesses including schizophrenia, depression, obsessive -compulsive disorders, autism, epilepsy and addiction. PMID:26793898

  1. Ligands for Ionotropic Glutamate Receptors

    NASA Astrophysics Data System (ADS)

    Swanson, Geoffrey T.; Sakai, Ryuichi

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory syn-aptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors.

  2. EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

    PubMed Central

    Allhorn, Maria; Olsén, Arne; Collin, Mattias

    2008-01-01

    Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. Results We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. Conclusion We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself. PMID:18182097

  3. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    NASA Astrophysics Data System (ADS)

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-01

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5‧-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  4. Glutamate Racemase Dimerization Inhibits Dynamic Conformational Flexibility and Reduces Catalytic Rates

    SciTech Connect

    Mehboob, Shahila; Guo, Liang; Fu, Wentao; Mittal, Anuradha; Yau, Tiffany; Truong, Kent; Johlfs, Mary; Long, Fei; Fung, Leslie W.-M.; Johnson, Michael E.

    2009-09-15

    Glutamate racemase (RacE) is a bacterial enzyme that converts L-glutamate to D-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with D-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the D-Glu to L-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.

  5. Activators of the Glutamate-Dependent Acid Resistance System Alleviate Deleterious Effects of YidC Depletion in Escherichia coli▿

    PubMed Central

    Yu, Zhong; Bekker, Martijn; Tramonti, Angela; Cook, Gregory M.; van Ulsen, Peter; Scheffers, Dirk-Jan; de Mattos, Joost Teixeira; De Biase, Daniela; Luirink, Joen

    2011-01-01

    The function of the essential inner membrane protein (IMP) YidC in Escherichia coli has been studied for a limited number of model IMPs and primarily using targeted approaches. These studies suggested that YidC acts at the level of insertion, folding, and quality control of IMPs, both in the context of the Sec translocon and as a separate entity. To further our understanding of YidC's role in IMP biogenesis, we screened a random overexpression library for factors that rescued the growth of cells upon YidC depletion. We found that the overexpression of the GadX and GadY regulators of the glutamate-dependent acid resistance system complemented the growth defect of YidC-depleted cells. Evidence is presented that GadXY overexpression counteracts the deleterious effects of YidC depletion on at least two fronts. First, GadXY prepares the cells for the decrease in respiratory capacity upon the depletion of YidC. Most likely, GadXY-regulated processes reduce the drop in proton-motive force that impairs the fitness of YidC-depleted cells. Second, in GadXY-overproducing cells increased levels of the general chaperone GroEL cofractionate with the inner membranes, which may help to keep newly synthesized inner membrane proteins in an insertion-competent state when YidC levels are limiting. PMID:21216990

  6. New phenylglycine derivatives with potent and selective antagonist activity at presynaptic glutamate receptors in neonatal rat spinal cord.

    PubMed

    Jane, D E; Pittaway, K; Sunter, D C; Thomas, N K; Watkins, J C

    1995-08-01

    The depression of the monosynaptic excitation of neonatal rat motoneurones produced by the metabotropic glutamate receptor (mGluR) agonists (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate (ACPD) or L-2-amino-4-phosphonobutyrate (L-AP4) was antagonized by three novel phenylglycine analogues: (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG), (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) and (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG). The potencies of all the new compounds were greater than that of the previously reported (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG). For L-AP4-sensitive presynaptic mGluRs, the order of antagonist potency found was MPPG > MSPG > MTPG > MCPG. In contrast, the order of antagonist potency found for (1S,3S)-ACPD-sensitive presynaptic mGluRs was MTPG > MPPG > MSPG > MCPG. To date, MPPG (KD 9.2 microM) is the most potent L-AP4-sensitive receptor antagonist yet tested on the neonatal rat spinal cord. In addition, MTPG (KD 77 microM) is the most potent antagonist yet tested for (1S,3S)-ACPD-sensitive receptors in this preparation. PMID:8532166

  7. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective. PMID:25676153

  8. Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

    NASA Technical Reports Server (NTRS)

    Belliveau, J. W.; Lanyi, J. K.

    1977-01-01

    Halobacterium halobium is known to contain sheets of bacteriorhodopsin, a pigment which upon exposure to light undergoes cyclic protonation and deprotonation, resulting in net H(+) translocation. In this paper, experiments were conducted to test H. halobium cell envelope vesicles for respiration-induced glutamate uptake. It is shown that glutamate transport in H. halobium cell envelope vesicles can occur as a result of respiration, as well as light acting on bacteriorhodopsin. Glutamate transport can be energized by the oxidation of dimethyl phenylenediamine, and the properties of the transport system are entirely analogous to those observed with illumination as the source of energy. In the case of respiration-dependent glutamate transport, the transportation is also driven by a Na(+) gradient, thereby confirming the existence of a single glutamate transport system independent of the source of energy. The analogy observed is indirect evidence that the cytochrome oxidase of H. halobium functions as a H(+) pump.

  9. A novel glutamate transport system in poly(γ-glutamic acid)-producing strain Bacillus subtilis CGMCC 0833.

    PubMed

    Wu, Qun; Xu, Hong; Zhang, Dan; Ouyang, Pingkai

    2011-08-01

    Bacillus subtilis CGMCC 0833 is a poly(γ-glutamic acid) (γ-PGA)-producing strain. It has the capacity to tolerate high concentration of extracellular glutamate and to utilize glutamate actively. Such a high uptake capacity was owing to an active transport system for glutamate. Therefore, a specific transport system for L-glutamate has been observed in this strain. It was a novel transport process in which glutamate was symported with at least two protons, and an inward-directed sodium gradient had no stimulatory effect on it. K(m) and V(m) for glutamate transport were estimated to be 67 μM and 152 nmol⁻¹ min⁻¹ mg⁻¹ of protein, respectively. The transport system showed structural specificity and stereospecificity and was strongly dependent on extracellular pH. Moreover, it could be stimulated by Mg²⁺, NH₄⁺, and Ca²⁺. In addition, the glutamate transporter in this strain was studied at the molecular level. As there was no important mutation of the transporter protein, it appeared that the differences of glutamate transporter properties between this strain and other B. subtilis strains were not due to the differences of the amino acid sequence and the structure of transporter protein. This is the first extensive report on the properties of glutamate transport system in γ-PGA-producing strain. PMID:21437781

  10. Expression of the survival of motor neuron (SMN) gene in primary neurons and increase in SMN levels by activation of the N-methyl-D-aspartate glutamate receptor.

    PubMed

    Andreassi, Catia; Patrizi, Anna Letizia; Monani, Umrao R; Burghes, A H M; Brahe, Christina; Eboli, Maria Luisa

    2002-03-01

    Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease caused by mutations of the survival of motor neuron (SMN) gene. The SMN protein is expressed ubiquitously as part of a 300-kilodalton multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. During the differentiation process of spinal motor neurons and cerebellar granule cells, the acquisition of mature electrophysiological and molecular properties is linked to the activation of the glutamate receptors of N-methyl-D-aspartate (NMDA) subtype. We have used primary cultures of rat cerebellar granules to study SMN expression during neuronal differentiation in vitro and in response to the activation of the NMDA receptor. We report that the expression of gems, the nuclear structures where SMN concentrates, is developmentally regulated. The highest expression is associated with the cell clustering phase and expression of NMDA receptors. Stimulation of the NMDA receptor induces an increase in gem number and in SMN transcription, through activation of its promoter. These results demonstrate that SMN levels are dependent on synaptic activity, implying that SMN may have important neuron-specific functions downstream of synaptic activation. PMID:12030329

  11. Topiramate antagonism of L-glutamate-induced paroxysms in planarians

    PubMed Central

    Raffa, Robert B.; Finno, Kristin E.; Tallarida, Christopher S.; Rawls, Scott M.

    2010-01-01

    We recently reported that NMDA (N-Methyl-D-aspartate) and AMPA (α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) induce concentration-dependent paroxysms in planarians (Dugesia dorotocephala). Since the postulated mechanisms of action of the sulfamate-substituted monosaccharide antiepileptic drug topiramate include inhibition of glutamate-activated ion channels, we tested the hypothesis that topiramate would inhibit glutamate-induced paroxysms in our model. We demonstrate that: (1) L-glutamate (1–10 mM), but not D-glutamate, induced dose-related paroxysms, and that (2) topiramate dose-relatedly (0.3–3 mM) inhibited L-glutamate-induced paroxysms. These results provide further evidence of a topiramate-sensitive glutamate receptor-mediated activity in this model. PMID:20863783

  12. Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme.

    PubMed

    Ling, V; Snedden, W A; Shelp, B J; Assmann, S M

    1994-08-01

    The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway. PMID:7919983

  13. Activation of glial glutamate transporter via MAPK p38 prevents enhanced and long-lasting non-evoked resting pain after surgical incision in rats.

    PubMed

    Reichl, Sylvia; Segelcke, Daniel; Keller, Viktor; Jonas, Robin; Boecker, Armin; Wenk, Manuel; Evers, Dagmar; Zahn, Peter K; Pogatzki-Zahn, Esther M

    2016-06-01

    Pain after surgery has recently become a major issue not only due to lack of treatment success in the acute phase; even more alarming is the large number of patients developing prolonged pain after surgery. Because spinal glutamate as well as spinal glia plays a major role in acute incisional pain, we investigated the role of the spinal glial glutamate transporters (GT), GLAST, GLT-1, for acute and prolonged pain and hyperalgesia caused by an incision. Spinal administration of the GT-inhibitor DL-TBOA increased non-evoked pain but not evoked pain behavior (hyperalgesia) up to 2 weeks after incision. In accordance, spinal GLAST (and to a lesser degree GLT-1) were upregulated after incision for several days. Long-term incision induced GT upregulation was prevented by long-lasting p38-inhibitor administration but not by long-lasting ERK1/2-inhibition after incision. In accordance, daily treatment with the p38-inhibitor (but not the ERK1/2 inhibitor) prolonged non-evoked but not evoked pain behavior after incision. In electrophysiological experiments, spontaneous activity of high threshold (HT) (but not wide dynamic range (WDR)) neurons known to transmit incision induced non-evoked pain was increased after prolonged treatment with the p38-inhibitor. In conclusion, our findings indicate a new spinal pathway by which non-evoked pain behavior after incision is modulated. The pathway is modality (non-evoked pain) and neuron (HT) specific and disturbance contributes to prolonged long-term pain after surgical incision. This may have therapeutic implications for the treatment of acute and - even more relevant - for prevention of chronic pain after surgery in patients. PMID:26920805

  14. [Glutamate neurotransmission, stress and hormone secretion].

    PubMed

    Jezová, D; Juránková, E; Vigas, M

    1995-11-01

    Glutamate neurotransmission has been investigated in relation to several physiological processes (learning, memory) as well as to neurodegenerative and other disorders. Little attention has been paid to its involvement in neuroendocrine response during stress. Penetration of excitatory amino acids from blood to the brain is limited by the blood-brain barrier. As a consequence, several toxic effects but also bioavailability for therapeutic purposes are reduced. A free access to circulating glutamate is possible only in brain structures lacking the blood-brain barrier or under conditions of its increased permeability. Excitatory amino acids were shown to stimulate the pituitary hormone release, though the mechanism of their action is still not fully understood. Stress exposure in experimental animals induced specific changes in mRNA levels coding the glutamate receptor subunits in the hippocampus and hypothalamus. The results obtained with the use of glutamate receptor antagonists indicate that a number of specific receptor subtypes contribute to the stimulation of ACTH release during stress. The authors provided also data on the role of NMDA receptors in the control of catecholamine release, particularly in stress-induced secretion of epinephrine. These results were the first piece of evidence on the involvement of endogenous excitatory amino acids in neuroendocrine activation during stress. Neurotoxic effects of glutamate in animals are well described, especially after its administration in the neonatal period. In men, glutamate toxicity and its use as a food additive are a continuous subject of discussions. The authors found an increase in plasma cortisol and norepinephrine, but not epinephrine and prolactin, in response to the administration of a high dose of glutamate. It cannot be excluded that these effects might be induced even by lower doses in situations with increased vulnerability to glutamate action (age, individual variability). (Tab. 1, Fig. 6, Ref. 44

  15. Exercise increases mitochondrial glutamate oxidation in the mouse cerebral cortex.

    PubMed

    Herbst, Eric A F; Holloway, Graham P

    2016-07-01

    The present study investigated the impact of acute exercise on stimulating mitochondrial respiratory function in mouse cerebral cortex. Where pyruvate-stimulated respiration was not affected by acute exercise, glutamate respiration was enhanced following the exercise bout. Additional assessment revealed that this affect was dependent on the presence of malate and did not occur when substituting glutamine for glutamate. As such, our results suggest that glutamate oxidation is enhanced with acute exercise through activation of the malate-aspartate shuttle. PMID:27184881

  16. Synthesis of a specific monolithic column with artificial recognition sites for L-glutamic acid via cryo-crosslinking of imprinted nanoparticles.

    PubMed

    Göktürk, Ilgım; Üzek, Recep; Uzun, Lokman; Denizli, Adil

    2016-06-01

    In this study, a new molecular imprinting (MIP)-based monolithic cryogel column was prepared using chemically crosslinked molecularly imprinted nanoparticles, to achieve a simplified chromatographic separation (SPE) for a model compound, L-glutamic acid (L-Glu). Cryogelation through crosslinking of imprinted nanoparticles forms stable monolithic cryogel columns. This technique reduces the leakage of nanoparticles and increases the surface area, while protecting the structural features of the cryogel for stable and efficient recognition of the template molecule. A non-imprinted monolithic cryogel column (NIP) was also prepared, using non-imprinted nanoparticles produced without the addition of L-Glu during polymerization. The molecularly imprinted monolithic cryogel column (MIP) indicates apparent recognition selectivity and a good adsorption capacity compared to the NIP. Also, we have achieved a significant increase in the adsorption capacity, using the advantage of high surface area of the nanoparticles. PMID:25749280

  17. Glutamate-induced sensitization of rat masseter muscle fibers.

    PubMed

    Cairns, B E; Gambarota, G; Svensson, P; Arendt-Nielsen, L; Berde, C B

    2002-01-01

    In rats, intradermal or intraarticular injection of glutamate or selective excitatory amino acid receptor agonists acting at peripheral excitatory amino acid receptors can decrease the intensity of mechanical stimulation required to evoke nocifensive behaviors, an indication of hyperalgesia. Since excitatory amino acid receptors have been found on the terminal ends of cutaneous primary afferent fibers, it has been suggested that increased tissue glutamate levels may have a direct sensitizing effect on primary afferent fibers, in particular skin nociceptors. However, less is known about the effects of glutamate on deep tissue afferent fibers. In the present study, a series of experiments were undertaken to investigate the effect of intramuscular injection of glutamate on the excitability and mechanical threshold of masseter muscle afferent fibers in anesthetized rats of both sexes. Injection of 1.0 M, but not 0.1 M glutamate evoked masseter muscle afferent activity that was significantly greater than that evoked by isotonic saline. The mechanical threshold of masseter muscle afferent fibers, which was assessed with a Von Frey hair, was reduced by approximately 50% for a period of 30 min after injection of 1.0 M glutamate, but was unaffected by injections of 0.1 M glutamate or isotonic saline. Injection of 25% dextrose, which has the same osmotic strength as 1.0 M glutamate, did not evoke significant activity in or decrease the mechanical threshold of masseter muscle afferent fibers. Magnetic resonance imaging experiments confirmed that injection of 25% dextrose and 1.0 M glutamate produced similar edema volumes in the masseter muscle tissue. Co-injection of 0.1 M kynurenate, an excitatory amino acid receptor antagonist, and 1.0 M glutamate attenuated glutamate-evoked afferent activity and prevented glutamate-induced mechanical sensitization. When male and female rats were compared, no difference in the baseline mechanical threshold or in the magnitude of glutamate

  18. Conformational coupling between the active site and residues within the KC-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase

    PubMed Central

    Ahn, Young O.; Mahinthichaichan, Paween; Lee, Hyun Ju; Ouyang, Hanlin; Kaluka, Daniel; Yeh, Syun-Ru; Arjona, Davinia; Rousseau, Denis L.; Tajkhorshid, Emad; Ädelroth, Pia; Gennis, Robert B.

    2014-01-01

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the KC-channel is a conserved glutamate in subunit III. However, the majority of the KC-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the KC-channel, was found to depend on the conformation of Y241Vc, located in subunit I at the interface with subunit III. Mutations of Y241Vc (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241Vc and the active site cross-linked Y255Vc, as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme. PMID:25288772

  19. Release of ATP and glutamate in the nucleus tractus solitarii mediate pulmonary stretch receptor (Breuer–Hering) reflex pathway

    PubMed Central

    Gourine, Alexander V; Dale, Nicholas; Korsak, Alla; Llaudet, Enrique; Tian, Faming; Huckstepp, Robert; Spyer, K Michael

    2008-01-01

    The Breuer–Hering inflation reflex is initiated by activation of the slowly adapting pulmonary stretch receptor afferents (SARs), which monosynaptically activate second-order relay neurones in the dorsal medullary nucleus of the solitary tract (NTS). Here we demonstrate that during lung inflation SARs release both ATP and glutamate from their central terminals to activate these NTS neurones. In anaesthetized and artificially ventilated rats, ATP- and glutamate-selective microelectrode biosensors placed in the NTS detected rhythmic release of both transmitters phase-locked to lung inflation. This release of ATP and glutamate was independent of the centrally generated respiratory rhythm and could be reversibly abolished during the blockade of the afferent transmission in the vagus nerve by topical application of local anaesthetic. Microionophoretic application of ATP increased the activity of all tested NTS second-order relay neurones which receive monosynaptic inputs from the SARs. Unilateral microinjection of ATP into the NTS site where pulmonary stretch receptor afferents terminate produced central apnoea, mimicking the effect of lung inflation. Application of P2 and glutamate receptor antagonists (pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid, suramin and kynurenic acid) significantly decreased baseline lung inflation-induced firing of the second-order relay neurones. These data demonstrate that ATP and glutamate are released in the NTS from the central terminals of the lung stretch receptor afferents, activate the second-order relay neurones and hence mediate the key respiratory reflex — the Breuer—Hering inflation reflex. PMID:18617567

  20. Exciting Times for Pancreatic Islets: Glutamate Signaling in Endocrine Cells.

    PubMed

    Otter, Silke; Lammert, Eckhard

    2016-03-01

    Glutamate represents a key excitatory neurotransmitter in the central nervous system, and also modulates the function and viability of endocrine cells in pancreatic islets. In insulin-secreting beta cells, glutamate acts as an intracellular messenger, and its transport into secretory granules promotes glucose- and incretin-stimulated insulin secretion. Mitochondrial degradation of glutamate also contributes to insulin release when glutamate dehydrogenase is allosterically activated. It also signals extracellularly via glutamate receptors (AMPA and NMDA receptors) to modulate glucagon, insulin and somatostatin secretion, and islet cell survival. Its degradation products, GABA and γ-hydroxybutyrate, are released and also influence islet cell behavior. Thus, islet glutamate receptors, such as the NMDA receptors, might serve as possible drug targets to develop new medications for adjunct treatment of diabetes. PMID:26740469

  1. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    DOE PAGESBeta

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active sitemore » metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.« less

  2. Monomeric Corynebacterium glutamicum N-acetyl glutamate kinase maintains sensitivity to L-arginine but has a lower intrinsic catalytic activity.

    PubMed

    Huang, Yuanyuan; Li, Cheng; Zhang, Hao; Liang, Shuli; Han, Shuangyan; Lin, Ying; Yang, Xiaorong; Zheng, Suiping

    2016-02-01

    N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine, and L-arginine-sensitive NAGK typically has hexameric architecture. Defining the relationship between this architecture and L-arginine inhibition can provide a foundation to identify the key amino acids involved in the allosteric regulation network of L-arginine. In the present study, the key amino acids in the N-terminal helix (N-helix) of Corynebacterium glutamicum (Cg) NAGK required for hexamer formation were determined using structural homology modeling and site-directed mutagenesis. It was also verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis, but that it is not the determinant of inhibition by L-arginine. Monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form, indicating that monomers contain an independent, sensitive signal transduction network of L-arginine to mediate allosteric regulation. Mutation studies of CgNAGKs also revealed that amino acid residues 18-23 of the N-helix are required for inhibition by L-arginine, and that E19 may be an essential amino acid influencing the apparent affinity of L-arginine. Collectively, these studies may illuminate the basic mechanism of metabolic homeostasis of C. glutamicum. PMID:26512006

  3. Astroglial glutamate transporters coordinate excitatory signaling and brain energetics.

    PubMed

    Robinson, Michael B; Jackson, Joshua G

    2016-09-01

    In the mammalian brain, a family of sodium-dependent transporters maintains low extracellular glutamate and shapes excitatory signaling. The bulk of this activity is mediated by the astroglial glutamate transporters GLT-1 and GLAST (also called EAAT2 and EAAT1). In this review, we will discuss evidence that these transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various 'energy-generating' systems, including the Na(+)/K(+)-ATPase, the Na(+)/Ca(2+) exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the 'fuel' to support glutamate uptake. Given the importance of glutamate uptake to maintaining synaptic signaling and preventing excitotoxicity, it should not be surprising that some of these systems appear to 'redundantly' support the energetic costs of glutamate uptake. Although the glutamate-glutamine cycle contributes to recycling of neurotransmitter pools of glutamate, this is an over-simplification. The ramifications of co-compartmentalization of glutamate transporters with mitochondria for glutamate metabolism are discussed. Energy consumption in the brain accounts for ∼20% of the basal metabolic rate and relies almost exclusively on glucose for the production of ATP. However, the brain does not possess substantial reserves of glucose or other fuels. To ensure adequate energetic supply, increases in neuronal activity are matched by increases in cerebral blood flow via a process known as 'neurovascular coupling'. While the mechanisms for this coupling are not completely resolved, it is generally agreed that astrocytes, with processes that extend to synapses and endfeet that surround blood vessels, mediate at least some of the signal that causes vasodilation. Several studies have shown that either genetic deletion or

  4. ATPase active-site electrostatic interactions control the global conformation of the 100 kDa SecA translocase

    PubMed Central

    Kim, Dorothy M.; Zheng, Haiyan; Huang, Yuanpeng J.; Montelione, Gaetano T.; Hunt, John F.

    2013-01-01

    SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in E. coli SecA and triggers it completely in B. subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its sidechain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes. PMID:23167435

  5. Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

    PubMed

    Sancho-Vaello, Enea; Fernández-Murga, M Leonor; Rubio, Vicente

    2008-04-01

    N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity. PMID:18319063

  6. Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor*

    PubMed Central

    Choi, Ucheor B.; Kazi, Rashek; Stenzoski, Natalie; Wollmuth, Lonnie P.; Uversky, Vladimir N.; Bowen, Mark E.

    2013-01-01

    The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating. PMID:23782697

  7. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  8. Impaired circadian photosensitivity in mice lacking glutamate transmission from retinal melanopsin cells.

    PubMed

    Gompf, Heinrich S; Fuller, Patrick M; Hattar, Samer; Saper, Clifford B; Lu, Jun

    2015-02-01

    Intrinsically photoreceptive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and convey retinal light inputs to the circadian system via the retinohypothalamic tract (RHT) projection to the suprachiasmatic nucleus (SCN). The principal neurotransmitter of this projection is glutamate, and ipRGCs use the vesicular glutamate transporter 2 (VGLUT2) to package glutamate into synaptic vesicles. However, these neurons contain other potential neurotransmitters, such as pituitary adenylate cyclase activating polypeptide (PACAP). To test the role of glutamate in mediating ipRGC light inputs into the SCN, we crossed mice in which Cre-recombinase expression is driven by the melanopsin promotor (Opn4(Cre/+)) with mice in which the second exon of VGLUT2 is flanked by loxP sites (VGLUT2(fl/fl)), producing ipRGCs that are unable to package glutamate into synaptic vesicles. Such mice had free-running circadian rhythms that did not entrain to a 12:12 light-dark (12:12 LD) cycle, nor did they show a phase delay after a 45-min light pulse administered at circadian time (CT) 14. A small subset of the mice did appear to entrain to the 12:12 LD cycle with a positive phase angle to lights-off; a similar entrainment pattern could be achieved in free-running mice if they were exposed to a 12:12 LD cycle with light of a greater intensity. Glutamate transmission from the ipRGCs is necessary for normal light entrainment of the SCN at moderate (0.35 W/m(2)) light levels, but residual transmission (possibly by PACAP in ipRGCs or by other RGCs) can weakly entrain animals, particularly at very high (6.53 W/m(2)) light levels, although it may be less effective at suppressing locomotor activity (light masking). PMID:25512304

  9. Distinct inhibition of acute cocaine-stimulated motor activity following microinjection of a group III metabotropic glutamate receptor agonist into the dorsal striatum of rats.

    PubMed

    Mao, L; Wang, J Q

    2000-09-01

    Group III metabotropic glutamate receptors (mGluRs) are negatively coupled to adenylate cyclase through G-proteins. Activation of this group of mGluRs shows an inhibition of dopaminergic transmission in the forebrain. To define the role of striatal group III mGluRs in the regulation of basal and dopamine-stimulated motor behavior, the recently developed agonist and antagonist relatively selective for group III mGluRs were utilized to pharmacologically enhance and reduce group III mGluR glutamatergic tone in the dorsal striatum of chronically cannulated rats. Bilateral injections of a group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), did not alter basal levels of motor activity at three doses surveyed (1, 10, and 100 nmol). Neither did intracaudate injection of a group III antagonist, alpha-methyl-4-phosphonophenylglycine (MPPG), at 10, 30, and 100 nmol. However, pretreatment with L-AP4 (10 and 100 nmol) dose dependently blocked hyperlocomotion induced by acute injection of cocaine (20 mg/kg, i.p.), amphetamine (2.5 mg/kg, i.p.), or apomorphine (1 mg/kg, s.c.). The behavioral activity induced by cocaine was much more sensitive to L-AP4 than that induced by amphetamine and apomorphine. At 100 nmol, L-AP4 completely blocked cocaine effect whereas amphetamine- and apomorphine-stimulated behaviors were blocked only by 28% and 31%, respectively. The blocking effect of L-AP4 on cocaine action was reversed by pretreatment with MPPG. MPPG itself did not modify behavioral responses to cocaine, amphetamine, or apomorphine. These data indicate that the glutamatergic tone on the group III mGluRs is not active in the regulation of basal and acute dopamine-stimulated motor activity. However, enhanced group III mGluR glutamatergic transmission by an exogenous ligand is capable of suppressing behavioral responses to acute exposure of dopamine stimulants. PMID:11113488

  10. Brain to blood glutamate scavenging as a novel therapeutic modality: a review.

    PubMed

    Boyko, Matthew; Gruenbaum, Shaun E; Gruenbaum, Benjamin F; Shapira, Yoram; Zlotnik, Alexander

    2014-08-01

    It is well known that abnormally elevated glutamate levels in the brain are associated with secondary brain injury following acute and chronic brain insults. As such, a tight regulation of brain glutamate concentrations is of utmost importance in preventing the neurodegenerative effects of excess glutamate. There has been much effort in recent years to better understand the mechanisms by which glutamate is reduced in the brain to non-toxic concentrations, and in how to safely accelerate these mechanisms. Blood glutamate scavengers such as oxaloacetate, pyruvate, glutamate-oxaloacetate transaminase, and glutamate-pyruvate transaminase have been shown to reduce blood glutamate concentrations, thereby increasing the driving force of the brain to blood glutamate efflux and subsequently reducing brain glutamate levels. In the past decade, blood glutamate scavengers have gained increasing international interest, and its uses have been applied to a wide range of experimental contexts in animal models of traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, epilepsy, migraine, and malignant gliomas. Although glutamate scavengers have not yet been used in humans, there is increasing evidence that their use may provide effective and exciting new therapeutic modalities. Here, we review the laboratory evidence for the use of blood glutamate scavengers. Other experimental neuroprotective treatments thought to scavenge blood glutamate, including estrogen and progesterone, beta-adrenergic activation, hypothermia, insulin and glucagon, and hemodialysis and peritoneal dialysis are also discussed. The evidence reviewed here will hopefully pave the way for future clinical trials. PMID:24623040

  11. Crystal structure and molecular mechanism of an aspartate/glutamate racemase from Escherichia coli O157.

    PubMed

    Liu, Xiuhua; Gao, Fei; Ma, Yinliang; Liu, Shuang; Cui, Yaqi; Yuan, Zenglin; Kang, Xianjiang

    2016-04-01

    EcL-DER, the aspartate/glutamate racemase from the pathogen Escherichia coli O157, exhibits racemase activity for l-aspartate and l-glutamate. This study reports the crystal structures of apo-EcL-DER, the EcL-DER-l-aspartate and the EcL-DER-d-aspartate complexes. The EcL-DER structure contains two domains, forming pseudo-mirror symmetry in the active site. A unique catalytic pair consisting of Thr(83) and Cys(197) exists in the active site. The characteristic conformations of l-Asp and d-Asp in the active site provide a straight structural evidence for the racemization mechanism of EcL-DER. In addition, the diversity of catalytic pairs implies that PLP-independent amino acid racemases adopt various catalytic mechanisms and are classified into different subgroups. PMID:27001440

  12. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  13. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  14. Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

    PubMed

    Xie, Wenjie; Dolder, Silvia; Siegrist, Mark; Wetterwald, Antoinette; Hofstetter, Willy

    2016-08-01

    Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response

  15. Classical Conditioning of the Rabbit Eyelid Response Increases Glutamate Receptor Binding in Hippocampal Synaptic Membranes

    NASA Astrophysics Data System (ADS)

    Mamounas, Laura A.; Thompson, Richard F.; Lynch, Gary; Baudry, Michel

    1984-04-01

    Hippocampal pyramidal neurons exhibit a rapid within-trial increase in firing frequency during classical conditioning of the rabbit eyelid response. It has been proposed that the cellular mechanisms responsible for hippocampal long-term potentiation (LTP) may also mediate this learning-dependent increase in neuronal activity. The induction of LTP in rat hippocampal slices results in an increase in the number of [3H]glutamate-binding sites in the potentiated region. The present study investigates the kinetics of [3H]glutamate binding to hippocampal synaptic membranes after eyelid conditioning in the rabbit. We report that the regional distribution of [3H]glutamate binding across the layers of rabbit hippocampus is compatible with a dendritic localization. The pharmacological and ionic properties of the binding suggest that it is associated with an excitatory amino acid receptor. After eyelid conditioning, the maximal number of hippocampal [3H]glutamate-binding sites is increased in animals receiving paired presentations of the tone conditioned stimulus and corneal air-puff unconditioned stimulus relative to that found in naive or unpaired control animals. These results strengthen the hypothesis that an LTP-like mechanism underlies the increase in hippocampal firing frequency during rabbit eyelid conditioning.

  16. The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

    PubMed

    Lee, Hae-Ri; Jeon, Jun Ho; Park, Ok-Kyu; Chun, Jeong-Hoon; Park, Jungchan; Rhie, Gi-Eun

    2015-12-01

    The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively. PMID:26350415

  17. Activation of group III metabotropic glutamate receptors inhibits basal and amphetamine-stimulated dopamine release in rat dorsal striatum: an in vivo microdialysis study.

    PubMed

    Mao, L; Lau, Y S; Wang, J Q

    2000-09-22

    Group III metabotropic glutamate (mGlu) receptors are negatively coupled to adenylate cyclase and are distributed pre-synaptically in the striatum. A behavioral study previously conducted in this laboratory shows that activation of this group of mGlu receptors attenuates acute amphetamine-stimulated motor activity. By administering a group III selective agonist or antagonist via the dialysis probe, the present study employed in vivo microdialysis to evaluate the capacity of the group III selective agents to alter extracellular levels of dopamine in the dorsal striatum of normal and amphetamine-treated rats. It was found that the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4) dose-dependently (1, 10 and 100 microM) reduced basal levels of extracellular dopamine. In contrast, the group III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG) dose-dependently (10, 50 and 250 microM) elevated the basal release of extracellular dopamine. This elevation was antagonized by co-perfusion of L-AP4. Perfusion of 5-microM amphetamine through the dialysis probe increased extracellular dopamine in the dorsal striatum. Co-perfusion of L-AP4 (100 microM) significantly reduced amphetamine-stimulated dopamine levels, whereas co-perfusion of L-AP4 (100 microM) and MPPG (100 microM) did not alter the capacity of amphetamine to elicit dopamine release. The data obtained from this study demonstrate the presence of a tonically active glutamatergic tone on group III mGlu receptors in the dorsal striatum to pre-synaptically regulate basal dopamine release in an inhibitory fashion. Moreover, activation of L-AP4-sensitive group III mGlu receptors can suppress the phasic release of dopamine induced by a dopamine stimulant amphetamine. PMID:10996594

  18. Silencing Glycogen Synthase Kinase-3β Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1*

    PubMed Central

    Shinohara, Mie; Ybanez, Maria D.; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2010-01-01

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver. PMID:20061376

  19. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  20. Activation of mGluR7s inhibits cocaine-induced reinstatement of drug-seeking behavior by a nucleus accumbens glutamate-mGluR2/3 mechanism in rats.

    PubMed

    Li, Xia; Li, Jie; Gardner, Eliot L; Xi, Zheng-Xiong

    2010-09-01

    The metabotropic glutamate receptor 7 (mGluR7) has been reported to be involved in cocaine and alcohol self-administration. However, the role of mGluR7 in relapse to drug seeking is unknown. Using a rat relapse model, we found that systemic administration of AMN082, a selective mGluR7 allosteric agonist, dose-dependently inhibits cocaine-induced reinstatement of drug-seeking behavior. Intracranial microinjections of AMN082 into the nucleus accumbens (NAc) or ventral pallidum, but not the dorsal striatum, also inhibited cocaine-primed reinstatement, an effect that was blocked by local co-administration of MMPIP, a selective mGluR7 antagonist. In vivo microdialysis demonstrated that cocaine priming significantly increased extracellular dopamine in the NAc, ventral pallidum and dorsal striatum, while increasing extracellular glutamate in the NAc only. AMN082 alone failed to alter extracellular dopamine, but produced a slow-onset long-lasting increase in extracellular glutamate in the NAc only. Pre-treatment with AMN082 dose-dependently blocked both cocaine-enhanced NAc glutamate and cocaine-induced reinstatement, an effect that was blocked by MMPIP or LY341497 (a selective mGluR2/3 antagonist). These data suggest that mGluR7 activation inhibits cocaine-induced reinstatement of drug-seeking behavior by a glutamate-mGluR2/3 mechanism in the NAc. The present findings support the potential use of mGluR7 agonists for the treatment of cocaine addiction. PMID:20534005

  1. The synthesis and characterization of poly(γ-glutamic acid)-coated magnetite nanoparticles and their effects on antibacterial activity and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Inbaraj, B. Stephen; Kao, T. H.; Tsai, T. Y.; Chiu, C. P.; Kumar, R.; Chen, B. H.

    2011-02-01

    Magnetite nanoparticles (MNPs) modified with sodium and calcium salts of poly(γ-glutamic acid) (NaPGA and CaPGA) were synthesized by the coprecipitation method, followed by characterization and evaluation of their antibacterial and cytotoxic effects. Superparamagnetic MNPs are particularly attractive for magnetic driving as well as bacterial biofilm and cell targeting in in vivo applications. Characterization of synthesized MNPs by the Fourier transform infrared spectra and magnetization curves confirmed the PGA coating on MNPs. The mean diameter of NaPGA- and CaPGA-coated MNPs as determined by transmission electron microscopy was 11.8 and 14 nm, respectively, while the x-ray diffraction pattern revealed the as-synthesized MNPs to be pure magnetite. Based on agar dilution assay, both NaPGA- and CaPGA-coated MNPs showed a lower minimum inhibitory concentration in Salmonella enteritidis SE 01 than the commercial antibiotics linezolid and cefaclor, but the former was effective against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 10832, whereas the latter was effective against Escherichia coli O157:H7 TWC 01. An in vitro cytotoxicity study in human skin fibroblast cells as measured by MTT assay implied the as-synthesized MNPs to be nontoxic. This outcome demonstrated that both γ-PGA-modified MNPs are cytocompatible and possess antibacterial activity in vitro, and thereby should be useful in in vivo studies for biomedical applications.

  2. Complex formation and catalytic activation by the PII signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942.

    PubMed

    Maheswaran, Mani; Urbanke, Claus; Forchhammer, Karl

    2004-12-31

    The signal transduction protein P(II) from the cyanobacterium Synechococcus elongatus strain PCC 7942 forms a complex with the key enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase (NAGK). Here we report the effect of complex formation on the catalytic properties of NAGK. Although pH and ion dependence are not affected, the catalytic efficiency of NAGK is strongly enhanced by binding of P(II), with K(m) decreasing by a factor of 10 and V(max) increasing 4-fold. In addition, arginine feedback inhibition of NAGK is strongly decreased in the presence of P(II), resulting in a tight control of NAGK activity under physiological conditions by P(II). Analysis of the NAGK-P(II) complex suggests that one P(II) trimer binds to one NAGK hexamer with a K(d) of approximately 3 nm. Complex formation is strongly affected by ATP and ADP. ADP is a strong inhibitor of complex formation, whereas ATP inhibits complex formation only in the absence of divalent cations or in the presence of Mg(2+) ions, together with increased 2-oxoglutarate concentrations. Ca(2+) is able to antagonize the negative effect of ATP and 2-oxoglutarate. ADP and ATP exert their adverse effect on NAGK-P(II) complex formation through binding to the P(II) protein. PMID:15502156

  3. On the regulative role of the glutamate receptor in mitochondria.

    PubMed

    Selin, Alexey A; Lobysheva, Natalia V; Nesterov, Semen V; Skorobogatova, Yulia A; Byvshev, Ivan M; Pavlik, Lyubov L; Mikheeva, Irina B; Moshkov, Dmitry A; Yaguzhinsky, Lev S; Nartsissov, Yaroslav R

    2016-05-01

    The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia. PMID:26812870

  4. Chronic Glutamate Toxicity in Neurodegenerative Diseases—What is the Evidence?

    PubMed Central

    Lewerenz, Jan; Maher, Pamela

    2015-01-01

    Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases. PMID:26733784

  5. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    PubMed

    Levite, Mia

    2014-08-01

    Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti-glutamate

  6. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  7. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  8. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  9. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  10. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  11. Kinemage of action - Proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

    SciTech Connect

    Sorensen, John L.; Stetefeld, Joerg

    2011-10-07

    Highlights: {yields} Inhibitors of tetrapyrrole cofactor biosynthesis may be useful antibiotics. {yields} Mechanism of critical enzyme, glutamate-1-semialdehyde aminomutase, is presented. {yields} Unique vitamin B6-dependant enzyme traps intermediate in active site. {yields} Molecular dynamics show that a re-orientation of the substrate is required. -- Abstract: Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an external cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.

  12. Human glutamate pyruvate transaminase (GPT): Localization to 8q24.3, cDNA and genomic sequences, and polymorphic sites

    SciTech Connect

    Sohocki, M.M.; Sullivan, L.S.; Daiger, S.P.

    1997-03-01

    Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a {open_quotes}half-YAC{close_quotes} from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this world-wide polymorphism. 22 refs., 3 figs.

  13. Modulation of intestinal L-glutamate transport by luminal leptin.

    PubMed

    Fanjul, Carmen; Barrenetxe, Jaione; Lostao, María Pilar; Ducroc, Robert

    2015-06-01

    Leptin is secreted into the digestive tract and contributes to the absorption of dietary molecules by regulating transporters activity. Here, we studied the effect of luminal leptin on the intestinal transport of L-glutamate, an important component of human diet. We examined the effect of leptin on L-glutamate uptake in rat intestine in vitro measuring glutamate-induced short-circuit current (Isc) in Ussing chambers and L-[(3)H (U)]-glutamate uptake in jejunal everted rings. Glutamate-induced Isc was only observed in Na(+)-free conditions. This Isc was concentration (1-60 mmol L(-1)) and pH dependent. Luminal leptin increased glutamate Isc (∼100 %). Dose-response curve showed a biphasic pattern, with maximal stimulations observed at 10(-13) and 10(-10) mmol L(-1), that were sensitive to leptin receptor antagonist. In everted rings, two glutamate transport mechanisms were distinguished: a Na(+)-dependent, H(+)-independent, that was inhibited by leptin (∼20 %), and a Na(+)-independent but H(+)-dependent, that was enhanced by leptin (∼20 %), in line with data obtained in Ussing chambers. Altogether, these data reveal original non-monotonic effect of luminal leptin in the intestine and demonstrate a new role for this hormone in the modulation of L-glutamate transport, showing that luminal active gut peptides can influence absorption of amino acids. PMID:25935421

  14. Neuronal vs glial glutamate uptake: Resolving the conundrum.

    PubMed

    Danbolt, N C; Furness, D N; Zhou, Y

    2016-09-01

    Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox. PMID:27235987

  15. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  16. Activating mutations in human c-Ha-ras-1 gene induced by reactive derivatives of safrole and the glutamic pyrolysis product, Glu-P-3.

    PubMed

    Ireland, C M; Cooper, C S; Marshall, C J; Hebert, E; Phillips, D H

    1988-09-01

    Foci of transformed NIH3T3 cells were observed after transfection of plasmids containing the c-Ha-ras-1 protooncogene modified in vitro either with the 3-N,N-acetoxyacetyl derivative (N-AcO-AGlu-P-3) of the mutagenic L-glutamic acid pyrolysis product 3-amino-4,6-dimethyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-3) or with 1'-acetoxysafrole (AcO-S), a reactive derivative of the carcinogen safrole. DNA isolated from these foci were used in a second round of transfection, and the DNA obtained from the secondary transformants was analysed to determine the nature of mutations responsible for activating the protooncogene. The polymerase chain reaction method was used to amplify sequences of the gene likely to contain activating mutations, and these regions were then subjected to selective hybridization with specific oligonucleotides to locate and identify the point mutations. Five out of six transformants induced by N-AcO-AGlu-P-3 contained mutations at codon 61. Three of the codon 61 mutations were at the first base and the other two were at the third base, all were GC----TA transversions. Two AcO-S-induced transformants contained a GC----TA transversion, in one case at the first base of codon 61, in the other at the first base of codon 12. Another AcO-S-induced transformant, and the sixth transformant induced by N-AcO-AGlu-P-3 were apparently not mutated in codon 12, 61 or 117. Both N-AcO-AGlu-P-3 and AcO-S react predominantly with guanine residues in DNA, and all the mutations identified here were at GC base pairs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3070278

  17. Mitochondrial Ca(2+) Processing by a Unit of Mitochondrial Ca(2+) Uniporter and Na(+)/Ca(2+) Exchanger Supports the Neuronal Ca(2+) Influx via Activated Glutamate Receptors.

    PubMed

    Strokin, Mikhail; Reiser, Georg

    2016-06-01

    The current study demonstrates that in hippocampal neurons mitochondrial Ca(2+) processing supports Ca(2+) influx via ionotropic glutamate (Glu) receptors. We define mitochondrial Ca(2+) processing as Ca(2+) uptake via mitochondrial Ca(2+) uniporter (MCU) combined with subsequent Ca(2+) release via mitochondrial Na(+)/Ca(2+) exchanger (NCX). Our tool is to measure the Ca(2+) influx rate in primary hippocampal co-cultures, i.e. neurons and astrocytes, by fluorescent digital microscopy, using a Fura-2-quenching method where we add small amounts of Mn(2+) in the superfusion medium. Thus, Ca(2+) influx is measured with Mn(2+) in the bath. Ru360 as inhibitor of mitochondrial Ca(2+) uptake through MCU strongly reduces the rate of Ca(2+) influx in Glu-stimulated primary hippocampal neurons. Similarly, the Ca(2+) influx rate in Glu-stimulated neurons declines after suppression of potential-dependent MCU, when we depolarize mitochondria with rotenone. With inhibition of Ca(2+) release from mitochondria via NCX using CGP-37157 the Ca(2+) influx via N-methyl-D-aspartate (NMDA)- and kainate-sensitive receptors is slowed down. Working jointly as mitochondrial Ca(2+) processing unit, MCU and NCX, apparently sustain the Ca(2+) throughput of activated Glu-sensitive receptors. Our results revise the role frequently attributed to mitochondria in neuronal Ca(2+) homeostasis, where mitochondria function mainly as Ca(2+) buffer, and prevent excessively high cytosolic Ca(2+) concentration increase during neuronal activity. The mechanism to control Ca(2+) influx in neurons, as discovered in this study, highlights mitochondrial Ca(2+) processing as a promising pharmacological target. We discuss this pathway in relation to the endoplasmic reticulum-related mechanisms of Ca(2+) processing. PMID:26842930

  18. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  19. Activation of Type 4 Metabotropic Glutamate Receptor Attenuates Oxidative Stress-Induced Death of Neural Stem Cells with Inhibition of JNK and p38 MAPK Signaling.

    PubMed

    Zhang, Zhichao; Ma, Wen; Wang, Li; Gong, Hanshi; Tian, Yumei; Zhang, Jianshui; Liu, Jianxin; Lu, Haixia; Chen, Xinlin; Liu, Yong

    2015-11-15

    Promoting both endogenous and exogenous neural stem cells' (NSCs) survival in the hostile host environments is essential to cell replacement therapy for central nervous system (CNS) disorders. Type 4 metabotropic glutamate receptor (mGluR4), one of the members of mGluRs, has been shown to protect neurons from acute and chronic excitotoxic insults in various brain damages. The present study investigated the preventive effects of mGluR4 on NSC injury induced by oxidative stress. Under challenge with H2O2, loss of cell viability was observed in cultured rat NSCs, and treatment with selective mGluR4 agonist VU0155041 conferred protective effects against the loss of cellular viability in a concentration-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Pretreatment of VU0155041 (30 μM) also inhibited the excessive NSC death induced by H2O2, and group III mGluRs antagonist (RS)-a-methylserine-O-phosphate (MSOP) or gene-targeted knockdown abolished the protective action of mGluR4, indicated by propidium iodide-Hoechst and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. Western blot assay demonstrated that mGluR4 activation reversed the decreased procaspase-8/9/3and the destructed Bcl-2/Bax expressing balance, and likewise, MSOP and mGluR4 knockdown abrogated the action of mGluR4 activity. Furthermore, inhibition of JNK and p38 mitogen-activated protein kinases (MAPKs) were observed after mGluR4 activation, and as paralleling control, JNK-specific inhibitor SP600125 and p38-specific inhibitor SB203580 significantly rescued the H2O2-mediated NSC apoptosis and cleavage of procaspase-3. We suggest that activation of mGluR4 prevents oxidative stress-induced NSC death and apoptotic-associated protein activities with involvement of inhibiting the JNK and p38 pathways in cell culture. Our findings may help to develop strategies for enhancing the resided and transplanted NSC survival

  20. Activation of Phosphatidylinositol-Linked Dopamine Receptors Induces a Facilitation of Glutamate-Mediated Synaptic Transmission in the Lateral Entorhinal Cortex

    PubMed Central

    Glovaci, Iulia; Chapman, C. Andrew

    2015-01-01

    The lateral entorhinal cortex receives strong inputs from midbrain dopamine neurons that can modulate its sensory and mnemonic function. We have previously demonstrated that 1 µM dopamine facilitates synaptic transmission in layer II entorhinal cortex cells via activation of D1-like receptors, increased cAMP-PKA activity, and a resulting enhancement of AMPA-receptor mediated currents. The present study assessed the contribution of phosphatidylinositol (PI)-linked D1 receptors to the dopaminergic facilitation of transmission in layer II of the rat entorhinal cortex, and the involvement of phospholipase C activity and release of calcium from internal stores. Whole-cell patch-clamp recordings of glutamate-mediated evoked excitatory postsynaptic currents were obtained from pyramidal and fan cells. Activation of D1-like receptors using SKF38393, SKF83959, or 1 µM dopamine induced a reversible facilitation of EPSCs which was abolished by loading cells with either the phospholipase C inhibitor U-73122 or the Ca2+ chelator BAPTA. Neither the L-type voltage-gated Ca2+ channel blocker nifedipine, nor the L/N-type channel blocker cilnidipine, blocked the facilitation of synaptic currents. However, the facilitation was blocked by blocking Ca2+ release from internal stores via inositol 1,4,5-trisphosphate (InsP3) receptors or ryanodine receptors. Follow-up studies demonstrated that inhibiting CaMKII activity with KN-93 failed to block the facilitation, but that application of the protein kinase C inhibitor PKC(19-36) completely blocked the dopamine-induced facilitation. Overall, in addition to our previous report indicating a role for the cAMP-PKA pathway in dopamine-induced facilitation of synaptic transmission, we demonstrate here that the dopaminergic facilitation of synaptic responses in layer II entorhinal neurons also relies on a signaling cascade dependent on PI-linked D1 receptors, PLC, release of Ca2+ from internal stores, and PKC activation which is likely dependent

  1. Compounds extracted from Phyllantus and Jatropha elliptica inhibit the binding of [3H]glutamate and [3H]GMP-PNP in rat cerebral cortex membrane.

    PubMed

    Martini, L H; Souza, C R; Marques, P B; Calixto, J B; Yunes, R A; Souza, D O

    2000-02-01

    Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [3H]glutamate and [3H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [3H]glutamate binding and geraniin inhibited [3H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds. PMID:10786704

  2. Neurotoxic Potential of Lunar and Martian Dust: Influence on Em, Proton Gradient, Active Transport, and Binding of Glutamate in Rat Brain Nerve Terminals

    PubMed Central

    Krisanova, Natalia; Kasatkina, Ludmila; Sivko, Roman; Borysov, Arseniy; Nazarova, Anastasiya; Slenzka, Klaus; Borisova, Tatiana

    2013-01-01

    Abstract The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[14C]glutamate, it was shown that there is an increase in l-[14C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na+] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation

  3. [Glutamate and malignant gliomas, from epilepsia to biological aggressiveness: therapeutic implications].

    PubMed

    Blecic, Serge; Rynkowski, Michal; De Witte, Olivier; Lefranc, Florence

    2013-09-01

    In this review article, we describe the unrecognized roles of glutamate and glutamate receptors in malignant glioma biology. The neurotransmitter glutamate released from malignant glioma cells in the extracellular matrix is responsible for seizure induction and at higher concentration neuronal cell death. This neuronal cell death will create vacated place for tumor growth. Glutamate also stimulates the growth and the migration of glial tumor cells by means of the activation of glutamate receptors on glioma cells in a paracrine and autocrine manner. The multitude of effects of glutamate in glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters in the adjuvant treatment of malignant gliomas in neurology and neuro-oncology. Using the website www.clinicaltrials.gov/ as a reference - a service developed by the National Library of Medicine for the National Health Institute in USA - we have evoked the few clinical trials completed and currently ongoing with therapies targeting the glutamate receptors. PMID:23883552

  4. The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid.

    PubMed Central

    Rasool, C G; Nicolaidis, S; Akhtar, M

    1976-01-01

    A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase. PMID:10889

  5. Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

    PubMed Central

    Grassini, Gaia; Pennacchietti, Eugenia; Cappadocio, Francesca; Occhialini, Alessandra; De Biase, Daniela

    2015-01-01

    In orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known “classical” Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved. PMID:25853037

  6. Structural Features of the Glutamate Transporter Family

    PubMed Central

    Slotboom, Dirk Jan; Konings, Wil N.; Lolkema, Juke S.

    1999-01-01

    Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary

  7. Mood disorders: regulation by metabotropic glutamate receptors.

    PubMed

    Pilc, Andrzej; Chaki, Shigeyuki; Nowak, Gabriel; Witkin, Jeffrey M

    2008-03-01

    Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling. PMID:18164691

  8. Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity, and Increases the Sensitivity of the Circadian System to Light

    PubMed Central

    Pritchett, David; Jagannath, Aarti; Brown, Laurence A.; Tam, Shu K. E.; Hasan, Sibah; Gatti, Silvia; Harrison, Paul J.; Bannerman, David M.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes Grm2 and Grm3). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of Grm2/3 double knockout (Grm2/3-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, Grm2/3-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, Grm2/3-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of Grm2/3-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained. PMID:25950516

  9. Lithium stimulates glutamate "release" and inositol 1,4,5-trisphosphate accumulation via activation of the N-methyl-D-aspartate receptor in monkey and mouse cerebral cortex slices.

    PubMed Central

    Dixon, J F; Los, G V; Hokin, L E

    1994-01-01

    Beginning at therapeutic concentrations (1-1.5 mM), the anti-manic-depressive drug lithium stimulated the release of glutamate, a major excitatory neurotransmitter in the brain, in monkey cerebral cortex slices in a time- and concentration-dependent manner, and this was associated with increased inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphoric acid (CPP), dizocilpine (MK-801), ketamine, and Mg(2+)-antagonists to the N-methyl-D-aspartate (NMDA) receptor/channel complex selectively inhibited lithium-stimulated Ins(1,4,5)P3 accumulation. Antagonists to cholinergic-muscarinic, alpha 1-adrenergic, 5-hydroxytryptamine2 (serotoninergic), and H1 histaminergic receptors had no effect. Antagonists to non-NMDA glutamate receptors had no effect on lithium-stimulated Ins(1,4,5)P3 accumulation. Possible reasons for this are discussed. Similar results were obtained in mouse cerebral cortex slices. Carbetapentane, which inhibits glutamate release, inhibited lithium-induced Ins(1,4,5)P3 accumulation in this model. It is concluded that the primary effect of lithium in the cerebral cortex slice model is stimulation of glutamate release, which, presumably via activation of the NMDA receptor, leads to Ca2+ entry. Ins(1,4,5)P3 accumulation increases due to the presumed increased influx of intracellular Ca2+, which activates phospholipase C. These effects may have relevance to the therapeutic action of lithium in the treatment of manic depression as well as its toxic effects, especially at lithium blood levels above 1.5 mM. Images PMID:8078888

  10. Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity, and Increases the Sensitivity of the Circadian System to Light.

    PubMed

    Pritchett, David; Jagannath, Aarti; Brown, Laurence A; Tam, Shu K E; Hasan, Sibah; Gatti, Silvia; Harrison, Paul J; Bannerman, David M; Foster, Russell G; Peirson, Stuart N

    2015-01-01

    Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes Grm2 and Grm3). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of Grm2/3 double knockout (Grm2/3-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, Grm2/3-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, Grm2/3-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of Grm2/3-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained. PMID:25950516

  11. The novel isoxazoline ectoparasiticide fluralaner: selective inhibition of arthropod γ-aminobutyric acid- and L-glutamate-gated chloride channels and insecticidal/acaricidal activity.

    PubMed

    Gassel, Michael; Wolf, Christian; Noack, Sandra; Williams, Heike; Ilg, Thomas

    2014-02-01

    Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide. PMID:24365472

  12. Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase.

    PubMed

    Vacca, R A; Giannattasio, S; Graber, R; Sandmeier, E; Marra, E; Christen, P

    1997-08-29

    Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct. PMID:9268327

  13. A review of glutamate's role in traumatic brain injury mechanisms

    NASA Astrophysics Data System (ADS)

    Good, Cameron H.

    2013-05-01

    Glutamate is the primary excitatory neurotransmitter used by the central nervous system (CNS) for synaptic communication, and its extracellular concentration is tightly regulated by glutamate transporters located on nearby astrocytes. Both animal models and human clinical studies have demonstrated elevated glutamate levels immediately following a traumatic brain event, with the duration and severity of the rise corresponding to prognosis. This rise in extracellular glutamate likely results from a combination of excessive neurotransmitter release from damaged neurons and down regulation of uptake mechanisms in local astrocytes. The immediate results of a traumatic event can lead to necrotic tissue in severely injured regions, while prolonged increases in excitatory transmission can cause secondary excitotoxic injury through activation of delayed apoptotic pathways. Initial TBI animal studies utilized a variety of broad glutamate receptor antagonists to successfully combat secondary injury mechanisms, but unfortunately this same strategy has proven inconclusive in subsequent human trials due to deleterious side effects and heterogeneity of injuries. More recent treatment strategies have utilized specific glutamate receptor subunit antagonists in an effort to minimize side effects and have shown promising results. Future challenges will be detecting the concentration and kinetics of the glutamate rise following injury, determining which patient populations could benefit from antagonist treatment based on their extracellular glutamate concentrations and when drugs should be administered to maximize efficacy.

  14. Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

    PubMed

    Leone, M J; Beaule, C; Marpegan, L; Simon, T; Herzog, E D; Golombek, D A

    2015-05-01

    The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system. PMID:25798929

  15. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  16. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  17. Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.

    PubMed

    Thomas, Ajit G; O'Driscoll, Cliona M; Bressler, Joseph; Kaufmann, Walter; Rojas, Camilo J; Slusher, Barbara S

    2014-01-01

    Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest. PMID:24269238

  18. Cocaine-induced neuroadaptations in the dorsal striatum: glutamate dynamics and behavioral sensitization.

    PubMed

    Parikh, Vinay; Naughton, Sean X; Shi, Xiangdang; Kelley, Leslie K; Yegla, Brittney; Tallarida, Christopher S; Rawls, Scott M; Unterwald, Ellen M

    2014-09-01

    Recent evidence suggests that diminished ability to control cocaine seeking arises from perturbations in glutamate homeostasis in the nucleus accumbens. However, the neurochemical substrates underlying cocaine-induced neuroadaptations in the dorsal striatum and how these mechanisms link to behavioral plasticity is not clear. We employed glutamate-sensitive microelectrodes and amperometry to study the impact of repeated cocaine administration on glutamate dynamics in the dorsolateral striatum of awake freely-moving rats. Depolarization-evoked glutamate release was robustly increased in cocaine-pretreated rats challenged with cocaine. Moreover, the clearance of glutamate signals elicited either by terminal depolarization or blockade of non-neuronal glutamate transporters slowed down dramatically in cocaine-sensitized rats. Repeated cocaine exposure also reduced the neuronal tone of striatal glutamate. Ceftriaxone, a β-lactam antibiotic that activates the astrocytic glutamate transporter, attenuated the effects of repeated cocaine exposure on synaptic glutamate release and glutamate clearance kinetics. Finally, the antagonism of AMPA glutamate receptors in the dorsolateral striatum blocked the development of behavioral sensitization to repeated cocaine administration. Collectively, these data suggest that repeated cocaine exposure disrupts presynaptic glutamate transmission and transporter-mediated clearance mechanisms in the dorsal striatum. Moreover, such alterations produce an over activation of AMPA receptors in this brain region leading to the sensitized behavioral response to repeated cocaine. PMID:24911954

  19. Astrocyte/neuron ratio and its importance on glutamate toxicity: an in vitro voltammetric study.

    PubMed

    Hacimuftuoglu, Ahmet; Tatar, Abdulgani; Cetin, Damla; Taspinar, Numan; Saruhan, Fatih; Okkay, Ufuk; Turkez, Hasan; Unal, Deniz; Stephens, Robert Louis; Suleyman, Halis

    2016-08-01

    The purpose of this study was to clarify the relationship between neuron cells and astrocyte cells in regulating glutamate toxicity on the 10th and 20th day in vitro. A mixed primary culture system from newborn rats that contain cerebral cortex neurons cells was employed to investigate the glutamate toxicity. All cultures were incubated with various glutamate concentrations, then viability tests and histological analyses were performed. The activities of glutamate transporters were determined by using in vitro voltammetry technique. Viable cell number was decreased significantly on the 10th day at 10(-7) M and at 10(-6) M glutamate applications, however, viable cell number was not decreased at 20th day. Astrocyte number was increased nearly six times on the 20th day as compared to the 10th day. The peak point of glutamate reuptake capacity was about 2 × 10(-4) M on the 10th day and 10(-3) M on the 20th day. According to our results, we suggested that astrocyte age was important to maintain neuronal survival against glutamate toxicity. Thus, we revealed activation or a trigger point of glutamate transporters on astrocytes due to time since more glutamate was taken up by astrocytes when glutamate transporters on the astrocyte were triggered with high exogenous glutamate concentrations. In conclusion, the present investigation is the first voltammetric study on the reuptake parameters of glutamate in vitro. PMID:26438331

  20. Glutamate release from astrocytic gliosomes under physiological and pathological conditions.

    PubMed

    Milanese, Marco; Bonifacino, Tiziana; Zappettini, Simona; Usai, Cesare; Tacchetti, Carlo; Nobile, Mario; Bonanno, Giambattista

    2009-01-01

    Glial subcellular particles (gliosomes) have been purified from rat cerebral cortex or mouse spinal cord and investigated for their ability to release glutamate. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins, such as GFAP and S-100 but not neuronal proteins, such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin, high KCl, veratrine, 4-aminopyridine, AMPA, or ATP by mechanisms involving extracellular Ca2+, Ca2+ release from intracellular stores as well as reversal of glutamate transporters. In addition, gliosomes can release glutamate also by a mechanism involving heterologous transporter activation (heterotransporters) located on glutamate-releasing and glutamate transporter-expressing (homotransporters) gliosomes. This glutamate release involves reversal of glutamate transporters and anion channel opening, but not exocytosis. Both the exocytotic and the heterotransporter-mediated glutamate release were more abundant in gliosomes prepared from the spinal cord of transgenic mice, model of amyotrophic lateral sclerosis, than in controls; suggesting the involvement of astrocytic glutamate release in the excitotoxicity proposed as a cause of motor neuron degeneration. The results support the view that gliosomes may represent a viable preparation that allows to study mechanisms of astrocytic transmitter release and its regulation in healthy animals and in animal models of brain diseases. PMID:19607977

  1. Activation of Metabotropic Glutamate Receptor Type 2/3 Supports the Involvement of the Hippocampal Mossy Fiber Pathway on Contextual Fear Memory Consolidation

    ERIC Educational Resources Information Center

    Daumas, Stephanie; Ceccom, Johnatan; Halley, Helene; Frances, Bernard; Lassalle, Jean-Michel

    2009-01-01

    Elucidating the functional properties of the dentate gyrus (DG), CA3, and CA1 areas is critical for understanding the role of the dorsal hippocampus in contextual fear memory processing. In order to specifically disrupt various hippocampal inputs, we used region-specific infusions of DCG-IV, the metabotropic glutamate receptor agonist, which…

  2. The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae.

    PubMed

    Ziegler, Anna B; Augustin, Hrvoje; Clark, Nathan L; Berthelot-Grosjean, Martine; Simonnet, Mégane M; Steinert, Joern R; Geillon, Flore; Manière, Gérard; Featherstone, David E; Grosjean, Yael

    2016-01-01

    Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner. PMID:26805723

  3. The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae

    PubMed Central

    Ziegler, Anna B.; Augustin, Hrvoje; Clark, Nathan L.; Berthelot-Grosjean, Martine; Simonnet, Mégane M.; Steinert, Joern R.; Geillon, Flore; Manière, Gérard; Featherstone, David E.; Grosjean, Yael

    2016-01-01

    Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner. PMID:26805723

  4. Neurotoxic potential of lunar and martian dust: influence on em, proton gradient, active transport, and binding of glutamate in rat brain nerve terminals.

    PubMed

    Krisanova, Natalia; Kasatkina, Ludmila; Sivko, Roman; Borysov, Arseniy; Nazarova, Anastasiya; Slenzka, Klaus; Borisova, Tatiana

    2013-08-01

    The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due

  5. The active site behaviour of electrochemically synthesised gold nanomaterials.

    PubMed

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  6. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  7. Multifunctional PLGA particles containing poly(l-glutamic acid)-capped silver nanoparticles and ascorbic acid with simultaneous antioxidative and prolonged antimicrobial activity.

    PubMed

    Stevanović, Magdalena; Bračko, Ines; Milenković, Marina; Filipović, Nenad; Nunić, Jana; Filipič, Metka; Uskoković, Dragan P

    2014-01-01

    A water-soluble antioxidant (ascorbic acid, vitamin C) was encapsulated together with poly(l-glutamic acid)-capped silver nanoparticles (AgNpPGA) within a poly(lactide-co-glycolide) (PLGA) polymeric matrix and their synergistic effects were studied. The PLGA/AgNpPGA/ascorbic acid particles synthesized by a physicochemical method with solvent/non-solvent systems are spherical, have a mean diameter of 775 nm and a narrow size distribution with a polydispersity index of 0.158. The encapsulation efficiency of AgNpPGA/ascorbic acid within PLGA was determined to be >90%. The entire amount of encapsulated ascorbic acid was released in 68 days, and the entire amount of AgNpPGAs was released in 87 days of degradation. The influence of PLGA/AgNpPGA/ascorbic acid on cell viability, generation of reactive oxygen species (ROS) in HepG2 cells, as well as antimicrobial activity against seven different pathogens was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of these PLGA/AgNpPGA/ascorbic acid particles. We measured the kinetics of ROS formation in HepG2 cells by a DCFH-DA assay, and found that PLGA/AgNpPGA/ascorbic acid caused a significant decrease in DCF fluorescence intensity, which was 2-fold lower than that in control cells after a 5h exposure. This indicates that the PLGA/AgNpPGA/ascorbic acid microspheres either act as scavengers of intracellular ROS and/or reduce their formation. Also, the results of antimicrobial activity of PLGA/AgNpPGA/ascorbic acid obtained by the broth microdilution method showed superior and extended activity of these particles. The samples were characterized using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, zeta potential and particle size analysis. This paper presents a new approach to the treatment of infection that at the same time offers a very pronounced antioxidant effect. PMID:23988864

  8. Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2001-03-01

    We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia. PMID:11259512

  9. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  10. Temperature Differentially Facilitates Spontaneous but Not Evoked Glutamate Release from Cranial Visceral Primary Afferents

    PubMed Central

    Fawley, Jessica A.; Hofmann, Mackenzie E.; Largent-Milnes, Tally M.; Andresen, Michael C.

    2015-01-01

    Temperature is fundamentally important to all biological functions including synaptic glutamate release. Vagal afferents from the solitary tract (ST) synapse on second order neurons in the nucleus of the solitary tract, and glutamate release at this first central synapse controls autonomic reflex function. Expression of the temperature-sensitive Transient Receptor Potential Vanilloid Type 1 receptor separates ST afferents into C-fibers (TRPV1+) and A-fibers (TRPV1-). Action potential-evoked glutamate release is similar between C- and A-fiber afferents, but TRPV1 expression facilitates a second form of synaptic glutamate release in C-fibers by promoting substantially more spontaneous glutamate release. The influence of temperature on different forms of glutamate release is not well understood. Here we tested how temperature impacts the generation of evoked and spontaneous release of glutamate and its relation to TRPV1 expression. In horizontal brainstem slices of rats, activation of ST primary afferents generated synchronous evoked glutamate release (ST-eEPSCs) at constant latency whose amplitude reflects the probability of evoked glutamate release. The frequency of spontaneous EPSCs in these same neurons measured the probability of spontaneous glutamate release. We measured both forms of glutamate from each neuron during ramp changes in bath temperature of 4–5°C. Spontaneous glutamate release from TRPV1+ closely tracked with these thermal changes indicating changes in the probability of spontaneous glutamate release. In the same neurons, temperature changed axon conduction registered as latency shifts but ST-eEPSC amplitudes were constant and independent of TRPV1 expression. These data indicate that TRPV1-operated glutamate release is independent of action potential-evoked glutamate release in the same neurons. Together, these support the hypothesis that evoked and spontaneous glutamate release originate from two pools of vesicles that are independently

  11. Polysaccharides from wolfberry antagonizes glutamate excitotoxicity in rat cortical neurons.

    PubMed

    Ho, Yuen-Shan; Yu, Man-Shan; Yik, Suet-Yi; So, Kwok-Fai; Yuen, Wai-Hung; Chang, Raymond Chuen-Chung

    2009-12-01

    Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer's disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 microg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-D-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H(2)O(2)) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H(2)O(2). Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate. PMID:19499323

  12. Prefrontal glutamate correlates of methamphetamine sensitization and preference.

    PubMed

    Lominac, Kevin D; Quadir, Sema G; Barrett, Hannah M; McKenna, Courtney L; Schwartz, Lisa M; Ruiz, Paige N; Wroten, Melissa G; Campbell, Rianne R; Miller, Bailey W; Holloway, John J; Travis, Katherine O; Rajasekar, Ganesh; Maliniak, Dan; Thompson, Andrew B; Urman, Lawrence E; Kippin, Tod E; Phillips, Tamara J; Szumlinski, Karen K

    2016-03-01

    Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA-drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated MA sensitized drug-induced glutamate release and lowered indices of N-methyl-d-aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low-dose MA is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency. PMID:26742098

  13. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  14. Molecular basis for convergent evolution of glutamate recognition by pentameric ligand-gated ion channels

    PubMed Central

    Lynagh, Timothy; Beech, Robin N.; Lalande, Maryline J.; Keller, Kevin; Cromer, Brett A.; Wolstenholme, Adrian J.; Laube, Bodo

    2015-01-01

    Glutamate is an indispensable neurotransmitter, triggering postsynaptic signals upon recognition by postsynaptic receptors. We questioned the phylogenetic position and the molecular details of when and where glutamate recognition arose in the glutamate-gated chloride channels. Experiments revealed that glutamate recognition requires an arginine residue in the base of the binding site, which originated at least three distinct times according to phylogenetic analysis. Most remarkably, the arginine emerged on the principal face of the binding site in the Lophotrochozoan lineage, but 65 amino acids upstream, on the complementary face, in the Ecdysozoan lineage. This combined experimental and computational approach throws new light on the evolution of synaptic signalling. PMID:25708000

  15. Miniaturized thin film glutamate and glutamine biosensors.

    PubMed

    Moser, I; Jobst, G; Aschauer, E; Svasek, P; Varahram, M; Urban, G; Zanin, V A; Tjoutrina, G Y; Zharikova, A V; Berezov, T T

    1995-01-01

    Integrated thin film biosensors were developed for the simultaneous measurement of L-glutamine and L-glutamate in a mu-flow cell. Due to a novel glutaminase with an activity optimum in the neutral pH range, direct monitoring of glutamine in a mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by co-immobilization of glutaminase with glutamate oxidase within a photopatterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response was linear in the concentration range of 50 mumol to 10 mmol glutamine/l. Additionally, a glutamate biosensor was integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor-chip could be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor-chip with an integrated flow cell provided the possibility of simultaneous measurement of four different parameters at a cell volume of 1 microliter. In order to complete the microsystem, and in order to obtain a "lab on chip", a battery operated surface mounted device (SMD) potentiostat was developed. PMID:7612205

  16. Glutamate carboxypeptidase II (NAALADase) inhibition as a novel therapeutic strategy.

    PubMed

    Thomas, Ajit G; Wozniak, Krystyna M; Tsukamoto, Takashi; Calvin, David; Wu, Ying; Rojas, Camilo; Vornov, James; Slusher, Barbara S

    2006-01-01

    GCP II inhibition decreases extracellular excitotoxic glutamate and increases extracellular NAAG, both of which provide neuroprotection. We have demonstrated with our potent and selective GCP II inhibitors efficacy in models of stroke, ALS and neuropathic pain. GCP II inhibition may have significant potential benefits over existing glutamate-based neuroprotection strategies. The upstream mechanism seems selective for excitotoxic induced glutamate release, as GCP II inhibitors in normal animals induced no change in basal glutamate. This suggestion has recently been corroborated by Lieberman and coworkers24 who found that both NAAG release and increase in GCP II activity appear to be induced by electrical stimulation in crayfish nerve fibers and that subsequent NAAG hydrolysis to glutamate contributes, at least in part, to subsequent NMDA receptor activation. Interestingly, even at relatively high doses of compounds, GCP II inhibition did not appear to be associated with learning/memory deficits in animals. Additionally, quantitative neurophysiological testing data and visual analog scales for 'psychedelic effects' in Phase I single dose and repeat dose studies showed GCP II inhibition to be safe and well tolerated by both healthy volunteers and diabetic patients. GCP II inhibition may represent a novel glutamate regulating strategy devoid of the side effects that have hampered the development of postsynaptic glutamate receptor antagonists. PMID:16802724

  17. Enzyme-Doped Thin Films and Optical Fiber Sensors for Glutamate

    NASA Astrophysics Data System (ADS)

    Rickus, Jenna L.; Tobin, Allan J.; Zink, Jeffrey I.; Dunn, Bruce S.

    2002-10-01

    Biomolecules encapsulated in porous silicate glass using the sol-gel process form optically transparent materials capable of biorecognition. We are working to design biosensors from these materials for the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. Previously we demonstrated the ability of glutamate dehydrogenase (GDH)-doped sol-gel bulk materials to measure glutamate at varying concentrations. Here we show that GDH can be encapsulated in a thin film while retaining its enzymatic activity. The films are likely to be reaction limited rather than diffusion limited, as the reaction rate at saturating glutamate concentrations varies linearly with enzyme loading. At a given enzyme loading, the film reaction rate increases with increasing glutamate concentration, demonstrating its potential as a glutamate sensor material. In addition we have shown that the enzyme-doped sol-gel glass can be deposited onto the tip of an optical fiber. The fiber is active and responds to the presence of glutamate.

  18. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids. PMID:26221781

  19. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-directed Mutagenesis, Alternate Substrates, and Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  20. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  1. Transport Mechanism of a Bacterial Homologue of Glutamate Transporters

    SciTech Connect

    Reyes, N.; Ginter, C; Boudker, O

    2009-01-01

    Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, GltPh, which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt{sub Ph} in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt{sub Ph} and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.

  2. Chronic at-level thermal hyperalgesia following rat cervical contusion spinal cord injury is accompanied by neuronal and astrocyte activation and loss of the astrocyte glutamate transporter, GLT1, in superficial dorsal horn.

    PubMed

    Putatunda, Rajarshi; Hala, Tamara J; Chin, Jeannie; Lepore, Angelo C

    2014-09-18

    Neuropathic pain is a form of pathological nociception that occurs in a significant portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. While many peripheral and central mechanisms have been implicated in neuropathic pain, central sensitization of dorsal horn spinothalamic tract (STT) neurons is a major underlying substrate. Furthermore, dysregulation of extracellular glutamate homeostasis and chronic astrocyte activation play important underlying roles in persistent hyperexcitability of these superficial dorsal horn neurons. To date, central sensitization and astrocyte changes have not been characterized in cervical SCI-induced neuropathic pain models, despite the fact that a major portion of SCI patients suffer contusion trauma to cervical spinal cord. In this study, we have characterized 2 rat models of unilateral cervical contusion SCI that behaviorally result in chronic persistence of thermal hyperalgesia in the ipsilateral forepaw. In addition, we find that STT neurons are chronically activated in both models when compared to laminectomy-only uninjured rats. Finally, persistent astrocyte activation and significantly reduced expression of the major CNS glutamate transporter, GLT1, in superficial dorsal horn astrocytes are associated with both excitability changes in STT neurons and the neuropathic pain behavioral phenotype. In conclusion, we have characterized clinically-relevant rodent models of cervical contusion-induced neuropathic pain that result in chronic activation of both STT neurons and astrocytes, as well as compromise in astrocyte glutamate transporter expression. These models can be used as important tools to further study mechanisms underlying neuropathic pain post-SCI and to test potential therapeutic interventions. PMID:24833066

  3. Increasing the Receptor Tyrosine Kinase EphB2 Prevents Amyloid-β-induced Depletion of Cell Surface Glutamate Receptors by a Mechanism That Requires the PDZ-binding Motif of EphB2 and Neuronal Activity*

    PubMed Central

    Miyamoto, Takashi; Kim, Daniel; Knox, Joseph A.; Johnson, Erik; Mucke, Lennart

    2016-01-01

    Diverse lines of evidence suggest that amyloid-β (Aβ) peptides causally contribute to the pathogenesis of Alzheimer disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wild-type or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity but not on its kinase activity. We further present evidence that the protective effect of EphB2 may be mediated by the AMPA-type glutamate receptor subunit GluA2, which can become associated with the PDZ-binding motif of EphB2 through PDZ domain-containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrPC), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPA-type glutamate receptors. PMID:26589795

  4. [Effect of vitamin B3-active compounds on the content of free and combined gamma-aminobutyric acid and glutamic acid in the brain of mice].

    PubMed

    Rozanov, V A; Reĭtarova, T E

    1983-01-01

    The bound and free GABA and glutamic acid content in the brain of F1 (CBA X C57B1/6) hybrid mice was investigated by the Eliott method. A tendency to a decrease of GABA and glutamate content in the brain with their practically constant bound/free ratio was observed 24 h after calcium-D-pantothenate injections (150 mumole/kg, 9 injections for 3 days). Calcium-D-homopantothenate injected in the same way caused a significant decrease in the GABA content, and a sharp drop of the bound/free GABA ratio. The effect is not associated with the influence of calcium ions in the composition of the injected compounds. PMID:6140785

  5. Translational neurophysiological markers for activity of the metabotropic glutamate receptor (mGluR2) modulator JNJ-40411813: Sleep EEG correlates in rodents and healthy men.

    PubMed

    Ahnaou, A; de Boer, P; Lavreysen, H; Huysmans, H; Sinha, V; Raeymaekers, L; Van De Casteele, T; Cid, J M; Van Nueten, L; Macdonald, G J; Kemp, J A; Drinkenburg, W H I M

    2016-04-01

    Alterations in rapid eye movement sleep (REM) have been suggested as valid translational efficacy markers: activation of the metabotropic glutamate receptor 2 (mGluR2) was shown to increase REM latency and to decrease REM duration. The present paper addresses the effects on vigilance states of the mGluR2 positive allosteric modulator (PAM) JNJ-40411813 at different circadian times in rats and after afternoon dosing in humans. Due to its dual mGluR2 PAM/serotonin 2A (5-HT2A) receptor antagonism in rodents, mGlu2R specificity of effects was studied in wild-type (WT) and mGluR2 (-/-) mice. 5-HT2A receptor occupancy was determined in humans using positron emission tomography (PET). Tolerance development was examined in rats after chronic dosing. EEG oscillations and network connectivity were assessed using multi-channel EEG. In rats, JNJ-40411813 increased deep sleep time and latency of REM onset but reduced REM time when administered 2 h after 'lights on' (CT2): this was sustained after chronic dosing. At CT5 similar effects were elicited, at CT10 only deep sleep was enhanced. Withdrawal resulted in baseline values, while re-administration reinstated drug effects. Parieto-occipital cortical slow theta and gamma oscillations were correlated with low locomotion. The specificity of functional response was confirmed in WT but not mGluR2 (-/-) mice. A double-blind, placebo-controlled polysomnographic study in healthy, elderly subjects showed that 500 mg of JNJ-40411813 consistently increased deep sleep time, but had no effect on REM parameters. This deep sleep effect was not explained by 5-HT2A receptor binding, as in the PET study even 700 mg only marginally displaced the tracer. JNJ-40411813 elicited comparable functional responses in rodents and men if circadian time of dosing was taken into account. These findings underscore the translational potential of sleep mechanisms in evaluating mGluR2 therapeutics when administered at the appropriate circadian time. PMID

  6. Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: Does a third metal binding site modulate endonuclease activity?

    PubMed Central

    Ho, Ming-Hsun; De Vivo, Marco; Peraro, Matteo Dal; Klein, Michael L.

    2010-01-01

    Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg2+ concentration. Moreover, in RNase H the glutamate residue E188 has been shown to be essential for full enzymatic activation regardless of the Mg2+ concentration. The catalytic center is known to contain two Mg2+ ions (Nowotny et al.) and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg2+. Importantly, the presence of a third Mg2+ ion, bound to the second-shell ligand E188, is persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations depending on the concentration of Mg2+. At standard concentration, a third Mg2+ is found in the catalytic pocket but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg2+ ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg2+ ions nearby the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A, and also supports the key role of E188 in localizing the third Mg2+ ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD

  7. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  8. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  9. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    PubMed

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that

  10. Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

    PubMed

    Sirithanakorn, Chaiyos; Jitrapakdee, Sarawut; Attwood, Paul V

    2016-08-01

    The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis. PMID:27379711

  11. Vitamin C neuroprotection against dose-dependent glutamate-induced neurodegeneration in the postnatal brain.

    PubMed

    Shah, Shahid Ali; Yoon, Gwang Ho; Kim, Hyun-Ok; Kim, Myeong Ok

    2015-05-01

    Glutamate-induced excitotoxicity due to over-activation of glutamate receptors and associated energy depletion (phosphorylation and activation of AMPK) results in neuronal cell death in various neurological disorders. Restoration of energy balance during an excitotoxic insult is critical for neuronal survival. Ascorbic acid (vitamin C), an essential nutrient with well-known antioxidant potential, protects the brain from oxidative damage in various models of neurodegeneration. In this study, we reported the therapeutic efficacy of vitamin C in response to glutamate-induced excitation, resulting in energy depletion and apoptosis in the hippocampus of the developing rat brain. A single subcutaneous injection of glutamate at two different concentrations (5 and 10 mg/kg) in postnatal day 7 rat pups increased brain glutamate levels and increased the protein expression of neuronal apoptotic markers. Both doses of glutamate upregulated the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, cytochrome-c release, caspase-3 activation and the expression of PARP-1. However, co-treatment of vitamin C (250 mg/kg) with glutamate decreased brain glutamate levels and reversed the changes induced by glutamate in the developing hippocampus. Interestingly, only a high dose of glutamate caused the phosphorylation and activation of AMPK and induced neuronal cell death, whereas a low dose of glutamate failed to mediate these effects. Vitamin C supplementation reduced the glutamate-induced phosphorylation of AMPK and attenuated neuronal cell death, as assessed morphologically by Fluoro Jade B in the hippocampal CA1 region of the developing brain. Taken together, our results indicated that glutamate in both concentrations is toxic to the immature rat brain, whereas vitamin C is pharmacologically effective against glutamate-induced neurodegeneration. PMID:25701025

  12. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  13. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  14. Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery.

    PubMed

    Poen, Sinothai; Nakatani, Yoshio; Opel-Reading, Helen K; Lassé, Moritz; Dobson, Renwick C J; Krause, Kurt L

    2016-05-01

    Glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria and has been a favoured target in pharmaceutical drug design efforts. It has recently been proven to be essential in Mycobacterium tuberculosis, the causative organism of tuberculosis, a disease for which new medications are urgently needed. In the present study, we have determined the protein crystal structures of MurI from both M. tuberculosis and Mycobacterium smegmatis in complex with D-glutamate to 2.3 Å and 1.8 Å resolution respectively. These structures are conserved, but reveal differences in their active site architecture compared with that of other MurI structures. Furthermore, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a new drug design effort will be needed to develop inhibitors. A new type of MurI dimer arrangement has been observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found to date. The mycobacterial MurI dimer is tightly associated, with a KD in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. We created triple mutants of this interface in the M. smegmatis glutamate racemase (D26R/R105A/G194R or E) that have appreciable activity (kcat=0.056-0.160 min(-1) and KM=0.26-0.51 mM) and can be utilized to screen proposed antimicrobial candidates for inhibition. PMID:26964898

  15. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

    SciTech Connect

    Sheng, Jun; Charng, Yee-yung; Preiss, J.

    1996-03-05

    Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.

  16. Mechanisms of glutamate toxicity in multiple sclerosis: biomarker and therapeutic opportunities.

    PubMed

    Macrez, Richard; Stys, Peter K; Vivien, Denis; Lipton, Stuart A; Docagne, Fabian

    2016-09-01

    Research advances support the idea that excessive activation of the glutamatergic pathway plays an important part in the pathophysiology of multiple sclerosis. Beyond the well established direct toxic effects on neurons, additional sites of glutamate-induced cell damage have been described, including effects in oligodendrocytes, astrocytes, endothelial cells, and immune cells. Such toxic effects could provide a link between various pathological aspects of multiple sclerosis, such as axonal damage, oligodendrocyte cell death, demyelination, autoimmunity, and blood-brain barrier dysfunction. Understanding of the mechanisms underlying glutamate toxicity in multiple sclerosis could help in the development of new approaches for diagnosis, treatment, and follow-up in patients with this debilitating disease. While several clinical trials of glutamatergic modulators have had disappointing results, our growing understanding suggests that there is reason to remain optimistic about the therapeutic potential of these drugs. PMID:27571160

  17. Sodium-Dependent Glutamate Uptake by an Alkaliphilic, Thermophilic Bacillus Strain, TA2.A1

    PubMed Central

    Peddie, Catherine J.; Cook, Gregory M.; Morgan, Hugh W.

    1999-01-01

    A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (ΔpNa) across the cell membrane. N,N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (ΔΨ), but only when sodium was present. In the absence of sodium, the rate of ΔΨ-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of ΔpNa alone (i.e., no ΔΨ). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 μM, and the Vmax was 0.7 nmol · min−1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues. PMID:10322019

  18. Effect of insulin on the compartmentation of glutamate for protein synthesis

    SciTech Connect

    Brown, A.B.; Mohan, C.; Bessman, S.P.

    1986-03-05

    The effect of insulin on the formation of CO/sub 2/ and incorporation of 1-/sup 14/C glutamine and U-/sup 14/C acetate into protein was studied in isolated rat hepatocytes. Insulin caused an 18% increase in /sup 14/CO/sub 2/ production from U-/sup 14/C acetate in comparison to a 10% increase from 1-/sup 14/C glutamate. Insulin caused a greater increase in the incorporation of tracer acetate carbons into hepatocyte protein. Hydrolysis of labeled protein and subsequent determination of glutamate specific activity revealed that incorporation of acetate carbons into protein as glutamate was about 52% greater in the presence of insulin. These results demonstrate the existence of two compartments of glutamate for protein synthesis: (i) glutamate generated in the Krebs cycle through transamination of a-ketoglutarate; (ii) cytosolic glutamate. Insulin had a greater stimulatory effect on the incorporation of glutamate generated in the Krebs cycle.

  19. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  20. Synthesis and studies of polypeptide materials: Enantioselective polymerization of gamma-benzyl glutamate-N-carboxyanhydride and synthesis of optically active poly(beta-peptides)

    NASA Astrophysics Data System (ADS)

    Cheng, Jianjun

    A class of zero-valent transition metal complexes have been developed by Deming et al for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs). This discovery provided a superior starting point for the development of enantioselective polymerizations of racemic alpha-NCAs. Bidentate chiral ligands were synthesized and tested for their abilities to induce enantioselective polymerization of gamma-benzyl-glutamate NCA (Glu NCA) when they were coordinated to zero-valent nickel complexes. When optically active 2-pyridinyl oxazoline ligands were mixed with bis(1,5-cyclooctadiene)nickel in THF, chiral nickel complexes were formed that selectively polymerized one enantiomer of Glu NCA over the other. The highest selectivity was observed with the nickel complex of (S)-4-tert-butyl-2-pyridinyl oxazoline, which gave a ratio of enantiomeric polymerization rate constants (kD/kL) of 5.2. It was found that subtle modification of this ligand by incorporation of additional substituents had a substantial impact on initiator enantioselectivities. In separate efforts, methodology was developed for the general synthesis of optically active beta-aminoacid-N-carboxyanhydrides (beta-NCAs) via cyclization of Nbeta-Boc- or Nbeta-Cbz-beta-amino acids using phosphorus tribromide. The beta-NCA molecules could be polymerized in good yields using strong bases or transition metal complexes to give optically active poly(beta-peptides) bearing proteinogenic side chains. The resulting poly(beta-peptides), which have moderate molecular weights, adopt stable helical conformations in solution. Poly(beta-homoglutamate and poly(beta-homolysine), the side-chain deprotected polymers, were found to display pH dependent helix-coil conformation transitions in aqueous solution, similar to their alpha-analogs. A novel method for poly(beta-aspartate) synthesis was developed via the polymerization of L-aspartate alkyl ester beta lactams using metal-amido complexes. Poly

  1. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  2. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

  3. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  4. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

    NASA Astrophysics Data System (ADS)

    Brew, Helen; Attwell, David

    1987-06-01

    Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

  5. Transport dynamics in a glutamate transporter homologue

    PubMed Central

    Akyuz, Nurunisa; Altman, Roger B.; Blanchard, Scott C.; Boudker, Olga

    2013-01-01

    Summary Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons1. Their mechanism involves transitions between extracellular- (outward-) and intracellular- (inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane2. This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold3. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging4, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods5,6. We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle. PMID:23792560

  6. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  7. Glutamate Receptor Ion Channels: Structure, Regulation, and Function

    PubMed Central

    Wollmuth, Lonnie P.; McBain, Chris J.; Menniti, Frank S.; Vance, Katie M.; Ogden, Kevin K.; Hansen, Kasper B.; Yuan, Hongjie; Myers, Scott J.; Dingledine, Ray

    2010-01-01

    The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors. PMID:20716669

  8. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

  9. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  10. Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of α-ketoglutarate in Yarrowia lipolytica.

    PubMed

    Guo, Hongwei; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen

    2016-01-01

    α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L(-1) in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts. PMID:26428234

  11. Protein kinase C -dependent regulation of synaptosomal glutamate uptake under conditions of hypergravity

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana; Krisanova, Natalia; Borisov, Arseniy; Sivko, Roman

    Glutamate is not only the main excitatory neurotransmitter in the mammalian CNS, but also a potent neurotoxin. Excessive concentration of ambient glutamate over activates glutamate receptors and causes neurotoxicity. Uptake of glutamate from the extracellular space into nerve cells was mediated by sodium-dependent glutamate transporters located in the plasma membrane. It was shown that the activity of glutamate transporters in rat brain nerve terminals was decreased after hypergravity (centrifugation of rats in special containers at 10 G for 1 hour). This decrease may result from the reduction in the number of glutamate transporters expressed in the plasma membrane of nerve terminals after hypergravity that was regulated by protein kinase C. The possibility of the involvement of protein kinase C in the regulation of the activity of glutamate transporters was assessed under conditions of hypergravity. The effect of protein kinase C inhibitor GF 109 203X on synaptosomal L-[14C]glutamate uptake was analysed. It was shown that the inhibitor decreased L-[14C]glutamate uptake by 15 % in control but did not influence it after hypergravity. In control, the initial velocity of L-[14C]glutamate uptake in the presence of the inhibitor decreased from 2.5 ± 0.2 nmol x min-1 x mg-1 of proteins to 2.17 ± 0.1 nmol x min-1 x mg-1 of proteins, whereas after hypergravity this value lowered from 2.05 ± 0.1 nmol x min-1 x mg-1 of proteins to 2.04 ± 0.1 nmol x min-1 x mg-1 of proteins. Thus, protein kinase C -dependent alteration in the cell surface expression of glutamate transporters may be one of the causes of a decrease in the activity of glutamate transporters after hypergravity.

  12. The role of glutamate signaling in pain processes and its regulation by GCP II inhibition.

    PubMed

    Wozniak, K M; Rojas, C; Wu, Y; Slusher, B S

    2012-01-01

    Glutamate is the predominant excitatory neurotransmitter used by primary afferent synapses and neurons in the spinal cord dorsal horn. Glutamate and glutamate receptors are also located in areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. Not surprisingly, glutamate receptors have been an attractive target for new pain therapies. However, their widespread distribution and array of function has often resulted in drugs targeting these sites having undesirable side-effects. This chapter will review, in general terms, the current knowledge of glutamate and its effects at various glutamate receptors with regards to nociception. In addition, we will briefly review the glutamatergic drugs currently in use as treatments for pain, as well as known novel candidates in various stages of clinical trial. Lastly, we will summarize the data supporting a novel target for pain intervention by way of GCPII inhibition, which appears devoid of the side-effects associated with direct glutamate receptor antagonists and thus holds major promise for future therapy. GCPII (glutamate carboxypeptidase II) cleaves the prevalent neuropeptide NAAG into NAA and glutamate and there is widespread evidence and belief that targeting the glutamate derived from this enzymatic action may be a promising therapeutic route. PMID:22304711

  13. Synthesis, structure-activity relationships and biological evaluation of 4,5,6,7-tetrahydropyrazolopyrazines as metabotropic glutamate receptor 5 negative allosteric modulators.

    PubMed

    Hirose, Wataru; Kato, Yoshihiro; Yamamoto, Takayoshi; Kassai, Momoe; Takata, Makoto; Hayashi, Shun; Arai, Yukiyo; Imai, Satoki; Yoshida, Kohzo

    2016-08-15

    The design, synthesis and SAR studies of novel 4,5,6,7-tetrahydropyrazolopyrazines as metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) are presented in this letter. Starting from a HTS hit compound (1, IC50=477nM), optimization of various groups led to the synthesis of a potent mGluR5 NAM (32, IC50=75nM) with excellent rat PK profile and good brain penetration. This compound produced oral antidepressant-like effect in a mouse tale suspension model (MED: 30mg/kg). PMID:27432763

  14. Structure-Activity Relationships in a Novel Series of 7-Substituted-Aryl Quinolines and 5-Substituted-Aryl Benzothiazoles at the Metabotropic Glutamate Receptor Subtype 5

    PubMed Central

    Zhang, Peng; Zou, Mu-Fa; Rodriguez, Alice L.; Conn, P. Jeffrey; Newman, Amy Hauck

    2010-01-01

    The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in numerous neuropsychiatric disorders including addiction. We have discovered that the rigid diaryl alkyne template, derived from the potent and selective noncompetitive mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), can serve to guide the design of novel quinoline analogues and pharmacophore optimization has resulted in potent mGluR5 noncompetitive antagonists (EC50 range 60–100 nM) in the quinoline series. PMID:20382541

  15. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  16. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  17. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  18. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  19. Glutamate Neurocircuitry: Theoretical Underpinnings in Schizophrenia

    PubMed Central

    Schwartz, Thomas L.; Sachdeva, Shilpa; Stahl, Stephen M.

    2012-01-01

    The Dopamine Hypothesis of Schizophrenia is actively being challenged by the NMDA Receptor Hypofunctioning Hypothesis of Schizophrenia. The latter hypothesis may actually be the starting point in neuronal pathways that ultimately modifies dopamine pathways involved in generating both positive and negative symptoms of schizophrenia postulated by the former hypothesis. The authors suggest that even this latter, NMDA receptor-based, hypothesis is likely too narrow and offer a review of typical glutamate and dopamine-based neurocircuitry, propose genetic vulnerabilities impacting glutamate neurocircuitry, and provide a broad interpretation of a possible etiology of schizophrenia. In conclusion, there is a brief review of potential schizophrenia treatments that rely on the etiologic theory provided in the body of the paper. PMID:23189055

  20. Computational Studies of Glutamate Transporters

    PubMed Central

    Setiadi, Jeffry; Heinzelmann, Germano; Kuyucak, Serdar

    2015-01-01

    Glutamate is the major excitatory neurotransmitter in the human brain whose binding to receptors on neurons excites them while excess glutamate are removed from synapses via transporter proteins. Determination of the crystal structures of bacterial aspartate transporters has paved the way for computational investigation of their function and dynamics at the molecular level. Here, we review molecular dynamics and free energy calculation methods used in these computational studies and discuss the recent applications to glutamate transporters. The focus of the review is on the insights gained on the transport mechanism through computational methods, which otherwise is not directly accessible by experimental probes. Recent efforts to model the mammalian glutamate and other amino acid transporters, whose crystal structures have not been solved yet, are included in the review. PMID:26569328

  1. Laser-scanning astrocyte mapping reveals increased glutamate-responsive domain size and disrupted maturation of glutamate uptake following neonatal cortical freeze-lesion

    PubMed Central

    Armbruster, Moritz; Hampton, David; Yang, Yongjie; Dulla, Chris G.

    2014-01-01

    Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL) model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM), which combines glutamate transport current (TC) recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes. PMID:25249939

  2. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  3. Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.

    PubMed Central

    Wu, T Y; Chang, Y C

    1994-01-01

    L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins. PMID:7516151

  4. Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.

    PubMed

    Wu, T Y; Chang, Y C

    1994-06-01

    L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate > N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins. PMID:7516151

  5. Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III

    SciTech Connect

    Hlouchova, Klara; Barinka, Cyril; Konvalinka, Jan; Lubkowski, Jacek

    2009-10-23

    Glutamate carboxypeptidase III (GCPIII) is a metalloenzyme that belongs to the transferrin receptor/glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) superfamily. GCPIII has been studied mainly because of its evolutionary relationship to GCPII, an enzyme involved in a variety of neuropathologies and malignancies, such as glutamatergic neurotoxicity and prostate cancer. Given the potential functional and pharmacological overlap between GCPIII and GCPII, studies addressing the structural and physiological properties of GCPIII are crucial for obtaining a deeper understanding of the GCPII/GCPIII system. In the present study, we report high-resolution crystal structures of the human GCPIII ectodomain in a 'pseudo-unliganded' state and in a complex with: (a) L-glutamate (a product of hydrolysis); (b) a phosphapeptide transition state mimetic, namely (2S,3'S)-{l_brace}[(3'-amino-3'-carboxy-propyl)-hydroxyphosphinoyl]methyl{r_brace}-pentanedioic acid; and (c) quisqualic acid, a glutamate biostere. Our data reveal the overall fold and quaternary arrangement of the GCPIII molecule, define the architecture of the GCPIII substrate-binding cavity, and offer an experimental evidence for the presence of Zn{sup 2+} ions in the bimetallic active site. Furthermore, the structures allow us to detail interactions between the enzyme and its ligands and to characterize the functional flexibility of GCPIII, which is essential for substrate recognition. A comparison of these GCPIII structures with the equivalent GCPII complexes reveals differences in the organization of specificity pockets, in surface charge distribution, and in the occupancy of the co-catalytic zinc sites. The data presented here provide information that should prove to be essential for the structurally-aided design of GCPIII-specific inhibitors and might comprise guidelines for future comparative GCPII/GCPIII studies.

  6. Genotoxicity of monosodium glutamate.

    PubMed

    Ataseven, Nazmiye; Yüzbaşıoğlu, Deniz; Keskin, Ayten Çelebi; Ünal, Fatma

    2016-05-01

    Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro. PMID:26929995

  7. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  8. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  9. Molecular signalling mediating the protective effect of A1 adenosine and mGlu3 metabotropic glutamate receptor activation against apoptosis by oxygen/glucose deprivation in cultured astrocytes.

    PubMed

    Ciccarelli, Renata; D'Alimonte, Iolanda; Ballerini, Patrizia; D'Auro, Mariagrazia; Nargi, Eleonora; Buccella, Silvana; Di Iorio, Patrizia; Bruno, Valeria; Nicoletti, Ferdinando; Caciagli, Francesco

    2007-05-01

    Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A(1) adenosine and mGlu3 metabotropic glutamate receptors with N(6)-chlorocyclopentyladenosine (CCPA) and (-)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-X(L), and were highly protective against apoptotic death, as shown by nuclear 4'-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A(1) and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways. PMID:17293559

  10. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

    PubMed

    Caffrey, C R; Hansell, E; Lucas, K D; Brinen, L S; Alvarez Hernandez, A; Cheng, J; Gwaltney, S L; Roush, W R; Stierhof, Y D; Bogyo, M; Steverding, D; McKerrow, J H

    2001-11-01

    Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine. PMID:11704274

  11. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  12. The Structure of RalF, an ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

    SciTech Connect

    Amor,J.; Swails, J.; Zhu, X.; Roy, C.; Nagai, H.; Ingmundson, A.; Cheng, X.; Kahn, R.

    2005-01-01

    The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the 'glutamic finger,' conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

  13. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  14. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  15. Metabotropic Glutamate Receptors for Parkinson's Disease Therapy

    PubMed Central

    Gasparini, Fabrizio; Di Paolo, Thérèse; Gomez-Mancilla, Baltazar

    2013-01-01

    Excessive glutamatergic signalling within the basal ganglia is implicated in the progression of Parkinson's disease (PD) and inthe emergence of dyskinesia associated with long-term treatment with L-DOPA. There is considerable research focus on the discovery and development of compounds that modulate glutamatergic signalling via glutamate receptors, as treatments for PD and L-DOPA-induced dyskinesia (LID). Although initial preclinical studies with ionotropic glutamate receptor antagonists showed antiparkinsonian and antidyskinetic activity, their clinical use was limited due to psychiatric adverse effects, with the exception of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, currently used to reduce dyskinesia in PD patients. Metabotropic receptor (mGlu receptor) modulators were considered to have a more favourable side-effect profile, and several agents have been studied in preclinical models of PD. The most promising results have been seen clinically with selective antagonists of mGlu5 receptor and preclinically with selective positive allosteric modulators of mGlu4 receptor. The growing understanding of glutamate receptor crosstalk also raises the possibility of more precise modulation of glutamatergic transmission, which may lead to the development of more effective agents for PD. PMID:23853735

  16. Molecular pharmacology of glutamate transporters, EAATs and VGLUTs.

    PubMed

    Shigeri, Yasushi; Seal, Rebecca P; Shimamoto, Keiko

    2004-07-01

    L-Glutamate serves as a major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is stored in synaptic vesicles by an uptake system that is dependent on the proton electrochemical gradient (VGLUTs). Following its exocytotic release, glutamate activates fast-acting, excitatory ionotropic receptors and slower-acting metabotropic receptors to mediate neurotransmission. Na+-dependent glutamate transporters (EAATs) located on the plasma membrane of neurons and glial cells rapidly terminate the action of glutamate and maintain its extracellular concentration below excitotoxic levels. Thus far, five Na+-dependent glutamate transporters (EAATs 1-5) and three vesicular glutamate transporters (VGLUTs 1-3) have been identified. Examination of EAATs and VGLUTs in brain preparations and by heterologous expression of the various cloned subtypes shows these two transporter families differ in many of their functional properties including substrate specificity and ion requirements. Alterations in the function and/or expression of these carriers have been implicated in a range of psychiatric and neurological disorders. EAATs have been implicated in cerebral stroke, epilepsy, Alzheimer's disease, HIV-associated dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma, while VGLUTs have been implicated in schizophrenia. To examine the physiological role of glutamate transporters in more detail, several classes of transportable and non-transportable inhibitors have been developed, many of which are derivatives of the natural amino acids, aspartate and glutamate. This review summarizes the development of these indispensable pharmacological tools, which have been critical to our understanding of normal and abnormal synaptic transmission. PMID:15210307

  17. Glutamate: the new frontier in pharmacotherapy for cocaine addiction.

    PubMed

    Uys, Joachim D; LaLumiere, Ryan T

    2008-11-01

    Considerable research into the neurobiology of cocaine addiction has shed light on the role of glutamate. Findings from models of relapse to cocaine-seeking indicate that the glutamatergic system is critically involved, as glutamate levels in the nucleus accumbens increase during reinstatement and glutamate receptor activation is necessary for reinstatement to drug-seeking. Thus, it would seem beneficial to block the increased glutamate release, but full antagonists of ionotropic glutamate receptors produce undesirable side effects. Therefore, modulation of glutamatergic transmission would be advantageous and provide novel pharmacotherapeutic avenues. Pharmacotherapies have been developed that have the potential to modulate excessive glutamatergic transmission through ionotropic and metabotropic (mGluR) glutamate receptors. Compounds that modulate glutamatergic transmission through ionotropic glutamate receptors include the non-competitive N-methyl-D-aspartic acid antagonists, amantadine and memantine, and the partial N-methyl-D-aspartic acid agonist d-cycloserine. They have shown promise in preclinical models of cocaine addiction. The mGluR2/3 agonist LY379268 is effective in inhibiting cocaine seeking in preclinical animal models and could decrease stress-induced relapse due to its anxiolytic effects. Similarly, the mGluR1/5 antagonists, 2-methyl-6-(phenylethynyl)pyridine and 3-[2-methyl-4-thiazolyl)ethynyl]pyridine, have shown to be effective in preclinical models of cocaine addiction. The cysteine pro-drug, N-acetylcysteine, restores the inhibitory tone on presynaptic glutamate receptors and has been effective in reducing cue-induced craving and cocaine use in humans. Furthermore, anticonvulsants, such as topiramate or lamotrigine, have shown efficacy in treating cocaine dependence or reducing relapse in humans. Future pharmacotherapy may focus on manipulating signal transduction proteins and pathways, which include Homer/N-methyl-D-aspartic acid complexes, to

  18. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  19. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  20. Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog.

    PubMed

    Zhao, Gengxiang; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-25

    N-Acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound. Here we reported the preparation of the bisubstrate analog, CoA-S-acetyl-L-glutamate, the crystal structure of ngNAGS with CoA-NAG bound, and kinetic studies of several active site mutants. The results are consistent with a one-step nucleophilic addition-elimination mechanism with Glu353 as the catalytic base and Ser392 as the catalytic acid. The structure of the ngNAGS-bisubstrate complex together with the previous ngNAGS structures delineates the catalytic reaction path for ngNAGS. PMID:23261468

  1. Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog

    PubMed Central

    ZHAO, GENGXIANG; ALLEWELL, NORMA M.; TUCHMAN, MENDEL; SHI, DASHUANG

    2013-01-01

    N -acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound. Here we reported the preparation of the bisubstrate analog, CoA-S-acetyl-L-glutamate, the crystal structure of ngNAGS with CoA-NAG bound, and kinetic studies of several active site mutants. The results are consistent with a one-step nucleophilic addition-elimination mechanism with Glu353 as the catalytic base and Ser392 as the catalytic acid. The structure of the ngNAGS-bisubstrate complex together with the previous ngNAGS structures delineates the catalytic reaction path for ngNAGS. PMID:23261468

  2. Shifts in striatal responsivity evoked by chronic stimulation of dopamine and glutamate systems.

    PubMed

    Canales, J J; Capper-Loup, C; Hu, D; Choe, E S; Upadhyay, U; Graybiel, A M

    2002-10-01

    Dopamine and glutamate are key neurotransmitters in cortico-basal ganglia loops affecting motor and cognitive function. To examine functional convergence of dopamine and glutamate neurotransmitter systems in the basal ganglia, we evaluated the long-term effects of chronic stimulation of each of these systems on striatal responses to stimulation of the other. First we exposed rats to chronic intermittent cocaine and used early-gene assays to test the responsivity of the striatum to subsequent acute motor cortex stimulation by application of the GABA(A) (gamma-aminobutyric acid alpha subunit) receptor antagonist, picrotoxin. Reciprocally, we studied the effects of chronic intermittent motor cortex stimulation on the capacity for subsequent acute dopaminergic treatments to induce early-gene activation in the striatum. Prior treatment with chronic intermittent cocaine induced motor sensitization and significantly potentiated the striatal expression of Fos-family early genes in response to stimulation of the motor cortex. Contrary to this, chronic intermittent stimulation of the motor cortex down-regulated cocaine-induced gene expression in the striatum, but enhanced striatal gene expression induced by a full D1 receptor agonist (SKF 81297) and did not change the early-gene response elicited by a D2 receptor antagonist (haloperidol). These findings suggests that repeated dopaminergic stimulation produces long-term enhancement of corticostriatal signalling from the motor cortex, amplifying cortically evoked modulation of the basal ganglia. By contrast, persistent stimulation of the motor cortex inhibits cocaine-stimulated signalling in the striatum, but not signalling mediated by individual dopamine receptor sites, suggesting that chronic cortical hyperexcitability produces long-term impairment of dopaminergic activity and compensation at the receptor level. These findings prompt a model of the basal ganglia function as being regulated by opposing homeostatic dopamine-glutamate

  3. Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death.

    PubMed

    Montoliu, C; Llansola, M; Monfort, P; Corbalan, R; Fernandez-Marticorena, I; Hernandez-Viadel, M L; Felipo, V

    2001-04-01

    Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as "scavengers" of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP. PMID:14715472

  4. Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

    2011-03-01

    Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

  5. Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

    2010-10-01

    Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

  6. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  7. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  8. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  9. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  10. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor

    SciTech Connect

    Sobolevsky, Alexander I.; Rosconi, Michael P.; Gouaux, Eric

    2010-02-02

    Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact ionotropic glutamate receptor are unknown. Here we report the crystal structure of the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 {angstrom} resolution in complex with a competitive antagonist. The receptor harbours an overall axis of two-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting four-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis, we suggest that GluN1 and GluN2A NMDA (N-methyl-D-aspartate) receptors have a similar architecture, with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers.

  11. Coupling of glutamate and glucose uptake in cultured Bergmann glial cells.

    PubMed

    Mendez-Flores, Orquidia G; Hernández-Kelly, Luisa C; Suárez-Pozos, Edna; Najimi, Mustapha; Ortega, Arturo

    2016-09-01

    Glutamate, the main excitatory neurotransmitter in the vertebrate brain, exerts its actions through specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of sodium-dependent, glutamate uptake transporters mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing neuronal death. The sustained sodium influx associated to glutamate removal in glial cells, activates the sodium/potassium ATPase restoring the ionic balance, additionally, glutamate entrance activates glutamine synthetase, both events are energy demanding, therefore glia cells increase their ATP expenditure favouring glucose uptake, and triggering several signal transduction pathways linked to proper neuronal glutamate availability, via the glutamate/glutamine shuttle. To further characterize these complex transporters interactions, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity, plasma membrane localization and protein levels of glucose transporters was detected upon d-aspartate exposure. Interestingly, this increase is the result of a protein kinase C-dependent signaling cascade. Furthermore, a glutamate-dependent glucose and glutamate transporters co-immunoprecipitation was detected. These results favour the notion that glial cells are involved in glutamatergic neuronal physiology. PMID:27184733

  12. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  13. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells

    PubMed Central

    Thomas, Ajit G.; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A.; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S.

    2015-01-01

    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  14. Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

    PubMed Central

    Roon, Robert J.; Even, Harvey L.; Larimore, Fred

    1974-01-01

    A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and α-ketoglutarate (Km values of 2.6 μM, 1.0 mM, and 140 μM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase. PMID:4362465

  15. Frontal glutamate and reward processing in adolescence and adulthood.

    PubMed

    Gleich, Tobias; Lorenz, Robert C; Pöhland, Lydia; Raufelder, Diana; Deserno, Lorenz; Beck, Anne; Heinz, Andreas; Kühn, Simone; Gallinat, Jürgen

    2015-11-01

    The fronto-limbic network interaction, driven by glutamatergic and dopaminergic neurotransmission, represents a core mechanism of motivated behavior and personality traits. Reward seeking behavior undergoes tremendous changes in adolescence paralleled by neurobiological changes of this network including the prefrontal cortex, striatum and amygdala. Since fronto-limbic dysfunctions also underlie major psychiatric diseases beginning in adolescence, this investigation focuses on network characteristics separating adolescents from adults. To investigate differences in network interactions, the brain reward system activity (slot machine task) together with frontal glutamate concentration (anterior cingulate cortex, ACC) was measured in 28 adolescents and 26 adults employing functional magnetic resonance imaging and magnetic resonance spectroscopy, respectively. An inverse coupling of glutamate concentrations in the ACC and activation of the ventral striatum was observed in adolescents. Further, amygdala response in adolescents was negatively correlated with the personality trait impulsivity. For adults, no significant associations of network components or correlations with impulsivity were found. The inverse association between frontal glutamate concentration and striatal activation in adolescents is in line with the triadic model of motivated behavior stressing the important role of frontal top-down inhibition on limbic structures. Our data identified glutamate as the mediating neurotransmitter of this inhibitory process and demonstrates the relevance of glutamate on the reward system and related behavioral traits like impulsivity. This fronto-limbic coupling may represent a vulnerability factor for psychiatric disorders starting in adolescence but not in adulthood. PMID:25009315

  16. Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

    PubMed Central

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  17. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    PubMed

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  18. Characterization of the target of ivermectin, the glutamate-gated chloride channel, from Anopheles gambiae

    PubMed Central

    Meyers, Jacob I.; Gray, Meg; Kuklinski, Wojtek; Johnson, Lucas B.; Snow, Christopher D.; Black, William C.; Partin, Kathryn M.; Foy, Brian D.

    2015-01-01

    ABSTRACT The use of insecticide-treated nets and indoor residual insecticides targeting adult mosquito vectors is a key element in malaria control programs. However, mosquito resistance to the insecticides used in these applications threatens malaria control efforts. Recently, the mass drug administration of ivermectin (IVM) has been shown to kill Anopheles gambiae mosquitoes and disrupt Plasmodium falciparum transmission in the field. We cloned the molecular target of IVM from A. gambiae, the glutamate-gated chloride channel (AgGluCl), and characterized its transcriptional patterns, protein expression and functional responses to glutamate and IVM. AgGluCl cloning revealed an unpredicted fourth splice isoform as well as a novel exon and splice site. The predicted gene products contained heterogeneity in the N-terminal extracellular domain and the intracellular loop region. Responses to glutamate and IVM were measured using two-electrode voltage clamp on Xenopus laevis oocytes expressing AgGluCl. IVM induced non-persistent currents in AgGluCl-a1 and did not potentiate glutamate responses. In contrast, AgGluCl-b was insensitive to IVM, suggesting that the AgGluCl gene could produce IVM-sensitive and -insensitive homomultimers from alternative splicing. AgGluCl isoform-specific transcripts were measured across tissues, ages, blood feeding status and sex, and were found to be differentially transcribed across these physiological variables. Lastly, we stained adult, female A. gambiae for GluCl expression. The channel was expressed in the antenna, Johnston's organ, supraesophageal ganglion and thoracic ganglia. In summary, we have characterized the first GluCl from a mosquito, A. gambiae, and described its unique activity and expression with respect to it as the target of the insecticide IVM. PMID:25994631

  19. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  20. Design and Synthesis of Systemically Active Metabotropic Glutamate Subtype-2 and -3 (mGlu2/3) Receptor Positive Allosteric Modulators (PAMs): Pharmacological Characterization and Assessment in a Rat Model of Cocaine Dependence

    PubMed Central

    2015-01-01

    As part of our ongoing small-molecule metabotropic glutamate (mGlu) receptor positive allosteric modulator (PAM) research, we performed structure–activity relationship (SAR) studies around a series of group II mGlu PAMs. Initial analogues exhibited weak activity as mGlu2 receptor PAMs and no activity at mGlu3. Compound optimization led to the identification of potent mGlu2/3 selective PAMs with no in vitro activity at mGlu1,4–8 or 45 other CNS receptors. In vitro pharmacological characterization of representative compound 44 indicated agonist-PAM activity toward mGlu2 and PAM activity at mGlu3. The most potent mGlu2/3 PAMs were characterized in assays predictive of ADME/T and pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu2/3 PAMs. On the basis of its overall profile, compound 74 was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in rats after intraperitoneal administration. These mGlu2/3 receptor PAMs have significant potential as small molecule tools for investigating group II mGlu pharmacology. PMID:24735492

  1. Glutamate receptors at atomic resolution

    SciTech Connect

    Mayer, Mark L.

    2010-12-03

    At synapses throughout the brain and spinal cord, the amino-acid glutamate is the major excitatory neurotransmitter. During evolution, a family of glutamate-receptor ion channels seems to have been assembled from a kit consisting of discrete ligand-binding, ion-channel, modulatory and cytoplasmic domains. Crystallographic studies that exploit this unique architecture have greatly aided structural analysis of the ligand-binding core, but the results also pose a formidable challenge, namely that of resolving the allosteric mechanisms by which individual domains communicate and function in an intact receptor.

  2. Vitamin E-Oligo(methyl diglycol l-glutamate) as a Biocompatible and Functional Surfactant for Facile Preparation of Active Tumor-Targeting PLGA Nanoparticles.

    PubMed

    Wu, Jintian; Zhang, Jian; Deng, Chao; Meng, Fenghua; Zhong, Zhiyuan

    2016-07-11

    Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles have attracted an enormous interest for controlled drug delivery. Their clinical applications are, however, partly hindered by lack of biocompatible, biodegradable and functional surfactants. Here, we designed and developed a novel biocompatible surfactant based on amphiphilic vitamin E-oligo(methyl diglycol l-glutamate) (VEOEG) for facile fabrication of robust and tumor-targeting PLGA-based nanomedicines. VEOEG was prepared with controlled Mn of 1.7-2.6 kg/mol and low molecular weight distribution (Đ = 1.04-1.16) via polymerization of methyl diglycol l-glutamate N-carboxyanhydride using vitamin E-ethylenediamine derivative (VE-NH2) as an initiator. VEOEG had a hydrophile-lipophile balance data of 13.8-16.1 and critical micellar concentration of 189.3-203.8 mg/L depending on lengths of oligopeptide. Using VEOEG as a surfactant, PLGA nanoparticles could be obtained via nanoprecipitation method with a small and uniform hydrodynamic size of 135 nm and positive surface charge of +26.6 mV, in accordance with presence of amino groups at the surface. The resulting PLGA nanoparticles could be readily coated with hyaluronic acid (HA) to form highly stable, small-sized (143 nm), monodisperse, and negatively charged nanoparticles (HA-PLGA NPs). Notably, paclitaxel-loaded HA-PLGA NPs (PTX-HA-PLGA NPs) exhibited better antitumor effects in CD44-positive MCF-7 breast tumor cells than Taxol (a clinical paclitaxel formulation). The in vivo pharmacokinetics assay in nude mice displayed that PTX-HA-PLGA NPs possessed a long plasma half-life of 3.14 h. The in vivo biodistribution studies revealed that PTX-HA-PLGA NPs had a high tumor PTX level of 8.4% ID/g, about 6 times better than that of Taxol. Interestingly, therapeutic studies showed that PTX-HA-PLGA NPs caused significantly more effective tumor growth inhibition, better survival rate and lower adverse effect than Taxol. VEOEG has emerged as a versatile and functional

  3. Vanilloids selectively sensitize thermal glutamate release from TRPV1 expressing solitary tract afferents.

    PubMed

    Hofmann, Mackenzie E; Andresen, Michael C

    2016-02-01

    Vanilloids, high temperature, and low pH activate the transient receptor potential vanilloid type 1 (TRPV1) receptor. In spinal dorsal root ganglia, co-activation of one of these gating sites on TRPV1 sensitized receptor gating by other modes. Here in rat brainstem slices, we examined glutamate synaptic transmission in nucleus of the solitary tract (NTS) neurons where most cranial primary afferents express TRPV1, but TRPV1 sensitization is unknown. Electrical shocks to the solitary tract (ST) evoked EPSCs (ST-EPSCs). Activation of TRPV1 with capsaicin (100 nM) increased spontaneous EPSCs (sEPSCs) but inhibited ST-EPSCs. High concentrations of the ultra-potent vanilloid resiniferatoxin (RTX, 1 nM) similarly increased sEPSC rates but blocked ST-EPSCs. Lowering the RTX concentration to 150 pM modestly increased the frequency of the sEPSCs without causing failures in the evoked ST-EPSCs. The sEPSC rate increased with raising bath temperature to 36 °C. Such thermal responses were larger in 150 pM RTX, while the ST-EPSCs remained unaffected. Vanilloid sensitization of thermal responses persisted in TTX but was blocked by the TRPV1 antagonist capsazepine. Our results demonstrate that multimodal activation of TRPV1 facilitates sEPSC responses in more than the arithmetic sum of the two activators, i.e. co-activation sensitizes TRPV1 control of spontaneous glutamate release. Since action potential evoked glutamate release is unaltered, the work provides evidence for cooperativity in gating TRPV1 plus a remarkable separation of calcium mechanisms governing the independent vesicle pools responsible for spontaneous and evoked release at primary afferents in the NTS. PMID:26471418

  4. Roles of subunit phosphorylation in regulating glutamate receptor function

    PubMed Central

    Wang, John Q.; Guo, Ming-Lei; Jin, Dao-Zhong; Xue, Bing; Fibuch, Eugene E.; Mao, Li-Min

    2014-01-01

    Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

  5. Glutamate in peripheral organs: Biology and pharmacology.

    PubMed

    Du, Jie; Li, Xiao-Hui; Li, Yuan-Jian

    2016-08-01

    Glutamate is a versatile molecule existing in both the central nervous system and peripheral organs. Previous studies have mainly focussed on the biological effect of glutamate in the brain. Recently, abundant evidence has demonstrated that glutamate also participates in the regulation of physiopathological functions in peripheral tissues, including the lung, kidney, liver, heart, stomach and immune system, where the glutamate/glutamate receptor/glutamate transporter system plays an important role in the pathogenesis of certain diseases, such as myocardial ischaemia/reperfusion injury and acute gastric mucosa injury. All these findings provide new insight into the biology and pharmacology of glutamate and suggest a potential therapeutic role of glutamate in non-neurological diseases. PMID:27164423

  6. Neuronal pyruvate carboxylation supports formation of transmitter glutamate.

    PubMed

    Hassel, B; Brâthe, A

    2000-02-15

    Release of transmitter glutamate implies a drain of alpha-ketoglutarate from neurons, because glutamate, which is formed from alpha-ketoglutarate, is taken up by astrocytes. It is generally believed that this drain is compensated by uptake of glutamine from astrocytes, because neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates, which requires pyruvate carboxylation. Here we show that cultured cerebellar granule neurons form releasable [(14)C]glutamate from H(14)CO(3)(-) and [1-(14)C]pyruvate via pyruvate carboxylation, probably mediated by malic enzyme. The activity of pyruvate carboxylation was calculated to be approximately one-third of the pyruvate dehydrogenase activity in neurons. Furthermore, intrastriatal injection of NaH(14)CO(3) or [1-(14)C]pyruvate labeled glutamate better than glutamine, showing that pyruvate carboxylation occurs in neurons in vivo. This means that neurons themselves to a large extent may support their release of glutamate, and thus entails a revision of the current view of glial-neuronal interactions and the importance of the glutamine cycle. PMID:10662824

  7. Single rodent mesohabenular axons release glutamate and GABA

    PubMed Central

    Root, David H.; Mejias-Aponte, Carlos; Zhang, Shiliang; Wang, Huiling; Hoffman, Alexander F.; Lupica, Carl R.; Morales, Marisela

    2016-01-01

    The lateral habenula (LHb) is involved in reward, aversion, addiction, and depression, through descending interactions with several brain structures, including the ventral tegmental area (VTA). VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb co-express markers for both glutamate-signaling (vesicular glutamate transporter 2, VGluT2) and GABA-signaling (glutamate decarboxylase, GAD; and vesicular GABA transporter, VGaT). A single axon from these mesohabenular neurons co-expresses VGluT2-protein and VGaT-protein, and surprisingly establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin-2 (ChR2) driven by VGluT2 or VGaT promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light-activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that co-transmits glutamate and GABA, and provides the majority of mesohabenular inputs. PMID:25242304

  8. System xc- regulates microglia and macrophage glutamate excitotoxicity in vivo

    PubMed Central

    Kigerl, Kristina A.; Ankeny, Daniel P.; Garg, Sanjay K.; Wei, Ping; Guan, Zhen; Lai, Wenmin; McTigue, Dana M.; Banerjee, Ruma; Popovich, Phillip G.

    2011-01-01

    It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system xc-) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system xc- and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system xc-, could be a novel approach for attenuating injurious neuroinflammatory cascades. PMID:22079587

  9. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  10. Mechanisms underlying hypertriglyceridemia in rats with monosodium L-glutamate-induced obesity: evidence of XBP-1/PDI/MTP axis activation.

    PubMed

    França, Lucas Martins; Freitas, Larissa Nara Costa; Chagas, Vinicyus Teles; Coêlho, Caio Fernando Ferreira; Barroso, Wermerson Assunção; Costa, Graciomar Conceição; Silva, Lucilene Amorim; Debbas, Victor; Laurindo, Francisco Rafael Martins; Paes, Antonio Marcus de Andrade

    2014-01-10

    Non-alcoholic fatty liver disease (NAFLD) is intimately associated with insulin resistance and hypertriglyceridemia, whereas many of the mechanisms underlying this association are still poorly understood. In the present study, we investigated the relationship between microsomal triglyceride transfer protein (MTP) and markers of endoplasmic reticulum (ER) stress in the liver of rats subjected to neonatal monosodium L-glutamate (MSG)-induced obesity. At age 120 days old, the MSG-obese animals exhibited hyperglycemia, hypertriglyceridemia, insulin resistance, and liver steatosis, while the control (CTR) group did not. Analysis using fast protein liquid chromatography of the serum lipoproteins revealed that the triacylglycerol content of the very low-density lipoprotein (VLDL) particles was twice as high in the MSG animals compared with the CTR animals. The expression of ER stress markers, GRP76 and GRP94, was increased in the MSG rats, promoting a higher expression of X-box binding protein 1 (XBP-1), protein disulfide isomerase (PDI), and MTP. As the XBP-1/PDI/MTP axis has been suggested to represent a significant lipogenic mechanism in the liver response to ER stress, our data indicate that hypertriglyceridemia and liver steatosis occurring in the MSG rats are associated with increased MTP expression. PMID:24333444

  11. Thirst Is Associated with Suppression of Habenula Output and Active Stress Coping: Is there a Role for a Non-canonical Vasopressin-Glutamate Pathway?

    PubMed Central

    Zhang, Limei; Hernández, Vito S.; Vázquez-Juárez, Erika; Chay, Freya K.; Barrio, Rafael A.

    2016-01-01

    Water-homeostasis is a fundamental physiological process for terrestrial life. In vertebrates, thirst drives water intake, but the neuronal circuits that connect the physiology of water regulation with emotional context are poorly understood. Vasopressin (VP) is a prominent messenger in this circuit, as well as L-glutamate. We have investigated the role of a VP circuit and interaction between thirst and motivational behaviors evoked by life-threatening stimuli in rats. We demonstrate a direct pathway from hypothalamic paraventricular VP-expressing, glutamatergic magnocellular neurons to the medial division of lateral habenula (LHbM), a region containing GABAergic neurons. In vivo recording and juxtacellular labeling revealed that GABAergic neurons in the LHbM had locally branching axons, and received VP-positive axon terminal contacts on their dendrites. Water deprivation significantly reduced freezing and immobility behaviors evoked by innate fear and behavioral despair, respectively, accompanied by decreased Fos expression in the lateral habenula. Our results reveal a novel VP-expressing hypothalamus to the LHbM circuit that is likely to evoke GABA-mediated inhibition in the LHbM, which promotes escape behavior during stress coping. PMID:27065810

  12. Therapeutic Effects of Glutamic Acid in Piglets Challenged with Deoxynivalenol

    PubMed Central

    Ren, Wenkai; Yin, Jie; Tan, Bie; Liu, Gang; Li, Lili; Nyachoti, Charles Martin; Xiong, Xia; Wu, Guoyao

    2014-01-01

    The mycotoxin deoxynivalenol (DON), one of the most common food contaminants, primarily targets the gastrointestinal tract to affect animal and human health. This study was conducted to examine the protective function of glutamic acid on intestinal injury and oxidative stress caused by DON in piglets. Twenty-eight piglets were assigned randomly into 4 dietary treatments (7 pigs/treatment): 1) uncontaminated control diet (NC), 2) NC+DON at 4 mg/kg (DON), 3) NC+2% glutamic acid (GLU), and 4) NC+2% glutamic acid + DON at 4 mg/kg (DG). At day 15, 30 and 37, blood samples were collected to determine serum concentrations of CAT (catalase), T-AOC (total antioxidant capacity), H2O2 (hydrogen peroxide), NO (nitric oxide), MDA (maleic dialdehyde), DAO (diamine oxidase) and D-lactate. Intestinal morphology, and the activation of Akt/mTOR/4EBP1 signal pathway, as well as the concentrations of H2O2, MDA, and DAO in kidney, liver and small intestine, were analyzed at day 37. Results showed that DON significantly (P<0.05) induced oxidative stress in piglets, while this stress was remarkably reduced with glutamic acid supplementation according to the change of oxidative parameters in blood and tissues. Meanwhile, DON caused obvious intestinal injury from microscopic observations and permeability indicators, which was alleviated by glutamic acid supplementation. Moreover, the inhibition of DON on Akt/mTOR/4EBP1 signal pathway was reduced by glutamic acid supplementation. Collectively, these data suggest that glutamic acid may be a useful nutritional regulator for DON-induced damage manifested as oxidative stress, intestinal injury and signaling inhibition. PMID:24984001

  13. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  14. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer.

    PubMed

    Dinpajooh, Mohammadhasan; Martin, Daniel R; Matyushov, Dmitry V

    2016-01-01

    Enzymes in biology's energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  15. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  16. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  17. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  18. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  19. Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

    PubMed Central

    Clerc, Pascaline; Young, Christina A.; Bordt, Evan A.; Grigore, Alina M.; Fiskum, Gary; Polster, Brian M.

    2013-01-01

    Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO4, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg2+ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO4 improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO4 or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO4 protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO4 protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate. PMID:24236167

  20. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  1. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  2. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  3. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  4. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and... GENERALLY RECOGNIZED AS SAFE Multiple Purpose GRAS Food Substances § 182.1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation. This substance is generally recognized...

  5. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  6. Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of Excitatory Amino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c -

    NASA Astrophysics Data System (ADS)

    Bridges, Richard J.; Patel, Sarjubhai A.

    As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates not only in standard fast synaptic communication, but also contributes to higher order signal processing, as well as neuropathology. Given this variety of functional roles, interest has been growing as to how the extracellular concentrations of l-glutamate surrounding neurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, each of which appears capable of influencing both the signaling and pathological actions of l-glutamate within the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystine exchanger, system x c - (Sx c -). While the family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequestering l-glutamate in neurons and glia, Sxc - provides a route for the export of glutamate from cells into the extracellular environment. The primary intent of this work is to provide an overview of the inhibitors and substrates that have been developed to delineate the pharmacological specificity of these transport systems, as well as be exploited as probes with which to selectively investigate function. Particular attention is paid to the development of small molecule templates that mimic the structural properties of the endogenous substrates, l-glutamate, l-aspartate and l-cystine and how strategic control of functional group position and/or the introduction of lipophilic R-groups can impact multiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrate activity.

  7. Exposure to altered gravity conditions results in hypoxia-related enhancement of the presynaptic transporter-mediated release of glutamate.

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana

    High-affinity Na+-dependent glutamate transporters locate in the plasma membrane and maintain the low concentration of glutamate in synaptic cleft by the uptake of glutamate into neurons. Under hypoxic conditions glutamate transporters contribute to the glutamate release due to functioning in reverse mode. The release of glutamate via reverse-operated Na+-dependent glutamate transporters was investigated in brain synaptosomes under conditions of centrifugeinduced hypergravity. Flow cytometric analisis revealed similarity in the size and cytoplasmic granularity of control and hypergravity synaptosomes. Protonophore FCCP dissipates the proton gradient across synaptic vesicle thus synaptic vesicles are not able to keep glutamate inside. 1 microM FCCP induced the release of 4. 8 ±1. 0 % and 8. 0 ±1. 0 % of total accumulated synaptosomal label in control and G-loaded animals, respectively. Ca 2+-independent high- KCl stimulated L-[14C]glutamate release from synaptosomes preliminary treated with FCCP increased considerably from 27. 0 ± 2. 2 % to 35. 0 ± 2. 3 % after centrifuge-induced hypergravity. No-transportable inhibitor of glutamate transporter DL-threo-beta-benzyloxyaspartate was found to inhibit high-KCl and FCCP-stimulated release of L-[14C]glutamate, thus the release was concluded to occur due to reversal of glutamate transporters. We have also found the inhibition of the activity of Na \\ K ATPase in the plasma membrane of synaptosomes after hypergravity that might also contribute to the enhancement of the transporter-mediated release of glutamate. These hypergravity-induced alterations in the transporter-mediated release of glutamate were suggested to correlate with the hypoxic injury of neurons. The changes we have revealed for the transporter-mediated release of glutamate may lead to mental disorders, upcoming seizures and neurotoxicity under hypergravity conditions.

  8. On the Role of Glutamate in Presynaptic Development: Possible Contributions of Presynaptic NMDA Receptors

    PubMed Central

    Fedder, Karlie N.; Sabo, Shasta L.

    2015-01-01

    Proper formation and maturation of synapses during development is a crucial step in building the functional neural circuits that underlie perception and behavior. It is well established that experience modifies circuit development. Therefore, understanding how synapse formation is controlled by synaptic activity is a key question in neuroscience. In this review, we focus on the regulation of excitatory presynaptic terminal development by glutamate, the predominant excitatory neurotransmitter in the brain. We discuss the evidence that NMDA receptor activation mediates these effects of glutamate and present the hypothesis that local activation of presynaptic NMDA receptors (preNMDARs) contributes to glutamate-dependent control of presynaptic development. Abnormal glutamate signaling and aberrant synapse development are both thought to contribute to the pathogenesis of a variety of neurodevelopmental disorders, including autism spectrum disorders, intellectual disability, epilepsy, anxiety, depression, and schizophrenia. Therefore, understanding how glutamate signaling and synapse development are linked is important for understanding the etiology of these diseases. PMID:26694480

  9. On the Role of Glutamate in Presynaptic Development: Possible Contributions of Presynaptic NMDA Receptors.

    PubMed

    Fedder, Karlie N; Sabo, Shasta L

    2015-01-01

    Proper formation and maturation of synapses during development is a crucial step in building the functional neural circuits that underlie perception and behavior. It is well established that experience modifies circuit development. Therefore, understanding how synapse formation is controlled by synaptic activity is a key question in neuroscience. In this review, we focus on the regulation of excitatory presynaptic terminal development by glutamate, the predominant excitatory neurotransmitter in the brain. We discuss the evidence that NMDA receptor activation mediates these effects of glutamate and present the hypothesis that local activation of presynaptic NMDA receptors (preNMDARs) contributes to glutamate-dependent control of presynaptic development. Abnormal glutamate signaling and aberrant synapse development are both thought to contribute to the pathogenesis of a variety of neurodevelopmental disorders, including autism spectrum disorders, intellectual disability, epilepsy, anxiety, depression, and schizophrenia. Therefore, understanding how glutamate signaling and synapse development are linked is important for understanding the etiology of these diseases. PMID:26694480

  10. Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy.

    PubMed

    Feng, Shengjie; Ma, Shaorong; Jia, Caixia; Su, Yujuan; Yang, Shenglian; Zhou, Kechun; Liu, Yani; Cheng, Ju; Lu, Dunguo; Fan, Liu; Wang, Yizheng

    2016-05-01

    Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gαi and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy. PMID:27113760

  11. Mechanisms for maintaining extracellular glutamate levels in the anoxic turtle striatum.

    PubMed

    Milton, Sarah L; Thompson, John W; Lutz, Peter L

    2002-05-01

    The turtle Trachemys scripta is one of a limited group of vertebrates that can withstand hours to days without oxygen. One facet of anoxic survival is the turtle's ability to maintain basal extracellular glutamate levels, whereas in most vertebrates, anoxia triggers massive excitotoxic glutamate release. We investigated glutamate release and reuptake in the anoxic turtle and the effects of adenosine and ATP-sensitive potassium (K(ATP)) channels on glutamate homeostasis. Striatal extracellular glutamate was measured in anesthetized T. scripta by microdialysis in normoxia and over 2-h anoxia. Glutamate release is decreased by 44% in the early anoxic turtle; this anoxia-induced decrease in glutamate release was prevented when K(ATP) channels and adenosine receptors were blocked simultaneously but not when either mechanism was blocked individually. We hypothesize that the continued release and reuptake of glutamate during anoxia help maintain neuronal tone and aid in the recovery of a functional neuronal network after long periods of anoxia, whereas activation of adenosine and/or K(ATP) conserves energy by reducing glutamate release and lowering transport costs. PMID:11959671

  12. On the defensive action of glutamate against the cytotoxicity and fibrogenicity of quartz dust.

    PubMed Central

    Morosova, K I; Aronova, G V; Katsnelson, B A; Velichkovski, B T; Genkin, A M; Elnichnykh, L N; Privalova, L I

    1982-01-01

    The cytotoxic action of quartz (DQ12) particles on cultures of rat peritoneal macrophages, as estimated by the inhibition of the TTC-reductase activity, is considerably reduced by preincubation with glutamic acid and by adding sodium glutamate (15 mg/ml) to the drinking water of the rats donating the macrophages. This increase in macrophage resistance under the influence of glutamate is the most probable cause of the delay in the development of silicotic fibrosis shown in several experiments on rats intratracheally injected with quartz and then treated by prolonged administration of glutamate. This effect is probably connected with the influence of glutamate on the stability of the macrophage membranes, which can in its turn be explained by different mechanisms, including the influence on the synthesis and phosphorylation of adenosine nucleotides. Such an influence was shown in rats receiving glutamate by the change of the ATP/ADP ratio in macrophages, but not in erythrocytes. The resistance of rat erythrocytes to the haemolytic action of quartz is also not influenced by the action of glutamate neither in vitro nor in vivo. Such differences in the influences of glutamate on two types of cells, equally susceptible to quartz cytotoxicity but considerably differing in the character of energy metabolism, is an indirect proof of the role of the latter in the realisation of the anticytotoxic, and thereby antifibrogenic, effect of glutamate. PMID:6124270

  13. Relationship between Zinc (Zn2+) and Glutamate Receptors in the Processes Underlying Neurodegeneration

    PubMed Central

    Pochwat, Bartłomiej; Nowak, Gabriel; Szewczyk, Bernadeta

    2015-01-01

    The results from numerous studies have shown that an imbalance between particular neurotransmitters may lead to brain circuit dysfunction and development of many pathological states. The significance of glutamate pathways for the functioning of the nervous system is equivocal. On the one hand, glutamate transmission is necessary for neuroplasticity, synaptogenesis, or cell survival, but on the other hand an excessive and long-lasting increased level of glutamate in the synapse may lead to cell death. Under clinical conditions, hyperactivity of the glutamate system is associated with ischemia, epilepsy, and neurodegenerative diseases such as Alzheimer's, Huntington's, and many others. The achievement of glutamate activity in the physiological range requires efficient control by endogenous regulatory factors. Due to the fact that the free pool of ion Zn2+ is a cotransmitter in some glutamate neurons; the role of this element in the pathophysiology of a neurodegenerative diseases has been intensively studied. There is a lot of evidence for Zn2+ dyshomeostasis and glutamate system abnormalities in ischemic and neurodegenerative disorders. However, the precise interaction between Zn2+ regulative function and the glutamate system is still not fully understood. This review describes the relationship between Zn2+ and glutamate dependent signaling pathways under selected pathological central nervous system (CNS) conditions. PMID:26106488

  14. Glutamate carboxypeptidase inhibition reduces the severity of chemotherapy-induced peripheral neurotoxicity in rat.

    PubMed

    Carozzi, Valentina A; Chiorazzi, Alessia; Canta, Annalisa; Lapidus, Rena G; Slusher, Barbara S; Wozniak, Krystyna M; Cavaletti, Guido

    2010-05-01

    Chemotherapy is the most common method to treat cancer. The use of certain antineoplastic drugs, however, is associated with the development of peripheral neuropathy that can be dose-limiting. Excitotoxic glutamate release, leading to excessive glutamatergic neurotransmission and activation of N-methyl-D-aspartate (NMDA) receptors, is associated with neuronal damage and death in several nervous system disorders. N-Acetyl-aspartyl-glutamate (NAAG) is an abundant neuropeptide widely distributed in the central and peripheral nervous system which is physiologically hydrolyzed by the enzyme glutamate carboxypeptidase into N-Acetyl-aspartyl (NAA) and glutamate. Pharmacological inhibition of glutamate carboxypeptidase results in decreased glutamate and increased endogenous NAAG and has been shown to provide neuroprotection in several preclinical models. Here, we report the neuroprotective effect of an orally available glutamate carboxypeptidase inhibitor on three well-established animal models of chemotherapy (cisplatin, paclitaxel, bortezomib)-induced peripheral neuropathy. In all cases, glutamate carboxypeptidase inhibition significantly improved the chemotherapy-induced nerve conduction velocity deficits. In addition, morphological and morphometrical alterations induced by cisplatin and bortezomib in dorsal root ganglia (DRG) were improved by glutamate carboxypeptidase inhibition. Our data support a novel approach for the treatment of chemotherapy-induced peripheral neuropathy. PMID:19763734

  15. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  16. Glutamate-1-semialdehyde aminotransferase from Sulfolobus solfataricus.

    PubMed

    Palmieri, G; Di Palo, M; Scaloni, A; Orru, S; Marino, G; Sannia, G

    1996-12-01

    Glutamate-1-semialdehyde aminotransferase (GSA-AT) from the extremely thermophilic bacterium Sulfolobus solfataricus has been purified to homogeneity and characterized. GSA-AT is the last enzyme in the C5 pathway for the conversion of glutamate into the tetrapyrrole precursor delta-aminolaevulinate (ALA) in plants, algae and several bacteria. The active form of GSA-AT from S. solfataricus seems to be a homodimer with a molecular mass of 87 kDa. The absorption spectrum of the purified aminotransferase is indicative of the presence of pyridoxamine 5'-phosphate (PMP) cofactor, and the catalytic activity of the enzyme is further stimulated by addition of PMP. 3-Amino-2,3-dihydrobenzoic acid is an inhibitor of the aminotransferase activity. The N-terminal amino acid sequence of GSA-AT from S. solfataricus was found to share significant similarity with the eukaryotic and eubacterial enzymes. Evidence is provided that ALA synthesis in S. solfataricus follows the C5 pathway characteristic of plants, algae, cyanobacteria and many other bacteria. PMID:8973563

  17. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  18. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  19. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  20. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  1. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus si