WorldWideScience

Sample records for determine antagonist binding

  1. Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

    Science.gov (United States)

    Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

    2017-11-22

    We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

  2. Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Mungalpara, J; Steen, A

    2014-01-01

    have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments. EXPERIMENTAL APPROACH: Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated...... activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. KEY RESULTS: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic......-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. CONCLUSIONS AND IMPLICATIONS: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes...

  3. Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

    International Nuclear Information System (INIS)

    Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

    1987-01-01

    The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [ 3 H]-antagonist as well as of the labeled natural neurotransmitter, [ 3 H]-acetylcholine [( 3 H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [ 3 H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [ 3 H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [ 3 H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [ 3 H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms

  4. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  5. Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding.

    Directory of Open Access Journals (Sweden)

    Henrik Keränen

    Full Text Available To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A(2A adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.

  6. Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

    Science.gov (United States)

    Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

    2017-09-01

    Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

  7. A DFT approach to discriminate the antagonist and partial agonist activity of ligands binding to the NMDA receptor

    Science.gov (United States)

    Haslak, Zeynep Pinar; Bozkurt, Esra; Dutagaci, Bercem; De Proft, Frank; Aviyente, Viktorya; De Vleeschouwer, Freija

    2018-02-01

    The activation of N-methyl-D-aspartate receptors is found to be intimately associated with neurodegenerative diseases which make them promising therapeutic targets. Despite the significantly increasing multidisciplinary interests centred on this ionotropic channel, design of new ligands with intended functional activity remains a great challenge. In this article, a computational study based on density functional theory is presented to understand the structural factors of ligands determining their function as antagonists and partial agonists. With this aim, the GluN1 subunit is chosen as being one of the essential components in the activation mechanism, and quantum chemical calculations are implemented for 30 antagonists and 30 partial agonists known to bind to this subunit with different binding affinities. Several quantum chemical descriptors are investigated which might unlock the difference between antagonists and partial agonists.

  8. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...

  9. Stereochemistry of quinoxaline antagonist binding to a glutamate receptor investigated by Fourier transform infrared spectroscopy.

    Science.gov (United States)

    Madden, D R; Thiran, S; Zimmermann, H; Romm, J; Jayaraman, V

    2001-10-12

    The stereochemistry of the interactions between quinoxaline antagonists and the ligand-binding domain of the glutamate receptor 4 (GluR4) have been investigated by probing their vibrational modes using Fourier transform infrared spectroscopy. In solution, the electron-withdrawing nitro groups of both compounds establish a resonance equilibrium that appears to stabilize the keto form of one of the cyclic amide carbonyl bonds. Changes in the 6,7-dinitro-2,3-dihydroxyquinoxaline vibrational spectra on binding to the glutamate receptor, interpreted within the framework of a published crystal structure, illuminate the stereochemistry of the interaction and suggest that the binding site imposes a more polarized electronic bonding configuration on this antagonist. Similar spectral changes are observed for 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline, confirming that its interactions with the binding site are highly similar to those of 6,7-dinitro-2,3-dihydroxyquinoxaline and leading to a model of the 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline-S1S2 complex, for which no crystal structure is available. Conformational changes within the GluR ligand binding domain were also monitored. Compared with the previously reported spectral changes seen on binding of the agonist glutamate, only a relatively small change is detected on antagonist binding. This correlation between the functional effects of different classes of ligand and the magnitude of the spectroscopic changes they induce suggests that the spectral data reflect physiologically relevant conformational processes.

  10. Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

    Science.gov (United States)

    Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

    2008-10-01

    GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

  11. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  12. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    International Nuclear Information System (INIS)

    Waser, Beatrice; Reubi, Jean Claude

    2014-01-01

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the 125 iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer 125 I-GLP-1(7-36)amide. Receptor autoradiography studies with 125 I-GLP-1(7-36)amide agonist or 125 I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist 125 I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer 125 I-GLP-1(7-36)amide. For comparison, 125 I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with 125 I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  13. The structure of a mixed GluR2 ligand-binding core dimer in complex with (S)-glutamate and the antagonist (S)-NS1209

    DEFF Research Database (Denmark)

    Kasper, Christina; Pickering, Darryl S; Mirza, Osman

    2006-01-01

    domains has been observed. (S)-NS1209 adopts a novel binding mode, including hydrogen bonding to Tyr450 and Gly451 of D1. Parts of (S)-NS1209 occupy new areas of the GluR2 ligand-binding cleft, and bind near residues that are not conserved among receptor subtypes. The affinities of (RS)-NS1209 at the Glu....... The thermodynamics of binding of the antagonists (S)-NS1209, DNQX and (S)-ATPO to the GluR2 ligand-binding core have been determined by displacement isothermal titration calorimetry. The displacement of (S)-glutamate by all antagonists was shown to be driven by enthalpy....

  14. ``In silico'' study of the binding of two novel antagonists to the nociceptin receptor

    Science.gov (United States)

    Della Longa, Stefano; Arcovito, Alessandro

    2018-02-01

    Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D1303,32 (upper indexes are Ballesteros-Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y1313,33, M1343,36, I2195,43, Q2806,52 and V2836,55) in the orthosteric site of NOP, accommodating either the pyridine-pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D1303,32 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y1313,33 and M1343,36. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a

  15. Mutational analysis of the antagonist-binding site of the histamine H(1) receptor.

    Science.gov (United States)

    Wieland, K; Laak, A M; Smit, M J; Kühne, R; Timmerman, H; Leurs, R

    1999-10-15

    We combined in a previously derived three-dimensional model of the histamine H(1) receptor (Ter Laak, A. M., Timmerman, H., Leurs, H., Nederkoorn, P. H. J., Smit, M. J., and Donne-Op den Kelder, G. M. (1995) J. Comp. Aid. Mol. Design. 9, 319-330) a pharmacophore for the H(1) antagonist binding site (Ter Laak, A. M., Venhorst, J., Timmerman, H., and Donné-Op de Kelder, G. M. (1994) J. Med. Chem. 38, 3351-3360) with the known interacting amino acid residue Asp(116) (in transmembrane domain III) of the H(1) receptor and verified the predicted receptor-ligand interactions by site-directed mutagenesis. This resulted in the identification of the aromatic amino acids Trp(167), Phe(433), and Phe(436) in transmembrane domains IV and VI of the H(1) receptor as probable interaction points for the trans-aromatic ring of the H(1) antagonists. Subsequently, a specific interaction of carboxylate moieties of two therapeutically important, zwitterionic H(1) antagonists with Lys(200) in transmembrane domain V was predicted. A Lys(200) --> Ala mutation results in a 50- (acrivastine) to 8-fold (d-cetirizine) loss of affinity of these zwitterionic antagonists. In contrast, the affinities of structural analogs of acrivastine and cetirizine lacking the carboxylate group, triprolidine and meclozine, respectively, are unaffected by the Lys(200) --> Ala mutation. These data strongly suggest that Lys(200), unique for the H(1) receptor, acts as a specific anchor point for these "second generation" H(1) antagonists.

  16. Preliminary Molecular Dynamic Simulations of the Estrogen Receptor Alpha Ligand Binding Domain from Antagonist to Apo

    Directory of Open Access Journals (Sweden)

    Adrian E. Roitberg

    2008-06-01

    Full Text Available Estrogen receptors (ER are known as nuclear receptors. They exist in the cytoplasm of human cells and serves as a DNA binding transcription factor that regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. The human estrogen receptor comes in two forms, alpha and beta. This work focuses on the alpha form of the estrogen receptor. The ERα is found in breast cancer cells, ovarian stroma cells, endometrium, and the hypothalamus. It has been suggested that exposure to DDE, a metabolite of DDT, and other pesticides causes conformational changes in the estrogen receptor. Before examining these factors, this work examines the protein unfolding from the antagonist form found in the 3ERT PDB crystal structure. The 3ERT PDB crystal structure has the estrogen receptor bound to the cancer drug 4-hydroxytamoxifen. The 4-hydroxytamoxifen ligand was extracted before the simulation, resulting in new conformational freedom due to absence of van der Waals contacts between the ligand and the receptor. The conformational changes that result expose the binding clef of the co peptide beside Helix 12 of the receptor forming an apo conformation. Two key conformations in the loops at either end of the H12 are produced resulting in the antagonist to apo conformation transformation. The results were produced over a 42ns Molecular Dynamics simulation using the AMBER FF99SB force field.

  17. Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants.

    Science.gov (United States)

    Jantan, I; Rafi, I A A; Jalil, J

    2005-01-01

    Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).

  18. Differential binding of urokinase and peptide antagonists to the urokinase receptor

    DEFF Research Database (Denmark)

    Engelholm, L H; Behrendt, N

    2001-01-01

    though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee u......PAR), in spite of the fact that uPAR from all of the four species cross-reacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding...

  19. Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

    International Nuclear Information System (INIS)

    Rahmani, N.H.; Gulati, A.; Bhargava, H.N.

    1991-01-01

    The binding of 3 H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3 H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3 H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (B max value) and apparent dissociation constant (K d value) values of 3 H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midgrain, cortex and spinal cord of WKY and SHR rates did not differ. The B max value of 3 H-naltrexone binding to membranes of hypothalamus of SHR rates was 518% higher than WKY rats but the K d values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3 H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats

  20. Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

    Energy Technology Data Exchange (ETDEWEB)

    Rahmani, N.H.; Gulati, A.; Bhargava, H.N. (Univ. of Illinois, Chicago (USA))

    1991-01-01

    The binding of {sup 3}H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. {sup 3}H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of {sup 3}H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (B{sub max}value) and apparent dissociation constant (K{sub d} value) values of {sup 3}H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midgrain, cortex and spinal cord of WKY and SHR rates did not differ. The B{sub max} value of {sup 3}H-naltrexone binding to membranes of hypothalamus of SHR rates was 518% higher than WKY rats but the K{sub d} values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with {sup 3}H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.

  1. New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding

    Science.gov (United States)

    Lupala, Cecylia S.; Gomez-Gutierrez, Patricia; Perez, Juan J.

    2016-01-01

    hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33 % success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in the present work.

  2. Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

    Science.gov (United States)

    Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

    1998-08-21

    Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical

  3. CC12, a high-affinity ligand for [3H]cimetidine binding, is an improgan antagonist

    NARCIS (Netherlands)

    Hough, L.H.; Nalwalk, J.B.; Phillips, J.R.; Kern, B.; Shan, Z.; Wentland, M.; de Esch, I.J.P.; Janssen, EA; Barr, T.; Stadel, R.

    2007-01-01

    Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [

  4. Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain

    DEFF Research Database (Denmark)

    Smith, Donald F; Dyve, Suzan; Minuzzi, Luciano

    2006-01-01

    Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain......Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain...

  5. Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

    Science.gov (United States)

    Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

    2015-01-01

    The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

  6. Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity

    Directory of Open Access Journals (Sweden)

    Douglas Bledsoe

    2017-05-01

    Full Text Available N-methyl D-aspartate receptors (NMDAR play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D and two GluN3 (A–B subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD and ligand binding domain (LBD, transmembrane domain (TMD and an intracellular C-terminal domain (CTD. Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

  7. Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

    Science.gov (United States)

    Hoyer, Inna; Haas, Ann-Karin; Kreuchwig, Annika; Schülein, Ralf; Krause, Gerd

    2013-02-01

    The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

  8. Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

    Science.gov (United States)

    Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

    2017-07-18

    Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

  9. Absence of age-related changes in the binding of the beta adrenergic antagonist 125I-iodohydroxybenzylpindolol in rat heart

    International Nuclear Information System (INIS)

    Tumer, N.; Bender, J.; Roberts, J.

    1987-01-01

    The effect of age on the density and the affinity of beta adrenergic receptors was determined in the hearts of Fischer 344 rats at three ages, 6, 12, and 24 months old. The binding of the beta adrenergic antagonist 125 I-iodohydroxybenzylpindolol (IHYP), was used to quantitate and characterize cardiac beta adrenergic receptors. The maximal number of binding sites (B/sub max/ = F moles/mg of protein) were 26.3 +/- 2.5, 25.4 +/- 0.99, and 24.5 +/- 2.4 and the dissociation constants (K/sub d/ = nM) were 0.166 +/- 0.014, 0.126 +/- 0.006, and 0.135 +/- 0.015 for 6, 12, and 24 months old animals, respectively. There were no significant differences among the three ages. These results support the contention that neither beta adrenergic receptor density of affinity changes with age in the ventricles of the rat heart

  10. Structure-based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists.

    Science.gov (United States)

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

  11. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  12. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    Science.gov (United States)

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  13. Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

    Science.gov (United States)

    Tatsinkam, Arnold Junior; Mulloy, Barbara; Rider, Christopher C

    2015-08-15

    Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers. © 2015 Authors; published by Portland Press Limited.

  14. Binding and functional pharmacological characteristics of gepant-type antagonists in rat brain and mesenteric arteries

    DEFF Research Database (Denmark)

    Sheykhzade, Majid; Amandi, Nilofar; Pla, Monica Vidal

    2017-01-01

    AIM: The neuropeptide calcitonin gene-related peptide (CGRP) is found in afferent sensory nerve fibers innervating the resistance arteries and plays a pivotal role in a number of neurovascular diseases such as migraine and subarachnoid bleedings. The present study investigates the binding and ant...

  15. Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

    International Nuclear Information System (INIS)

    Chang, R.S.; Lotti, V.J.; Chen, T.B.; Kunkel, K.A.

    1986-01-01

    [3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [ 3 H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [ 3 H]-(+/-)-L-364,718 correlated with their ability to displace [ 125 I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [ 125 I]CCK-8 than [ 3 H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [ 3 H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl 2 . [ 3 H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [ 3 H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [ 3 H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions

  16. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  17. Characteristics of the binding of [3H]nitrendipine to rabbit ventricular membranes: modification by other Ca++ channel antagonists and by the Ca++ channel agonist Bay K 8644

    International Nuclear Information System (INIS)

    Janis, R.A.; Sarmiento, J.G.; Maurer, S.C.; Bolger, G.T.; Triggle, D.J.

    1984-01-01

    This study was carried out to characterize [ 3 H]nitrendipine binding to cardiac membranes and to test the hypothesis that high affinity binding of Ca++ channel antagonists and agonists is to Ca++ channels. Binding was specific, rapid, reversible and stereoselective. The relative order of potency of nifedipine analogs for inhibition of binding was the same as that for inhibition of smooth and cardiac muscle contraction. Results with diltiazem, verapamil and lidoflazine were consistent with the hypothesis that nondihydropyridine Ca++ channel antagonists act at one or more sites allosterically linked to the 1,4-dihydropyridine site in cardiac cells. The Ca++ channel agonist Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] displaced specifically bound [ 3 H]nitrendipine in an apparently competitive manner with an IC50 value of 5 nM. The results suggest that organic antagonists do not act by physically blocking the Ca++ channel. The data also support the hypothesis that the high affinity binding sites for [ 3 H]nitrendipine in isolated cardiac membranes are associated with Ca++ channels that are inactivated or are otherwise unavailable for opening

  18. Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site

    DEFF Research Database (Denmark)

    Kvist, Trine; Steffensen, Thomas Bielefeldt; Greenwood, Jeremy R

    2013-01-01

    NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glyci...... screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design....

  19. Pharmacogenetic Aspects of the Interaction of AT1 Receptor Antagonists With ATP-Binding Cassette Transporter ABCG2

    Directory of Open Access Journals (Sweden)

    Anne Ripperger

    2018-05-01

    Full Text Available The ATP-binding cassette transporter ABCG2 (BCRP and MXR is involved in the absorption, distribution, and elimination of numerous drugs. Thus, drugs that are able to reduce the activity of ABCG2, e.g., antihypertensive AT1 receptor antagonists (ARBs, may cause drug-drug interactions and compromise drug safety and efficacy. In addition, genetic variability within the ABCG2 gene may influence the ability of the transporter to interact with ARBs. Thus, the aim of this study was to characterize the ARB-ABCG2 interaction in the light of naturally occurring variations (F489L, R482G or amino acid substitutions with in silico-predicted relevance for the ARB-ABCG2 interaction (Y469A; M483F; Y570A. For this purpose, ABCG2 variants were expressed in HEK293 cells and the impact of ARBs on ABCG2 activity was studied in vitro using the pheophorbide A (PhA efflux assay. First, we demonstrated that both the F489L and the Y469A substitution, respectively, reduced ABCG2 protein levels in these cells. Moreover, both substitutions enhanced the inhibitory effect of candesartan cilexetil, irbesartan, losartan, and telmisartan on ABCG2-mediated PhA efflux, whereas the R482G substitution blunted the inhibitory effect of candesartan cilexetil and telmisartan in this regard. In contrast, the ARB-ABCG2 interaction was not altered in cells expressing either the M483F or the Y570A variant, respectively. In conclusion, our data indicate that the third transmembrane helix and adjacent regions of ABCG2 may be of major importance for the interaction of ARBs with the ABC transporter. Moreover, we conclude from our data that individuals carrying the F489L polymorphism may be at increased risk of developing ABCG2-related drug-drug interactions in multi-drug regimens involving ARBs.

  20. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions.

    Directory of Open Access Journals (Sweden)

    Ruyin Cao

    Full Text Available Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD study of the human adenosine receptor type 2A (hA(2AR in complex with caffeine--a system of high neuro-pharmacological relevance--within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system, often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA(2AR and possibly other GPCRs.

  1. [3H]AVP binding to rat renal tubular receptors during long-term treatment with an antagonist of arginine vasopressin

    International Nuclear Information System (INIS)

    Mah, S.C.; Whitebread, S.E.; De Gasparo, M.; Hofbauer, K.G.

    1988-01-01

    The interaction of an antagonist of arginine vasopressin (AVP), d(CH2)5-D-Tyr(Et)VAVP, with renal tubular V2 receptors were studied in medullary membrane preparations from kidneys of Sprague-Dawley and Brattleboro rats. In both rat strains, V2 receptors had comparable KD and Bmax values for binding of [3H]AVP. In vitro studies revealed that the V2-antagonist was more potent than cold AVP in displacing [3H]AVP. In vivo treatment of Sprague-Dawley rats with the antagonist over one week resulted only in a transient state of diabetes insipidus (DI). No specific [3H]AVP binding was detectable throughout the period of administration. Chronic treatment of Brattleboro rats resulted in a complete normalization of water intake. This agonistic effect was also associated with undetectable [3H]AVP binding. After stopping the infusion of d(CH2)5-D-Tyr(Et)VAVP, Bmax values tended to rise but had still not reached base line values after 6 days. In contrast, the chronic infusion of AVP in Brattleboro rats resulted in a reduction in water intake which was accompanied by a decreased Bmax. [3H]AVP binding remained detectable during the entire treatment period. Thereafter Bmax was restored to base line values within 2 days of stopping the infusion. These results suggest that d(CH2)5-D-Tyr(Et)VAVP has a high affinity for V2 receptors in both Sprague-Dawley and Brattleboro rats. Its rate of dissociation from the receptor appears to be much slower than that of AVP. In Brattleboro rats, the binding of d(CH2)5-D-Tyr(Et)VAVP leads to an antidiuretic response. In Sprague-Dawley rats, a transient diuretic response is followed by a progressive normalization in water intake. This occurs despite persistent and complete blockade of renal medullary V2 receptors

  2. Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

    Science.gov (United States)

    Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

    2016-04-01

    Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1-3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

  3. [3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

    International Nuclear Information System (INIS)

    Marquis, O.; Robaut, C.; Cavero, I.

    1988-01-01

    In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

  4. Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability

    Science.gov (United States)

    The benztropine analog JHW 007 displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks effects of cocaine,including its self-administration. To determine sites responsible for the cocaine-antagonist effects of JHW 007, ...

  5. DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

    DEFF Research Database (Denmark)

    Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

    1995-01-01

    antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

  6. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The expe...

  7. Synthesis of 11C-SCH 23390, a dopamine D-1 receptor antagonist, for use in in vivo receptor binding studies with PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Stone-Elander, Sharon; Farde, Lars; Ehrin, Erling; Fasth, Karl-Johan; Langstroem, Bengt; Sedvall, Goeran; Karolinska Hospital, Stockholm; Uppsala Univ.

    1986-01-01

    Central dopamine receptors are generally accepted to exist in at least two distinct subtypes: D-1 and D-2. Recently a benzazepine, SCH 23390, was reported to be a selective D-1 dopaminergic antagonist. PET studies of the radio-brominated 76 Br-SCH 23390 reported by Friedman, et al. indicated that the analog exhibits specific binding in the striatum of the monkey brain. Here we report the synthesis of 11 C-SCH 23390 suitable for the in vivo study of dopamine D-1 receptors in the human brain. (author)

  8. Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

    Science.gov (United States)

    Cioffi, Christopher L; Dobri, Nicoleta; Freeman, Emily E; Conlon, Michael P; Chen, Ping; Stafford, Douglas G; Schwarz, Daniel M C; Golden, Kathy C; Zhu, Lei; Kitchen, Douglas B; Barnes, Keith D; Racz, Boglarka; Qin, Qiong; Michelotti, Enrique; Cywin, Charles L; Martin, William H; Pearson, Paul G; Johnson, Graham; Petrukhin, Konstantin

    2014-09-25

    Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.

  9. Molecular determinants of non-competitive antagonist binding to the mouse GPRC6A receptor

    DEFF Research Database (Denmark)

    Faure, Helene; Gorojankina, Tatiana; Rice, Nadejda

    2009-01-01

    GPRC6A displays high sequence homology to the Ca2+-sensing receptor (CaSR). Here we report that the calcimimetic Calindol and the calcilytic NPS2143 antagonize increases in inositol phosphate elicited by L-ornithine-induced activation of mouse GPRC6A after transient coexpression with Galpha(qG66D...... demonstrated to interact with calcilytics or calcimimetics. The mutations F666A(3.32), F670A(3.36), W797A(6.48) caused a loss of L-ornithine ability to activate GPRC6A mutants. The F800A(6.51) mutant was not implicated in either Calindol or NPS 2143 recognition. The E816Q(7.39) mutation led to a loss...

  10. Fighting obesity with a sugar-based library: discovery of novel MCH-1R antagonists by a new computational-VAST approach for exploration of GPCR binding sites.

    Science.gov (United States)

    Heifetz, Alexander; Barker, Oliver; Verquin, Geraldine; Wimmer, Norbert; Meutermans, Wim; Pal, Sandeep; Law, Richard J; Whittaker, Mark

    2013-05-24

    Obesity is an increasingly common disease. While antagonism of the melanin-concentrating hormone-1 receptor (MCH-1R) has been widely reported as a promising therapeutic avenue for obesity treatment, no MCH-1R antagonists have reached the market. Discovery and optimization of new chemical matter targeting MCH-1R is hindered by reduced HTS success rates and a lack of structural information about the MCH-1R binding site. X-ray crystallography and NMR, the major experimental sources of structural information, are very slow processes for membrane proteins and are not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of these methods to impact the drug discovery process for GPCR targets in "real-time", and hence, there is an urgent need for other practical and cost-efficient alternatives. We present here a conceptually pioneering approach that integrates GPCR modeling with design, synthesis, and screening of a diverse library of sugar-based compounds from the VAST technology (versatile assembly on stable templates) to provide structural insights on the MCH-1R binding site. This approach creates a cost-efficient new avenue for structure-based drug discovery (SBDD) against GPCR targets. In our work, a primary VAST hit was used to construct a high-quality MCH-1R model. Following model validation, a structure-based virtual screen yielded a 14% hit rate and 10 novel chemotypes of potent MCH-1R antagonists, including EOAI3367472 (IC50 = 131 nM) and EOAI3367474 (IC50 = 213 nM).

  11. Quantitative light microscopic autoradiographic localization of binding sites labelled with [3H]vasopressin antagonist d(CH2)5Tyr(Me)VP in the rat brain, pituitary and kidney

    International Nuclear Information System (INIS)

    Leeuwen, F.W. van; Beek, E.M. van der; Heerikhuize, J.J. van; Wolters, P.; Meulen, G. van der; Wan, Yieh-Ping

    1987-01-01

    Binding sites for the vasopressin (VP) antagonist d(CH 2 ) 5 Tyr(Me)VP, were located in various brain areas (e.g. the lateral septum, amygdala, choroid plexus and nucleus of the solitary tract) using light microscopic autoradiography. A number of areas (e.g. suprachiasmatic and arcuate nucleus, pineal gland) which previously showed no VP binding were labelled in the present study. The olfactory nucleus and ventromedial hypothalamic nucleus were not labelled. It therefore appears that d(CH 2 ) 5 Tyr(Me)VP is capable of discriminating between VP and oxytocin binding sites and more sensitive means of detecting VP binding sites than VP alone. (Author)

  12. Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    N. Kumar

    2005-10-01

    Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

  13. The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

    Science.gov (United States)

    Zhang, Adrianna P P; Bornholdt, Zachary A; Liu, Tong; Abelson, Dafna M; Lee, David E; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

    2012-02-01

    Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

  14. The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

    Directory of Open Access Journals (Sweden)

    Adrianna P P Zhang

    2012-02-01

    Full Text Available Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan and nonpathogenic to humans (Reston. These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

  15. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    International Nuclear Information System (INIS)

    Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

    1987-01-01

    N/sup α/-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-[ 125 I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125 I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme

  16. Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

    Science.gov (United States)

    Müller, C E; Maurinsh, J; Sauer, R

    2000-01-01

    The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

  17. Characteristics of the binding of (/sup 3/H)nitrendipine to rabbit ventricular membranes: modification by other Ca++ channel antagonists and by the Ca++ channel agonist Bay K 8644

    Energy Technology Data Exchange (ETDEWEB)

    Janis, R.A.; Sarmiento, J.G.; Maurer, S.C.; Bolger, G.T.; Triggle, D.J.

    1984-10-01

    This study was carried out to characterize (/sup 3/H)nitrendipine binding to cardiac membranes and to test the hypothesis that high affinity binding of Ca++ channel antagonists and agonists is to Ca++ channels. Binding was specific, rapid, reversible and stereoselective. The relative order of potency of nifedipine analogs for inhibition of binding was the same as that for inhibition of smooth and cardiac muscle contraction. Results with diltiazem, verapamil and lidoflazine were consistent with the hypothesis that nondihydropyridine Ca++ channel antagonists act at one or more sites allosterically linked to the 1,4-dihydropyridine site in cardiac cells. The Ca++ channel agonist Bay K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate) displaced specifically bound (/sup 3/H)nitrendipine in an apparently competitive manner with an IC50 value of 5 nM. The results suggest that organic antagonists do not act by physically blocking the Ca++ channel. The data also support the hypothesis that the high affinity binding sites for (/sup 3/H)nitrendipine in isolated cardiac membranes are associated with Ca++ channels that are inactivated or are otherwise unavailable for opening.

  18. Transport Gap and exciton binding energy determination in organic semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Stefan; Schoell, Achim; Reinert, Friedrich; Umbach, Eberhard [University of Wuerzburg (Germany). Experimental Physics II; Casu, Benedetta [Inst. f. Physik. u. Theor. Chemie, Tuebingen (Germany)

    2008-07-01

    The transport gap of an organic semiconductor is defined as the energy difference between the HOMO and LUMO levels in the presence of a hole or electron, respectively, after relaxation has occurred. Its knowledge is mandatory for the optimisation of electronic devices based on these materials. UV photoelectron spectroscopy (UPS) and inverse photoelectron spectroscopy (IPES) are routinely applied to measure these molecular levels. However, the precise determination of the transport gap on the basis of the respective data is not an easy task. It involves fundamental questions about the properties of organic molecules and their condensates, about their reaction on the experimental probe, and on the evaluation of the spectroscopic data. In particular electronic relaxation processes, which occur on the time scale of the photo excitation, have to be considered adequately. We determined the transport gap for the organic semiconductors PTCDA, Alq3, DIP, CuPc, and PBI-H4. After careful data analysis and comparison to the respective values for the optical gap we obtain values for the exciton binding energies between 0.1-0.5 eV. This is considerably smaller than commonly believed and indicates a significant delocalisation of the excitonic charge over various molecular units.

  19. Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

    Science.gov (United States)

    Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

    2010-04-22

    Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

  20. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  1. Novel and validated titrimetric method for determination of selected angiotensin-II-receptor antagonists in pharmaceutical preparations and its comparison with UV spectrophotometric determination

    Directory of Open Access Journals (Sweden)

    Shrikant H. Patil

    2012-12-01

    Full Text Available A novel and simple titrimetric method for determination of commonly used angiotensin-II-receptor antagonists (ARA-IIs is developed and validated. The direct acid base titration of four ARA-IIs, namely eprosartan mesylate, irbesartan, telmisartan and valsartan, was carried out in the mixture of ethanol:water (1:1 as solvent using standardized sodium hydroxide aqueous solution as titrant, either visually using phenolphthalein as an indicator or potentiometrically using combined pH electrode. The method was found to be accurate and precise, having relative standard deviation of less than 2% for all ARA-IIs studied. Also, it was shown that the method could be successfully applied to the assay of commercial pharmaceuticals containing the above-mentioned ARA-IIs. The validity of the method was tested by the recovery studies of standard addition to pharmaceuticals and the results were found to be satisfactory. Results obtained by this method were found to be in good agreement with those obtained by UV spectrophotometric method. For UV spectrophotometric analysis ethanol was used as a solvent and wavelength of 233 nm, 246 nm, 296 nm, and 250 nm was selected for determination of eprosartan mesylate, irbesartan, telmisartan, and valsartan respectively. The proposed titrimetric method is simple, rapid, convenient and sufficiently precise for quality control purposes. Keywords: Angiotensin-II-receptor antagonists, Titrimetric assay, UV spectrophotometry, Validation

  2. Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination.

    Directory of Open Access Journals (Sweden)

    Yuna Kim

    2006-06-01

    Full Text Available The genes encoding members of the wingless-related MMTV integration site (WNT and fibroblast growth factor (FGF families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9 and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch--Sry in the case of mammals--is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways.

  3. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  4. Determination of binding affinities of some approved drugs to ...

    African Journals Online (AJOL)

    Ascaris MRFR was obtained as pdb file (3vra) from the Protein Data Bank and further prepared for docking simulations using both Chimera v. 1.8.1 and AutoDock tools v. 1.5.6. In order to validate the docking protocol, the binding of atpenin, an experimental anthelmintic compound, to MRFR was successfully reproduced in ...

  5. Structural Determinants of Specific Lipid Binding to Potassium Channels

    NARCIS (Netherlands)

    Weingarth, M.H.|info:eu-repo/dai/nl/330985655; Prokofyev, A.; van der Cruijsen, E.A.W.|info:eu-repo/dai/nl/330826743; Nand, D.|info:eu-repo/dai/nl/337731403; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Pongs, O.; Baldus, M.|info:eu-repo/dai/nl/314410864

    2013-01-01

    We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsAKv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while

  6. Determination of some histamine H1-receptor antagonists in dosage forms.

    Science.gov (United States)

    Gazy, Azza A; Mahgoub, Hoda; El-Yazbi, F A; El-Sayed, M A; Youssef, Rasha M

    2002-10-15

    Three simple and accurate methods are presented for determination of Cetirizine, Fexofenadine, Loratadine and Acrivastine in pure form and commercial dosage forms. The first method is based on the reaction of the above cited drugs with bromocresol purple dye to form ion-pair complex extractable with chloroform and subsequently measured spectrophotometrically. Secondly, eosin gives with these drugs ion-pair complex, measurable directly without extraction both spectrophotometrically and spectrofluorimetrically. The last method involves the base-catalysed condensation of mixed anhydrides of organic acids (citric acid/acetic anhydride) where as the tertiary amino group in the above-cited drugs acts as the basic catalyst. The product of condensation is measured spectrophotometrically. All the reaction conditions for the proposed methods have been studied. Copyright 2002 Elsevier Science B.V.

  7. Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

    Science.gov (United States)

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

    2012-03-14

    An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

  8. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  9. Two selective HPTLC methods for determination of some angiotensin II receptor antagonists in tablets and biological fluids.

    Science.gov (United States)

    Salah, Gamal A; Abd El-Wadood, Hanaa M; Khairy, Mohamed; Khorshed, Ahmed A

    2017-07-01

    Two simple, selective, precise and highly sensitive high-performance thin-layer chromatography (HPTLC) methods have been developed and validated for analysis of five angiotensin II receptor antagonists, namely losartan, irbesartan valsartan, candesartan and olmesartan, which are widely used in clinical practice. HPTLC of the drugs was performed on pre-coated silica gel HPTLC plates 60 F 254 by development using a mobile phase composed of chloroform-acetone-glacial acetic acid (7.8:1.5:0.7m v/v/v), which was suitable for all of the studied drugs. The first method depended on utilizing reflectance/fluorescence mode for detection while the second method depended on using 2,3,5,6-tetrachloro-1,4-benzoquinone as spraying reagent for the first time to form orange spots scanned at 460 nm. A good linear relationship was obtained over the concentration ranges of 1.2-60 and 360-3000 ng/band while detection and quantification limits were in the ranges of 0.07-0.43, 45.2-140.49 and 0.21-1.29, 137.05-425.74 ng/band for reflectance/fluorescence and reflectance/absorbance methods respectively. The developed methods were applied successfully for their determination in tablets and spiked human plasma for reflectance/fluorescence method with good accuracy and precision, and so can be applied in the pharmacokinetic and bioavailability studies. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Experimental and theoretical determination of optical binding forces

    Czech Academy of Sciences Publication Activity Database

    Brzobohatý, Oto; Čižmár, T.; Karásek, Vítězslav; Šiler, Martin; Dholakia, K.; Zemánek, Pavel

    2010-01-01

    Roč. 18, č. 24 (2010), s. 25389-25402 ISSN 1094-4087 R&D Projects: GA ČR GA202/09/0348; GA MŠk OC08034; GA MŠk(CZ) LC06007; GA MŠk ED0017/01/01 Institutional research plan: CEZ:AV0Z20650511 Keywords : optical binding forces * micro-particles * inter-particle effects * optical manipulation * optical tweezers Subject RIV: BH - Optics, Masers, Lasers Impact factor: 3.749, year: 2010

  11. Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

    DEFF Research Database (Denmark)

    Meier, Sebastian; Beeren, Sophie

    2014-01-01

    We describe a simple method for the simultaneous determination of association constants for a guest binding to seven different hosts in a mixture of more than 20 different oligosaccharides. If the binding parameters are known for one component in the mixture, a single NMR titration suffices...

  12. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  13. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  14. Structural Determinants for the Binding of Morphinan Agonists to the μ-Opioid Receptor.

    Directory of Open Access Journals (Sweden)

    Xiaojing Cong

    Full Text Available Atomistic descriptions of the μ-opioid receptor (μOR noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP and hydromorphone (HMP, are investigated using molecular dynamics (MD simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

  15. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

    Flock, Tilman; Hauser, Alexander S; Lund, Nadia

    2017-01-01

    of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation...

  16. Binding determinants in the interplay between porcine aminopeptidase N and enterotoxigenic Escherichia coli F4 fimbriae.

    Science.gov (United States)

    Xia, Pengpeng; Quan, Guomei; Yang, Yi; Zhao, Jing; Wang, Yiting; Zhou, Mingxu; Hardwidge, Philip R; Zhu, Jianzhong; Liu, Siguo; Zhu, Guoqiang

    2018-02-26

    The binding of F4 + enterotoxigenic Escherichia coli (ETEC) and the specific receptor on porcine intestinal epithelial cells is the initial step in F4 + ETEC infection. Porcine aminopeptidase N (APN) is a newly discovered receptor for F4 fimbriae that binds directly to FaeG adhesin, which is the major subunit of the F4 fimbriae variants F4ab, F4ac, and F4ad. We used overlapping peptide assays to map the APN-FaeG binding sites, which has facilitated in the identifying the APN-binding amino acids that are located in the same region of FaeG variants, thereby limiting the major binding regions of APN to 13 peptides. To determine the core sequence motif, a panel of FaeG peptides with point mutations and FaeG mutants were constructed. Pull-down and binding reactivity assays using piglet intestines determined that the amino acids G159 of F4ab, N209 and L212 of F4ac, and A200 of F4ad were the critical residues for APN binding of FaeG. We further show using ELISA and confocal microscopy assay that amino acids 553-568, and 652-670 of the APN comprise the linear epitope for FaeG binding in all three F4 fimbriae variants.

  17. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  18. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  19. Determination of binding-dioxygen in dioxygen complexes by headspace gas chromatography.

    Science.gov (United States)

    Wang, Wei; Feng, Shun; Li, Ya-ni; Wu, Meiying; Wang, Jide

    2008-06-06

    Dioxygen complexes play important roles in organisms' bodies, so the determination of binding-dioxygen has practical significance. A simple and robust method based on headspace gas chromatography was proposed to determine the binding-dioxygen in dioxygen complexes. By measuring the content change of nitrogen gas in a vial, the amount of oxygen released from dixoygen complexes can be determined. The method was validated using potassium chlorate as model sample, and the results exhibited good recoveries (90-99%) with the relative standard deviation less than 8%. It was also used to analyze dioxygen complex of cobalt bis(salicylaldehyde) ethylenediimine and polyamine cobalt complexes prepared by solid-phase reaction.

  20. Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications.

    Directory of Open Access Journals (Sweden)

    Jose L Ortega Roldan

    Full Text Available SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.

  1. Benzodiazepine receptor antagonists for hepatic encephalopathy

    DEFF Research Database (Denmark)

    Als-Nielsen, B; Gluud, L L; Gluud, C

    2004-01-01

    Hepatic encephalopathy may be associated with accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition. Benzodiazepine receptor antagonists may have a beneficial effect on patients with hepatic encephalopathy.......Hepatic encephalopathy may be associated with accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition. Benzodiazepine receptor antagonists may have a beneficial effect on patients with hepatic encephalopathy....

  2. Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

    Science.gov (United States)

    Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

    2007-07-01

    Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

  3. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  4. GABAB antagonists

    DEFF Research Database (Denmark)

    Frydenvang, Karla Andrea; Hansen, J J; Krogsgaard-Larsen, P

    1994-01-01

    chromatographic techniques. The absolute stereochemistry of (-)-(R)-phaclofen was established by X-ray crystallographic analysis. (-)-(R)-Phaclofen was shown to inhibit the binding of [3H]-(R)-baclofen to GABAB receptor sites on rat cerebellar membranes (IC50 = 76 +/- 13 microM), whereas (+)-(S...

  5. In vivo binding, pharmacokinetics and metabolism of the selective M2 muscarinic antagonists [3H]AF-DX 116 and [3H]AF-DX 384 in the anesthetized rat

    International Nuclear Information System (INIS)

    Mickala, Patrick; Boutin, Herve; Bellanger, Cecile; Chevalier, Cyril; MacKenzie, Eric T.; Dauphin, Francois

    1996-01-01

    The pharmacokinetics, in vivo binding and metabolism of two M 2 muscarinic receptor antagonists, [ 3 H]AF-DX 116 and [ 3 H]AF-DX 384, were studied in anesthetized rats, which received either the tracer alone or following a saturating injection of atropine. Both radioligands were cleared from the circulation with distribution half-lives of 17 and 14 sec and elimination half-lives of 17 and 40 min for [ 3 H]AF-DX 116 and [ 3 H]AF-DX 384, respectively. A radioactive distribution, predominant in peripheral organs when compared to brain, was found at each time studied after tracer injection. Atropine-displaceable tracer uptake was evidenced at 20-40 min in brain (31%), submandibular glands (26%), spleen (37%) and notably heart (55%) for [ 3 H]AF-DX 116 but only in heart (50%) for [ 3 H]AF-DX 384 at 10-20 min. Regional brain sampling revealed a relatively uniform distribution of [ 3 H]AF-DX 384 and a -45% atropine saturation effect (i.e., specific binding) in the thalamus 20 min after injection. Sequential thin-layer chromatographic studies performed on tissue extracts demonstrated the rapid appearance of labeled metabolites of both radiotracers in brain (but less so in liver) and especially in cardiac tissues, where almost 70% of total radioactivity still corresponded to authentic tracer 40 min after injection. Thus, based on their low blood-brain barrier permeability and the high presence of labeled metabolites in the central nervous system, AF-DX 116 and AF-DX 384 might be more helpful in the study of M 2 muscarinic receptors present in heart rather than brain. Labeled with positron emittors, these M 2 antagonists might be applicable to the pathophysiological study of disease states, such as cardiomyopathies

  6. Characterization of glucagon-like peptide-1 receptor-binding determinants.

    Science.gov (United States)

    Xiao, Q; Jeng, W; Wheeler, M B

    2000-12-01

    Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

  7. A new graphic plot analysis for determination of neuroreceptor binding in positron emission tomography studies.

    Science.gov (United States)

    Ito, Hiroshi; Yokoi, Takashi; Ikoma, Yoko; Shidahara, Miho; Seki, Chie; Naganawa, Mika; Takahashi, Hidehiko; Takano, Harumasa; Kimura, Yuichi; Ichise, Masanori; Suhara, Tetsuya

    2010-01-01

    In positron emission tomography (PET) studies with radioligands for neuroreceptors, tracer kinetics have been described by the standard two-tissue compartment model that includes the compartments of nondisplaceable binding and specific binding to receptors. In the present study, we have developed a new graphic plot analysis to determine the total distribution volume (V(T)) and nondisplaceable distribution volume (V(ND)) independently, and therefore the binding potential (BP(ND)). In this plot, Y(t) is the ratio of brain tissue activity to time-integrated arterial input function, and X(t) is the ratio of time-integrated brain tissue activity to time-integrated arterial input function. The x-intercept of linear regression of the plots for early phase represents V(ND), and the x-intercept of linear regression of the plots for delayed phase after the equilibrium time represents V(T). BP(ND) can be calculated by BP(ND)=V(T)/V(ND)-1. Dynamic PET scanning with measurement of arterial input function was performed on six healthy men after intravenous rapid bolus injection of [(11)C]FLB457. The plot yielded a curve in regions with specific binding while it yielded a straight line through all plot data in regions with no specific binding. V(ND), V(T), and BP(ND) values calculated by the present method were in good agreement with those by conventional non-linear least-squares fitting procedure. This method can be used to distinguish graphically whether the radioligand binding includes specific binding or not.

  8. Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

    Science.gov (United States)

    Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

    2013-02-15

    PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

  9. ACTH antagonists

    Directory of Open Access Journals (Sweden)

    Adrian John Clark

    2016-08-01

    Full Text Available ACTH acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1 Cushing’s disease and ectopic ACTH syndrome – especially whilst preparing for definitive treatment of a causative tumour, or in refractory cases, or (2 congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role.

  10. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    International Nuclear Information System (INIS)

    Öberg, Christine; Belikov, Sergey

    2012-01-01

    Highlights: ► wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, ΔN-hH1.4, were compared. ► Both histones bind to chromatin, however, ΔN-hH1.4 displays lower binding affinity. ► Interaction of ΔN-hH1.4 with chromatin includes a significant unspecific component. ► N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). The ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that ΔN-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  11. Determination of ABA-binding proteins contents in subcellular fractions isolated from cotton seedlings using radioimmunoanalysis

    International Nuclear Information System (INIS)

    Tursunkhodjayeva, F.M.

    2004-01-01

    Full text: Knowledge of plants' hormone receptor sites is essential to understanding of the principles of phytohormone action in cells and tissues. The hormone abscisic acid (ABA) takes part in many important physiological processes of plants, including water balance and resistance to salt stress. The detection of salt tolerance in the early stages of ontogenesis is desirable for effective cultivation of cotton. Usually such characteristics are determined visually after genetic analysis of hybrids over several generations. This classic method of genetics requires a long time to grow several generations of cotton plants. In this connection we study ABA-binding protein contents in subcellular fractions isolated from seedlings of several kinds of cotton with different tolerance to salt stress. The contents of ABA-binding protein in nuclei and chloroplasts fractions isolated from cotton seedlings were determined using radioimmunoanalysis. The subcellular fractions were prepared by ultracentrifugation in 0,25 - 2,2 M sucrose gradient. ABA-binding protein was isolated from cotton seedlings by affinity chromatography. The antibodies against ABA-binding protein of cotton were developed in rabbits according standard protocols. Than the antibodies were labelled by radioisotope J 125 according Greenwood et al. It was shown, that the nuclei and chloroplasts fractions isolated from cotton with high tolerance to salt stress contain ABA-binding protein up to 1,5-1,8 times more, than the same fractions from cotton with low tolerance to salt stress. So, the ABA-binding protein contents in cotton seedlings may be considered as a marker for screening of cotton kinds, which may potentially have high tolerance to salt stress

  12. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Sanders

    Full Text Available The epidermal growth factor receptor (EGFR is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of

  13. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  14. Simultaneous determination of moxifloxacin and H2 receptor antagonist in pharmaceutical dosage formulations by RP-HPLC: application to in vitro drug interactions

    Directory of Open Access Journals (Sweden)

    Najma Sultana

    2011-01-01

    Full Text Available Simultaneous determination of moxifloxacin (MOX and H2-antagonists was first time developed in bulk and formulations. Purospher STAR C18 (250 x 4.6 mm, 5 μm column was used. The mobile phase (methanol: water: ACN, 60:45:5 v/v/v, pH 2.7 was delivered at a flow rate of 1.0 mL min-1, eluent was monitored at 236, 270 and 310 nm for cimetidine, famotidine and ranitidine, respectively. The proposed method is specific, accurate (98-103%, precise (intra-day and inter-day variation 0.098-1.970% and linear (r>0.998. The LOD and LOQ were 0.006-0.018 and 0.019-0.005 μg mL-1, respectively. The statistical parameters were applied to verify the results. The method is applicable to routine analysis of formulations and interaction of MOX with H2-antagonist.

  15. Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry.

    Science.gov (United States)

    Hansen, Lee D; Transtrum, Mark K; Quinn, Colette; Demarse, Neil

    2016-05-01

    Isothermal calorimetry allows monitoring of reaction rates via direct measurement of the rate of heat produced by the reaction. Calorimetry is one of very few techniques that can be used to measure rates without taking a derivative of the primary data. Because heat is a universal indicator of chemical reactions, calorimetry can be used to measure kinetics in opaque solutions, suspensions, and multiple phase systems and does not require chemical labeling. The only significant limitation of calorimetry for kinetic measurements is that the time constant of the reaction must be greater than the time constant of the calorimeter which can range from a few seconds to a few minutes. Calorimetry has the unique ability to provide both kinetic and thermodynamic data. This article describes the calorimetric methodology for determining reaction kinetics and reviews examples from recent literature that demonstrate applications of titration calorimetry to determine kinetics of enzyme-catalyzed and ligand binding reactions. A complete model for the temperature dependence of enzyme activity is presented. A previous method commonly used for blank corrections in determinations of equilibrium constants and enthalpy changes for binding reactions is shown to be subject to significant systematic error. Methods for determination of the kinetics of enzyme-catalyzed reactions and for simultaneous determination of thermodynamics and kinetics of ligand binding reactions are reviewed. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

    Directory of Open Access Journals (Sweden)

    Nathan Steere

    Full Text Available The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages, which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby, a highly conserved antagonist of the Wnt/β-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx, which thereby abrogated the inhibitory effect of Cby on β-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

  17. A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

    Science.gov (United States)

    Steere, Nathan; Chae, Vanessa; Burke, Michael; Li, Feng-Qian; Takemaru, Ken-ichi; Kuriyama, Ryoko

    2012-01-01

    The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages), which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby), a highly conserved antagonist of the Wnt/β-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx), which thereby abrogated the inhibitory effect of Cby on β-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

  18. Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Caitlin L Rowe

    Full Text Available Rabies virus P-protein is expressed as five isoforms (P1-P5 which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP-recognised nuclear localization sequence in the N-terminal region (N-NLS, the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES. However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2, and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P

  19. Calcium antagonist binding sites in the rat brain: Quantitative autoradiographic mapping using the 1, 4-Dihydropyridines [3H]PN 200-110 and [3H]PY 108-068

    International Nuclear Information System (INIS)

    Cortes, R.; Supavilai, P.; Karobath, M.; Palacios, J.M.

    1984-01-01

    An in vitro autoradiographic technique has been used for the quantitative mapping of calcium antagonist binding sites (CABS) in the rat brain, using the 1, 4-dihydropyridines [ 3 H]PN 200-110 and [ 3 H]PY 108-068 as ligands. CABS were distributed throughout the brain in a highly heterogeneous fashion. The highest densities of CABS were observed in the olfactory bulb, hippocampus and parts of the amygdala. The neocortex was also rich in CABS. The basal ganglia, thalamus and hypothalamus presented intermediate levels of CABS while low densities of sites were seen in areas such as the cerebellum, pons and white matter tracts. The distributions of CABS in brain does not correlate with indexes of brain blood flow, regional glucose utilization or the distributions of receptor binding sites for drugs and neurotransmitters analyzed until now. No correlation exists between CABS distribution and that of any neurotransmitter or brain enzyme described so far. The heterogeneous distributions of CABS is suggestive of a neuronal localization, an idea supported by lesion experiments. (Author)

  20. Calcium antagonist binding sites in the rat brain: Quantitative autoradiographic mapping using the 1, 4-dihydropyridines (TH)PN 200-110 and (TH)PY 108-068

    Energy Technology Data Exchange (ETDEWEB)

    Cortes, R.; Supavilai, P.; Karobath, M.; Palacios, J.M.

    1984-01-01

    An in vitro autoradiographic technique has been used for the quantitative mapping of calcium antagonist binding sites (CABS) in the rat brain, using the 1, 4-dihydropyridines (TH)PN 200-110 and (TH)PY 108-068 as ligands. CABS were distributed throughout the brain in a highly heterogeneous fashion. The highest densities of CABS were observed in the olfactory bulb, hippocampus and parts of the amygdala. The neocortex was also rich in CABS. The basal ganglia, thalamus and hypothalamus presented intermediate levels of CABS while low densities of sites were seen in areas such as the cerebellum, pons and white matter tracts. The distributions of CABS in brain does not correlate with indexes of brain blood flow, regional glucose utilization or the distributions of receptor binding sites for drugs and neurotransmitters analyzed until now. No correlation exists between CABS distribution and that of any neurotransmitter or brain enzyme described so far. The heterogeneous distributions of CABS is suggestive of a neuronal localization, an idea supported by lesion experiments. (Author).

  1. The use of isothermal titration calorimetry to determine the thermodynamics of metal ion binding to low-cost sorbents

    International Nuclear Information System (INIS)

    Karlsen, Vigdis; Heggset, Ellinor Baevre; Sorlie, Morten

    2010-01-01

    The thermodynamics of Al 3+ , Cr 3+ , and Pb 2+ binding to the abundant biopolymer chitin have been determined using isothermal titration calorimetry (ITC) and compared to what is observed for binding to activated carbon. The use of ITC enables the detection of two distinct binding sites on chitin for all three metal ions. For the relative strong binding sites, free energy changes ranges from -37.6 kJ/mol to -41.8 kJ/mol while the same values are from -30.1 kJ/mol to -31.8 kJ/mol for the relative weak binding sites. All binding reactions to chitin are entropically driven. Interactions of the metal ions to activated carbon are best fitted as a single-site binding with relative weak binding with free energy changes from -26.3 kJ/mol to -26.8 kJ/mol.

  2. Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration.

    Science.gov (United States)

    Kahle, Claudia; Holzgrabe, Ulrike

    2004-10-01

    Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

  3. Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

    Directory of Open Access Journals (Sweden)

    Tatyana Mamonova

    Full Text Available Na+/H+ Exchanger Regulatory Factor-1 (NHERF1 is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A, uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD simulations in combination with biological mutagenesis, fluorescent polarization (FP binding assays, and isothermal titration calorimetry (ITC, we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.

  4. Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

    Science.gov (United States)

    Qian, Cheng; Kovalchik, Kevin A; MacLennan, Matthew S; Huang, Xiaohua; Chen, David D Y

    2017-06-01

    Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype.

    Science.gov (United States)

    Roy, Kunal; Leonard, J Thomas; Sengupta, Chandana

    2004-07-16

    Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.

  6. Separate and simultaneous binding effects of aspirin and amlodipine to human serum albumin based on fluorescence spectroscopic and molecular modeling characterizations: A mechanistic insight for determining usage drugs doses

    International Nuclear Information System (INIS)

    Abdollahpour, Nooshin; Asoodeh, Ahmad; Saberi, Mohammad Reza; Chamani, JamshidKhan

    2011-01-01

    The binding of aspirin (ASA) and amlodipine (AML) to human serum albumin (HSA) in aqueous solution was investigated by multiple techniques such as fluorescence quenching, resonance light scattering (RLS), three-dimensional fluorescence spectroscopy, FT-IR and zeta-potential measurements in an aqueous solution at pH=7.4. For the protein-ligand association reaction, fluorescence measurements can give important clues as to the binding of ligands to proteins, e.g., the binding mechanism, binding mode, binding constants, binding sites, etc. Fluorescence spectroscopy showed that ASA and AML could quench the HSA fluorescence spectra, and this quenching effect became more significant when both ASA and AML coexisted. The results pointed at the interaction between HSA and both drugs as ternary systems decreasing the binding constant and binding stability of the HSA-drug complex as a binary system. Therefore, by reducing the amount of drugs transported to their targets, the free drug concentration of the target would be reduced, lowering the efficacy of the drugs. It was demonstrated that there exists antagonistic behavior between the two drugs when it comes to binding of HSA. Furthermore, the fluorescence results also showed that the quenching mechanism of HSA-drug complexes as binary and ternary systems is a static procedure. The number of binding sites of HSA-ASA, (HSA-AML)ASA, HSA-AML and (HSA-ASA) AML were 1.31, 0.92, 1 and 0.93, respectively. Due to the existence of the antagonistic action between ASA and AML, the binding distance r was reduced. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that the antagonistic action between ASA and AML would alter the micro-environment around Trp and Tyr residues. Moreover, the simultaneous presence of ASA and AML during binding to HSA should be taken into account in multidrug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic

  7. Separate and simultaneous binding effects of aspirin and amlodipine to human serum albumin based on fluorescence spectroscopic and molecular modeling characterizations: A mechanistic insight for determining usage drugs doses

    Energy Technology Data Exchange (ETDEWEB)

    Abdollahpour, Nooshin [Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad (Iran, Islamic Republic of); Asoodeh, Ahmad [Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Saberi, Mohammad Reza [Department of Medical Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Chamani, JamshidKhan, E-mail: chamani@ibb.ut.ac.ir [Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad (Iran, Islamic Republic of)

    2011-09-15

    The binding of aspirin (ASA) and amlodipine (AML) to human serum albumin (HSA) in aqueous solution was investigated by multiple techniques such as fluorescence quenching, resonance light scattering (RLS), three-dimensional fluorescence spectroscopy, FT-IR and zeta-potential measurements in an aqueous solution at pH=7.4. For the protein-ligand association reaction, fluorescence measurements can give important clues as to the binding of ligands to proteins, e.g., the binding mechanism, binding mode, binding constants, binding sites, etc. Fluorescence spectroscopy showed that ASA and AML could quench the HSA fluorescence spectra, and this quenching effect became more significant when both ASA and AML coexisted. The results pointed at the interaction between HSA and both drugs as ternary systems decreasing the binding constant and binding stability of the HSA-drug complex as a binary system. Therefore, by reducing the amount of drugs transported to their targets, the free drug concentration of the target would be reduced, lowering the efficacy of the drugs. It was demonstrated that there exists antagonistic behavior between the two drugs when it comes to binding of HSA. Furthermore, the fluorescence results also showed that the quenching mechanism of HSA-drug complexes as binary and ternary systems is a static procedure. The number of binding sites of HSA-ASA, (HSA-AML)ASA, HSA-AML and (HSA-ASA) AML were 1.31, 0.92, 1 and 0.93, respectively. Due to the existence of the antagonistic action between ASA and AML, the binding distance r was reduced. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that the antagonistic action between ASA and AML would alter the micro-environment around Trp and Tyr residues. Moreover, the simultaneous presence of ASA and AML during binding to HSA should be taken into account in multidrug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic

  8. Assay method for organic calcium antagonist drugs and a kit for such an assay

    International Nuclear Information System (INIS)

    Snyder, S. H.; Gould, R. J.

    1985-01-01

    A method for measuring the level of organic calcium antagonist drug in a body fluid comprises preparing a mixture of a radioactive calcium antagonist drug, a body fluid containing a calcium antagonist drug and a calcium antagonist receptor material, measuring the radioactivity of the radioactive calcium antagonist drug bound to said calcium antagonist receptor material and deriving the concentration of the calcium antagonist drug in the body fluid from a standard curve indicating the concentration of calcium antagonist drug versus inhibition of binding of said radioactive calcium antagonist drug to said receptor sites caused by the calcium antagonist drug in said body fluid. A kit for measuring the level of an organic calcium drug comprises a receptacle containing a radioactive calcium antagonist drug, a calcium antagonist receptor material and a standard amount of a nonradioactive calcium antagonist drug

  9. Utilization of ICP/OES for the determination of trace metal binding to different humic fractions.

    Science.gov (United States)

    de la Rosa, G; Peralta-Videa, J R; Gardea-Torresdey, J L

    2003-02-28

    In this study, the use of inductively coupled plasma/optical emission spectrometry (ICP/OES) to determine multi-metal binding to three biomasses, Sphagnum peat moss, humin and humic acids is reported. All the investigations were performed under part per billion (ppb) concentrations. Batch pH profile experiments were performed using multi-metal solutions of Cd(II), Cu(II), Pb(II), Ni(II), Cr(III) and Cr(VI). The results showed that at pH 2 and 3, the metal affinity of the three biomasses exposed to the multi-metal solution that included Cr(III) presented the following order: Cu(II), Pb(II)>Ni(II)>Cr(III)>Cd(II). On the other hand, when Cr(VI) was in the heavy metal mixture, Sphagnum peat moss and humin showed the following affinity: Cu(II), Pb(II)>Ni(II)>Cr(VI)>Cd(II); however, the affinity of the humic acids was: Cu(II)>Pb(II), Cr(VI)>Ni(II)>Cd(II). The results demonstrated that pH values of 4 and 5 were the most favorable for the heavy metal binding process. At pH 5, all the metals, except for Cr(VI), were bound between 90 and 100% to the three biomasses. However, the binding capacity of humic acids decreased at pH 6 in the presence of Cr(VI). The results showed that the ICP/OES permits the determination of heavy metal binding to organic matter at ppb concentration. These results will be very useful in understanding the role of humic substances in the fate and transport of heavy metals, and thus could provide information to develop new methodologies for the removal of low concentrations of toxic heavy metals from contaminated waters.

  10. Recovery of stress-impaired social behavior by an antagonist of the CRF binding protein, CRF6-33, in the bed nucleus of the stria terminalis of male rats.

    Science.gov (United States)

    Vasconcelos, Mailton; Stein, Dirson J; Albrechet-Souza, Lucas; Miczek, Klaus A; de Almeida, Rosa Maria M

    2018-01-09

    Social stress is recognized to promote the development of neuropsychiatric and mood disorders. Corticotropin releasing factor (CRF) is an important neuropeptide activated by social stress, and it contributes to neural and behavioral adaptations, as indicated by impaired social interactions and anhedonic effects. Few studies have focused on the role of the CRF binding protein (CRFBP), a component of the CRF system, and its activity in the bed nucleus of stria terminalis (BNST), a limbic structure connecting amygdala and hypothalamus. In this study, animals' preference for sweet solutions was examined as an index of stress-induced anhedonic responses in Wistar rats subjected to four brief intermittent episodes of social defeat. Next, social approach was assessed after local infusions of the CRFBP antagonist, CRF fragment 6-33 (CRF 6-33 ) into the BNST. The experience of brief episodes of social defeat impaired social approach behaviors in male rats. However, intra-BNST CRF 6-33 infusions restored social approach in stressed animals to the levels of non-stressed rats. CRF 6-33 acted selectively on social interaction and did not alter general exploration in nether stressed nor non-stressed rats. These findings suggest that BNST CRFBP is involved in the modulation of anxiety-like responses induced by social stress. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Second Harmonic Correlation Spectroscopy: Theory and Principles for Determining Surface Binding Kinetics.

    Science.gov (United States)

    Sly, Krystal L; Conboy, John C

    2017-06-01

    A novel application of second harmonic correlation spectroscopy (SHCS) for the direct determination of molecular adsorption and desorption kinetics to a surface is discussed in detail. The surface-specific nature of second harmonic generation (SHG) provides an efficient means to determine the kinetic rates of adsorption and desorption of molecular species to an interface without interference from bulk diffusion, which is a significant limitation of fluorescence correlation spectroscopy (FCS). The underlying principles of SHCS for the determination of surface binding kinetics are presented, including the role of optical coherence and optical heterodyne mixing. These properties of SHCS are extremely advantageous and lead to an increase in the signal-to-noise (S/N) of the correlation data, increasing the sensitivity of the technique. The influence of experimental parameters, including the uniformity of the TEM00 laser beam, the overall photon flux, and collection time are also discussed, and are shown to significantly affect the S/N of the correlation data. Second harmonic correlation spectroscopy is a powerful, surface-specific, and label-free alternative to other correlation spectroscopic methods for examining surface binding kinetics.

  12. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  13. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    Science.gov (United States)

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  14. Excitatory amino acid receptor antagonists

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B

    1997-01-01

    We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation......)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments......, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation...

  15. Unusual estrogen-binding liver protein: additional data on the structural determinants of androgenic ligands

    International Nuclear Information System (INIS)

    Smirnov, A.N.; Shchelkunova, T.A.; Rozen, V.B.

    1986-01-01

    The relative competitive activity of a number of androstane derivatives was determined according to the 50% displacement of [ 3 H] estradiol from complexes with an unusual estrogen-binding protein (UEBP) of the liver of male rats. It was shown that: (1) the bulk of the energy of the bond of the steroid to protein is due to hydrophobic interactions; (2) the real ability to form specific complexes with the UEBP at androgen concentrations close to the physiological is determined by the 17β-hydroxyl and is enhanced by the 3α- or 2α-hydroxy group; (3) the 3- and 17-keto groups weaken the interaction of androgens with the UEBP; (4) cis-coupling of the A and B rings in the molecule of androgens does not prevent the binding of the steroids to protein. These data substantially refine the concepts of the mechanisms of the interaction of androgens with the UEBP and may promote an elucidation of the physiological function of this protein

  16. Attenuation of antagonist-induced impairment of dopamine receptors by L-prolyl-L-leucyl-glycinamide

    International Nuclear Information System (INIS)

    Saleh, M.I.M.

    1988-01-01

    The present study was undertaken in order to determine whether chronic,long-term postnatal challenge of rat pups per se, with specific dopamine D1 and D2 receptor antagonists, would modify the ontogeny of the respective receptor types. Since the neuropeptide L-prolyl-L-leucyl-glycinamide (PLG) attenuates the effect of haloperidol on dopamine D2 receptors in adult rats it was of interest to determine whether PLG would modulate antagonists-induced alterations in the ontogeny of striatal dopamine D1 and D2 receptors. Half of the rats were treated daily for 32 days from birth with SCH-23390, a selective dopamine D1 antagonist; or spiroperidol, a selective dopamine D2 antagonists; or both SCH-23390 and spiroperidol; or saline. The other half of the litters were treated with PLG, in combination with the other treatments. Animals were decapitated at 5, 8, and 12 weeks from birth for neurochemical analysis of the striatum. Chronic SCH-23390 treatment produced a 70-80% decrease in the binding of [ 3 H] SCH-23390 to striatal homogenates. The alteration at 5 weeks was associated with a 78% decrease in the Bmax for [ 3 H] SCH-23390 binding, and no change in the K D . Similarly, at 5, 8, and 12 weeks, chronic spiroperidol treatment reduced the binding of [ 3 H] spiroperidol to striatal homogenates by 70-80%

  17. Structural determination of functional units of the nucleotide binding domain (NBD94 of the reticulocyte binding protein Py235 of Plasmodium yoelii.

    Directory of Open Access Journals (Sweden)

    Ardina Grüber

    2010-02-01

    Full Text Available Invasion of the red blood cells (RBC by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94 of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547, NBD94(566-663 and NBD94(674-793, respectively. Using fluorescence correlation spectroscopy NBD94(444-547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663 and NBD94(674-793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663 consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793 in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the

  18. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    Science.gov (United States)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  19. Use of a specific cholecystokinin receptor antagonist (L-364,718) to determine the role of cholecystokinin on feeding and body weight in rats with obstructive jaundice.

    Science.gov (United States)

    Tangoku, A; Doi, R; Chowdhury, P; Pasley, J N; McKay, D W; Rayford, P L

    1992-01-01

    We conducted a study to examine the role of cholecystokinin in feeding behavior and weight change in rats with obstructive jaundice. Daily food and water intake, body weight, and short-term food intake were determined in two groups of rats with surgically induced obstructive jaundice and in control rats. One group of rats with obstructive jaundice was given L-364,718, a selective cholecystokinin receptor antagonist. Plasma bilirubin and cholecystokinin levels were measured in each rat before and 7 days after surgery. Daily food intake and body weight were decreased in obstructive jaundice rats compared with control rats during the first week after surgery (P less than .05); however, obstructive jaundice rats treated with L-364,718 had increased food intake and body weight (P less than .05). Short-term food intake measured for 30 minutes and 120 minutes in food-deprived obstructive jaundice rats was decreased when compared with control rats (P less than .05), but the obstructive jaundice rats given L-364,718 had increased short-term food intake (P less than .05). Water intake was similar between the two groups of rats. Plasma levels of cholecystokinin and bilirubin were increased in obstructive jaundice rats with and without L-364,718 treatment (P less than .05). The results support the concept that endogenously elevated levels of plasma cholecystokinin play an important role in decreased food intake and subsequent loss of body weight in rats with obstructive jaundice.

  20. Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Biswaranjan; Williams, Martin L.; Doak, Bradley C.; Vazirani, Mansha; Ilyichova, Olga [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); Wang, Geqing [La Trobe University, La Trobe Institute for Molecular Bioscience (Australia); Bermel, Wolfgang [Bruker Biospin GmbH (Germany); Simpson, Jamie S.; Chalmers, David K. [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); King, Glenn F. [The University of Queensland, Institute for Molecular Bioscience (Australia); Mobli, Mehdi, E-mail: m.mobli@uq.edu.au [The University of Queensland, Centre for Advanced Imaging (Australia); Scanlon, Martin J., E-mail: martin.scanlon@monash.edu [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia)

    2016-11-15

    We describe a general approach to determine the binding pose of small molecules in weakly bound protein–ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met {sup ε}CH{sub 3} assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-{sup 13}C,{sup 15}N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein–ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

  1. DNA-binding determinants promoting NHEJ by human Polμ.

    Science.gov (United States)

    Martin, Maria Jose; Juarez, Raquel; Blanco, Luis

    2012-12-01

    Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

  2. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yezhong [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhou Bo [College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhang Xiaoping; Huang Ping [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); Li Chaohong [Education Ministry Key Laboratory for Oral Biomedical Engineering, School of Stomatology, Wuhan University, Wuhan 430072 (China); Liu Yi [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China) and College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)], E-mail: prof.liuyi@263.net

    2009-04-30

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K{sub a}) between MG and BSA at four different temperatures were obtained to be 3.734 x 10{sup 4}, 3.264 x 10{sup 4}, 2.718 x 10{sup 4}, and 2.164 x 10{sup 4} L mol{sup -1}, respectively. The enthalpy change ({delta}H) and entropy change ({delta}S) were calculated to be -27.25 kJ mol{sup -1} and -11.23 J mol{sup -1} K{sup -1}, indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the {alpha}-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.

  3. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    International Nuclear Information System (INIS)

    Zhang Yezhong; Zhou Bo; Zhang Xiaoping; Huang Ping; Li Chaohong; Liu Yi

    2009-01-01

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K a ) between MG and BSA at four different temperatures were obtained to be 3.734 x 10 4 , 3.264 x 10 4 , 2.718 x 10 4 , and 2.164 x 10 4 L mol -1 , respectively. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -27.25 kJ mol -1 and -11.23 J mol -1 K -1 , indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the α-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules

  4. A New Method for Determining Structure Ensemble: Application to a RNA Binding Di-Domain Protein.

    Science.gov (United States)

    Liu, Wei; Zhang, Jingfeng; Fan, Jing-Song; Tria, Giancarlo; Grüber, Gerhard; Yang, Daiwen

    2016-05-10

    Structure ensemble determination is the basis of understanding the structure-function relationship of a multidomain protein with weak domain-domain interactions. Paramagnetic relaxation enhancement has been proven a powerful tool in the study of structure ensembles, but there exist a number of challenges such as spin-label flexibility, domain dynamics, and overfitting. Here we propose a new (to our knowledge) method to describe structure ensembles using a minimal number of conformers. In this method, individual domains are considered rigid; the position of each spin-label conformer and the structure of each protein conformer are defined by three and six orthogonal parameters, respectively. First, the spin-label ensemble is determined by optimizing the positions and populations of spin-label conformers against intradomain paramagnetic relaxation enhancements with a genetic algorithm. Subsequently, the protein structure ensemble is optimized using a more efficient genetic algorithm-based approach and an overfitting indicator, both of which were established in this work. The method was validated using a reference ensemble with a set of conformers whose populations and structures are known. This method was also applied to study the structure ensemble of the tandem di-domain of a poly (U) binding protein. The determined ensemble was supported by small-angle x-ray scattering and nuclear magnetic resonance relaxation data. The ensemble obtained suggests an induced fit mechanism for recognition of target RNA by the protein. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Potentiodynamic formation of gold nanoparticles film on glassy carbon electrode using aminophenyl diazonium cations grafted gold nanoparticles: Determination of histamine H2 receptor antagonist

    International Nuclear Information System (INIS)

    Kesavan, Srinivasan; Revin, S. Brillians; John, S. Abraham

    2014-01-01

    Graphical abstract: - Highlights: • Grafting based AuNPs were synthesized in aqueous medium by spontaneous grafting. • GC/ITO electrode was modified with AuNPs film by potentiodynamic method. • AuNPs film modified electrode was characterized by XPS, AFM and CV. • Simultaneous determination of ranitidine and paracetamol was demonstrated. • Practical application was demonstrated in commercial drugs. - Abstract: The aminophenyl (AP) functionalized AuNPs (AP-AuNPs) were synthesized in aqueous medium by spontaneous grafting method and were used for the formation of AuNPs film on glassy carbon (GC) and indium tin oxide (ITO) surfaces by potentiodynamic method. The formed AP-AuNPs film modified electrodes were characterized by cyclic voltammetry (CV), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS), and X-ray photoelectron spectroscopy (XPS). EIS studies show that the electron transfer reaction of [Fe(CN) 6 ] 3-/4− was higher at the AP-AuNPs film modified electrode (1.58 × 10 −4 cm s −1 ) than at bare (3.78 × 10 −5 cm s −1 ) GC electrode. The surface coverage of the AP-AuNPs film modified electrode was found to be 4.4 × 10 −10 mol cm −2 . The film formation takes place via -NH 2 groups of AP-AuNPs, which was confirmed by XPS from the observed peaks corresponding to =N-H (396.7 eV), -N-H (399.2 eV), -N = N- (400.2 eV) and -N + -H (403.3 eV). The AP-AuNPs film modified electrode was successfully utilized for the determination of histamine H 2 receptor antagonist ranitidine (RA). Further, the AP-AuNPs film modified electrode was effectively used for the selective determination of RA in the presence of 40-fold excess paracetamol. The present method was successfully used to determine the concentration of RA in commercial drugs

  6. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  7. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  8. Lignite utilisation for binding of radionuclides and uranium determination by adsorptive stripping chronopotentiometry

    International Nuclear Information System (INIS)

    Zavodska, L.

    2010-01-01

    This work in its first part deals with non-energetic use of young brown coal - lignite. The goal of the work was to perform a complex physical and chemical characterisation of the Slovak lignite (deposit Cary) which could contribute to rts use in binding of radionuclides in the environment. The characteristics of Slovak lignite were compared to leonardite (Gascoyne, USA) and Czech lignite (the area of Mikulcice). In the samples the residual humidity was determined. as well. as the content of ashes. representation of selected inorganic (As, Ba, Cd, Co, Cr, Cu, Ni, Pb and Zn) as well as organo-elements (C, H, N, 0, S) and crystalline forms of inorganic phase in lignite. The specific surface (BET isotherm) of lignite was determined, representation of total and free humic acids and their cation exchange capacity. The samples underwent al.so gamma-spectrometric analysis ( 40 K, 226 Ra, 228 Ac, 137 Cs and 60 Co). Using the radio-indicator method ( 85 Sr and 137 Cs) the kinetics of sorption of Cs + and Sr + ions in Slovak lignite was studied. The efficiency of sorption for both cations on lignite was evaluated on the basis of constructed sorption isotherms. The sorption of Cs + ions on Slovak lignite is more suitable for the Freundlich type of isotherm, in case of Sr + ions the sorption is better described by Langmuir isotherm. The results of sorption experiments on lignite in comparison with clinoptilolite and montmorillonite K-10 showed a more efficient sorption for Sr Ions. despite the more coarse-grained fraction of this matrix. This finding supports the possibility of lignite application for binding of a quite mobile radiostrontium, which may be applied for construction of radioactive waste repositories or for remediation of contaminated soils. The second part of the work deals with the development and validation of new fast and reliable electrochemical method of uranium determination in natural waters. It is based on physical adsorption of created complex of uranium

  9. Clinical value of determination of TSH-binding inhibiting immunoglobulins (TBII) by a radioreceptor assay

    International Nuclear Information System (INIS)

    Heberling, H.J.; Bierwolf, B.; Lohmann, D.

    1986-01-01

    The clinical value of a commercial kit for determination of TBII was evaluated. 50 patients with untreated Graves' disease, 21 patients with Graves' disease before and during medical therapy, 18 patients after finishing medical therapy and 10 patients after surgical treatment were examined. Besides these, 41 patients with other thyroid diseases and 36 patients without any thyroid disorder were included. In 47 (94%) of 50 patients with untreated Graves' disease TBII were detectable in serum using a TSH standard curve. Binding activities exceeding 10 U/l TSH equivalents were regarded as positive. In other thyroid diseases TBII were negative with the exception of 3 of 22 patient with autonomously functioning thyroid nodules. After 12 months of antithyroid drug treatment of 19 patients the incidence of positive antibody findings was 26%. During follow-up after medical therapy (1-9 years) 7 of 18 patients had increased TBII in correlation with clinical and functional findings. The determination of TBII by TRAK assay proved to be a sensitive and specific method. The assay can be used to differentiate between hyperthyroidism of autoimmune or non-immunogenic origin. Thus the method seems to be helpful for the follow-up under medical treatment of patients with Graves' disease. (author)

  10. Determination of free and bound riboflavin in cow's milk using a novel flavin-binding protein.

    Science.gov (United States)

    Koop, Julia; Monschein, Stefanie; Pauline Macheroux, E; Knaus, Tanja; Macheroux, Peter

    2014-03-01

    A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151μg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Application of quantitative structure-activity relationship to the determination of binding constant based on fluorescence quenching

    Energy Technology Data Exchange (ETDEWEB)

    Wen Yingying [Department of Applied Chemistry, Yantai University, Yantai 264005 (China); Liu Huitao, E-mail: liuht-ytu@163.co [Department of Applied Chemistry, Yantai University, Yantai 264005 (China); Luan Feng; Gao Yuan [Department of Applied Chemistry, Yantai University, Yantai 264005 (China)

    2011-01-15

    Quantitative structure-activity relationship (QSAR) model was used to predict and explain binding constant (log K) determined by fluorescence quenching. This method allowed us to predict binding constants of a variety of compounds with human serum albumin (HSA) based on their structures alone. Stepwise multiple linear regression (MLR) and nonlinear radial basis function neural network (RBFNN) were performed to build the models. The statistical parameters provided by the MLR model (R{sup 2}=0.8521, RMS=0.2678) indicated satisfactory stability and predictive ability while the RBFNN predictive ability is somewhat superior (R{sup 2}=0.9245, RMS=0.1736). The proposed models were used to predict the binding constants of two bioactive components in traditional Chinese medicines (isoimperatorin and chrysophanol) whose experimental results were obtained in our laboratory and the predicted results were in good agreement with the experimental results. This QSAR approach can contribute to a better understanding of structural factors of the compounds responsible for drug-protein interactions, and can be useful in predicting the binding constants of other compounds. - Research Highlights: QSAR models for binding constants of some compounds to HSA were developed. The models provide a simple and straightforward way to predict binding constant. QSAR can give some insight into structural features related to binding behavior.

  12. Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations.

    Science.gov (United States)

    Solomentsev, Gleb; Diehl, Carl; Akke, Mikael

    2018-03-06

    FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15 N and 2 H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

  13. Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

    2010-01-01

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

  14. Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

    2010-01-15

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

  15. Determination of copper binding in Pseudomonas putida CZ1 by chemical modifications and X-ray absorption spectroscopy.

    Science.gov (United States)

    Chen, XinCai; Shi, JiYan; Chen, YingXu; Xu, XiangHua; Chen, LiTao; Wang, Hui; Hu, TianDou

    2007-03-01

    Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.

  16. [3H]naloxone as an opioid receptor label: Analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues

    International Nuclear Information System (INIS)

    Schnittler, M.; Repke, H.; Liebmann, C.; Schrader, U.; Schulze, H.P.; Neubert, K.

    1990-01-01

    The nonselective antagonist [ 3 H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opiod ligands as well as various β-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between μ 1 and μ 2 binding sites. The affinities to the μ sites obtained with [ 3 H]naloxone as label are in a good agreement with those from experiments with the μ selective radioligand [ 3 H]DAGO. The μ 1 site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity μ 1 subtype. (author)

  17. Evidence that diclofenac and celecoxib are thyroid hormone receptor beta antagonists.

    Science.gov (United States)

    Zloh, Mire; Perez-Diaz, Noelia; Tang, Leslie; Patel, Pryank; Mackenzie, Louise S

    2016-02-01

    Long term use of NSAIDs is linked to side effects such as gastric bleeding and myocardial infarction. Use of in silico methods and pharmacology to investigate the potential for NSAIDs diclofenac, celecoxib and naproxen to bind to nuclear receptors. In silico screening predicted that both diclofenac and celecoxib has the potential to bind to a number of different nuclear receptors; docking analysis confirmed a theoretical ability for diclofenac and celecoxib but not naproxen to bind to TRβ. Results from TRβ luciferase reporter assays confirmed that both diclofenac and celecoxib display TRβ antagonistic properties; celecoxib, IC50 3.6 × 10(-6)M, and diclofenac IC50 5.3 × 10(-6)M, comparable to the TRβ antagonist MLS (IC50 3.1 × 10(-6)M). In contrast naproxen, a cardio-sparing NSAID, lacked TRβ antagonist effects. In order to determine the effects of NSAIDs in whole organ in vitro, we used isometric wire myography to measure the changes to Triiodothyronine (T3) induced vasodilation of rat mesenteric arteries. Incubation of arteries in the presence of the TRβ antagonist MLS000389544 (10(-5)M), as well as diclofenac (10(-5)M) and celecoxib (10(-5)M) but not naproxen significantly inhibited T3 induced vasodilation compared to controls. These results highlight the benefits of computational chemistry methods used to retrospectively analyse well known drugs for side effects. Using in silico and in vitro methods we have shown that both celecoxib and diclofenac but not naproxen exhibit off-target TRβ antagonist behaviour, which may be linked to their detrimental side effects. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Pharmacological analysis of calcium antagonist receptors

    International Nuclear Information System (INIS)

    Reynolds, I.J.

    1987-01-01

    This work focuses on two aspects of the action of calcium antagonist drugs, namely, the interaction of drugs with receptors for verapamil-like calcium antagonists, and the interactions of drugs with voltage-sensitive calcium fluxes in rat brain synaptosomes. From binding studies I have found that the ligand of choice for labeling the verapamil receptor is (-)[ 3 H]desmethoxy-verapamil. This drug labels potently, reversibly and stereoselectively two receptors in membranes prepared from rat brain and rabbit skeletal muscle tissues. In equilibrium studies dihydropyridine calcium antagonists interact in a non-competitive fashion, while many non-DHPs are apparently competitive. In-depth kinetic studies in skeletal muscle membranes indicate that the two receptors are linked in a negative heterotropic fashion, and that low-affinity binding of (-) [ 3 H]desmethoxy-verapamil may be to the diltiazem receptor. However, these studies were not able to distinguish between the hypothesis that diltiazem binds to spatially separate, allosterically coupled receptors, and the hypothesis that diltiazem binds to a subsite of the verapamil receptor

  19. Accurate determination of the binding energy of the formic acid dimer: The importance of geometry relaxation

    Science.gov (United States)

    Kalescky, Robert; Kraka, Elfi; Cremer, Dieter

    2014-02-01

    The formic acid dimer in its C2h-symmetrical cyclic form is stabilized by two equivalent H-bonds. The currently accepted interaction energy is 18.75 kcal/mol whereas the experimental binding energy D0 value is only 14.22 ±0.12 kcal/mol [F. Kollipost, R. W. Larsen, A. V. Domanskaya, M. Nörenberg, and M. A. Suhm, J. Chem. Phys. 136, 151101 (2012)]. Calculation of the binding energies De and D0 at the CCSD(T) (Coupled Cluster with Single and Double excitations and perturbative Triple excitations)/CBS (Complete Basis Set) level of theory, utilizing CCSD(T)/CBS geometries and the frequencies of the dimer and monomer, reveals that there is a 3.2 kcal/mol difference between interaction energy and binding energy De, which results from (i) not relaxing the geometry of the monomers upon dissociation of the dimer and (ii) approximating CCSD(T) correlation effects with MP2. The most accurate CCSD(T)/CBS values obtained in this work are De = 15.55 and D0 = 14.32 kcal/mol where the latter binding energy differs from the experimental value by 0.1 kcal/mol. The necessity of employing augmented VQZ and VPZ calculations and relaxing monomer geometries of H-bonded complexes upon dissociation to obtain reliable binding energies is emphasized.

  20. The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

    Directory of Open Access Journals (Sweden)

    Matthias Stein

    Full Text Available Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

  1. Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

    Science.gov (United States)

    Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

    2015-10-07

    Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

  2. Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

    DEFF Research Database (Denmark)

    Møller, Charlotte; Sprenger, Richard R.; Stürup, Stefan

    2011-01-01

    Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis...... and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges...... were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached...

  3. Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

    Science.gov (United States)

    Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

    2003-01-01

    The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

  4. Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

    Science.gov (United States)

    Schwaighofer, Andreas; Pechlaner, Maria; Oostenbrink, Chris; Kotlowski, Caroline; Araman, Can; Mastrogiacomo, Rosa; Pelosi, Paolo; Knoll, Wolfgang; Nowak, Christoph; Larisika, Melanie

    2014-04-18

    Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

    DEFF Research Database (Denmark)

    Madsen, Kenneth L; Beuming, Thijs; Niv, Masha Y

    2005-01-01

    PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (te...

  6. Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

    Science.gov (United States)

    Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

    2014-03-14

    Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

  7. USING 19F-NMR SPECTROSCOPY TO DETERMINE TRIFLURALIN BINDING TO SOIL

    Science.gov (United States)

    Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soil-bound trifluralin residues, 14

  8. Protein binding studies with radiolabeled compounds containing radiochemical impurities. Equilibrium dialysis versus dialysis rate determination

    DEFF Research Database (Denmark)

    Honoré, B

    1987-01-01

    The influence of radiochemical impurities in dialysis experiments with high-affinity ligands is investigated. Albumin binding of labeled decanoate (97% pure) is studied by two dialysis techniques. It is shown that equilibrium dialysis is very sensitive to the presence of impurities resulting...

  9. Determination of Vanadium Binding Mode on Seawater-Contacted Polyamidoxime Adsorbents

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhicheng [Lawrence Berkeley National Laboratory (LBNL); Rao, Linfeng [Lawrence Berkeley National Laboratory (LBNL); Abney, Carter W. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Bryantsev, Vyacheslav [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Ivanov, Aleksandr [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2017-09-01

    Adsorbents developed for the recovery of uranium from seawater display poor selectivity over other transition metals present in the ocean, with vanadium particularly problematic. To improve selectivity, an indispensable step is the positive identification of metal binding environments following actual seawater deployment. In this work we apply x-ray absorption fine structure (XAFS) spectroscopy to directly investigate the vanadium binding environment on seawater-deployed polyamidoxime adsorbents. Comparison of the x-ray absorption near edge spectra (XANES) reveal marked similarities to recently a reported non-oxido vanadium (V) structure formed upon binding with cyclic imidedioxime, a byproduct of generating amidoxime functionalities. Density functional theory (DFT) calculations provided a series of putative vanadium binding environments for both vanadium (IV) and vanadium (V) oxidation states, and with both amidoxime and cyclic imidedioxime. Fits of the extended XAFS (EXAFS) data confirmed vanadium (V) is bound exclusively by the cyclic imidedioxime moiety in a 1:2 metal:ligand fashion, though a modest structural distortion is also observed compared to crystal structure data and computationally optimized geometries which is attributed to morphology effects from the polymer graft chain and the absence of crystal packing interactions. These results demonstrate that improved selectivity for uranium over vanadium can be achieved by suppressing the formation of cyclic imidedioxime during preparation of polyamidoxime adsorbents for seawater uranium recovery.

  10. Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

    Directory of Open Access Journals (Sweden)

    Daniel eClayton

    2015-01-01

    Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

  11. Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking.

    Directory of Open Access Journals (Sweden)

    Maria E Palm-Espling

    Full Text Available Cisplatin (CisPt is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15 are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4, indicative of a possible drug detoxification mechanism.

  12. Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

    Energy Technology Data Exchange (ETDEWEB)

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1985-10-07

    Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

  13. Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

    Science.gov (United States)

    A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind and trigger unwanted immune responses, RUC-2 does not disturb the configuration of its larger partner.

  14. Interfacial binding of cutinase rather than its catalytic activity determines the steady state interfacial tension during oil drop lipid hydrolysis.

    Science.gov (United States)

    Flipsen, J A; van Schaick, M A; Dijkman, R; van der Hijden, H T; Verheij, H M; Egmond, M R

    1999-02-01

    Hydrolysis of triglycerides by cutinase from Fusarium solani pisi causes in oil drop tensiometer experiments a decrease of the interfacial tension. A series of cutinase variants with amino acid substitutions at its molecular surface yielded different values of the steady state interfacial tension. This tension value poorly correlated with the specific activity as such nor with the total activity (defined as the specific activity multiplied by the amount of enzyme bound) of the cutinase variants. Moreover, it appeared that at activity levels above 15% of that of wild type cutinase the contribution of hydrolysis to the decrease of the tension is saturating. A clear positive correlation was found between the interfacial tension plateau value and the interfacial binding of cutinase, as determined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). These results indicate that the interfacial steady state level is not determined by the rate of hydrolysis, but mainly by the interfacial binding of cutinase.

  15. Goitre in girls at the age of puberty. Part 2. Determination of thyroid iodine uptake and triiodothyronine-binding index

    International Nuclear Information System (INIS)

    Mach, W.

    1977-01-01

    Thyroid iodine uptake and 125 I-triiodothyronine-binding index (T 3 BC) were determined in 114 girls with juvenile goitre and the results were related to the size of the goitre and its duration. Measurements of thyroid iodine uptake showed a gradual decrease of its value with increasing size of goitre and its longer duration without reaching, however, the values regarded as normal. T 3 BC showed no significant changes in relation to the size of goitre and its duration. (author)

  16. Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

    OpenAIRE

    Stephanie J. Ruiz; Gea K. Schuurman-Wolters; Bert Poolman

    2016-01-01

    BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC) importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related co...

  17. Simple method for determining binding energies of fullerene and complex atomic negative ions

    Science.gov (United States)

    Felfli, Zineb; Msezane, Alfred

    2017-04-01

    A robust potential which embeds fully the vital core polarization interaction has been used in the Regge pole method to explore low-energy electron scattering from C60, Eu and Nb through the total cross sections (TCSs) calculations. From the characteristic dramatically sharp resonances in the TCSs manifesting negative ion formation in these systems, we extracted the binding energies for the C60, Euand Nbanions they are found to be in outstanding agreement with the measured electron affinities of C60, Eu and Nb. Common among these considered systems, including the standard atomic Au is the formation of their ground state negative ions at the second Ramsauer-Townsend (R-T) minima of their TCSs. Indeed, this is a signature of all the fullerenes and complex atoms considered thus far. Shape resonances, R-T minima and binding energies of the resultant anions are presented. This work was supported by U.S. DOE, Basic Energy Sciences, Office of Energy Research.

  18. Experiments on the radioimmunological determination of the triiodothyronine binding capacity of human blood serum

    International Nuclear Information System (INIS)

    Traub, H.W.J.

    1980-01-01

    Bovine serum antibodies have been obtained from rabbits. Constant amounts of antibodies have been incubated with increasing amounts of antigens. The maximum value found for T 3 binding to the antigen-antibody complex was 66 μg RA/mg antigen. Human serum samples containing 0,05 ng 125 J-T 3 /μl were incubated with 600 μg RA, and the antigen was precipitated with 8,4 mg anti-RA. The mean value obtained for 30 enthyreotic test persons was a binding to RA of 7.9 +- 0,52% 125 J-T 3 . Comparative measurements of patient and standard serum samples shared only slight differences. This means that the measured data do not reflect the thyroid function. (orig./MG) [de

  19. Binding of Mn(II) ions to lecithin bilayers as determined by ESR and NMR investigations

    International Nuclear Information System (INIS)

    Sabatini, G.; Tiezzi, E.; Valensin, G.

    1983-01-01

    The Mn(II)-lecithin system was investigated by means of paramagnetic relaxation studies. Unsonicated and sonicated aqueous dispersions were considered at various temperatures and pH values. Information was derived from both the frequency dependence of the ESR line shape and the paramagnetic contributions to the water proton relaxation rates. A dynamic equilibrium was suggested, by taking into account the role of the through-water cation binding in the metal-lipid interaction

  20. Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

    Directory of Open Access Journals (Sweden)

    Stephanie J. Ruiz

    2016-12-01

    Full Text Available BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related compounds have failed to demonstrate transport mediated by BilE. The putative substrate-binding domain (SBD of BilE was expressed in isolation and the crystal structure solved at 1.5 Å. Although the overall fold is characteristic of SBDs, the binding site varies considerably relative to the well-characterized homologs ProX from Archaeoglobus fulgidus and OpuBC and OpuCC from Bacillus subtilis. This suggests that BilE may bind an as-yet unknown ligand. Elucidation of the natural substrate of BilE could reveal a novel bile resistance mechanism.

  1. What Shapes the Phylogenetic Structure of Anuran Communities in a Seasonal Environment? The Influence of Determinism at Regional Scale to Stochasticity or Antagonistic Forces at Local Scale.

    Science.gov (United States)

    Martins, Clarissa de Araújo; Roque, Fabio de Oliveira; Santos, Bráulio A; Ferreira, Vanda Lúcia; Strüssmann, Christine; Tomas, Walfrido Moraes

    2015-01-01

    Ecological communities are structured by both deterministic and stochastic processes. We investigated phylogenetic patterns at regional and local scales to understand the influences of seasonal processes in shaping the structure of anuran communities in the southern Pantanal wetland, Brazil. We assessed the phylogenetic structure at different scales, using the Net Relatedness Index (NRI), the Nearest Taxon Index (NTI), and phylobetadiversity indexes, as well as a permutation test, to evaluate the effect of seasonality. The anuran community was represented by a non-random set of species with a high degree of phylogenetic relatedness at the regional scale. However, at the local scale the phylogenetic structure of the community was weakly related with the seasonality of the system, indicating that oriented stochastic processes (e.g. colonization, extinction and ecological drift) and/or antagonist forces drive the structure of such communities in the southern Pantanal.

  2. Structure of noncoding RNA is a determinant of function of RNA binding proteins in transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Oyoshi Takanori

    2012-01-01

    Full Text Available Abstract The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs, resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT activity in human cells. Transcription regulator EWS (Ewing's sarcoma, which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

  3. Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.

    Science.gov (United States)

    Ito, Takashi; Takahashi, Masayuki; Okazaki, Osamu; Sugiyama, Yuichi

    2010-08-02

    The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.

  4. Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor.

    Science.gov (United States)

    Hannon, Clare; Cruz-Migoni, Abimael; Platonova, Olga; Owen, Robin L; Nettleship, Joanne E; Miller, Ami; Carr, Stephen B; Harris, Gemma; Rabbitts, Terence H; Phillips, Simon E V

    2018-03-01

    Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P2 1 , with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.

  5. Inverse gas chromatography as a method for determination of surface properties of binding materials

    Science.gov (United States)

    Yu, Jihai; Lu, Xiaolei; Yang, Chunxia; Du, Baoli; Wang, Shuxian; Ye, Zhengmao

    2017-09-01

    Inverse gas chromatography (IGC) is a promising measurement technique for investigating the surface properties of binding materials, which are the major influence element for the adsorption performance of superplasticizer. In this work, using the IGC method, blast furnace slag (BFS), sulphoaluminate cement (SAC) and portland cement (P·O) are employed to systematically evaluate the corresponding dispersive component (γsd), specific surface free energy (γsab), and acid-base properties. The obtained results show that γsd contributes to a major section of the surface free energy in the three binding materials, suggesting they are of a relatively low polarity. Compared to the two kinds of cements, the BFS possesses the highest dispersive and specific surface free energies (the values are 45.01 mJ/m2 and 11.68 mJ/m2, respectively), and also exhibits a wider distribution range of γsd, indicating their surfaces are heterogeneous. For acid-base properties, the results indicate the surfaces of three samples are basic in nature. In addition, the adsorption investigation shows that per unit surface of BFS adsorbs the most superplasticizer molecules, which indicates the higher surface free energies is beneficial to the superplasticizer adsorption.

  6. Autoradiographic determination of catechol estrogen binding sites in brain, pituitary and uterus

    International Nuclear Information System (INIS)

    Parvizi, N.; Sar, M.; Duncan, G.E.; Stumpf, W.E.

    1985-01-01

    The anatomical pattern of nuclear binding of 2-OH[6,9- 3 H]estradiol ([ 3 H]2-OHE 2 ) in brain, pituitary and uterus have been studied autoradiographically. Autoradiograms of forebrain, pituitary and uterus show nuclear concentrations of radioactivity in certain cells. This nuclear concentration is abolished when unlabelled 2-OHE 2 or E 2 was injected prior to the injection of [ 3 H]2-OHE 2 . In the brain nuclear labelling is observed in the septal-preoptic region, in the anterior hypothalamic area, and in the central hypothalamic area. Some estrogen-sensitive nuclear groups, such as lateral septum and hippocampus, do not show accumulation of radioactivity. In the uterus, luminal and glandular epithelium, stromal cell and muscle cells are labelled. A comparison of the quantitative nuclear uptake of radioactivity and of the different time intervals after the injection of different doses shows similar uptake of nuclear radioactivity. This is comparable to data obtained after [6,7- 3 H]estradiol ([ 3 H]E 2 ) injection. The results provide clear evidence for nuclear binding of catechol estrogens of the same magnitude as [ 3 H]E 2 after in vivo treatment. (Auth.).0

  7. DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis

    International Nuclear Information System (INIS)

    Wiesehahn, G.; Hearst, J.E.

    1978-01-01

    Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength uv light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28 0 +- 4 0 . For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 29 0 +- 3 0 . Assuming an unwinding angle of 28 0 for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4'-aminomethyltrioxsalen were 300 to 1400 M -1 in NaCl at 0.2 to 0.05 M and 300 to 2500 M -1 in Mg 2+ at 4 to 0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg 2+ was determined to be 70 M -1

  8. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  9. Primary and secondary structural determinants in the receptor binding sequence β-(38-57) from human luteinizing hormone

    International Nuclear Information System (INIS)

    Keutmann, H.T.; Charlesworth, M.C.; Kitzmann, K.; Mason, K.A.; Johnson, L.; Ryan, R.J.

    1988-01-01

    The intercysteine loop sequence 38-57 in the β subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG). Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, the authors have used analogues of hLHβ-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125 I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. Far-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% α-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. These results indicate that the 38-57 sequence is a relatively rigid and structurally autonomous region, not merely a series of residues constrained passively into a loop by a disulfide linkage. It includes segments of ordered structure, probably including both amphipathic helical and turn sequences. Evidence from studies of other hormones suggests that this region may be important to binding and specificity in the glycoprotein hormones as a group

  10. Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

    International Nuclear Information System (INIS)

    Roman-Lopez, C.R.; Goodson, J.; Allred, J.B.

    1987-01-01

    Conditions are described under which monomeric [ 14 C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [ 14 C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria

  11. Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

    Science.gov (United States)

    Parody, Nuria; Fuertes, Miguel Angel; Alonso, Carlos; Pico de Coaña, Yago

    2013-01-01

    The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

  12. Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry

    DEFF Research Database (Denmark)

    Jørgensen, Thomas J D; Gårdsvoll, Henrik; Danø, Keld

    2004-01-01

    Using amide hydrogen exchange combined with electrospray ionization mass spectrometry, we have in this study determined the number of amide hydrogens on several peptides that become solvent-inaccessible as a result of their high-affinity interaction with the urokinase-type plasminogen activator...... receptor (uPAR). These experiments reveal that at least six out of eight amide hydrogens in a synthetic nine-mer peptide antagonist (AE105) become sequestered upon engagement in uPAR binding. Various uPAR mutants with decreased affinity for this peptide antagonist gave similar results, thereby indicating...... that deletion of the favorable interactions involving the side chains of these residues in uPAR does not affect the number of hydrogen bonds established by the main chain of the peptide ligand. The isolated growth factor-like domain (GFD) of the cognate serine protease ligand for uPAR showed 11 protected amide...

  13. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

  14. Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

    Science.gov (United States)

    Lawson, D M; Williams, C E; Mitchenall, L A; Pau, R N

    1998-12-15

    . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

  15. Dual antagonists of integrins.

    Science.gov (United States)

    Nadrah, K; Dolenc, M Sollner

    2005-01-01

    The roles of integrins in pathologies have been studied intensively and only partially explained. This has resulted in the development of several nanomolar antagonists to certain integrins. In most cases, the aim was to produce compounds which are highly selective towards specific integrins. This paradigm has recently shifted a little. Targeting two or more integrins with one compound has become a very attractive concept, especially since it has become clear that several severe disorders, such as pathological angiogenesis, cannot be treated just with highly specific integrin antagonists. This review is aimed to elucidate some aspects regarding the design of drugs with dual activity towards integrins. Integrin structure and tissue distribution will first be described, in order to provide the basis for their functions in various pathologies which will follow. Inhibitors of several pairs of integrins will be described.

  16. Infrared Dielectric Screening Determines the Low Exciton Binding Energy of Metal-Halide Perovskites.

    Science.gov (United States)

    Umari, Paolo; Mosconi, Edoardo; De Angelis, Filippo

    2018-02-01

    The performance of lead-halide perovskites in optoelectronic devices is due to a unique combination of factors, including highly efficient generation, transport, and collection of photogenerated charge carriers. The mechanism behind efficient charge generation in lead-halide perovskites is still largely unknown. Here, we investigate the factors that influence the exciton binding energy (E b ) in a series of metal-halide perovskites using accurate first-principles calculations based on solution of the Bethe-Salpeter equation, coupled to ab initio molecular dynamics simulations. We find that E b is strongly modulated by screening from low-energy phonons, which account for a factor ∼2 E b reduction, while dynamic disorder and rotational motion of the organic cations play a minor role. We calculate E b = 15 meV for MAPbI 3 , in excellent agreement with recent experimental estimates. We then explore how different material combinations (e.g., replacing Pb → Pb:Sn→ Sn; and MA → FA → Cs) may lead to different E b values and highlight the mechanisms underlying E b tuning.

  17. Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

    Science.gov (United States)

    Bizarro, C. V.; Alemany, A.; Ritort, F.

    2012-01-01

    RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na+]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg2+ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs. PMID:22492710

  18. Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

    Science.gov (United States)

    Valko, Klara; Nunhuck, Shenaz; Bevan, Chris; Abraham, Michael H; Reynolds, Derek P

    2003-11-01

    A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites. Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2236-2248, 2003

  19. Full domain closure of the ligand-binding core of the ionotropic glutamate receptor iGluR5 induced by the high affinity agonist dysiherbaine and the functional antagonist 8,9-dideoxyneodysiherbaine

    DEFF Research Database (Denmark)

    Frydenvang, Karla Andrea; Lash, L Leanne; Naur, Peter

    2009-01-01

    The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and...

  20. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. (National Institute of Mental Health, Bethesda, MD (USA))

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  1. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C.

    1991-01-01

    Equilibrium binding studies with the sigma receptor ligand [ 3 H]1,3-di(2-tolyl)guanidine ([ 3 H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [ 3 H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [ 3 H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [ 3 H]DTG from site 2, suggesting an association of this binding site with calcium channels

  2. Evaluation of antagonistic fungi against charcoal rot of sunflower ...

    African Journals Online (AJOL)

    In vitro, sensitivity of Macrophomina phaseolina (Tassi) Goid determined through inhibition zone technique to various antagonistic fungi viz., Aspergillus niger, Aspergillus flavus, Trichoderma viride, Trichoderma harzianum and Penicillium capsulatum amended into PDA medium. All the antagonists reduced the colony ...

  3. Carbon adaptation influence the antagonistic ability of ...

    African Journals Online (AJOL)

    Influences of carbon adaptation on antagonistic activities of three Pseudomonas aeruginosa strains V4, V7 and V10 against Fusarium oxysporum f. sp. melonis were determined in this study. Results from this study showed that the P. aeruginosa strains and their adapted strains significantly inhibited the growth of mycelium ...

  4. Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry

    Science.gov (United States)

    Santos, Inês C.; Waybright, Veronica B.; Fan, Hui; Ramirez, Sabra; Mesquita, Raquel B. R.; Rangel, António O. S. S.; Fryčák, Petr; Schug, Kevin A.

    2015-07-01

    Described is a new method based on the concept of controlled band dispersion, achieved by hyphenating flow injection analysis with ESI-MS for noncovalent binding determinations. A continuous stirred tank reactor (CSTR) was used as a FIA device for exponential dilution of an equimolar host-guest solution over time. The data obtained was treated for the noncovalent binding determination using an equimolar binding model. Dissociation constants between vancomycin and Ac-Lys(Ac)-Ala-Ala-OH peptide stereoisomers were determined using both the positive and negative ionization modes. The results obtained for Ac- L-Lys(Ac)- D-Ala- D-Ala (a model for a Gram-positive bacterial cell wall) binding were in reasonable agreement with literature values made by other mass spectrometry binding determination techniques. Also, the developed method allowed the determination of dissociation constants for vancomycin with Ac- L-Lys(Ac)- D-Ala- L-Ala, Ac- L-Lys(Ac)- L-Ala- D-Ala, and Ac- L-Lys(Ac)- L-Ala- L-Ala. Although some differences in measured binding affinities were noted using different ionization modes, the results of each determination were generally consistent. Differences are likely attributable to the influence of a pseudo-physiological ammonium acetate buffer solution on the formation of positively- and negatively-charged ionic complexes.

  5. Direct demonstration of D1 dopamine receptors in the bovine parathyroid gland using the D1 selective antagonist [125I]-SCH 23982

    International Nuclear Information System (INIS)

    Monsma, F.J. Jr.; Sibley, D.R.

    1989-01-01

    The presence of D1 dopamine receptors in the parathyroid gland has been proposed based on the demonstration of dopaminergic regulation of adenylate cyclase activity and parathyroid hormone release in dispersed bovine parathyroid cells. Using a radioiodinated D1 selective antagonist [125I]-SCH 23982, we have now directly labeled and characterized the D1 dopamine receptors in bovine parathyroid gland membranes. [125I]-SCH 23982 binds in a saturable manner with high affinity and low nonspecific binding to membranes prepared from bovine parathyroid glands. D1 dopamine receptors are present in this preparation at a concentration of approximately 130 fMoles/mg protein and [125I]-SCH 23982 binding increases with increasing protein concentration in a linear fashion. Determination of the Kd using the association (k1) and dissociation (k-1) rate constants revealed good agreement with the Kd determined by saturation analysis (390 pM vs. 682 pM, respectively). Inhibition of 0.3 nM [125I]-SCH 23982 binding by a series of dopaminergic antagonists verified the D1 nature of this binding site, exhibiting appropriate affinities and rank order of potency. The competition curves of all antagonists exhibited Hill coefficients that were not significantly different from 1. Inhibition of [125I]-SCH 23982 binding by dopamine and other dopaminergic agonists revealed the presence of high and low affinity agonist binding sites. Addition of 200 microM GppNHp effected a complete conversion of high affinity dopamine binding sites to a homogeneous population of low affinity dopamine sites. The D1 receptors identified in the parathyroid gland with [125I]-SCH 23982 appear to be pharmacologically identical with those previously characterized in the central nervous system

  6. Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

    Science.gov (United States)

    Tabachnick, M; Perret, V

    1987-08-01

    [125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

  7. Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1.

    Science.gov (United States)

    Geithe, Christiane; Protze, Jonas; Kreuchwig, Franziska; Krause, Gerd; Krautwurst, Dietmar

    2017-11-01

    Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

  8. The 5-HT2A receptor binding pattern in the human brain is strongly genetically determined

    DEFF Research Database (Denmark)

    Pinborg, Lars H; Arfan, Haroon; Haugbol, Steven

    2007-01-01

    With the appropriate radiolabeled tracers, positron emission tomography (PET) enables in vivo human brain imaging of markers for neurotransmission, including neurotransmitter synthesis, receptors, and transporters. Whereas structural imaging studies have provided compelling evidence that the human...... brain anatomy is largely genetically determined, it is currently unknown to what degree neuromodulatory markers are subjected to genetic and environmental influence. Changes in serotonin 2A (5-HT(2A)) receptors have been reported to occur in various neuropsychiatric disorders and an association between...

  9. Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

    Science.gov (United States)

    Newman-Tancredi, A; Verrièle, L; Touzard, M; Millan, M J

    2001-10-05

    5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

  10. Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

    OpenAIRE

    Shishmarev, Dmitry; Wang, Yao; Mason, Claire E.; Su, Xun-Cheng; Oakley, Aaron J.; Graham, Bim; Huber, Thomas; Dixon, Nicholas E.; Otting, Gottfried

    2013-01-01

    Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron den...

  11. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    International Nuclear Information System (INIS)

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J.

    1990-01-01

    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed

  12. Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

    Science.gov (United States)

    Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

    1998-11-06

    The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

  13. TCR-contacting residues orientation and HLA-DRβ* binding preference determine long-lasting protective immunity against malaria

    International Nuclear Information System (INIS)

    Alba, Martha P.; Suarez, Carlos F.; Varela, Yahson; Patarroyo, Manuel A.; Bermudez, Adriana; Patarroyo, Manuel E.

    2016-01-01

    Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DRβ1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DRβ3*, β4*, β5* alleles. Complete PPII L 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DRβ1*PBR pockets 1 and 9, a gauche − rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. This data, in association with previously-described p3 and p7 apolar residues having gauche + orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. - Highlights: • Stereo-electronic and topochemical rules associated with FPLLI immunological memory. • Presence of very high long-lasting antibody titres against Plasmodium falciparum Spz. • Protective memory induction associated with a binding capacity to HLA-DRβ1*. • gauche − rotamer orientation in p8 polar residue is related to is related to immunological memory.

  14. TCR-contacting residues orientation and HLA-DRβ* binding preference determine long-lasting protective immunity against malaria

    Energy Technology Data Exchange (ETDEWEB)

    Alba, Martha P.; Suarez, Carlos F. [Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá D. C. (Colombia); Universidad del Rosario, Bogotá D. C. (Colombia); Universidad de Ciencias Aplicadas y Ambientales (UDCA), Bogotá (Colombia); Varela, Yahson [Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá D. C. (Colombia); Patarroyo, Manuel A.; Bermudez, Adriana [Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá D. C. (Colombia); Universidad del Rosario, Bogotá D. C. (Colombia); Patarroyo, Manuel E., E-mail: mepatarr@gmail.com [Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá D. C. (Colombia); Universidad Nacional de Colombia, Bogotá D. C. (Colombia)

    2016-09-02

    Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DRβ1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DRβ3*, β4*, β5* alleles. Complete PPII{sub L} 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DRβ1*PBR pockets 1 and 9, a gauche{sup −} rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. This data, in association with previously-described p3 and p7 apolar residues having gauche{sup +} orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. - Highlights: • Stereo-electronic and topochemical rules associated with FPLLI immunological memory. • Presence of very high long-lasting antibody titres against Plasmodium falciparum Spz. • Protective memory induction associated with a binding capacity to HLA-DRβ1*. • gauche{sup −} rotamer orientation in p8 polar residue is related to is related to immunological memory.

  15. Benzimidazoles as benzamide replacements within cyclohexane-based CC chemokine receptor 2 (CCR2) antagonists.

    Science.gov (United States)

    Cherney, Robert J; Mo, Ruowei; Meyer, Dayton T; Pechulis, Anthony D; Guaciaro, Michael A; Lo, Yvonne C; Yang, Gengjie; Miller, Persymphonie B; Scherle, Peggy A; Zhao, Qihong; Cvijic, Mary Ellen; Barrish, Joel C; Decicco, Carl P; Carter, Percy H

    2012-10-01

    We describe the design, synthesis, and evaluation of benzimidazoles as benzamide replacements within a series of trisubstituted cyclohexane CCR2 antagonists. 7-Trifluoromethylbenzimidazoles displayed potent binding and functional antagonism of CCR2 while being selective over CCR3. These benzimidazoles were also incorporated into lactam-containing antagonists, thus completely eliminating the customary bis-amide. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Possible site of action of CGRP antagonists in migraine

    DEFF Research Database (Denmark)

    Tfelt-Hansen, Peer; Olesen, Jes

    2011-01-01

    The calcitonin gene-related peptide (CGRP) receptor antagonists olcegepant and telcagepant are very potent drugs. Both are effective in migraine but in doses much higher than would be predicted from receptor binding and other in vitro results. This could perhaps suggest an effect of CGRP antagoni...

  17. Possible site of action of CGRP antagonists in migraine

    DEFF Research Database (Denmark)

    Tfelt-Hansen, Peer; Olesen, Jes

    2011-01-01

    The calcitonin gene-related peptide (CGRP) receptor antagonists olcegepant and telcagepant are very potent drugs. Both are effective in migraine but in doses much higher than would be predicted from receptor binding and other in vitro results. This could perhaps suggest an effect of CGRP...

  18. Benzodiazepine receptor antagonists for acute and chronic hepatic encephalopathy

    DEFF Research Database (Denmark)

    Als-Nielsen, B; Kjaergard, L L; Gluud, C

    2001-01-01

    The pathogenesis of hepatic encephalopathy is unknown. It has been suggested that liver failure leads to the accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition which may progress to coma. Several trials have assessed benzodiazepine receptor...... antagonists for hepatic encephalopathy, but the results are conflicting....

  19. Atomistic modeling determination of placeholder binding energy of Ti, C, and N atoms on a-Fe (100) surfaces

    International Nuclear Information System (INIS)

    Wei, X J; Liu, Y P; Han, S P

    2015-01-01

    A Fe(100) surface containing Ti, C, and N was constructed and optimized to study the placeholder binding energy of the Ti, C, and N surface atoms; this was achieved by searching the transition state with the LST (linear synchronous transit) method of the CASTEP (Cambridge Serial Total Energy Package) module. Also, the authors analyzed electron structures to determine how Ti, C, and N atoms strengthen the Fe(100) surface. The results show that when Ti, C, or N atoms take placeholder alone, or simultaneously at the Fe(100) surface, the structure stability is at its best. When including Ti, C, and N as solid solutions on the Fe(100) surface, orbital electrons of Fe3d, Ti3d, C2p, and N2p hybridize near the Fermi level; the number of electronic bonding peaks increase and bonding capacity enhances. Also, a large amount of covalent bonds formed. Covalent bonds and metallic bond coexisted. (paper)

  20. [Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

    Science.gov (United States)

    Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

    2013-05-01

    A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

  1. A Simple, Fast, Low Cost, HPLC/UV Validated Method for Determination of Flutamide: Application to Protein Binding Studies.

    Science.gov (United States)

    Esmaeilzadeh, Sara; Valizadeh, Hadi; Zakeri-Milani, Parvin

    2016-06-01

    The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r(2) > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide.

  2. Species differences in mGluR5 binding sites in mammalian central nervous system determined using in vitro binding with [{sup 18}F]F-PEB

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Shil [Department of Research Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)], E-mail: shailendra_patel@merck.com; Hamill, Terence G.; Connolly, Brett; Jagoda, Elaine; Li Wenping; Gibson, Raymond E. [Department of Research Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)

    2007-11-15

    Binding of [{sup 18}F]3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([{sup 18}F]F-PEB) was evaluated in membranes and tissue sections prepared from rat, rhesus and human brain. Saturation equilibrium binding experiments with frozen brain cortex and caudate-putamen membranes of young adult rhesus and human and with cortex and striatum from rat yielded data indicative of specific high-affinity binding (K{sub D}=0.1-0.15 nM, n{>=}3) to a saturable site previously shown to be metabotropic glutamate receptor 5 (mGluR5; Patel S, Ndubizu O, Hamill T, Chaudhary A, Burns HD, Hargreaves RJ, Gibson RE. Screening cascade and development of potential positron emission tomography radiotracers for mGluR5: in vitro and in vivo characterization. Mol Imaging Biol 2005;7:314-323). High-affinity binding of [{sup 18}F]F-PEB was also detected in cerebellum membranes from rat, rhesus and human. The density of binding sites (B{sub max}) measured using [{sup 18}F]F-PEB followed the rank order cortex{approx}caudate-putamen/striatum>cerebellum for all three species, with the cerebellum B{sub max} being significantly lower than that observed in the other regions. Receptor autoradiography studies in tissue sections confirmed that the regional distribution of [{sup 18}F]F-PEB in mammalian central nervous system is consistent with that of mGluR5 and that a small but specific mGluR5 signal is observed in rhesus and human cerebellum. A small and quantifiable specific signal could also be observed in rat cerebellum using this radiotracer. Immunohistochemical analysis in brain sections revealed a rank order of staining in rhesus and human brain of cortex{approx}caudate-putamen>cerebellum. Rat brain immunohistochemistry followed the same rank order, although the staining in the cerebellum was significantly lower. Using a 'no-wash' wipe assay, the development of a specific signal within 20 min of incubation of tissue brain sections (>60% in the cortex and striatum; 36-49% in the cerebellum

  3. Species differences in mGluR5 binding sites in mammalian central nervous system determined using in vitro binding with [18F]F-PEB

    International Nuclear Information System (INIS)

    Patel, Shil; Hamill, Terence G.; Connolly, Brett; Jagoda, Elaine; Li Wenping; Gibson, Raymond E.

    2007-01-01

    Binding of [ 18 F]3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([ 18 F]F-PEB) was evaluated in membranes and tissue sections prepared from rat, rhesus and human brain. Saturation equilibrium binding experiments with frozen brain cortex and caudate-putamen membranes of young adult rhesus and human and with cortex and striatum from rat yielded data indicative of specific high-affinity binding (K D =0.1-0.15 nM, n≥3) to a saturable site previously shown to be metabotropic glutamate receptor 5 (mGluR5; Patel S, Ndubizu O, Hamill T, Chaudhary A, Burns HD, Hargreaves RJ, Gibson RE. Screening cascade and development of potential positron emission tomography radiotracers for mGluR5: in vitro and in vivo characterization. Mol Imaging Biol 2005;7:314-323). High-affinity binding of [ 18 F]F-PEB was also detected in cerebellum membranes from rat, rhesus and human. The density of binding sites (B max ) measured using [ 18 F]F-PEB followed the rank order cortex∼caudate-putamen/striatum>cerebellum for all three species, with the cerebellum B max being significantly lower than that observed in the other regions. Receptor autoradiography studies in tissue sections confirmed that the regional distribution of [ 18 F]F-PEB in mammalian central nervous system is consistent with that of mGluR5 and that a small but specific mGluR5 signal is observed in rhesus and human cerebellum. A small and quantifiable specific signal could also be observed in rat cerebellum using this radiotracer. Immunohistochemical analysis in brain sections revealed a rank order of staining in rhesus and human brain of cortex∼caudate-putamen>cerebellum. Rat brain immunohistochemistry followed the same rank order, although the staining in the cerebellum was significantly lower. Using a 'no-wash' wipe assay, the development of a specific signal within 20 min of incubation of tissue brain sections (>60% in the cortex and striatum; 36-49% in the cerebellum) from all three species confirmed previous in vivo

  4. Determination of natural organic matter and iron binding capacity in fen samples

    Science.gov (United States)

    Kügler, Stefan; Cooper, Rebecca E.; Frieder Mohr, Jan; Wichard, Thomas; Küsel, Kirsten

    2017-04-01

    illustrated by van Krevelen plots, which indicate the presence of different substance classes including condensed aromatics, lignins and tannins known to complex iron. Our results indicate a variety of potential Fe-DOM-complexes present in the PWE samples when iron is incorporated into the elemental composition search. Using DPP we determine the complexation capacity of iron in the natural matrix of the fen along with the identification of ligands in order to estimate the iron bioavailability for bacteria. As the microbial redox system of the fen is impacted by other metals in the environment, we perform comprehensive analysis of the entirety of metal ions and concentrations in the water samples. Dialysis chambers are currently installed in the iron-rich fen from which pore water samples will be collected at 1 cm increments between 0-20 cm depth to determine the depth profiles of Fe(II)- and Fe(III)-concentration and evaluate the influence of the depth profiles on the interplay between microorganism comprising the natural microbial redox system of the fen. We have shown that metal-DOM-pH interactions affect the bioavailable metal concentration in fen water systems. This information will pave the way for a better understanding of the bacterial recruitment of trace elements and microbial redox reactions.

  5. Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding

    Directory of Open Access Journals (Sweden)

    Silverman B David

    2007-11-01

    Full Text Available Abstract Background Comparison of different protein x-ray structures has previously been made in a number of different ways; for example, by visual examination, by differences in the locations of secondary structures, by explicit superposition of structural elements, e.g. α-carbon atom locations, or by procedures that utilize a common symmetry element or geometrical feature of the structures to be compared. Results A new approach is applied to determine the structural changes that an antibody protein domain experiences upon its interaction with an antigenic target. These changes are determined with the use of two different, however comparable, sets of principal axes that are obtained by diagonalizing the second-order tensors that yield the moments-of-geometry as well as an ellipsoidal characterization of domain shape, prior to and after interaction. Determination of these sets of axes for structural comparison requires no internal symmetry features of the domains, depending solely upon their representation in three-dimensional space. This representation may involve atomic, Cα, or residue centroid coordinates. The present analysis utilizes residue centroids. When the structural changes are minimal, the principal axes of the domains, prior to and after interaction, are essentially comparable and consequently may be used for structural comparison. When the differences of the axes cannot be neglected, but are nevertheless slight, a smaller relatively invariant substructure of the domains may be utilized for comparison. The procedure yields two distance metrics for structural comparison. First, the displacements of the residue centroids due to antigenic binding, referenced to the ellipsoidal principal axes, are noted. Second, changes in the ellipsoidal distances with respect to the non-interacting structure provide a direct measure of the spatial displacements of the residue centroids, towards either the interior or exterior of the domain

  6. Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

    Science.gov (United States)

    Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

    2018-06-01

    Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

  7. Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

    Science.gov (United States)

    Duffy, Noah H; Lester, Henry A; Dougherty, Dennis A

    2012-10-19

    The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

  8. Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

    Science.gov (United States)

    Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

    2012-01-01

    The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

  9. Naloxone : actions of an antagonist

    NARCIS (Netherlands)

    Dorp, Eveline Louise Arianna van

    2009-01-01

    The opioid antagonist naloxone has a special place in pharmacology – it has no intrinsic action of its own, but it is able to save lives in the case of life threatening side-effects caused by other drugs. Naloxone is an antagonist for all opioid receptors, but most specifically for the μ-opioid

  10. Flexible Acyclic Polyol-Chloride Anion Complexes and Their Characterization by Photoelectron Spectroscopy and Variable Temperature Binding Constant Determinations

    Energy Technology Data Exchange (ETDEWEB)

    Shokri, Alireza; Wang, Xue B.; Wang, Yangping; O' Doherty, George A.; Kass, Steven R.

    2016-03-17

    Flexible acyclic alcohols with 1–5 hydroxyl groups were bound to chloride anion and these complexes were interrogated by negative ion photoelectron spectroscopy and companion density functional theory computations. The resulting vertical detachment energies are reproduced on average to 0.10 eV by M06-2X/aug-cc-pVTZ predictions and range from 4.45 – 5.96 eV. These values are 0.84 – 2.35 eV larger than the adiabatic detachment energy of Cl– as a result of the larger hydrogen bond networks in the bigger polyols. Adiabatic detachment energies of the alcohol–Cl– clusters are more difficult to determine both experimentally and computationally. This is due to the large geometry changes that occur upon photodetachment and the large bond dissociation energy of H–Cl which enables the resulting chlorine atom to abstract a hydrogen from any of the methylene (CH2) or methine (CH) positions. Both ionic and non-ionic hydrogen bonds (i.e., OH•••Cl– and OH•••OH•••Cl–) form in the larger polyols complexes, and are found to be energetically comparable. Subtle structural differences, consequently can lead to the formation of different types of hydrogen bonds and maximizing the ionic ones is not always preferred. Solution equilibrium binding constants between the alcohols and tetrrabuylammonium chloride (TBACl) in acetonitrile at -24.2, 22.0, and 53.6 °C were also determined. The free energies of association are nearly identical for all of the substrates (i.e., ΔG° = -2.8 ± 0.7 kcal mol–1). Compensating enthalpy and entropy values reveal, contrary to expectation and the intrinsic gas-phase preferences, that the bigger systems with more hydroxyl groups are entropically favored and enthalpically disfavored relative to the smaller species. This suggests that more solvent molecules are released upon binding TBACl to alcohols with more hydroxyl groups and is consistent with the measured negative heat capacities. These quantities increase with

  11. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  12. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

    Science.gov (United States)

    Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

    1997-03-01

    The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

  13. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  14. Carbon adaptation influence the antagonistic ability of ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... INTRODUCTION. The use of antagonistic bacteria to control soil-borne ... plant was used to evaluate the antifungal activities of antagonistic bacteria. ..... antagonistic bacteria and cloning of its phenazine carboxylic acid genes.

  15. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    Science.gov (United States)

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  16. Fluctuating capacity and advance decision-making in Bipolar Affective Disorder - Self-binding directives and self-determination.

    Science.gov (United States)

    Gergel, Tania; Owen, Gareth S

    2015-01-01

    For people with Bipolar Affective Disorder, a self-binding (advance) directive (SBD), by which they commit themselves to treatment during future episodes of mania, even if unwilling, can seem the most rational way to deal with an imperfect predicament. Knowing that mania will almost certainly cause enormous damage to themselves, their preferred solution may well be to allow trusted others to enforce treatment and constraint, traumatic though this may be. No adequate provision exists for drafting a truly effective SBD and efforts to establish such provision are hampered by very valid, but also paralysing ethical, clinical and legal concerns. Effectively, the autonomy and rights of people with bipolar are being 'protected' through being denied an opportunity to protect themselves. From a standpoint firmly rooted in the clinical context and experience of mania, this article argues that an SBD, based on a patient-centred evaluation of capacity to make treatment decisions (DMC-T) and grounded within the clinician-patient relationship, could represent a legitimate and ethically coherent form of self-determination. After setting out background information on fluctuating capacity, mania and advance directives, this article proposes a framework for constructing such an SBD, and considers common objections, possible solutions and suggestions for future research. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Determination of the pattern of nuclear binding from the data on the lepton-nucleus deep inelastic scattering

    International Nuclear Information System (INIS)

    Smirnov, G.I.

    1999-01-01

    Nucleon structure function ratios r A (x) = F 2 A (x) / F 2 D (x) measured in the range of atomic masses A ≥ 4 are analyzed with the aim to determine the pattern of the x and A dependence of F 2 (x) modifications caused by nuclear environment. It is found that the x and A dependence of the deviations of the r A (x) from unity can be factorized in the entire range of x. The characteristic feature of the factorization is represented with the three cross-over points x i , i = 1 - 3 in which r A (x) = 1 independently of A. In the range x > 0.7 the pattern of r A (x) is fixed with x 3 = 0.84 ± 0.01. The pattern of the x dependence is compared with theoretical calculations of Burov, Molochkov and Smirnov to demonstrate that evolution of the nucleon structure as a function of A occurs in two steps, first for A ≤ 4 and second for A > 4. The long-standing problem of the origin of the EMC effect is understood as the modification of the nucleon structure in the field responsible for the binding forces in a three-nucleon system

  18. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    KAUST Repository

    Park, Soohyung; Mutz, Niklas; Schultz, Thorsten; Blumstengel, Sylke; Han, Ali; Aljarb, Areej; Li, Lain-Jong; List-Kratochvil, Emil J W; Amsalem, Patrick; Koch, Norbert

    2018-01-01

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron–hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  19. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    KAUST Repository

    Park, Soohyung

    2018-01-03

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron–hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  20. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    Science.gov (United States)

    Park, Soohyung; Mutz, Niklas; Schultz, Thorsten; Blumstengel, Sylke; Han, Ali; Aljarb, Areej; Li, Lain-Jong; List-Kratochvil, Emil J. W.; Amsalem, Patrick; Koch, Norbert

    2018-04-01

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron-hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  1. Glutamine 57 at the complementary binding site face is a key determinant of morantel selectivity for {alpha}7 nicotinic receptors.

    Science.gov (United States)

    Bartos, Mariana; Price, Kerry L; Lummis, Sarah C R; Bouzat, Cecilia

    2009-08-07

    Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal alpha7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of alpha7 receptors. The EC(50) for activation by morantel of both alpha7 and alpha7-5HT(3A) receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate alpha7-5HT(3A) channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of alpha7 and alpha7-5HT(3A), and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln(57) in alpha7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (epsilonG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp(149) of the principal face of the binding site, whereas the other cyclic group is proximal to Gln(57) of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for alpha7. These results provide new information for further progress in drug design.

  2. Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe.

    Science.gov (United States)

    Shvadchak, Volodymyr V; Falomir-Lockhart, Lisandro J; Yushchenko, Dmytro A; Jovin, Thomas M

    2011-04-15

    Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

  3. Anthranilate phosphoribosyltransferase: Binding determinants for 5'-phospho-alpha-d-ribosyl-1'-pyrophosphate (PRPP) and the implications for inhibitor design.

    Science.gov (United States)

    Evans, Genevieve L; Furkert, Daniel P; Abermil, Nacim; Kundu, Preeti; de Lange, Katrina M; Parker, Emily J; Brimble, Margaret A; Baker, Edward N; Lott, J Shaun

    2018-02-01

    Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a "bianthranilate"-like moiety are inhibitors with IC 50 values of 10-60μM, and K i values of 1.3-15μM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. 5-HT1A receptor antagonists reduce food intake and body weight by reducing total meals with no conditioned taste aversion.

    Science.gov (United States)

    Dill, M Joelle; Shaw, Janice; Cramer, Jeff; Sindelar, Dana K

    2013-11-01

    Serotonin acts through receptors controlling several physiological functions, including energy homeostasis regulation and food intake. Recent experiments demonstrated that 5-HT1A receptor antagonists reduce food intake. We sought to examine the microstructure of feeding with 5-HT1A receptor antagonists using a food intake monitoring system. We also examined the relationship between food intake, inhibition of binding and pharmacokinetic (PK) profiles of the antagonists. Ex vivo binding revealed that, at doses used in this study to reduce food intake, inhibition of binding of a 5-HT1A agonist by ~40% was reached in diet-induced obese (DIO) mice with a trend for higher binding in DIO vs. lean animals. Additionally, PK analysis detected levels from 2 to 24h post-compound administration. Male DIO mice were administered 5-HT1A receptor antagonists LY439934 (10 or 30 mg/kg, p.o.), WAY100635 (3 or 10mg/kg, s.c.), SRA-333 (10 or 30 mg/kg, p.o.), or NAD-299 (3 or 10mg/kg, s.c.) for 3 days and meal patterns were measured. Analyses revealed that for each antagonist, 24-h food intake was reduced through a specific decrease in the total number of meals. Compared to controls, meal number was decreased 14-35% in the high dose. Average meal size was not changed by any of the compounds. The reduction in food intake reduced body weight 1-4% compared to Vehicle controls. Subsequently, a conditioned taste aversion (CTA) assay was used to determine whether the feeding decrease might be an indicator of aversion, nausea, or visceral illness caused by the antagonists. Using a two bottle preference test, it was found that none of the compounds produced a CTA. The decrease in food intake does not appear to be a response to nausea or malaise. These results indicate that 5-HT1A receptor antagonist suppresses feeding, specifically by decreasing the number of meals, and induce weight loss without an aversive side effect. © 2013 Elsevier Inc. All rights reserved.

  5. The Influence of Spatial Variation in Chromatin Density Determined by X-Ray Tomograms on the Time to Find DNA Binding Sites

    Science.gov (United States)

    Larabell, Carolyn A.; Le Gros, Mark A.; McQueen, David M.; Peskin, Charles S.

    2014-01-01

    In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process. PMID:23955281

  6. Correction: Mesoporous titania thin films as efficient enzyme carriers for paraoxon determination/detoxification: effects of enzyme binding and pore hierarchy on the biocatalyst activity and reusability.

    Science.gov (United States)

    Frančič, N; Bellino, M G; Soler-Illia, G J A A; Lobnik, A

    2016-07-07

    Correction for 'Mesoporous titania thin films as efficient enzyme carriers for paraoxon determination/detoxification: effects of enzyme binding and pore hierarchy on the biocatalyst activity and reusability' by N. Frančičet al., Analyst, 2014, 139, 3127-3136.

  7. Experimental Determination of pK[subscript a] Values and Metal Binding for Biomolecular Compounds Using [superscript 31]P NMR Spectroscopy

    Science.gov (United States)

    Swartz, Mason A.; Tubergen, Philip J.; Tatko, Chad D.; Baker, Rachael A.

    2018-01-01

    This lab experiment uses [superscript 31]P NMR spectroscopy of biomolecules to determine pK[subscript a] values and the binding energies of metal/biomolecule complexes. Solutions of adenosine nucleotides are prepared, and a series of [superscript 31]P NMR spectra are collected as a function of pH and in the absence and presence of magnesium or…

  8. ANTAGONISTIC POTENTIAL OF FLUORESCENT Pseudomonas ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    GROWTH OF TOMATO CHALLENGED WITH PHTOPATHOGENS ... This study focused on the antagonistic potential of fluorescent Pseudomonas in vitro, and its inoculation effect on growth .... the 5 days old culture in starch agar with Lugol's.

  9. Antagonistic targeting of the histamine H3 receptor decreases caloric intake in higher mammalian species.

    Science.gov (United States)

    Malmlöf, Kjell; Hastrup, Sven; Wulff, Birgitte Schellerup; Hansen, Barbara C; Peschke, Bernd; Jeppesen, Claus Bekker; Hohlweg, Rolf; Rimvall, Karin

    2007-04-15

    The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9). The energy intake recorded following administration of vehicle to the same animals served as control for the effect of the compound. In addition, rhesus monkey and pig histamine H(3) receptors were cloned from hypothalamic tissues and expressed in mammalian cell lines. The in vitro antagonist potencies of NNC 38-1202 at the H(3) receptors were determined using a functional GTPgammaS binding assay. Porcine and human H(3) receptors were found to have 93.3% identity at the amino acid level and the close homology between the monkey and human H(3) receptors (98.4% identity) was confirmed. The antagonist potencies of NNC 38-1202 at the porcine, monkey and human histamine H(3) receptors were high as evidenced by K(i)-values being clearly below 20 nM, whereas the K(i)-value on the rat H(3) receptor was significantly higher (56+/-6.0 nM). NNC 38-1202, given to pigs in a dose of 15 mg/kg, produced a significant (p<0.05) reduction (55%) of calorie intake compared with vehicle alone, (132.6+/-10.0 kcal/kgday versus 59.7+/-10.2 kcal/kgday). In rhesus monkeys administration of 0.1 and 1mg/kg decreased (p<0.05) average calorie intakes by 40 and 75%, respectively. In conclusion, the present study demonstrates that antagonistic targeting of the histamine H(3) receptor decreases caloric intake in higher mammalian species.

  10. Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist DHβE suggests a unique mode of antagonism.

    Directory of Open Access Journals (Sweden)

    Azadeh Shahsavar

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed, activated (open, and desensitized (closed states. The acetylcholine binding protein (AChBP is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls in complex with dihydro-β-erythroidine (DHβE, which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.

  11. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  12. Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette

    2014-01-01

    MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide...... library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices...... representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide...

  13. Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

    Directory of Open Access Journals (Sweden)

    Sami Rissanen

    2017-05-01

    Full Text Available Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo or raft, and liquid-disordered (Ld or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1, whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data.

  14. Characterization and design of antagonistic shape memory alloy actuators

    International Nuclear Information System (INIS)

    Georges, T; Brailovski, V; Terriault, P

    2012-01-01

    Antagonistic shape memory actuators use opposing shape memory alloy (SMA) elements to create devices capable of producing differential motion paths and two-way mechanical work in a very efficient manner. There is no requirement for additional bias elements to ‘re-arm’ the actuators and allow repetitive actuation. The work generation potential of antagonistic shape memory actuators is determined by specific SMA element characteristics and their assembly conditions. In this study, the selected SMA wires are assembled in antagonistic configuration and characterized using a dedicated test bench to evaluate their stress–strain characteristics as a function of the number of cycles. Using these functional characteristics, a so-called ‘working envelope’ is built to assist in the design of such an actuator. Finally, the test bench is used to simulate a real application of an antagonistic actuator (case study). (paper)

  15. The localization of key Bacillus subtilis penicillin binding proteins during cell growth is determined by substrate availability

    NARCIS (Netherlands)

    Lages, Marta Carolina Afonso; Beilharz, Katrin; Angeles, Danae Morales; Veening, Jan-Willem; Scheffers, Dirk-Jan

    2013-01-01

    The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for

  16. Selected Reaction Monitoring method to determine the species origin of blood-based binding agents in meats: a collaborative study

    NARCIS (Netherlands)

    Grundy, H.; Read, W.A.; Macarthur, R.; Alewijn, M.

    2013-01-01

    Binding products or food ‘glues’ are used throughout the food industry to increase the meat use rate or to augment economic efficiency. Some of these binders contain thrombin from bovine and porcine blood. The European parliament has recently banned thrombin-based additives and labelling legislation

  17. Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

    DEFF Research Database (Denmark)

    Rissanen, Sami; Grzybek, Michal; Orłowski, Adam

    2017-01-01

    membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane...

  18. Evidence that kidney function but not type 2 diabetes determines retinol-binding protein 4 serum levels

    DEFF Research Database (Denmark)

    Henze, Andrea; Frey, Simone K; Raila, Jens

    2008-01-01

    It has been suggested that retinol-binding protein 4 (RBP4) links adiposity, insulin resistance, and type 2 diabetes. However, circulating RBP4 levels are also affected by kidney function. Therefore, the aim of this study was to test whether RBP4 serum levels are primarily associated with kidney...... function or type 2 diabetes....

  19. Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

    Science.gov (United States)

    Patra, Digambara

    2010-01-15

    A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

  20. Screening of chemokine receptor CCR4 antagonists by capillary zone electrophoresis

    Directory of Open Access Journals (Sweden)

    Zhe Sun

    2011-11-01

    Full Text Available CC chemokine receptor 4 (CCR4 is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-(2-(4-chloro-phenyl-5-{[(naphthalen-1-ylmethyl-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl-N-(3-morpholin-4-yl-propyl-acetamide (S009 and the N-terminal extracellular tail (ML40 of CCR4 has been validated to be high affinity by capillary zone electrophoresis (CZE. The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-11, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases. Keywords: Capillary zone electrophoresis, CCR4 antagonist, 2-(2-(4-chloro-phenyl-5-{[(naphthalen-1-ylmethyl-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl-N-(3-morpholin-4-yl-propyl-acetamide, Interactions, Structural modification

  1. PK of immunoconjugate anticancer agent CMD-193 in rats: ligand-binding assay approach to determine in vivo immunoconjugate stability.

    Science.gov (United States)

    Hussain, Azher; Gorovits, Boris; Leal, Mauricio; Fluhler, Eric

    2014-01-01

    Antibody-drug conjugates (ADCs) are a new generation of anticancer therapeutics. The objective of this manuscript is to propose a methodology that can be used to assess the stability of the ADCs by using the PK data obtained by ligand-binding assays that measure various components of ADCs. The ligand-binding assays format of different components of ADCs provided unique valuable PK information. The mathematical manipulation of the bioanalytical data provided an insight into the in vivo integrity, indicating that the loading of the calicheamicin on the G193 antibody declines in an apparent slow first-order process. This report demonstrates the value of analyzing various components of the ADC and their PK profiles to better understand the disposition and in vivo stability of ADCs.

  2. In vivo binding of a radioiodinated derivative of SCH 23390 (SCH)

    International Nuclear Information System (INIS)

    McQuade, R.D.; Iorio, L.C.; Chipkin, R.E.; Barnett, A.

    1986-01-01

    The in vitro binding properties of SCH have been extensively characterized, however, little research has been conducted on its in vivo binding profile. This report is on the characterization of the in vivo binding of 7- 125 I-SCH (71 SCH), which possesses a specific radioactivity of 2200 Ci/mmole and exhibits in vitro binding properties similar to those of 3 H-SCH. Sprague-Dawley rats were injected with 0.02 nmoles of 7ISH, in the absence and presence of 30 nmoles of unlabeled SCH, and were sacrificed at 1,2 and 4 hours post-treatment. The striatum and cerebellum were removed, weighed and the radioactivity was counted. Specific binding was defined as cpm in the absence of SCH less cpm in the presence of SCH. One hour after injection, the striatum of the rats treated with 7ISCH contained 170,000 cpm/g tissue, of which 105,000 cpm/g tissue were specific. The cerebellum of the rats contained 75% fewer cpm/g tissue and did not exhibit specifically bound in the striatum, but not in the cerebellum. Sacrifice after longer periods of time resulted in a 35% decrease in specific binding between 1 and 2 h, and a 70% decrease between 1 and 4 h. The pharmacological profile of the in vivo binding of 7ISCH was determined by the ability of dopaminergic antagonists to inhibit specific binding in the rat striatum 1 h after treatment. Thirty nmoles of SCH caused a 55% inhibition of the specific binding to striatum, while an equal amount of SCH 23388, the inactive S isomer of SCH, resulted in no inhibition. Haloperidol, a D-2 antagonist, did not inhibit the specific binding of 7ISCH to the striatum at doses up to 3000 nmoles. These data indicate that 7ISCH is specifically bound, in vivo, to D-1 receptors in the rat striatum

  3. Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

    Science.gov (United States)

    Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

    2015-03-01

    The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Determining the effect of malondialdehyde on the IgE-binding capacity of shrimp tropomyosin upon in vitro digestion.

    Science.gov (United States)

    Lv, Liangtao; Lin, Hong; Li, Zhenxing; Ahmed, Ishfaq; Chen, Guanzhi

    2017-10-01

    Stability in simulated gastric fluids is considered an important parameter for the estimation of food allergenicity. Moreover, proteins in food are highly susceptible to lipid oxidation during processing and preservation. In this study, the change in the IgE-binding capacity of malondialdehyde (MDA)-treated shrimp tropomyosin (TM) following in vitro digestion was investigated by SDS-PAGE and western blot. Shrimp TM treated with different concentrations of MDA was slightly degraded and became increasingly resistant to pepsin digestion over time. While untreated TM was rapidly degraded, MDA-treated TM showed some resistance and was degraded by trypsin only after increasing the digestion time. Results of immunoblotting studies on IgE using sera from patients allergic to shrimp indicated that the IgE-binding capacity of TM and MDA (50 mmol L -1 )-treated TM decreased slightly after pepsin digestion and significantly decreased after trypsin digestion. The study indicated that the resistance of TM to degradation increased after oxidation. The treatment with proteases, especially trypsin, is quite effective in decreasing the IgG/IgE-binding capacity of shrimp TM. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses.

    Directory of Open Access Journals (Sweden)

    Hadar Israeli

    2017-04-01

    Full Text Available Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.

  6. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

    Science.gov (United States)

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

    2012-09-11

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  7. SSTR-Mediated Imaging in Breast Cancer: Is There a Role for Radiolabeled Somatostatin Receptor Antagonists?

    Science.gov (United States)

    Dalm, Simone U; Haeck, Joost; Doeswijk, Gabriela N; de Blois, Erik; de Jong, Marion; van Deurzen, Carolien H M

    2017-10-01

    Recent studies have shown enhanced tumor targeting by novel somatostatin receptor (SSTR) antagonists compared with clinically widely used agonists. However, these results have been obtained mostly in neuroendocrine tumors, and only limited data are available for cancer types with lower SSTR expression, including breast cancer (BC). To date, two studies have reported higher binding of the antagonist than the agonist in BC, but in both studies only a limited number of cases were evaluated. In this preclinical study, we further investigated whether the application of an SSTR antagonist can improve SSTR-mediated BC imaging in a large panel of BC specimens. We also generated an in vivo BC mouse model and performed SPECT/MRI and biodistribution studies. Methods: Binding of 111 In-DOTA-Tyr 3 -octreotate (SSTR agonist) and 111 In-DOTA-JR11 (SSTR antagonist) to 40 human BC specimens was compared using in vitro autoradiography. SSTR2 immunostaining was performed to confirm SSTR2 expression of the tumor cells. Furthermore, binding of the radiolabeled SSTR agonist and antagonist was analyzed in tissue material from 6 patient-derived xenografts. One patient-derived xenograft, the estrogen receptor-positive model T126, was chosen to generate in vivo mouse models containing orthotopic breast tumors for in vivo SPECT/MRI and biodistribution studies after injection with 177 Lu-DOTA-Tyr 3 -octreotate or 177 Lu-DOTA-JR11. Results: 111 In-DOTA-JR11 binding to human BC tissue was significantly higher than 111 In-DOTA-Tyr 3 -octreotate binding ( P < 0.001). The median ratio of antagonist binding versus agonist binding was 3.39 (interquartile range, 2-5). SSTR2 immunostaining confirmed SSTR2 expression on the tumor cells. SPECT/MRI of the mouse model found better tumor visualization with the antagonist. This result was in line with the significantly higher tumor uptake of the radiolabeled antagonist than of the agonist as measured in biodistribution studies 285 min after radiotracer

  8. Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

    International Nuclear Information System (INIS)

    Curtis, B.M.

    1986-01-01

    Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [ 3 H]nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed α(Mr 135,000), β(Mr 50,000), and γ(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The α and γ subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the α, β, and γ subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel

  9. Determination of the binding mode for anti-inflammatory natural product xanthohumol with myeloid differentiation protein 2

    Directory of Open Access Journals (Sweden)

    Fu W

    2016-01-01

    Full Text Available Weitao Fu,1,* Lingfeng Chen,1,* Zhe Wang,1 Chengwei Zhao,1 Gaozhi Chen,1 Xing Liu,1 Yuanrong Dai,2 Yuepiao Cai,1 Chenglong Li,1,3 Jianmin Zhou,1 Guang Liang1 1Chemical Biology Research Center, School of Pharmaceutical Sciences, 2Department of Respiratory Medicine, the Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 3Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH, USA *These authors contributed equally to this work Abstract: It is recognized that myeloid differentiation protein 2 (MD-2, a coreceptor of toll-like receptor 4 (TLR4 for innate immunity, plays an essential role in activation of the lipopolysaccharide signaling pathway. MD-2 is known as a neoteric and suitable therapeutical target. Therefore, there is great interest in the development of a potent MD-2 inhibitor for anti-inflammatory therapeutics. Several studies have reported that xanthohumol (XN, an anti-inflammatory natural product from hops and beer, can block the TLR4 signaling by binding to MD-2 directly. However, the interaction between MD-2 and XN remains unknown. Herein, our work aims at characterizing interactions between MD-2 and XN. Using a combination of experimental and theoretical modeling analysis, we found that XN can embed into the hydrophobic pocket of MD-2 and form two stable hydrogen bonds with residues ARG-90 and TYR-102 of MD-2. Moreover, we confirmed that ARG-90 and TYR-102 were two necessary residues during the recognition process of XN binding to MD-2. Results from this study identified the atomic interactions between the MD-2 and XN, which will contribute to future structural design of novel MD-2-targeting molecules for the treatment of inflammatory diseases. Keywords: myeloid differentiation 2, xanthohumol, binding mode, inflammation, molecular dynamics simulation 

  10. Characterization of a novel non-steroidal glucocorticoid receptor antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qun-Yi; Zhang, Meng [The National Center for Drug Screening, Shanghai (China); State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai (China); Hallis, Tina M.; DeRosier, Therese A. [Cell Systems Division, Invitrogen, Madison, WI (United States); Yue, Jian-Min; Ye, Yang [State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai (China); Mais, Dale E. [The National Center for Drug Screening, Shanghai (China); MPI Research, Mattawan, MI (United States); Wang, Ming-Wei, E-mail: wangmw@mail.shcnc.ac.cn [The National Center for Drug Screening, Shanghai (China); State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai (China)

    2010-01-15

    Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K{sub i} = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

  11. Characterization of a novel non-steroidal glucocorticoid receptor antagonist

    International Nuclear Information System (INIS)

    Li, Qun-Yi; Zhang, Meng; Hallis, Tina M.; DeRosier, Therese A.; Yue, Jian-Min; Ye, Yang; Mais, Dale E.; Wang, Ming-Wei

    2010-01-01

    Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K i = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

  12. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

    DEFF Research Database (Denmark)

    Barington, Line; Rummel, Pia C; Lückmann, Michael

    2016-01-01

    and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...

  13. Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a

  14. AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

    OpenAIRE

    Terry W. Moody; Nicole Tashakkori; Samuel A. Mantey; Paola Moreno; Irene Ramos-Alvarez; Marcello Leopoldo; Robert T. Jensen

    2017-01-01

    While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar ...

  15. Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

    DEFF Research Database (Denmark)

    Rist, Oystein; Grimstrup, Marie; Receveur, Jean-Marie

    2009-01-01

    Structure-activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists....... Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7nM and functional...

  16. Central actions of a novel and selective dopamine antagonist

    International Nuclear Information System (INIS)

    Schulz, D.W.

    1985-01-01

    Receptors for the neurotransmitter dopamine traditionally have been divided into two subgroups: the D 1 class, which is linked to the stimulation of adenylate cyclase-activity, and the D 2 class which is not. There is much evidence suggesting that it is the D 2 class which is not. There is much evidence suggesting that it is the D 2 dopamine receptor that mediates the physiological and behavioral actions of dopamine in the intact animal. However, the benzazepine SCH23390 is a dopamine antagonist which has potent behavioral actions while displaying apparent neurochemical selectivity for the D 1 class of dopamine receptors. The purpose of this dissertation was to (1) confirm and characterize this selectivity, and (2) test certain hypothesis related to possible modes of action of SCH233390. The inhibition of adenylate cyclase by SCH23390 occurred via an action at the dopamine receptor only. A radiolabeled analog of SCH23390 displayed the receptor binding properties of a specific high-affinity ligand, and regional receptor densities were highly correlated with dopamine levels. The subcellular distribution of [ 3 H]-SCH23390 binding did not correspond completely with that of dopamine-stimulated adenylate cyclase. The neurochemical potency of SCH23390 as a D 1 receptor antagonist was preserved following parental administration. A variety of dopamine agonists and antagonists displayed a high correlation between their abilities to compete for [ 3 H]-SCH23390 binding in vitro and to act at an adenylate cyclase-linked receptor. Finally, the relative affinities of dopamine and SCH23390 for both D 1 receptors and [ 3 H]-SCH23390 binding sites were comparable. It is concluded that the behavioral effects of SCH23390 are mediated by actions at D 1 dopamine receptors only, and that the physiological importance of this class of receptors should be reevaluated

  17. New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

    Science.gov (United States)

    Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

    2017-12-01

    Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

  18. Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

    Science.gov (United States)

    Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2017-06-06

    Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

  19. Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus

    Science.gov (United States)

    Asfor, Amin S.; Upadhyaya, Sasmita; Knowles, Nick J.; King, Donald P.; Paton, David J.

    2014-01-01

    Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design. PMID:24584474

  20. Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

    Directory of Open Access Journals (Sweden)

    Lauren A. Cooper

    2018-04-01

    Full Text Available In clustered regularly interspaced short palindromic repeat (CRISPR-Cas (CRISPR-associated immunity systems, short CRISPR RNAs (crRNAs are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.

  1. Potential Clinical Implications of the Urotensin II Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Emilie Kane

    2011-07-01

    Full Text Available Urotensin-II (UII, which binds to its receptor UT, plays an important role in the heart, kidneys, pancreas, adrenal gland and CNS. In the vasculature, it acts as a potent endothelium-independent vasoconstrictor and endothelium-dependent vasodilator. In disease states, this constriction-dilation equilibrium is disrupted. There is an upregulation of the UII system in heart disease, metabolic syndrome and kidney failure. The increase in UII release and UT expression suggest that UII system may be implicated in the pathology and pathogenesis of these diseases by causing an increase in ACAT-1 activity leading to SMC proliferation and foam cell infiltration, insulin resistance (DMII, as well as inflammation, high blood pressure and plaque formation. Recently, UT antagonists such as SB-611812, palosuran, and most recently a piperazino-isoindolinone based antagonist have been developed in the hope of better understanding the UII system and treating its associated diseases.

  2. Interaction between Ca++-channel antagonists and α2-adrenergic receptors in rabbit ileal cell membrane

    International Nuclear Information System (INIS)

    Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

    1986-01-01

    An interaction between Ca ++ -channel antagonists and the α 2 -adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an α 2 -agonist, stimulated NaCl absorption apparently by Ca ++ -channel antagonism since it inhibited 45 Ca ++ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca ++ -channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of 3 H-yohimbine, a specific α 2 -adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd ++ inhibited the specific binding of 3 H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca ++ -channels and α 2 -adrenergic receptors in ileal basolateral membranes. Some Ca ++ -channel antagonists alter α 2 -adrenergic binding to the receptor and α 2 -agonist binding leads to changes in Ca ++ entry. A close spatial relationship between the Ca ++ -channel and the α 2 -receptor could explain the data

  3. Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

    Science.gov (United States)

    Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

    2005-05-20

    The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

  4. Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

    Science.gov (United States)

    Kai, Zhen-Peng; Zhu, Jing-Jing; Deng, Xi-Le; Yang, Xin-Ling; Chen, Shan-Shan

    2018-04-03

    Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C -terminal amidated octapeptide Manse-AT (6-13). We identified three residues essential for bioactivity (Thr⁴, Arg6 and Phe⁸) by assaying alanine-replacement analogs of Manse-AT (6-13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10-13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10-13) validated our hypothesis. The IC 50 value of antagonist Manse-AT (10-13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10-13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

  5. Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model

    Directory of Open Access Journals (Sweden)

    Zhen-Peng Kai

    2018-04-01

    Full Text Available Insect G protein coupled receptors (GPCRs have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C-terminal amidated octapeptide Manse-AT (6–13. We identified three residues essential for bioactivity (Thr4, Arg6 and Phe8 by assaying alanine-replacement analogs of Manse-AT (6–13. Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10–13, we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10–13 validated our hypothesis. The IC50 value of antagonist Manse-AT (10–13 is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10–13 was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

  6. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  7. Determination of membrane disruption and genomic DNA binding of cinnamaldehyde to Escherichia coli by use of microbiological and spectroscopic techniques.

    Science.gov (United States)

    He, Tian-Fu; Zhang, Zhi-Hong; Zeng, Xin-An; Wang, Lang-Hong; Brennan, Charles S

    2018-01-01

    This work was aimed to investigate the antibacterial action of cinnamaldehyde (CIN) against Escherichia coli ATCC 8735 (E. coli) based on membrane fatty acid composition analysis, alterations of permeability and cell morphology as well as interaction with genomic DNA. Analysis of membrane fatty acids using gas chromatography-mass spectrometry (GC-MS) revealed that the proportion of unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were the major fatty acids in plasmic membrane, and their levels were significantly changed after exposure of E. coli to CIN at low concentrations. For example, the proportion of UFA decreased from 39.97% to 20.98%, while the relative content of SFA increased from 50.14% to 67.80% as E. coli was grown in increasing concentrations of CIN (from 0 to 0.88mM). Scanning electron microscopy (SEM) showed that the morphology of E. coli cells to be wrinkled, distorted and even lysed after exposure to CIN, which therefore decreased the cell viability. The binding of CIN to genomic DNA was probed using fluorescence, UV-Visible absorption spectra, circular dichroism, molecular modeling and atomic force microscopy (AFM). Results indicated that CIN likely bound to the minor groove of genomic DNA, and changed the secondary structure and morphology of this biomacromolecule. Therefore, CIN can be deem as a kind of natural antimicrobial agents, which influence both cell membrane and genomic DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Antiallergic effects of H1-receptor antagonists.

    Science.gov (United States)

    Baroody, F M; Naclerio, R M

    2000-01-01

    The primary mechanism of antihistamine action in the treatment of allergic diseases is believed to be competitive antagonism of histamine binding to cellular receptors (specifically, the H1-receptors), which are present on nerve endings, smooth muscles, and glandular cells. This notion is supported by the fact that structurally unrelated drugs antagonize the H1-receptor and provide clinical benefit. However, H1-receptor antagonism may not be their sole mechanism of action in treating allergic rhinitis. On the basis of in vitro and animal experiments, drugs classified as H1-receptor antagonists have long been recognized to have additional pharmacological properties. Most first-generation H1-antihistamines have anticholinergic, sedative, local anaesthetic, and anti-5-HT effects, which might favourably affect the symptoms of the allergic response but also contribute to side-effects. These additional properties are not uniformly distributed among drugs classified as H1-receptor antagonists. Azatadine, for example, inhibits in vitro IgE-mediated histamine and leukotriene (LT) release from mast cells and basophils. In human challenge models, terfenadine, azatadine, and loratadine reduce IgE-mediated histamine release. Cetirizine reduces eosinophilic infiltration at the site of antigen challenge in the skin, but not the nose. In a nasal antigen challenge model, cetirizine pretreatment did not affect the levels of histamine and prostaglandin D2 recovered in postchallenge lavages, whereas the levels of albumin, N-tosyl-L-arginine methyl ester (TAME) esterase activity, and LTs were reduced. Terfenadine, cetirizine, and loratadine blocked allergen-induced hyperresponsiveness to methacholine. In view of the complexity of the pathophysiology of allergy, a number of H1 antagonists with additional properties are currently under development for allergic diseases. Mizolastine, a new H1-receptor antagonist, has been shown to have additional actions that should help reduce the

  9. Evodiamine as a novel antagonist of aryl hydrocarbon receptor

    International Nuclear Information System (INIS)

    Yu, Hui; Tu, Yongjiu; Zhang, Chun; Fan, Xia; Wang, Xi; Wang, Zhanli; Liang, Huaping

    2010-01-01

    Research highlights: → Evodiamine interacted with the AhR. → Evodiamine inhibited the specific binding of [ 3 H]-TCDD to the AhR. → Evodiamine acts as an antagonist of the AhR. -- Abstract: Evodiamine, the major bioactive alkaloid isolated from Wu-Chu-Yu, has been shown to interact with a wide variety of proteins and modify their expression and activities. In this study, we investigated the interaction between evodiamine and the aryl hydrocarbon receptor (AhR). Molecular modeling results revealed that evodiamine directly interacted with the AhR. Cytosolic receptor binding assay also provided the evidence that evodiamine could interact with the AhR with the K i value of 28.4 ± 4.9 nM. In addition, we observed that evodiamine suppressed the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced nuclear translocation of the AhR and the expression of CYP1A1 dose-dependently. These results suggested that evodiamine was able to bind to the AhR as ligand and exhibit antagonistic effects.

  10. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  11. Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye-binding method.

    Science.gov (United States)

    Müller, Kerstin; Brunnberg, Leo

    2010-03-01

    In reptile medicine, plasma chemistry analysis is widely used for the evaluation of an individual's health status. The standard method for the determination of plasma albumin concentration is protein electrophoresis combined with the determination of total protein concentration, but the bromcresol green (BCG) dye-binding method is also used. The reliability of the BCG method for the measurement of albumin concentration in reptiles is unknown. The aim of this study was to compare the plasma albumin values of turtles obtained by protein electrophoresis and the BCG method. Between March 2008 and September 2008, heparinized plasma samples from 16 clinically healthy and 10 diseased turtles of different species were collected. Plasma albumin concentrations were measured by protein electrophoresis and by the BCG method. The results of the 2 methods were compared using Passing-Bablok regression and Bland-Altman plots. Albumin concentration measured by BCG was weakly correlated with the corresponding protein electrophoretic values in all turtles (r(s)=.610, Palbumin concentration measured with the 2 different methods differed significantly in all turtles (P=.009; Wilcoxon's test) and in healthy turtles (P=.005) but not in diseased animals (P=.241). In the Bland-Altman plot a systematic error was found between the 2 methods in diseased turtles. Measurement of albumin by the BCG dye-binding method may lead to inaccurate results for plasma albumin concentration, especially in ill turtles. Therefore, for health assessment in turtles, albumin should be measured by protein electrophoresis.

  12. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    International Nuclear Information System (INIS)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-01-01

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 (micro)g of DNA. This sensitivity may allow the method to be used for clinical applications

  13. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-12-29

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

  14. Muscarinic Receptor Agonists and Antagonists

    Directory of Open Access Journals (Sweden)

    David R. Kelly

    2001-02-01

    Full Text Available A comprehensive review of pharmacological and medical aspects of the muscarinic class of acetylcholine agonists and antagonists is presented. The therapeutic benefits of achieving receptor subtype selectivity are outlined and applications in the treatment of Alzheimer’s disease are discussed. A selection of chemical routes are described, which illustrate contemporary methodology for the synthesis of chiral medicinal compounds (asymmetric synthesis, chiral pool, enzymes. Routes to bicyclic intrannular amines and intramolecular Diels-Alder reactions are highlighted.

  15. Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

    NARCIS (Netherlands)

    Ruiz, Stephanie J.; Schuurman-Wolters, Gea K.; Poolman, Bert

    2016-01-01

    BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence

  16. Synthesis and pharmacological evaluation of DHβE analogs as neuronal nicotinic acetylcholine receptor antagonists

    DEFF Research Database (Denmark)

    Jepsen, Tue H.; Jensen, Anders A.; Lund, Mads Henrik

    2014-01-01

    Dihydro-β-erythroidine (DHβE) is a member of the Erythrina family of alkaloids and a potent competitive antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors (nAChRs). Guided by an X-ray structure of DHβE in complex with an ACh binding protein, we detail the design, synthesis...

  17. SELECTIVITY PROFILE OF SOME RECENT MUSCARINIC ANTAGONISTS IN BOVINE AND GUINEA-PIG TRACHEA AND HEART

    NARCIS (Netherlands)

    ROFFEL, AF; HAMSTRA, JJ; ELZINGA, CRS; ZAAGSMA, J

    1994-01-01

    The functional affinities of some recently developed subtype-selective muscarinic antagonists towards bovine tracheal smooth muscle muscarinic M(3) receptors were established and compared to binding affinities for bovine cardiac M(2) and functional affinities for guinea-pig tracheal smooth muscle

  18. Prevalence, specificity and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments.

    Directory of Open Access Journals (Sweden)

    Ylva Ivarsson

    Full Text Available BACKGROUND: PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs, important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited. METHODOLOGY/PRINCIPAL FINDINGS: We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of in vivo cell-localization studies and in vitro dot blot and Surface Plasmon Resonance (SPR experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. In vitro specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands. CONCLUSIONS/SIGNIFICANCE: Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands.

  19. Determination of the ratio between mercaptalbumin and nonmercaptalbumin by HPLC with fluorescence probe specifically binding to albumin.

    Science.gov (United States)

    Ohyama, Kaname; Kishikawa, Naoya; Matsuo, Aya; Imazato, Takahiro; Ueki, Yukitaka; Wada, Mitsuhiro; Nakashima, Kenichiro; Kuroda, Naotaka

    2014-01-01

    A simple and selective HPLC-fluorescence (FL) method with FL probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM), for simultaneous determination of mercaptalbumin (HMA) and nonmercaptalbumin (HNA1) was developed. After HMA and HNA1 were separated on an ion-exchange column, they were on-line and post-column mixed with DAPIM. The DAPIM-albumin complex produces FL (λex 370nm and λem 510nm); however, DAPIM solution never gives the FL. Based on this mechanism, selective determination of HMA and HNA1 were achieved without any pretreatment and interfering peak. The proposed method was applied to the measurement of HMA and HNA1 in human serum of healthy volunteers and diabetes mellitus patients. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

    Science.gov (United States)

    Qin, M; Oie, S

    1994-11-01

    The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

  1. GABAA receptor partial agonists and antagonists

    DEFF Research Database (Denmark)

    Krall, Jacob; Balle, Thomas; Krogsgaard-Larsen, Niels

    2015-01-01

    to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown...... antagonists have been essential in defining the tonic current but both remaining issues concerning the GABAARs involved and the therapeutic possibilities of modulating tonic inhibition underline the need for GABAAR antagonists with improved selectivity....

  2. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    Science.gov (United States)

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-09

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

  3. Twisted gastrulation, a BMP antagonist, exacerbates podocyte injury.

    Directory of Open Access Journals (Sweden)

    Sachiko Yamada

    Full Text Available Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7 in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1, a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

  4. Ring-to-chain conformation may be a determining factor in the ability of xanthophylls to bind to the bulk light-harvesting complex of plants

    Science.gov (United States)

    Young, Andrew J.; Phillip, Denise M.; Hashimoto, Hideki

    2002-12-01

    The binding of xanthophylls to the main light-harvesting complex (LHC) of higher plants has been studied using the technique of in vitro reconstitution. This demonstrated that the carotenoid diol lactucaxanthin (native to many LHC) would not support the assembly of LHC whilst other diols, notably zeaxanthin and lutein would. Analysis of the most stable forms of the carotenoid end-groups found in xanthophylls native to higher plant LHC (as determined by theoretical calculations) revealed profound differences in the adiabatic potential energy curves for the C5-C6-C7-C8-torsion angle for the ɛ end-groups in lactucaxanthin (6-s- trans), in comparison to carotenoids possessing a 3-hydroxy β end-group (zeaxanthin; 6-s- cis), 3-hydroxy-4-keto β end-group (astaxanthin, 6-s- cis) or a 3-hydroxy-5,6-epoxy end-group (violaxanthin, distorted 6-s- cis). The (ɛ end-groups of other carotenoids studied were 6-s- trans. We examine the possible relationship between carotenoid ring-to-chain conformation and binding to LHC.

  5. [Serum thyroxine-binding protein for determining the functional state of the thyroid gland in pregnant women with endemic goiter].

    Science.gov (United States)

    Korol'kova, O A; Cheremukhin, V I

    1975-01-01

    A determination was made of the hormone-forming capacity of the thyroid gland in pregnent women under conditions of goiter endemic at various periods of pregnancy by trimesters (123-in healthy pregnant women, 206-with euthyroid goiter of the I degree, 271-or II degree, 90-of the II degree, and 4-of the IV degree). A method of zonal electrophoresis in the medinal-veronal buffer was applied. Thyrofixin with I131 isotope (made in the USSR) was used. With increase of the periods of pregnancy and the degree of euthyroid hyperplasia of the thyroid gland and goiter the thyroid gland function became elevated irrespective of age.

  6. Identification and characterization of porcine mannan-binding lectin A (pMBL-A), and determination of serum concentration heritability

    DEFF Research Database (Denmark)

    Juul-Madsen, H. R.; Krogh-Meibom, T.; Henryon, M.

    2006-01-01

    affinity, ion exchange, and size exclusion chromatography and determined many of its characteristics. Based on the N-terminal sequence, multiple sequence alignment, and relative affinities to various carbohydrate ligands, we propose that the MBL purified in this study is pMBL-A. We have generated...... antibodies to this protein and established an immunoassay to quantify pMBL-A in serum. Using this assay, we found breed differences in pMBL-A concentration distributions and heritability estimates. In the Duroc breed (n=588), pMBL-A concentrations show a unimodal distribution with a mean of 9,125 ng...

  7. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity-labeling

    International Nuclear Information System (INIS)

    Baxter, R.C.; Martin, J.L.

    1989-01-01

    To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit

  8. Unraveling a Hotspot for TCR Recognition on HLA-A2: Evidence Against the Existence of Peptide-independent TCR Binding Determinants

    International Nuclear Information System (INIS)

    Gagnon, Susan J.; Borbulevych, Oleg Y.; Davis-Harrison, Rebecca L.; Baxter, Tiffany K.; Clemens, John R.; Armstrong, Kathryn M.; Turner, Richard V.; Damirjian, Marale; Biddison, William E.; Baker, Brian M.

    2005-01-01

    T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 α1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

  9. Thyroxine determination in serum by radioimmunoassay and competitive protein-binding analysis (simultaneous study using four test kits)

    International Nuclear Information System (INIS)

    Wagner, K.

    1978-01-01

    Three commercial test kits for radioimmunological T 4 -determination have been tested (T 4 -RIA Abbott, RIA-mat T 4 , T 4 -RIA Henning). The RIA method has also been compared with a CPBA test (Tetralute) for routine application. 1) The results show that CPBA (Tetralute) is superior over the three radioimmuno-assays with regard to intra-assay variance. The T 4 -RIA Abbott has been inferior to the two other RIA under test and to the Tetralute with regard to inter-assay variance. 2) The four test kits show significant differences and considerable deviations from the given nominal values in comparison with the mean values of two control sera. A control for correctness does not seem to be appropriate as it cannot be decided which of the test kits yields the true value of T 4 content. No systematic difference has been observed between RIA and CPBA in the determination of T 4 in control sera. 3) No detectable difference has been found among the RIA-T 4 test kits and comparing them with the CBPA test regarding their diagnostic precision. A good correlation of T 4 values of euthyroid patients comparing the Tetralute and the three radioimmunological test kits. The mean values of euthyroid sera obtained by the three radioimmuno-assays and the Tetralute are not in agreement with each other. (orig.) [de

  10. (-)[125I]-iodopindolol, a new highly selective radioiodinated beta-adrenergic receptor antagonist: measurement of beta-receptors on intact rat astrocytoma cells

    International Nuclear Information System (INIS)

    Barovsky, K.; Brooker, G.

    1980-01-01

    (-)-Pindolol, one of the most potent beta-adrenergic receptor antagonists, was radioiodinated using chloramine-T oxidation of carrier-free Na 125I and separated from unreacted pindolol to yield 2200 Ci/mmole (-)-[125I]-iodopindolol ((-)-[125I]-IPin). Mass and ultraviolet spectra confirmed that the iodination occurred on the indole ring, presumably at the 3 position. The binding of radiolabeled (-)-[125I]-IPin to beta-adrenergic receptors has been studied using intact C6 rat astrocytoma cells (2B subclone) grown in monolayer cultures. Binding of (-)[125IPin was saturable with time and concentration. Using 13 pM (-)-[125I]IPin, binding equilibrium was reached in 90 min at 21-22 degrees C. The reverse rate constant was 0.026 min-1 at 21 0 C. Specific binding (expressed as 1 microM(-)-propranolol displaceable counts) of (-)-[125I]-IPin was 95% of total binding. Scatchard analysis of (-)-[125I]-I]Pin binding revealed approximately 4300 receptors/cell and a dissociation constant of 30 pM. This was in excellent agreement with the kinetically determined dissociation constant of 35 pM. Displacement by propranolol and isoproterenol showed that (-)-[125I]-IPin binding sites were pharmacologically and stereospecifically selective. These results indicate that (-)-[125I]-IPin, a pure (-)-stereoisomer, high specific activity radioligand, selectively binds to beta-adrenergic receptors in whole cells with a high percentage of specific binding and should therefore be useful in the study and measurement of cellular beta-adrenergic receptors

  11. Identification of Bexarotene as a PPARγ Antagonist with HDX

    Directory of Open Access Journals (Sweden)

    David P. Marciano

    2015-01-01

    Full Text Available The retinoid x receptors (RXRs are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL. The RXRs form heterodimers with several nuclear receptors (NRs, including peroxisome proliferator-activated receptor gamma (PPARγ, to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.

  12. Receptors for luteinizing hormone-releasing hormone (LHRH) in benign prostatic hyperplasia (BPH) as potential molecular targets for therapy with LHRH antagonist cetrorelix.

    Science.gov (United States)

    Rozsa, Bernadett; Nadji, Mehrdad; Schally, Andrew V; Dezso, Balazs; Flasko, Tibor; Toth, Gyorgy; Mile, Melinda; Block, Norman L; Halmos, Gabor

    2011-04-01

    The majority of men will develop symptoms of benign prostatic hyperplasia (BPH) after 70 years of age. Various studies indicate that antagonists of LHRH, such as cetrorelix, exert direct inhibitory effects on BPH mediated by specific LHRH receptors. Our aim was to investigate the mRNA for LHRH and LHRH receptors and the expression of LHRH receptors in specimens of human BPH. The expression of mRNA for LHRH (n=35) and LHRH receptors (n=55) was investigated by RT-PCR in surgical specimens of BPH, using specific primers. The characteristics of binding sites for LHRH on 20 samples were determined by ligand competition assays. The LHRH receptor expression was also examined in 64 BPH specimens by immunohistochemistry. PCR products for LHRH were found in 18 of 35 (51%) BPH tissues and mRNA for LHRH receptors was detected in 39 of 55 (71%) BPH specimens. Eighteen of 20 (90%) samples showed a single class of high affinity binding sites for [D-Trp(6) ]LHRH with a mean K(d) of 4.04 nM and a mean B(max) of 527.6 fmol/mg membrane protein. LHRH antagonist cetrorelix showed high affinity binding to LHRH receptors in BPH. Positive immunohistochemical reaction for LHRH receptors was present in 42 of 64 (67%) BPH specimens. A high incidence of LHRH receptors in BPH supports the use of LHRH antagonists such as cetrorelix, for treatment of patients with lower urinary tract symptoms from BPH. Copyright © 2010 Wiley-Liss, Inc.

  13. Interleukin-1 antagonists for diabetes

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, Thomas

    2013-01-01

    pathways. The testing of specific anti-inflammatory biologics targeting single pro-inflammatory cytokines has provided clinical proof-of-concept. EXPERT OPINION: IL-1 antagonists have so far failed to meet primary end points in recent-onset type 1 diabetes in Phase IIa, and promising Phase I and IIa trials......INTRODUCTION: Diabetes is a currently incurable, epidemically growing global health concern. Contemporary symptomatic treatment targets acute and chronic metabolic consequences of relative or absolute insulin deficiency. Intensive multifactorial therapy is required to attenuate morbidity...... and mortality from late micro- and macrovascular complications, and despite current best clinical practice diabetes is still associated with shortened lifespan. There is an unmet need for interventions targeting pathogenetic mechanisms in diabetes, and the market for such therapies is huge. AREAS COVERED...

  14. Value of the radiolabelled GLP-1 receptor antagonist exendin(9-39) for targeting of GLP-1 receptor-expressing pancreatic tissues in mice and humans

    International Nuclear Information System (INIS)

    Waser, Beatrice; Reubi, Jean Claude

    2011-01-01

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans. Receptor autoradiography studies with 125 I-GLP-1(7-36)amide agonist or 125 I-Bolton-Hunter-exendin(9-39) antagonist radioligands were performed in mice pancreas and insulinomas as well as in human insulinomas; competition experiments were performed in the presence of increasing concentration of GLP-1(7-36)amide or exendin(9-39). The antagonist 125 I-Bolton-Hunter-exendin(9-39) labels mouse pancreatic β-cells and mouse insulinomas, but it does not label human pancreatic β-cells and insulinomas. High affinity displacement (IC 50 approximately 2 nM) is observed in mouse β-cells and insulinomas with either the exendin(9-39) antagonist or GLP-1(7-36)amide agonist. For comparison, the agonist 125 I-GLP-1(7-36)amide intensively labels mouse pancreatic β-cells, mouse insulinoma and human insulinomas; high affinity displacement is observed for the GLP-1(7-36)amide in all tissues; however, a 5 and 20 times lower affinity is found for exendin(9-39) in the mouse and human tissues, respectively. This study reports a species-dependent behaviour of the GLP-1 receptor antagonist exendin(9-39) that can optimally target GLP-1 receptors in mice but not in human tissue. Due to its overly low binding affinity, this antagonist is an inadequate targeting agent for human GLP-1 receptor-expressing tissues, as opposed to the GLP-1 receptor agonist, GLP-1(7-36)amide. (orig.)

  15. Value of the radiolabelled GLP-1 receptor antagonist exendin(9-39) for targeting of GLP-1 receptor-expressing pancreatic tissues in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, P.O. Box 62, Bern (Switzerland)

    2011-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-Bolton-Hunter-exendin(9-39) antagonist radioligands were performed in mice pancreas and insulinomas as well as in human insulinomas; competition experiments were performed in the presence of increasing concentration of GLP-1(7-36)amide or exendin(9-39). The antagonist {sup 125}I-Bolton-Hunter-exendin(9-39) labels mouse pancreatic {beta}-cells and mouse insulinomas, but it does not label human pancreatic {beta}-cells and insulinomas. High affinity displacement (IC{sub 50} approximately 2 nM) is observed in mouse {beta}-cells and insulinomas with either the exendin(9-39) antagonist or GLP-1(7-36)amide agonist. For comparison, the agonist {sup 125}I-GLP-1(7-36)amide intensively labels mouse pancreatic {beta}-cells, mouse insulinoma and human insulinomas; high affinity displacement is observed for the GLP-1(7-36)amide in all tissues; however, a 5 and 20 times lower affinity is found for exendin(9-39) in the mouse and human tissues, respectively. This study reports a species-dependent behaviour of the GLP-1 receptor antagonist exendin(9-39) that can optimally target GLP-1 receptors in mice but not in human tissue. Due to its overly low binding affinity, this antagonist is an inadequate targeting agent for human GLP-1 receptor-expressing tissues, as opposed to the GLP-1 receptor agonist, GLP-1(7-36)amide. (orig.)

  16. Design, synthesis and biological activity of 6-substituted carbamoyl benzimidazoles as new nonpeptidic angiotensin II AT₁ receptor antagonists.

    Science.gov (United States)

    Zhang, Jun; Wang, Jin-Liang; Zhou, Zhi-Ming; Li, Zhi-Huai; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

    2012-07-15

    A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic angiotensin II AT(1) receptor antagonists. The preliminary pharmacological evaluation revealed a nanomolar AT(1) receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6g is an orally active AT(1) receptor antagonist with low toxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

    Science.gov (United States)

    Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

    2016-03-24

    Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

  18. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  19. In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

    Science.gov (United States)

    Dupuis, Arnaud; Heim, Véronique; Ohlmann, Philippe; Gachet, Christian

    2015-12-08

    The P2Y₁₂/ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y₁₂ receptor defect and for evaluating P2Y₁₂ receptor agonists and antagonists. In the absence of suitable anti-P2Y₁₂ antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y₁₂ receptors with [³H]PSB-0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y₁₂ receptor density on cells, as well as the potencies and affinities of test agents at this site. Copyright © 2015 John Wiley & Sons, Inc.

  20. General anaesthesia does not improve outcome in opioid antagonist detoxification treatment: a randomised controlled trial

    NARCIS (Netherlands)

    Jong, C.A.J. de; Laheij, R.J.F.; Krabbe, P.F.M.

    2005-01-01

    Aim  Opioid detoxification by administering opioid-antagonists under general anaesthesia has caused considerable controversy. This study is conducted to determine whether rapid detoxification under general anaesthesia results in higher levels of opioid abstinence than rapid detoxification without

  1. General anaesthesia does not improve outcome in opioid antagonist detoxification treatment : a randomized controlled trial

    NARCIS (Netherlands)

    De Jong, Cor A J; Laheij, Robert J F; Krabbe, Paul F M

    AIM: Opioid detoxification by administering opioid-antagonists under general anaesthesia has caused considerable controversy. This study is conducted to determine whether rapid detoxification under general anaesthesia results in higher levels of opioid abstinence than rapid detoxification without

  2. General anaesthesia does not improve outcome in opioid antagonist detoxification treatment: a randomized controlled trial.

    NARCIS (Netherlands)

    Jong, C.A.J. de; Laheij, R.J.F.; Krabbe, P.F.M.

    2005-01-01

    AIM: Opioid detoxification by administering opioid-antagonists under general anaesthesia has caused considerable controversy. This study is conducted to determine whether rapid detoxification under general anaesthesia results in higher levels of opioid abstinence than rapid detoxification without

  3. Application of pressure ultrafiltration in determining the binding capacity of drugs to human albumin and to plasma proteins of intact and irradiated rat females

    International Nuclear Information System (INIS)

    Zima, M.

    1976-01-01

    The significance of the binding of drugs to plasma proteins has repeatedly been demonstrated and draws the interest of many pharmacologists. The described experiments served to study the binding of isoniazid (INH) to human albumin of various dilution and to whole plasma proteins of irradiated (on the Oth, 3rd and 6th day after exposure to 154.8 mC/kg=600 R) and non-irradiated rats using the technique of modified accelerated ultrafiltration through cellophane. The total characteristics of the binding and its changes were demonstrated by the equilibrium constant, the numbers of binding sites and the changes of free binding energy. The results show that the dilution of human albumin affects the strength of the INH binding on this albumin and further that the normally weak INH binding is diminished even more in irradiated rats. This cannot be explained by the change in the percentage composition of the rat plasma. (author)

  4. Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

    Science.gov (United States)

    Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

    2015-11-01

    In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach.

    Directory of Open Access Journals (Sweden)

    Sneha P

    Full Text Available Maturity-onset diabetes of the young type 3 (MODY3 is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A with MODY3. Missense mutations in the POU homeodomain (POUH of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203 in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD simulations (50ns revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important

  6. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

    Directory of Open Access Journals (Sweden)

    M.H. Sonobe

    2007-01-01

    Full Text Available A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test. In this study, the serum titers (values between 1.0 and 19.5 IU measured by a modified TOBI test (Modi-TOBI test and toxin neutralization assays were correlated (P < 0.0001. Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001 and for diphtheria (r² = 0.93, P < 0.0001 between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  7. Semi-empirical spectrophotometric (SESp) method for the indirect determination of the ratio of cationic micellar binding constants of counterions X⁻ and Br⁻(K(X)/K(Br)).

    Science.gov (United States)

    Khan, Mohammad Niyaz; Yusof, Nor Saadah Mohd; Razak, Norazizah Abdul

    2013-01-01

    The semi-empirical spectrophotometric (SESp) method, for the indirect determination of ion exchange constants (K(X)(Br)) of ion exchange processes occurring between counterions (X⁻ and Br⁻) at the cationic micellar surface, is described in this article. The method uses an anionic spectrophotometric probe molecule, N-(2-methoxyphenyl)phthalamate ion (1⁻), which measures the effects of varying concentrations of inert inorganic or organic salt (Na(v)X, v = 1, 2) on absorbance, (A(ob)) at 310 nm, of samples containing constant concentrations of 1⁻, NaOH and cationic micelles. The observed data fit satisfactorily to an empirical equation which gives the values of two empirical constants. These empirical constants lead to the determination of K(X)(Br) (= K(X)/K(Br) with K(X) and K(Br) representing cationic micellar binding constants of counterions X and Br⁻). This method gives values of K(X)(Br) for both moderately hydrophobic and hydrophilic X⁻. The values of K(X)(Br), obtained by using this method, are comparable with the corresponding values of K(X)(Br), obtained by the use of semi-empirical kinetic (SEK) method, for different moderately hydrophobic X. The values of K(X)(Br) for X = Cl⁻ and 2,6-Cl₂C6H₃CO₂⁻, obtained by the use of SESp and SEK methods, are similar to those obtained by the use of other different conventional methods.

  8. Striatal pre- and postsynaptic profile of adenosine A(2A receptor antagonists.

    Directory of Open Access Journals (Sweden)

    Marco Orru

    2011-01-01

    Full Text Available Striatal adenosine A(2A receptors (A(2ARs are highly expressed in medium spiny neurons (MSNs of the indirect efferent pathway, where they heteromerize with dopamine D(2 receptors (D(2Rs. A(2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1 receptors (A(1Rs. It has been hypothesized that postsynaptic A(2AR antagonists should be useful in Parkinson's disease, while presynaptic A(2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261 showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2AR-D(2R and A(1R-A(2AR heteromers to determine possible differences in the affinity of these compounds for different A(2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2AR when co-expressed with D(2R than with A(1R. KW-6002 showed the best relative affinity for A(2AR co-expressed with D(2R than co-expressed with A(1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile

  9. Antagonistic Phenomena in Network Dynamics

    Science.gov (United States)

    Motter, Adilson E.; Timme, Marc

    2018-03-01

    Recent research on the network modeling of complex systems has led to a convenient representation of numerous natural, social, and engineered systems that are now recognized as networks of interacting parts. Such systems can exhibit a wealth of phenomena that not only cannot be anticipated from merely examining their parts, as per the textbook definition of complexity, but also challenge intuition even when considered in the context of what is now known in network science. Here, we review the recent literature on two major classes of such phenomena that have far-reaching implications: (a) antagonistic responses to changes of states or parameters and (b) coexistence of seemingly incongruous behaviors or properties - both deriving from the collective and inherently decentralized nature of the dynamics. They include effects as diverse as negative compressibility in engineered materials, rescue interactions in biological networks, negative resistance in fluid networks, and the Braess paradox occurring across transport and supply networks. They also include remote synchronization, chimera states, and the converse of symmetry breaking in brain, power-grid, and oscillator networks as well as remote control in biological and bioinspired systems. By offering a unified view of these various scenarios, we suggest that they are representative of a yet broader class of unprecedented network phenomena that ought to be revealed and explained by future research.

  10. Beta 1- and beta 2-adrenergic 125I-pindolol binding sites in the interpeduncular nucleus of the rat: Normal distribution and the effects of deafferentation

    International Nuclear Information System (INIS)

    Battisti, W.P.; Artymyshyn, R.P.; Murray, M.

    1989-01-01

    The plasticity of the beta 1- and beta 2-adrenergic receptor subtypes was examined in the interpeduncular nucleus (IPN) of the adult rat. The beta-adrenergic receptor antagonist 125I-pindolol (125I-PIN) was used in conjunction with the selective subtype antagonists ICI 118,551 and ICI 89,406 to determine the subnuclear distribution of beta 1- and beta 2-adrenergic receptors in this nucleus and to correlate the receptor distribution with the distribution of both noradrenergic afferents from the locus coeruleus (LC) and non-noradrenergic afferents from the fasiculus retroflexus (FR). The density of these binding sites was examined following lesions that decreased (LC lesions) or increased (FR lesions) the density of the noradrenergic projection in the IPN. Quantitative radioautography indicated that beta 1-labeled binding sites account for the larger percentage of binding sites in the IPN. The beta 1-binding sites are densest in those subnuclei that receive a noradrenergic projection from the LC: the central, rostral, and intermediate subnuclei. beta 1-binding sites are algo homogeneously distributed throughout the lateral subnuclei, where there is no detectable noradrenergic innervation. beta 2-binding sites have a more restricted distribution. They are concentrated in the ventral half of the lateral subnuclei, where they account for 70% of total 125I-PIN binding sites. beta 2-binding sites are also present along the ventral border of the IPN. Some of this labeling extends into the central and intermediate subnuclei. Bilateral lesions of the LC, which selectively remove noradrenergic innervation to the IPN, result in an increase in the beta 1-binding sites. Bilateral lesions of the FR, which remove the major cholinergic and peptidergic input from the IPN, elicit an increase in noradrenergic projections and a decrease in beta 1-binding sites

  11. Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

    Science.gov (United States)

    Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

    2011-03-01

    Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  12. Gentamicin binds to the megalin receptor as a competitive inhibitor using the common ligand binding motif of complement type repeats

    DEFF Research Database (Denmark)

    Dagil, Robert; O'Shea, Charlotte; Nykjær, Anders

    2013-01-01

    megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described...... to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists....

  13. Evidence that the angiotensin at 2-receptor agonist compound 21 is also a low affinity thromboxane TXA2-receptor antagonist

    DEFF Research Database (Denmark)

    Fredgart, M.; Leurgans, T.; Stenelo, M.

    2015-01-01

    Objective: The objective of this study was to test whether Compound 21 (C21), a high-affinity, non-peptide angiotensinAT2-receptor agonist, is also an antagonist of thromboxane A2 (TXA2) receptors thus reducing both vasoconstriction and platelet aggregation. Design and method: Binding of C21...... to the TXA2 receptor was determined by TBXA2R Arrestin Biosensor Assay. Mouse mesenteric arteries were mounted in wire myographs, and responses to increasing concentrations of C21 (1nM- 10muM) were recorded during submaximal contractions with 0.1muM U46619 (TXA2 analogue) or 1muMphenylephrine. To control for......AT2-receptor specificity, arteries were pre-incubated with the AT2-receptor antagonist PD123319 (10muM), or mesenteric arteries from AT2-receptor knock-out (AT2R-/y) mice were used. An inhibitory effect of C21 (100nM - 10muM) on U46619 (0,3muM) induced platelet aggregation was examined in whole human...

  14. Human Interleukine-1 receptor antagonist:Cloning, Expression and Optimization in E.coli Host

    Directory of Open Access Journals (Sweden)

    Gh. Barati

    2014-07-01

    Full Text Available Introduction & Objective: Interleukine-1 receptor antagonist (IL-1RA is a powerful anti-inflammatory cytokine which limits the biological effects of IL-1. Due to structural similarity between IL-1 and its antagonist, IL-1RA competitively binds to IL-1 receptor which leads to no signal transduction. Therefore , it is applied in the treatment of patients with inflammatory diseases such as Rheumatoid Arthritis. The aim of this study is cloning, expression and op-timization of IL-1RA in E. coli. Materials & Methods: In this experimental study synthetically prepared cDNA was amplified by PCR. After double digestion with NdeI and XhoI restriction enzymes, this gene was cloned in pET28a expression vector. Expression of desired gene was analyzed at RNA level by RT-PCR and at protein level by SDS-PAGE and followed by western blot to confirm SDS-PAGE results. Optimization of recombinant protein expression was performed in dif-ferent IPTG concentrations and harvesting times after induction. Results: The presence of gene in pET28a was determined by colony-PCR and confirmed by restriction digestion. Transcription of cloned gene and expression of high yield recombinant protein were shown by RT-PCR and SDS-PAGE, respectively. The result of SDS-PAGE was confirmed by western blot. Expression was optimized in different induction time and IPTG concentrations Conclusion: The result of this study demonstrated expression of this recombinant protein at high level in E.coli system by pET28a expression vector. This study also showed a direct as-sociation between the increased level of expression and time of induction . Therefore, an overnight induction time with 0.1 mM IPTG concentration is recommended for a high level expression. (Sci J Hamadan Univ Med Sci 2014; 21 (2:145-151

  15. Structural aspects of inotropic bipyridine binding. Crystal structure determination to 1.9 A of the human serum transthyretin-milrinone complex.

    Science.gov (United States)

    Wojtczak, A; Luft, J R; Cody, V

    1993-03-25

    The crystal structure of human transthyretin (TTR) complexed with milrinone (2-methyl-5-cyano-3,4'-bipyridin-6(1H)-one), a positive inotropic cardiac agent, has been refined to R = 17.4% for 8-1.9-A resolution data. This report provides the first detailed description of protein interactions for an inotropic bipyridine agent which is an effective thyroid hormone binding competitor to transthyretin. Milrinone is bound along the 2-fold axis in the binding site with its substituted pyridone ring located deep within the channel of the two identical binding domains of the TTR tetramer. In this orientation the 5-cyano group occupies the same site as the 3'-iodine in the TTR complex with 3,3'-diiodothyronine (Wojtczak, A., Luft, J., and Cody, V. (1992) J. Biol. Chem. 267, 353-357), which is 3.5 A deeper in the channel than thyroxine (Blake, C. C. F., and Oately, S. J., (1977) Nature 268, 115-120). These structural results confirm computer modeling studies of milrinone structural homology with thyroxine and its TTR binding interactions and explain the effectiveness of milrinone competition for thyroxine binding to TTR. To understand the weaker binding affinity of the parent inotropic drug, amrinone (5-amino-3,4'-bipyridin-6(1H)-one), modeling studies of its TTR binding were carried out which indicate that the 5-amino group cannot participate in strong interactions with TTR and the lack of the 2-methyl further weakens amrinone binding.

  16. Nonpeptidic angiotensin II AT₁ receptor antagonists derived from 6-substituted aminocarbonyl and acylamino benzimidazoles.

    Science.gov (United States)

    Zhang, Jun; Wang, Jin-Liang; Yu, Wei-Fa; Zhou, Zhi-Ming; Tao, Wen-Chang; Wang, Yi-Cheng; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

    2013-11-01

    Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12 showed nanomolar AT₁ receptor binding affinity and high AT₁ receptor selectivity over AT₂ receptor in a preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT₁ IC₅₀ = 3 nM, AT₂ IC₅₀ > 10,000 nM, PA₂ = 8.51) and 11g (AT₁ IC₅₀ = 0.1 nM, AT₂ IC₅₀ = 149 nM, PA₂ = 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addition, they were orally active AT₁ receptor antagonists in spontaneous hypertensive rats. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Growth Hormone Receptor Antagonist Treatment Reduces Exercise Performance in Young Males

    DEFF Research Database (Denmark)

    Goto, K.; Doessing, S.; Nielsen, R.H.

    2009-01-01

    between the groups in terms of changes in serum free fatty acids, glycerol, (V) over dotO(2), or relative fat oxidation. Conclusion: GH might be an important determinant of exercise capacity during prolonged exercise, but GHR antagonist did not alter fat metabolism during exercise. (J Clin Endocrinol......Context: The effects of GH on exercise performance remain unclear. Objective: The aim of the study was to examine the effects of GH receptor (GHR) antagonist treatment on exercise performance. Design: Subjects were treated with the GHR antagonist pegvisomant or placebo for 16 d. After the treatment...... period, they exercised to determine exercise performance and hormonal and metabolic responses. Participants: Twenty healthy males participated in the study. Intervention: Subjects were treated with the GHR antagonist (n = 10; 10 mg/d) or placebo (n = 10). After the treatment period, they performed...

  18. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Genhai; Jensen, R.G. (Univ. of Arizona, Tucson (USA))

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  19. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chunmao [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China); Yu, You [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Jiang, Yongqiang, E-mail: jiangyq@bmi.ac.cn [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China)

    2015-10-23

    Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. We speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.

  20. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

    International Nuclear Information System (INIS)

    Zhang, Chunmao; Yu, You; Yang, Maojun; Jiang, Yongqiang

    2015-01-01

    Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. We speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.

  1. Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

    Science.gov (United States)

    Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

    2008-12-01

    The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

  2. Calcium antagonists for aneurysmal subarachnoid haemorrhage

    NARCIS (Netherlands)

    Dorhout Mees, S. M.; Rinkel, G. J. E.; Feigin, V. L.; Algra, A.; van den Bergh, W. M.; Vermeulen, M.; van Gijn, J.

    2007-01-01

    BACKGROUND: Secondary ischaemia is a frequent cause of poor outcome in patients with subarachnoid haemorrhage (SAH). Its pathogenesis has been incompletely elucidated, but vasospasm probably is a contributing factor. Experimental studies have suggested that calcium antagonists can prevent or reverse

  3. 125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors

    International Nuclear Information System (INIS)

    Linden, J.; Patel, A.; Earl, C.Q.; Craig, R.H.; Daluge, S.M.

    1988-01-01

    A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine derivatives were labeled with 125 I and evaluated as A1 receptor radioligands. The new radioligands bound to A1 receptors with KD values of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies with these antagonists and isotope dilution with the agonist [ 125 I]N6-(4-amino-3-iodobenzyl)adenosine, multiple agonist affinity states of A1 receptors have been identified

  4. Using docking and alchemical free energy approach to determine the binding mechanism of eEF2K inhibitors and prioritizing the compound synthesis.

    Science.gov (United States)

    Wang, Qiantao; Edupuganti, Ramakrishna; Tavares, Clint D J; Dalby, Kevin N; Ren, Pengyu

    2015-01-01

    A-484954 is a known eEF2K inhibitor with submicromolar IC50 potency. However, the binding mechanism and the crystal structure of the kinase remains unknown. Here, we employ a homology eEF2K model, docking and alchemical free energy simulations to probe the binding mechanism of eEF2K, and in turn, guide the optimization of potential lead compounds. The inhibitor was docked into the ATP-binding site of a homology model first. Three different binding poses, hypothesis 1, 2, and 3, were obtained and subsequently applied to molecular dynamics (MD) based alchemical free energy simulations. The calculated relative binding free energy of the analogs of A-484954 using the binding pose of hypothesis 1 show a good correlation with the experimental IC50 values, yielding an r (2) coefficient of 0.96 after removing an outlier (compound 5). Calculations using another two poses show little correlation with experimental data, (r (2) of less than 0.5 with or without removing any outliers). Based on hypothesis 1, the calculated relative free energy suggests that bigger cyclic groups, at R1 e.g., cyclobutyl and cyclopentyl promote more favorable binding than smaller groups, such as cyclopropyl and hydrogen. Moreover, this study also demonstrates the ability of the alchemical free energy approach in combination with docking and homology modeling to prioritize compound synthesis. This can be an effective means of facilitating structure-based drug design when crystal structures are not available.

  5. Determination of total arsenic using a novel Zn-ferrite binding gel for DGT techniques: Application to the redox speciation of arsenic in river sediments.

    Science.gov (United States)

    Gorny, Josselin; Lesven, Ludovic; Billon, Gabriel; Dumoulin, David; Noiriel, Catherine; Pirovano, Caroline; Madé, Benoît

    2015-11-01

    A new laboratory-made Zn-ferrite (ZnFe2O4) binding gel is fully tested using Diffusive Gradient in Thin films (DGT) probes to measure total As [including inorganic As(III) and As(V), as well as MonoMethyl Arsenic Acid (MMAA(V)) and DiMethyl Arsenic Acid (DMAA(V))] in river waters and sediment pore waters. The synthesis of the binding gel is easy, cheap and its insertion into the acrylamide gel is not problematic. An important series of triplicate tests have been carried out to validate the use of the Zn-ferrite binding gel in routine for several environmental matrixes studies, in order to test: (i) the effect of pH on the accumulation efficiency of inorganic As species; (ii) the reproducibility of the results; (iii) the accumulation efficiency of As species; (iv) the effects of the ionic strength and possible competitive anions; and (v) the uptake and the elution efficiency of As species after accumulation in the binding gel. All experimental conditions have been reproduced using two other existing binding gels for comparison: ferrihydrite and Metsorb® HMRP 50. We clearly demonstrate that the Zn-ferrite binding gel is at least as good as the two other binding gels, especially for pH values higher than 8. In addition, by taking into consideration the diffusion rates of As(III) and As(V) in the gel, combining the 3-mercaptopropyl [accumulating only As(III)] with the Zn-ferrite binding gels allows for performing speciation studies. An environmental study along the Marque River finally illustrates the ability of the new binding gel to be used for field studies. Copyright © 2015. Published by Elsevier B.V.

  6. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor α subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    International Nuclear Information System (INIS)

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M.

    1991-01-01

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) αsubunit forms a binding site for α-bungarotoxin (α-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR α1 subunits were tested for their ability to bind 125 I-α-BTX, and differences in α-BTX affinity were determined by using solution (IC 50 s) and solid-phase (K d s) assays. Panels of overlapping peptides corresponding to the complete α1 subunit of mouse and human were also tested for α-BTX binding, but other sequence segments forming the α-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased α-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/α1-subunit interface a vicinal disulfide bound was not required for α-BTX binding

  7. Carbobenzoxy amino acids: Structural requirements for cholecystokinin receptor antagonist activity

    International Nuclear Information System (INIS)

    Maton, P.N.; Sutliff, V.E.; Jensen, R.T.; Gardner, J.D.

    1985-01-01

    The authors used dispersed acini prepared from guinea pig pancreas to examine 28 carbobenzoxy (CBZ) amino acids for their abilities to function as cholecystokinin receptor antagonists. All amino acid derivatives tested, except for CBZ-alanine, CBZ-glycine, and N alpha-CBZ- lysine, were able to inhibit the stimulation of amylase secretion caused by the C-terminal octapeptide of cholecystokinin. In general, there was a good correlation between the ability of a carbobenzoxy amino acid to inhibit stimulated amylase secretion and the ability of the amino acid derivative to inhibit binding of 125 I-cholecystokinin. The inhibition of cholecystokinin-stimulated amylase secretion was competitive, fully reversible, and specific for those secretagogues that interact with the cholecystokinin receptor. The potencies with which the various carbobenzoxy amino acids inhibited the action of cholecystokinin varied 100-fold and CBZ-cystine was the most potent cholecystokinin receptor antagonist. This variation in potency was primarily but not exclusively a function of the hydrophobicity of the amino acid side chain

  8. Genetically determined serum levels of mannose-binding lectin correlate negatively with common carotid intima-media thickness in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Garred, Peter; Christiansen, Buris

    2010-01-01

    Patients with systemic lupus erythematosus (SLE) have excess cardiovascular morbidity and mortality due to accelerated atherosclerosis that cannot be attributed to traditional cardiovascular risk factors alone. Variant alleles of the mannose-binding lectin gene (MBL2) causing low serum...

  9. Functional antagonistic properties of clozapine at the 5-HT3 receptor.

    Science.gov (United States)

    Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

    1996-08-23

    The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

  10. Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions

    Energy Technology Data Exchange (ETDEWEB)

    MacArthur, Stewart; Li, Xiao-Yong; Li, Jingyi; Brown, James B.; Chu, Hou Cheng; Zeng, Lucy; Grondona, Brandi P.; Hechmer, Aaron; Simirenko, Lisa; Keranen, Soile V.E.; Knowles, David W.; Stapleton, Mark; Bickel, Peter; Biggin, Mark D.; Eisen, Michael B.

    2009-05-15

    BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.

  11. Synthesis, CMC Determination, Antimicrobial Activity and Nucleic Acid Binding of A Surfactant Copper(II) Complex Containing Phenanthroline and Alanine Schiff-Base.

    Science.gov (United States)

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-03-01

    A new water-soluble surfactant copper(II) complex [Cu(sal-ala)(phen)(DA)] (sal-ala = salicylalanine, phen = 1,10-phenanthroline, DA = dodecylamine), has been synthesized and characterized by physico-chemical and spectroscopic methods. The critical micelle concentration (CMC) values of this surfactant-copper(II) complex in aqueous solution were obtained from conductance measurements. Specific conductivity data (at 303, 308, 313. 318 and 323 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔG(0)m, ΔH(0)m and ΔS(0)m). The interaction of this complex with nucleic acids (DNA and RNA) has been explored by using electronic absorption spectral titration, competitive binding experiment, cyclic voltammetry, circular dichroism (CD) spectra, and viscosity measurements. Electronic absorption studies have revealed that the complex can bind to nucleic acids by the intercalative binding mode which has been verified by viscosity measurements. The DNA binding constants have also been calculated (Kb = 1.2 × 10(5) M(-1) for DNA and Kb = 1.6 × 10(5) M(-1) for RNA). Competitive binding study with ethidium bromide (EB) showed that the complex exhibits the ability to displace the DNA-bound-EB indicating that the complex binds to DNA in strong competition with EB for the intercalative binding site. The presence of hydrophobic ligands, alanine Schiff-base, phenanthroline and long aliphatic chain amine in the complex were responsible for this strong intercalative binding. The surfactant-copper (II) complex was screened for its antibacterial and antifungal activities against various microorganisms. The results were compared with the standard drugs, amikacin(antibacterial) and ketokonazole(antifungal).

  12. High-Throughput Screening of Small Molecules Identifies Hepcidin Antagonists

    Science.gov (United States)

    Fung, Eileen; Sugianto, Priscilla; Hsu, Jason; Damoiseaux, Robert; Ganz, Tomas

    2013-01-01

    Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)–approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC50 in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction. PMID:23292796

  13. Studies on the antagonistic action between chloramphenicol and quinolones with presence of bovine serum albumin by fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liu Baosheng, E-mail: lbs@hbu.edu.c [Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Center of Physics and Chemistry, Hebei University, Baoding 071002 (China); Zhao Fengli; Xue Chunli; Wang Jing; Lu Yunkai [Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Center of Physics and Chemistry, Hebei University, Baoding 071002 (China)

    2010-05-15

    Chloramphenicol (CHL) and quinolone drugs like ofloxacin (OFLX), lomefloxacin (LMX) and ciprofloxacin (CPFX) can all quench the fluorescence of bovine serum albumin (BSA) in the aqueous solution of pH=7.40. This quenching effect becomes more significant when CHL and quinolone drugs coexist. Based on this, further studies on the interactions between CHL and quinolone drugs using fluorescence spectrum are established. The results showed that the interaction between the drugs would increase the binding constant and binding stability of the drug and protein, thus reducing the amount of drugs transported to their targets. Therefore, free drug concentration at targets would decrease, reducing the efficacy of the drugs. It indicated that there exists antagonistic action between drugs. The results also showed that the quenching mechanism of BSA by the drugs is a static procedure. The number of binding sites is 1 in various systems. Due to the existence of the antagonistic action between drugs, the binding distance r is reduced. Studies utilizing synchronous spectra showed that the antagonistic action between the drugs would affect the conformation of BSA, making protein molecules extend and hydrophobic decrease. The order of antagonistic action between CHL and quinolone drugs is: CPFX>OFLX>LMX with presence of BSA.

  14. Studies on the antagonistic action between chloramphenicol and quinolones with presence of bovine serum albumin by fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Liu Baosheng; Zhao Fengli; Xue Chunli; Wang Jing; Lu Yunkai

    2010-01-01

    Chloramphenicol (CHL) and quinolone drugs like ofloxacin (OFLX), lomefloxacin (LMX) and ciprofloxacin (CPFX) can all quench the fluorescence of bovine serum albumin (BSA) in the aqueous solution of pH=7.40. This quenching effect becomes more significant when CHL and quinolone drugs coexist. Based on this, further studies on the interactions between CHL and quinolone drugs using fluorescence spectrum are established. The results showed that the interaction between the drugs would increase the binding constant and binding stability of the drug and protein, thus reducing the amount of drugs transported to their targets. Therefore, free drug concentration at targets would decrease, reducing the efficacy of the drugs. It indicated that there exists antagonistic action between drugs. The results also showed that the quenching mechanism of BSA by the drugs is a static procedure. The number of binding sites is 1 in various systems. Due to the existence of the antagonistic action between drugs, the binding distance r is reduced. Studies utilizing synchronous spectra showed that the antagonistic action between the drugs would affect the conformation of BSA, making protein molecules extend and hydrophobic decrease. The order of antagonistic action between CHL and quinolone drugs is: CPFX>OFLX>LMX with presence of BSA.

  15. NMDA receptor antagonists inhibit catalepsy induced by either dopamine D1 or D2 receptor antagonists.

    Science.gov (United States)

    Moore, N A; Blackman, A; Awere, S; Leander, J D

    1993-06-11

    In the present study, we investigated the ability of NMDA receptor antagonists to inhibit catalepsy induced by haloperidol, or SCH23390 and clebopride, selective dopamine D1 and D2 receptor antagonists respectively. Catalepsy was measured by recording the time the animal remained with its forepaws placed over a rod 6 cm above the bench. Pretreatment with either the non-competitive NMDA receptor antagonist, MK-801 (0.25-0.5 mg/kg i.p.) or the competitive antagonist, LY274614 (10-20 mg/kg i.p.) reduced the cataleptic response produced by haloperidol (10 mg/kg), SCH23390 (2.5-10 mg/kp i.p.) or clebopride (5-20 mg/kg i.p.). This demonstrates that NMDA receptor antagonists will reduce both dopamine D1 and D2 receptor antagonist-induced catalepsy. Muscle relaxant doses of chlordiazepoxide (10 mg/kg i.p.) failed to reduce the catalepsy induced by haloperidol, suggesting that the anticataleptic effect of the NMDA receptor antagonists was not due to a non-specific action. These results support the hypothesis that NMDA receptor antagonists may have beneficial effects in disorders involving reduced dopaminergic function, such as Parkinson's disease.

  16. Different behaviour of radioiodinated human recombinant interleukin-1 and its receptor antagonist in an animal model of infection

    International Nuclear Information System (INIS)

    Laken, C.J. van der; Boerman, O.C.; Oyen, W.J.G.; Ven, M.T.P. van den; Claessens, R.A.M.J.; Meer, J.W.M. van der; Corstens, F.H.M.

    1996-01-01

    Recently, we demonstrated that radiolabelled interleukin-1α (IL-1) specifically accumulates in focal infection in mice through interaction with its receptor. Unfortunately, systemic side-effects of IL-1 limit its clinical application. We investigated whether this problem could be circumvented by using the interleukin-1 receptor antagonist (IL-1ra), an equally sized protein that binds to the same receptors as IL-1 without induction of biological effects. Biodistribution of 125 I-IL-1 and 125 I-IL-1ra was determined in Swiss mice with Staphylococcus aureus-induced abscesses in the left calf muscle at 4, 12, 24 and 48 h after injection of either 0.4 MBq 125 I-IL-1 or 0.4 MBq 125 I-IL-1ra. In vitro, the proteins displayed similar binding characteristics. High-performance liquid chromatographic analysis revealed a tendency for IL-1ra to associate with serum proteins. Both proteins rapidly cleared from most organs. However, the abscess uptake of 125 I-IL-1ra was significantly lower than that of 125 I-IL-1 at all time points (48 h p.i.: 0.06±0.01%ID/g vs 0.60±0.04%ID/g; P 125 I-IL-1ra, while the ratios for 125 I-IL-1 reached 46.9±5.7 at 48 h p.i. Despite similar in vitro receptor binding, the abscess uptake of IL-1ra was much lower than that of IL-1. The interaction of IL-1ra with serum proteins in vivo may reduce its availability for receptor binding in the infection. Although on theoretical grounds IL-1ra is very interesting, these characteristics will prevent its development as a clinically useful radiopharmaceutical to image infection. (orig.). With 4 figs., 2 tabs

  17. Synthesis and biological evaluation of guanylhydrazone coactivator binding inhibitors for the estrogen receptor.

    Science.gov (United States)

    LaFrate, Andrew L; Gunther, Jillian R; Carlson, Kathryn E; Katzenellenbogen, John A

    2008-12-01

    Most patients with hormone-responsive breast cancer eventually develop resistance to traditional antiestrogens such as tamoxifen, and this has become a major obstacle in their treatment. We prepared and characterized the activity of a series of 16 guanylhydrazone small molecules that are designed to block estrogen receptor (ER) activity through a non-traditional mechanism, by directly interfering with coactivator binding to agonist-liganded ER. The inhibitory activity of these compounds was determined in cell-based transcription assays using ER-responsive reporter gene and mammalian two-hybrid assays. Several of the compounds gave IC(50) values in the low micromolar range. Two secondary assays were used to confirm that these compounds were acting through the proposed non-traditional mode of estrogen inhibitory action and not as conventional antagonists at the ligand binding site.

  18. Determining time-weighted average concentrations of nitrate and ammonium in freshwaters using DGT with ion exchange membrane-based binding layers

    DEFF Research Database (Denmark)

    Huang, Jianyin; Bennett, William W.; Welsh, David T.

    2016-01-01

    Commercially-available AMI-7001 anion exchange and CMI-7000 cation exchange membranes were utilised as binding layers for DGT measurements of NO3-N and NH4-N in freshwaters. These ion exchange membranes are easier to prepare and handle than DGT binding layers consisting of hydrogels cast with ion...... exchange resins. The membranes showed good uptake and elution efficiencies for both NO3-N and NH4-N. The membrane-based DGTs are suitable for pH 3.5-8.5 and ionic strength ranges (0.0001-0.014 and 0.0003-0.012 mol L−1 as NaCl for the AMI-7001 and CMI-7000 membrane, respectively) typical of most natural...... freshwaters. The binding membranes had high intrinsic binding capacities for NO3-N and NH4-N of 911 ± 88 μg and 3512 ± 51 μg, respectively. Interferences from the major competing ions for membrane-based DGTs are similar to DGTs employing resin-based binding layers but with slightly different selectivity...

  19. Polyester monomers lack ability to bind and activate both androgenic and estrogenic receptors as determined by in vitro and in silico methods.

    Science.gov (United States)

    Osimitz, Thomas G; Welsh, William J; Ai, Ni; Toole, Colleen

    2015-01-01

    The paper presents results from the screening of seven monomers used by Eastman Chemical to make various polymers. Ethylene glycol, diethylene glycol, polytetramethylene glycol, isophthalic acid, monosodium-5-sulfoisophthalic acid, 1,4-cyclohexanedicarboxylic acid, and dimethylcyclohexanedicarboxylate were screened for potential androgenicity or estrogenicity. The following studies were conducted: QSAR for binding to the AR and ER, in vitro Androgen Receptor Binding Assay, in vitro Estrogen Receptor Binding Assays (alpha and beta isoforms), in vitro Androgen Receptor Transactivation Assay in human cells, and in vitro Estrogen Receptor Transactivation Assay in human cells. None of the QSAR models predicted that any of the monomers possessed appreciable binding affinity for either AR or ER. Binding assays showed no evidence of interaction with either the AR or the alpha or beta ER receptors. Similarly, the AR and ER transactivation assays were negative. Moreover, six of the seven monomers have been subjected to 13-week and developmental toxicity studies in rats with no androgen- or estrogen-related effects being noted. Given the negative results of the in vitro screening assays (except PMG which demonstrated cytotoxicity) as well as available repeated dose and developmental and reproductive studies, the data suggest that none of the monomers tested exhibit androgenic or estrogenic hazards. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities

    Science.gov (United States)

    Wong, Megan J. Q.; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian

    2016-01-01

    ABSTRACT In host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist, despite significant antagonistic interactions between species, are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through the delivery of toxic effectors. It is well established that intraspecies protection is conferred by immunity proteins that neutralize effector toxicities. In contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS-active antagonistic bacterial species, Aeromonas hydrophila and Vibrio cholerae, to demonstrate that interspecies protection is dependent on effectors. A. hydrophila and V. cholerae do not share conserved immunity genes but could coexist equally in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the competing wild-type strain. Time-lapse microscopic analyses showed that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interactions inside each cluster and restricting the interactions to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulated our experimental observations. These results provide mechanistic insights regarding the general role of microbial weapons in determining the structures of complex multispecies communities. IMPORTANCE Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in

  1. Microbial herd protection mediated by antagonistic interaction in polymicrobial communities.

    Science.gov (United States)

    Wong, Megan; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian; Dong, Tao G

    2016-09-16

    In the host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist - despite significant antagonistic interactions between species - are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through delivery of toxic effectors. It is well established that intra-species protection is conferred by immunity proteins that neutralize effector toxicities. By contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS active antagonistic bacteria, Aeromonas hydrophila (AH) and Vibrio cholerae (VC), to demonstrate that interspecies protection is dependent on effectors. AH and VC do not share conserved immunity genes but could equally co-exist in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the other competing wild type. Time-lapse microscopy analyses show that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interaction inside each cluster and restricting it to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulate our experimental observation. These results provide mechanistic insights for the general role of microbial weapons in determining the structures of complex multispecies communities. Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and exchange of genetic materials

  2. The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

    Science.gov (United States)

    Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

    2016-12-01

    Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  3. Identification of a novel antagonist of the ErbB1 receptor capable of inhibiting migration of human glioblastoma cells

    DEFF Research Database (Denmark)

    Staberg, Mikkel; Riemer, Christian; Xu, Ruodan

    2013-01-01

    B1 targeting peptide, termed Herfin-1, was designed based on a model of the tertiary structure of the EGF-EGFR ternary complex. The binding kinetics of this peptide were determined employing surface plasmon resonance analyses. ErbB1-4 expression and phosphorylation in human glioblastoma cell lines U...... processing. RESULTS: The present study shows that Herfin-1 functions as an ErbB1 antagonist. It binds to the extracellular domain of ErbB1 with a KD value of 361 nM. In U87 and U118 cells, both expressing high levels of ErbB1, Herfin-1 inhibits EGF-induced ErbB1 phosphorylation and cell migration....... Additionally, Herfin-1 was found to increase neurite outgrowth in cerebellar granule neurons, likely through the inhibition of a sustained weak ErbB1 activation. CONCLUSIONS: Targeting the ErbB1 receptor dimerization interface is a promising strategy to inhibit receptor activation in ErbB1-expressing glioma...

  4. Pseudomonas orientalis F9: A Potent Antagonist against Phytopathogens with Phytotoxic Effect in the Apple Flower

    Directory of Open Access Journals (Sweden)

    Veronika Zengerer

    2018-02-01

    Full Text Available In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora, the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

  5. Pseudomonas orientalis F9: A Potent Antagonist against Phytopathogens with Phytotoxic Effect in the Apple Flower.

    Science.gov (United States)

    Zengerer, Veronika; Schmid, Michael; Bieri, Marco; Müller, Denise C; Remus-Emsermann, Mitja N P; Ahrens, Christian H; Pelludat, Cosima

    2018-01-01

    In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora , the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430 Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

  6. Identification of amino acid residues in the ligand-binding domain of the aryl hydrocarbon receptor causing the species-specific response to omeprazole: possible determinants for binding putative endogenous ligands.

    Science.gov (United States)

    Shiizaki, Kazuhiro; Ohsako, Seiichiroh; Kawanishi, Masanobu; Yagi, Takashi

    2014-02-01

    Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

  7. Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

    International Nuclear Information System (INIS)

    Prachayasittikul, Virapong; Isarankura Na Ayudhya, Chartchalerm; Tantimongcolwat, Tanawut; Galla, Hans-Joachim

    2005-01-01

    Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development

  8. Determination of hydrophobic coenzyme a esters and other lipids using a biosensor comprising a modified coenzyme a- and acyl-coa binding protein (acbp)

    DEFF Research Database (Denmark)

    2002-01-01

    , food and feed preparations, tissue extracts, acyl-CoA synthetase reaction media and various laboratory conditions using a modified Coenzyme A- and acyl-CoA binding protein (ACBP) is provided. Furthermore the invention relates to a construct comprising a peptide and a signal moiety for performing...

  9. Interaction of α-cyperone with human serum albumin: Determination of the binding site by using Discovery Studio and via spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Qing; He, Jiawei; Wu, Di; Wang, Jing; Yan, Jin; Li, Hui, E-mail: lihuilab@sina.com

    2015-08-15

    α-Cyperone, as the main constituent of Cyperus rotundus, is a sesquiterpene ketone. In this work, LigandFit and CDOCKER docking programs of Discovery Studio 3.1 were used to preliminarily estimate and further confirm the binding sites of α-cyperone. LigandFit results showed that α-cyperone is mainly bound in subdomain IIA. This finding was further confirmed by CDOCKER results. Site marker competitive experimental results also suggested that α-cyperone contains the same binding site as warfarin. Software simulation results further revealed that α-cyperone is mainly bound in subdomain IIA. Site marker competitive experiment results are consistent with simulation results. 3D fluorescence and CD spectroscopy results indicated that the native conformation of HSA molecule is affected by the presence of α-cyperone. - Highlights: • This work carried out by adopting molecular docking and spectroscopic studies. • Discovery studio 3.1 was used for estimating the binding sites. • The insertion of α-cyperone molecule caused the microenvironment of HSA changed. • The native conformation of HSA was changed during binding with α-cyperone.

  10. Interaction of α-cyperone with human serum albumin: Determination of the binding site by using Discovery Studio and via spectroscopic methods

    International Nuclear Information System (INIS)

    Wang, Qing; He, Jiawei; Wu, Di; Wang, Jing; Yan, Jin; Li, Hui

    2015-01-01

    α-Cyperone, as the main constituent of Cyperus rotundus, is a sesquiterpene ketone. In this work, LigandFit and CDOCKER docking programs of Discovery Studio 3.1 were used to preliminarily estimate and further confirm the binding sites of α-cyperone. LigandFit results showed that α-cyperone is mainly bound in subdomain IIA. This finding was further confirmed by CDOCKER results. Site marker competitive experimental results also suggested that α-cyperone contains the same binding site as warfarin. Software simulation results further revealed that α-cyperone is mainly bound in subdomain IIA. Site marker competitive experiment results are consistent with simulation results. 3D fluorescence and CD spectroscopy results indicated that the native conformation of HSA molecule is affected by the presence of α-cyperone. - Highlights: • This work carried out by adopting molecular docking and spectroscopic studies. • Discovery studio 3.1 was used for estimating the binding sites. • The insertion of α-cyperone molecule caused the microenvironment of HSA changed. • The native conformation of HSA was changed during binding with α-cyperone

  11. The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

    Science.gov (United States)

    Sean, Polen; Nguyen, Joseph H C; Semler, Bert L

    2008-09-01

    Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

  12. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    International Nuclear Information System (INIS)

    Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

    1987-01-01

    In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 μM, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. 45 Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated 45 Ca outflux. BPP was also capable of displacing the specific binding of [ 3 H]-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 μM) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant

  13. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Directory of Open Access Journals (Sweden)

    Jessica L Hastie

    2016-09-01

    Full Text Available σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP. In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme.

  14. Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang; Katritch, Vsevolod; Han, Gye Won; Hanson, Michael A.; Shi, Lei; Newman, Amy Hauck; Javitch, Jonathan A.; Cherezov, Vadim; Stevens, Raymond C. (Cornell); (Scripps); (NIDA); (Columbia); (UCSD); (Receptos)

    2010-11-30

    Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

  15. Antagonistic properties of microogranisms associated with cassava ...

    African Journals Online (AJOL)

    The antagonistic properties of indigenous microflora from cassava starch, flour and grated cassava were investigated using the conventional streak, novel ring and well diffusion methods. Antagonism was measured by zone of inhibition between the fungal plug and bacterial streak/ring. Bacillus species were more effective ...

  16. Small molecule antagonists of integrin receptors.

    Science.gov (United States)

    Perdih, A; Dolenc, M Sollner

    2010-01-01

    The complex and widespread family of integrin receptors is involved in numerous physiological processes, such as tissue remodeling, angiogenesis, development of the immune response and homeostasis. In addition, their key role has been elucidated in important pathological disorders such as cancer, cardiovascular diseases, osteoporosis, autoimmune and inflammatory diseases and in the pathogenesis of infectious diseases, making them highly important targets for modern drug design campaigns. In this review we seek to present a concise overview of the small molecule antagonists of this diverse and highly complex receptor family. Integrin antagonists are classified according to the targeted integrin receptor and are discussed in four sections. First we present the fibrinogen alpha(IIb)beta3 and the vitronectin alpha (V)beta(3) receptor antagonists. The remaining selective integrin antagonists are examined in the third section. The final section is dedicated to molecules with dual or multiple integrin activity. In addition, the use of antibodies and peptidomimetic approaches to modulate the integrin receptors are discussed, as well providing the reader with an overall appreciation of the field.

  17. Guanidine-acylguanidine bioisosteric approach in the design of radioligands: synthesis of a tritium-labeled N(G)-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist.

    Science.gov (United States)

    Keller, Max; Pop, Nathalie; Hutzler, Christoph; Beck-Sickinger, Annette G; Bernhardt, Günther; Buschauer, Armin

    2008-12-25

    Synthesis and characterization of (R)-N(alpha)-(2,2-diphenylacetyl)-N-(4-hydroxybenzyl)-N(omega)-([2,3-(3)H]-propanoyl)argininamide ([(3)H]-UR-MK114), an easily accessible tritium-labeled NPY Y(1) receptor (Y(1)R) antagonist (K(B): 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The radioligand binds with high affinity (K(D), saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells) and selectivity for Y(1)R over Y(2), Y(4), and Y(5) receptors. The title compound is a useful pharmacological tool for the determination of Y(1)R ligand affinities, quantification of Y(1)R binding sites, and autoradiography.

  18. Kinetic properties of 'dual' orexin receptor antagonists at OX1R and OX2R orexin receptors.

    Directory of Open Access Journals (Sweden)

    Gabrielle Elizabeth Callander

    2013-12-01

    Full Text Available Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various ‘dual’ orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [3H]-BBAC ((S-N-([1,1'-biphenyl]-2-yl-1-(2-((1-methyl-1H-benzo[d]imidazol-2-ylthioacetylpyrrolidine-2-carboxamide. In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-ylmethyl-9-(4-methoxypyrimidin-2-yl-2,9-diazaspiro[5.5]undecan-1-one bind rapidly and reach equilibrium very quickly in both binding and / or functional assays. Overall, the dual antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the dual antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.

  19. Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches.

    Science.gov (United States)

    Jiang, Tuo-Ying; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi; Shi, Jie-Hua

    2018-04-01

    Molecular interaction of atenolol, a selective β 1 receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K b ) were determined by the UV-vis absorption titration, and were found to be in the order of 10 3  M -1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH 0 ), entropy change (ΔS 0 ). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.

  20. Genetically determined high serum levels of mannose-binding lectin and agalactosyl IgG are associated with ischemic heart disease in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Garred, Peter; Madsen, Hans O.

    2007-01-01

    Patients with rheumatoid arthritis (RA) have excess morbidity and mortality due to ischemic heart disease. It has been suggested that high serum levels of mannose-binding lectin (MBL) and agalactosyl IgG (IgG-G0) are associated with increased inflammation in RA. MBL also enhances inflammation......-mediated tissue injury during postischemic reperfusion. This study was undertaken to examine whether these factors are associated with increased risk of ischemic heart disease in RA....

  1. Discovery of Natural Products as Novel and Potent FXR Antagonists by Virtual Screening

    Science.gov (United States)

    Diao, Yanyan; Jiang, Jing; Zhang, Shoude; Li, Shiliang; Shan, Lei; Huang, Jin; Zhang, Weidong; Li, Honglin

    2018-04-01

    Farnesoid X receptor (FXR) is a member of nuclear receptor family involved in multiple physiological processes through regulating specific target genes. The critical role of FXR as a transcriptional regulator makes it a promising target for diverse diseases, especially those related to metabolic disorders such as diabetes and cholestasis. However, the underlying activation mechanism of FXR is still a blur owing to the absence of proper FXR modulators. To identify potential FXR modulators, an in-house natural product database (NPD) containing over 4000 compounds was screened by structure-based virtual screening strategy and subsequent hit-based similarity searching method. After the yeast two-hybrid (Y2H) assay, six natural products were identified as FXR antagonists which blocked the CDCA-induced SRC-1 association. The IC50 values of compounds 2a, a diterpene bearing polycyclic skeleton, and 3a, named daphneone with chain scaffold, are as low as 1.29 μM and 1.79 μM, respectively. Compared to the control compound guggulsterone (IC50 = 6.47 μM), compounds 2a and 3a displayed 5-fold and 3-fold higher antagonistic activities against FXR, respectively. Remarkably, the two representative compounds shared low topological similarities with other reported FXR antagonists. According to the putative binding poses, the molecular basis of these antagonists against FXR was also elucidated in this report.

  2. Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor

    Directory of Open Access Journals (Sweden)

    Huanzhong Cui

    2016-10-01

    Full Text Available A series of antagonists specifically targeting growth hormone receptors (GHR in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH. Therefore, in this study, we developed and characterized a porcine GHR (pGHR antibody antagonist (denoted by AN98 via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1 secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

  3. The role of extra-atomic relaxation in determining Si2p binding energy shifts at silicon/silicon oxide interfaces

    International Nuclear Information System (INIS)

    Zhang, K.Z.; Greeley, J.N.; Banaszak Holl, M.M.; McFeely, F.R.

    1997-01-01

    The observed binding energy shift for silicon oxide films grown on crystalline silicon varies as a function of film thickness. The physical basis of this shift has previously been ascribed to a variety of initial state effects (Si endash O ring size, strain, stoichiometry, and crystallinity), final state effects (a variety of screening mechanisms), and extrinsic effects (charging). By constructing a structurally homogeneous silicon oxide film on silicon, initial state effects have been minimized and the magnitude of final state stabilization as a function of film thickness has been directly measured. In addition, questions regarding the charging of thin silicon oxide films on silicon have been addressed. From these studies, it is concluded that initial state effects play a negligible role in the thickness-dependent binding energy shift. For the first ∼30 Angstrom of oxide film, the thickness-dependent binding energy shift can be attributed to final state effects in the form of image charge induced stabilization. Beyond about 30 Angstrom, charging of the film occurs. copyright 1997 American Institute of Physics

  4. Human myometrial adrenergic receptors during pregnancy: identification of the alpha-adrenergic receptor by [3H] dihydroergocryptine binding

    International Nuclear Information System (INIS)

    Jacobs, M.M.; Hayashida, D.; Roberts, J.M.

    1985-01-01

    The radioactive alpha-adrenergic antagonist [ 3 H] dihydroergocryptine binds to particulate preparations of term pregnant human myometrium in a manner compatible with binding to the alpha-adrenergic receptor (alpha-receptor). [ 3 H] Dihydroergocryptine binds with high affinity (KD = 2 nmol/L and low capacity (receptor concentration = 100 fmol/mg of protein). Adrenergic agonists compete for [ 3 H] dihydroergocryptine binding sites stereo-selectively ([-]-norepinephrine is 100 times as potent as [+]-norepinephrine) and in a manner compatible with alpha-adrenergic potencies (epinephrine approximately equal to norepinephrine much greater than isoproterenol). Studies in which prazosin, an alpha 1-antagonist, and yohimbine, and alpha 2-antagonist, competed for [ 3 H] dihydroergocryptine binding sites in human myometrium indicated that approximately 70% are alpha 2-receptors and that 30% are alpha 1-receptors. [ 3 H] dihydroergocryptine binding to human myometrial membrane particulate provides an important tool with which to study the molecular mechanisms of uterine alpha-adrenergic response

  5. antagonistic effect of native bacillus isolates against black root rot

    African Journals Online (AJOL)

    ACSS

    A number of fungi and bacteria are known to be very effective .... Round. Convex. Smooth. Wrinkled. Slow. BS024. Irregular and spreading. Flat. Wavy .... Antibiotic effect of bacterial antagonist ..... antagonistic Bacillus and Trichoderma isolates ...

  6. Extended N-Arylsulfonylindoles as 5-HT6 Receptor Antagonists: Design, Synthesis & Biological Evaluation

    Directory of Open Access Journals (Sweden)

    Gonzalo Vera

    2016-08-01

    Full Text Available Based on a known pharmacophore model for 5-HT6 receptor antagonists, a series of novel extended derivatives of the N-arylsulfonyindole scaffold were designed and identified as a new class of 5-HT6 receptor modulators. Eight of the compounds exhibited moderate to high binding affinities and displayed antagonist profile in 5-HT6 receptor functional assays. Compounds 2-(4-(2-methoxyphenylpiperazin-1-yl-1-(1-tosyl-1H-indol-3-ylethanol (4b, 1-(1-(4-iodophenylsulfonyl-1H-indol-3-yl-2-(4-(2-methoxyphenylpiperazin-1-ylethanol (4g and 2-(4-(2-methoxyphenylpiperazin-1-yl-1-(1-(naphthalen-1-ylsulfonyl-1H-indol-3-ylethanol (4j showed the best binding affinity (4b pKi = 7.87; 4g pKi = 7.73; 4j pKi = 7.83. Additionally, compound 4j was identified as a highly potent antagonist (IC50 = 32 nM in calcium mobilisation functional assay.

  7. Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

    Science.gov (United States)

    Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

    2016-01-01

    CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

  8. AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Terry W. Moody

    2017-07-01

    Full Text Available While peptide antagonists for the gastrin-releasing peptide receptor (BB2R, neuromedin B receptor (BB1R, and bombesin (BB receptor subtype-3 (BRS-3 exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM. AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

  9. AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

    Science.gov (United States)

    Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

    2017-01-01

    While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

  10. AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

    Science.gov (United States)

    Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

    2017-01-01

    While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

  11. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

    Directory of Open Access Journals (Sweden)

    Eva Martínez-Pinilla

    2017-10-01

    Full Text Available The mechanism of action of cannabidiol (CBD, the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs; however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  12. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

    Science.gov (United States)

    Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

    2017-01-01

    The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  13. Displacement of DL-[3H]-2-amino-4-phosphonobutanoic acid ( [3H]APB) binding with methyl-substituted APB analogues and glutamate agonists

    International Nuclear Information System (INIS)

    Robinson, M.B.; Crooks, S.L.; Johnson, R.L.; Koerner, J.F.

    1985-01-01

    The binding of the excitatory amino acid antagonist DL-2-amino-4-phosphonobutanoic acid (DL-APB) to rat brain synaptic plasma membranes was characterized. As determined by Scatchard analysis, the binding was saturable and homogeneous with a Kd = 6.0 microM and Bmax = 380 pmol/mg of protein. The binding was dependent on the presence of Ca 2+ and Cl - ions and was diminished upon freezing. The association rate constant was 6.8 X 10(-3) microM -1 min -1 , and the dissociation rate constant was 2.0 X 10(-2) min -1 . The L isomers of APB, glutamate, and aspartate were more potent as displacers of APB binding than the D isomers. With the exception of kynurenic acid, all compounds examined in both systems were more potent as displacers of APB binding than as inhibitors of synaptic transmission. This difference in potency was most pronounced for agonists at dentate granule cells. L-Glutamate, D-glutamate, and L-glutamate tetrazole were between 140- and 7500-fold more potent as displacers of DL-APB binding than as inhibitors of synaptic transmission. D-2-Amino-5-phosphonopentanoic acid and alpha-methyl-APB were between 10- and 20-fold more potent as displacers of binding

  14. Improved Prediction of Bovine Leucocyte Antigens (BoLA) Presented Ligands by Use of Mass-Spectrometry-Determined Ligand and in Vitro Binding Data

    DEFF Research Database (Denmark)

    Nielsen, Morten; Connelley, Tim; Ternette, Nicola

    2018-01-01

    Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predic...... available at http://www.cbs.dtu.dk/services/NetMHCpan/NetBoLApan . The approach has here been applied to the BoLA-I system, but the pipeline is readily applicable to MHC systems in other species....

  15. Investigation of the Binding Site of CCR2 using 4-Azetidinyl-1-aryl-cyclohexane Derivatives: A Membrane Modeling and Molecular Dynamics Study

    Energy Technology Data Exchange (ETDEWEB)

    Kothandan, Gugan; Gadhe, Changdev G.; Cho, Seung Joo [Chosun Univ., Gwangju (Korea, Republic of)

    2013-11-15

    Chemokine receptor (CCR2) is a G protein-coupled receptor that contains seven transmembrane helices. Recent pharmaceutical research has focused on the antagonism of CCR2 and candidate drugs are currently undergoing clinical studies for the treatment of diseases like arthritis, multiple sclerosis, and type 2 diabetes. In this study, we analyzed the time dependent behavior of CCR2 docked with a potent 4-azetidinyl-1-aryl-cyclohexane (4AAC) derivative using molecular dynamics simulations (MDS) for 20 nanoseconds (ns). Homology modeling of CCR2 was performed and the 4AAC derivative was docked into this binding site. The docked model of selected conformations was then utilized to study the dynamic behavior of the 4AAC enzyme complexes inside lipid membrane. MDS of CCR2-16b of 4AAC complexes allowed us to refine the system since binding of an inhibitor to a receptor is a dynamic process and identify stable structures and better binding modes. Structure activity relationships (SAR) for 4AAC derivatives were investigated and reasons for the activities were determined. Probable binding pose for some CCR2 antagonists were determined from the perspectives of binding site. Initial modeling showed that Tyr49, Trp98, Ser101, Glu291, and additional residues are crucial for 4AAC binding, but MDS analysis showed that Ser101 may not be vital. 4AAC moved away from Ser101 and the hydrogen bonding between 4AAC and Ser101 vanished. The results of this study provide useful information regarding the structure-based drug design of CCR2 antagonists and additionally suggest key residues for further study by mutagenesis.

  16. Inhibition of CPU0213, a Dual Endothelin Receptor Antagonist, on Apoptosis via Nox4-Dependent ROS in HK-2 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2016-06-01

    Full Text Available Background/Aims: Our previous studies have indicated that a novel endothelin receptor antagonist CPU0213 effectively normalized renal function in diabetic nephropathy. However, the molecular mechanisms mediating the nephroprotective role of CPU0213 remain unknown. Methods and Results: In the present study, we first detected the role of CPU0213 on apoptosis in human renal tubular epithelial cell (HK-2. It was shown that high glucose significantly increased the protein expression of Bax and decreased Bcl-2 protein in HK-2 cells, which was reversed by CPU0213. The percentage of HK-2 cells that showed Annexin V-FITC binding was markedly suppressed by CPU0213, which confirmed the inhibitory role of CPU0213 on apoptosis. Given the regulation of endothelin (ET system to oxidative stress, we determined the role of redox signaling in the regulation of CPU0213 on apoptosis. It was demonstrated that the production of superoxide (O2-. was substantially attenuated by CPU0213 treatment in HK-2 cells. We further found that CPU0213 dramatically inhibited expression of Nox4 protein, which gene silencing mimicked the role of CPU0213 on the apoptosis under high glucose stimulation. We finally examined the role of CPU0213 on ET-1 receptors and found that high glucose-induced protein expression of endothelin A and B receptors was dramatically inhibited by CPU0213. Conclusion: Taken together, these results suggest that this Nox4-dependenet O2- production is critical for the apoptosis of HK-2 cells in high glucose. Endothelin receptor antagonist CPU0213 has an anti-apoptosis role through Nox4-dependent O2-.production, which address the nephroprotective role of CPU0213 in diabetic nephropathy.

  17. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  18. Antagonistic parent-offspring co-adaptation.

    Directory of Open Access Journals (Sweden)

    Mathias Kölliker

    2010-01-01

    Full Text Available In species across taxa, offspring have means to influence parental investment (PI. PI thus evolves as an interacting phenotype and indirect genetic effects may strongly affect the co-evolutionary dynamics of offspring and parental behaviors. Evolutionary theory focused on explaining how exaggerated offspring solicitation can be understood as resolution of parent-offspring conflict, but the evolutionary origin and diversification of different forms of family interactions remains unclear.In contrast to previous theory that largely uses a static approach to predict how "offspring individuals" and "parental individuals" should interact given conflict over PI, we present a dynamic theoretical framework of antagonistic selection on the PI individuals obtain/take as offspring and the PI they provide as parents to maximize individual lifetime reproductive success; we analyze a deterministic and a stochastic version of this dynamic framework. We show that a zone for equivalent co-adaptation outcomes exists in which stable levels of PI can evolve and be maintained despite fast strategy transitions and ongoing co-evolutionary dynamics. Under antagonistic co-adaptation, cost-free solicitation can evolve as an adaptation to emerging preferences in parents.We show that antagonistic selection across the offspring and parental life-stage of individuals favors co-adapted offspring and parental behavior within a zone of equivalent outcomes. This antagonistic parent-offspring co-adaptation does not require solicitation to be costly, allows for rapid divergence and evolutionary novelty and potentially explains the origin and diversification of the observed provisioning forms in family life.

  19. Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

    Science.gov (United States)

    Rivier, Jean E; Rivier, Catherine L

    2014-04-01

    Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Radiolabeling with fluorine-18 of a protein, interleukin-1 receptor antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Prenant, C., E-mail: cprenant@cyclopharma.f [Wolfson Molecular Imaging Centre, University of Manchester, Manchester (United Kingdom); Cawthorne, C. [Academic Department of Radiation Oncology, Christie NHS Foundation Trust, Manchester (United Kingdom); Fairclough, M. [Wolfson Molecular Imaging Centre, University of Manchester, Manchester (United Kingdom); Rothwell, N.; Boutin, H. [Faculty of Life Sciences, University of Manchester, Manchester (United Kingdom)

    2010-09-15

    IL-1RA is a naturally occurring antagonist of the pro-inflammatory cytokine interleukin-1 (IL-1) with high therapeutic promise, but its pharmacokinetic remains poorly documented. In this report, we describe the radiolabeling of recombinant human interleukin-1 receptor antagonist (rhIL-1RA) with fluorine-18 to allow pharmacokinetic studies by positron emission tomography (PET). rhIL-1RA was labeled randomly by reductive alkylation of free amino groups (the {epsilon}-amino group of lysine residues or amino-terminal residues) using [{sup 18}F]fluoroacetaldehyde under mild reaction conditions. Radiosyntheses used a remotely controlled experimental rig within 100 min and the radiochemical yield was in the range 7.1-24.2% (decay corrected, based on seventeen syntheses). We showed that the produced [{sup 18}F]fluoroethyl-rhIL-1ra retained binding specificity by conducting an assay on rat brain sections, allowing its pharmakokinetic study using PET.

  1. Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

    Science.gov (United States)

    Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

    2003-01-01

    Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

  2. Medicinal Chemistry of Competitive Kainate Receptor Antagonists

    Science.gov (United States)

    2010-01-01

    Kainic acid (KA) receptors belong to the group of ionotropic glutamate receptors and are expressed throughout in the central nervous system (CNS). The KA receptors have been shown to be involved in neurophysiological functions such as mossy fiber long-term potentiation (LTP) and synaptic plasticity and are thus potential therapeutic targets in CNS diseases such as schizophrenia, major depression, neuropathic pain and epilepsy. Extensive effort has been made to develop subtype-selective KA receptor antagonists in order to elucidate the physiological function of each of the five subunits known (GluK1−5). However, to date only selective antagonists for the GluK1 subunit have been discovered, which underlines the strong need for continued research in this area. The present review describes the structure−activity relationship and pharmacological profile for 10 chemically distinct classes of KA receptor antagonists comprising, in all, 45 compounds. To the medicinal chemist this information will serve as reference guidance as well as an inspiration for future effort in this field. PMID:22778857

  3. Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

    Science.gov (United States)

    Bazzicalupi, Carla; Ferraroni, Marta; Papi, Francesco; Massai, Lara; Bertrand, Benoît; Messori, Luigi; Gratteri, Paola; Casini, Angela

    2016-03-18

    The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Solution structure of the 45-residue ATP-binding peptide of adenylate kinase as determined by 2-D NMR, FTIR, and CD spectroscopy

    International Nuclear Information System (INIS)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, E.M.; Kuby, S.A.; Mildyan, A.S.

    1986-01-01

    In the X-ray structure of adenylate kinase residues 1-45 exist as 47% α-helix, 29% β-structure (strands and turns) and 24% coil. The solution structure of a synthetic peptide corresponding to residues 1-45, which constitutes the MgATP binding site was studied by 3 independent spectroscopic methods. Globularity of the peptide was shown by its broad NMR resonances which narrow upon denaturation, and by its ability to bind MgATP with similar affinity and conformation as the intact enzyme does. COSY and NOESY NMR methods at 250 and 500 MHz reveal proximities among NH, Cα, and Cβ protons indicative of >20% α-helix, and >20% β-structure. Correlation of regions of secondary structure with the primary sequence by 2D NMR indicates at least one α-helix (res. 23 to 29) and two β-strands (res. 12 to 15 and 34 to 38). The broad amide I band in the deconvoluted FTIR spectrum could be fit as the sum of 4 peaks due to specific secondary structures, yielding ≤=45% α-helix, ≤=40% β-structure and ≥=15% coil. The CD spectrum, from 185-250 nm, interpreted with a 3-parameter basis set, yielded 20 +/- 5% α=helix, and ≤=20% β-structure. The solution structure of peptide 1-45 thus approximates that of residues 1-45 in the crystal

  5. The relative potency of inverse opioid agonists and a neutral opioid antagonist in precipitated withdrawal and antagonism of analgesia and toxicity.

    Science.gov (United States)

    Sirohi, Sunil; Dighe, Shveta V; Madia, Priyanka A; Yoburn, Byron C

    2009-08-01

    Opioid antagonists can be classified as inverse agonists and neutral antagonists. In the opioid-dependent state, neutral antagonists are significantly less potent in precipitating withdrawal than inverse agonists. Consequently, neutral opioid antagonists may offer advantages over inverse agonists in the management of opioid overdose. In this study, the relative potency of three opioid antagonists to block opioid analgesia and toxicity and precipitate withdrawal was examined. First, the potency of two opioid inverse agonists (naltrexone and naloxone) and a neutral antagonist (6beta-naltrexol) to antagonize fentanyl-induced analgesia and lethality was determined. The order of potency to block analgesia was naltrexone > naloxone > 6beta-naltrexol (17, 4, 1), which was similar to that to block lethality (13, 2, 1). Next, the antagonists were compared using withdrawal jumping in fentanyl-dependent mice. The order of potency to precipitate withdrawal jumping was naltrexone > naloxone 6beta-naltrexol (1107, 415, 1). The relative potencies to precipitate withdrawal for the inverse agonists compared with the neutral antagonist were dramatically different from that for antagonism of analgesia and lethality. Finally, the effect of 6beta-naltrexol pretreatment on naloxone-precipitated jumping was determined in morphine and fentanyl-dependent mice. 6beta-Naltrexol pretreatment decreased naloxone precipitated withdrawal, indicating that 6beta-naltrexol is a neutral antagonist. These data demonstrate that inverse agonists and neutral antagonists have generally comparable potencies to block opioid analgesia and lethality, whereas the neutral opioid antagonist is substantially less potent in precipitating opioid withdrawal. These results support suggestions that neutral antagonists may have advantages over inverse agonists in the management of opioid overdose.

  6. Determining

    Directory of Open Access Journals (Sweden)

    Bahram Andarzian

    2015-06-01

    Full Text Available Wheat production in the south of Khuzestan, Iran is constrained by heat stress for late sowing dates. For optimization of yield, sowing at the appropriate time to fit the cultivar maturity length and growing season is critical. Crop models could be used to determine optimum sowing window for a locality. The objectives of this study were to evaluate the Cropping System Model (CSM-CERES-Wheat for its ability to simulate growth, development, grain yield of wheat in the tropical regions of Iran, and to study the impact of different sowing dates on wheat performance. The genetic coefficients of cultivar Chamran were calibrated for the CSM-CERES-Wheat model and crop model performance was evaluated with experimental data. Wheat cultivar Chamran was sown on different dates, ranging from 5 November to 9 January during 5 years of field experiments that were conducted in the Khuzestan province, Iran, under full and deficit irrigation conditions. The model was run for 8 sowing dates starting on 25 October and repeated every 10 days until 5 January using long-term historical weather data from the Ahvaz, Behbehan, Dezful and Izeh locations. The seasonal analysis program of DSSAT was used to determine the optimum sowing window for different locations as well. Evaluation with the experimental data showed that performance of the model was reasonable as indicated by fairly accurate simulation of crop phenology, biomass accumulation and grain yield against measured data. The normalized RMSE were 3%, 2%, 11.8%, and 3.4% for anthesis date, maturity date, grain yield and biomass, respectively. Optimum sowing window was different among locations. It was opened and closed on 5 November and 5 December for Ahvaz; 5 November and 15 December for Behbehan and Dezful;and 1 November and 15 December for Izeh, respectively. CERES-Wheat model could be used as a tool to evaluate the effect of sowing date on wheat performance in Khuzestan conditions. Further model evaluations

  7. Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog

    International Nuclear Information System (INIS)

    Reubi, Jean Claude; Cescato, Renzo; Waser, Beatrice; Erchegyi, Judit; Rivier, Jean E.

    2010-01-01

    Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties. We report here on a somatostatin antagonist that switched to an agonist upon coupling to a DOTA chelator. Two novel somatostatin analogs, 406-040-15 and its DOTA-coupled counterpart 406-051-20, with and without cold Indium labeling, were tested for their somatostatin receptor subtypes 1-5 (sst 1 -sst 5 ) binding affinity using receptor autoradiography. Moreover, they were tested functionally for their ability to affect sst 2 and sst 3 internalization in vitro in HEK293 cells stably expressing the human sst 2 or sst 3 receptor, using an immunofluorescence microscopy-based internalization assay. All three compounds were characterized as pan-somatostatin analogs having a high affinity for all five sst. In the sst 2 internalization assay, all three compounds showed an identical behavior, namely, a weak agonistic effect complemented by a weak antagonistic effect, compatible with the behavior of a partial agonist. Conversely, in the sst 3 internalization assay, 406-040-15 was a full antagonist whereas its DOTA-coupled counterpart, 406-051-20, with and without Indium labeling, switched to a full agonist. Adding the DOTA chelator to the somatostatin analog 406-040-15 triggers a switch at sst 3 receptor from an antagonist to an agonist. This indicates that potential radioligands for tumor targeting should always be tested functionally before further development, in particular if a chelator is added. (orig.)

  8. Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

    Science.gov (United States)

    Schiffer, Lina; Müller, Anne-Rose; Hobler, Anna; Brixius-Anderko, Simone; Zapp, Josef; Hannemann, Frank; Bernhardt, Rita

    2016-10-01

    Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Evaluation of in vivo selective binding of [11C]doxepin to histamine H1 receptors in five animal species

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Tsukada, Hideo; Harada, Norihiro; Mochizuki, Hideki; Kimura, Yuichi; Ishii, Kenji; Iwata, Ren; Yanai, Kazuhiko

    2004-01-01

    The specific binding of [ 11 C]doxepin, which has been used as a radioligand for mapping histamine H 1 receptors in human brain by positron emission tomography, was evaluated in five animal species. In mice the [ 11 C]doxepin uptake was reduced by treatment with cold doxepin and two H 1 receptor antagonists, but not with H 2 /H 3 antagonists. The specific binding evaluated with treatment with (+)-chlorpheniramine (H 1 antagonist) was in the range of 10-30% in mouse, rat, rabbit, and monkey, but was not detected in guinea pig

  10. Ligand binding affinity at the insulin receptor isoform A (IR-A and subsequent IR-A tyrosine phosphorylation kinetics are important determinants of mitogenic biological outcomes.

    Directory of Open Access Journals (Sweden)

    Harinda eRajapaksha

    2015-07-01

    Full Text Available The insulin receptor (IR is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A arises from alternative splicing of exon 11 and has different ligand binding and signalling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival and migration by activating some unique signalling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signalling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signalling (MAPK and Akt and receptor internalisation rates (related to mitogenic signalling. We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic ([His4, Tyr15, Thr49, Ile51] IGF-I, qIGF-I or metabolic (S597 peptide biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signalling through the IR-A. The 3-fold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316 and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and the kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

  11. Dopamine antagonists during parturition disrupt maternal care and the retention of maternal behavior in rats.

    Science.gov (United States)

    Byrnes, Elizabeth M; Rigero, Beth A; Bridges, Robert S

    2002-11-01

    Brief contact with pups at parturition enables the female rat to establish and retain the full repertoire of maternal behaviors, allowing her to respond rapidly to pups in the future. To determine whether the dopamine system is involved in the retention of maternal behavior, females were continuously infused with dopamine antagonists during the periparturitional period and then allowed either a brief interaction period with pups (3 h) or no interaction with pups (pups removed as they were born). Females were exposed to either the D1-like antagonist SCH 23390 (0.1 or 1.0 mg/kg/day) or the D2-like antagonist clebopride (0.5 or 1.0 mg/kg/day). The high dose of either DA antagonist resulted in significant attenuation of maternal care immediately postpartum. When tested for the retention of maternal behavior 7 days later, however, only the females exposed to the D2 antagonist displayed a delayed response to shown full maternal behavior (FMB) towards donor pups. Thus, while both dopamine receptor subtypes appear necessary for the full and rapid expression of maternal behavior during the early postpartum period, only the D2 receptor subtype appears to be involved in the retention of this behavior.

  12. Screening and Mechanism of Trapping Ligand Antagonist Peptide ...

    African Journals Online (AJOL)

    HP

    Flow cytometry was used to determine intracellular concentrations of Ca2+, and the possible role ... via binding membrane-associated CXCL1. (fractalkine). ... tested using FACSCalibur flow cytometer after filtration. All results were expressed as mean ± standard deviation (SD). Cross-linking of polypeptide H9 with receptors ...

  13. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

    Science.gov (United States)

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

    2015-09-01

    HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF

  14. MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

    Science.gov (United States)

    Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

    2012-01-17

    The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor

  15. GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

    International Nuclear Information System (INIS)

    Mukai, Eri; Toyoda, Kentaro; Kimura, Hiroyuki; Kawashima, Hidekazu; Fujimoto, Hiroyuki; Ueda, Masashi; Temma, Takashi; Hirao, Konomu; Nagakawa, Kenji; Saji, Hideo; Inagaki, Nobuya

    2009-01-01

    We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic β-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [ 125 I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [ 125 I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120 min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [ 125 I]BH-exendin(9-39) injection into transgenic mice with pancreatic β-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic β-cell imaging.

  16. In vivo and in vitro studies on a muscarinic presynaptic antagonist and postsynaptic agonist: BM-5

    International Nuclear Information System (INIS)

    Nordstrom, O.; Bartafi, T.; Frieder, B.; Grimm, V.; Ladinsky, H.; Unden, A.

    1986-01-01

    This paper reports on in vitro and in vivo studies with compound BM-5 which, at proper dosage, could have great potential since it could enhance cholinergic transmission by being a presynaptic antagonist and postsynaptic agonist. Binding studies are described in which tritium-4-NMPB, a muscarinic antagonist, was displaced by compound BM-5 in membranes from striatum, cerebral cortex, cerebellum and hippocampus. The binding data are summarized, which for each brain area involved 86-92 data points evaluated by means of nonlinear regression methods. Compound BM-5 recognized in each brain region a population of high and a population of low affinity binding sites; both of which were labelled with tritium-4-NMPB. It is shown that compound BM-5 causes muscarinic cholinergic agonist-like effects such as redness of the eye, increased motility in the gut, and impairment of locomotor behavior. It also produces muscarinic super-sensitivity upon chronic treatment, and decreases rat striatial ACh content by acute treatment

  17. Combination decongestion therapy in hospitalized heart failure: loop diuretics, mineralocorticoid receptor antagonists and vasopressin antagonists.

    Science.gov (United States)

    Vaduganathan, Muthiah; Mentz, Robert J; Greene, Stephen J; Senni, Michele; Sato, Naoki; Nodari, Savina; Butler, Javed; Gheorghiade, Mihai

    2015-01-01

    Congestion is the most common reason for admissions and readmissions for heart failure (HF). The vast majority of hospitalized HF patients appear to respond readily to loop diuretics, but available data suggest that a significant proportion are being discharged with persistent evidence of congestion. Although novel therapies targeting congestion should continue to be developed, currently available agents may be utilized more optimally to facilitate complete decongestion. The combination of loop diuretics, natriuretic doses of mineralocorticoid receptor antagonists and vasopressin antagonists represents a regimen of currently available therapies that affects early and persistent decongestion, while limiting the associated risks of electrolyte disturbances, hemodynamic fluctuations, renal dysfunction and mortality.

  18. Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

    Science.gov (United States)

    Cannon, J R; Eacho, P I

    1991-01-01

    Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation. Images Fig. 2. PMID:1747111

  19. Specific Binding of Adamantane Drugs and Direction of their Polar Amines in the Pore of the Influenza M2 Transmembrane Domain in Lipid Bilayers and Dodecylphosphocholine Micelles Determined by NMR Spectroscopy

    Science.gov (United States)

    Cady, Sarah D.; Wang, Jun; Wu, Yibing; DeGrado, William F.; Hong, Mei

    2011-01-01

    The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins. PMID:21381693

  20. National Systematic Legal Review of State Policies on Emergency Medical Services Licensure Levels' Authority to Administer Opioid Antagonists.

    Science.gov (United States)

    Kinsman, Jeremiah M; Robinson, Kathy

    2018-02-27

    Previous research conducted in November 2013 found there were a limited number of states and territories in the United States (US) that authorize emergency medical technicians (EMTs) and emergency medical responders (EMRs) to administer opioid antagonists. Given the continued increase in the number of opioid-related overdoses and deaths, many states have changed their policies to authorize EMTs and EMRs to administer opioid antagonists. The goal of this study is to provide an updated description of policy on EMS licensure levels' authority to administer opioid antagonists for all 50 US states, the District of Columbia (DC), and the Commonwealth of Puerto Rico (PR). State law and scopes of practice were systematically reviewed using a multi-tiered approach to determine each state's legally-defined EMS licensure levels and their authority to administer an opioid antagonist. State law, state EMS websites, and state EMS scope of practice documents were identified and searched using Google Advanced Search with Boolean Search Strings. Initial results of the review were sent to each state office of EMS for review and comment. As of September 1, 2017, 49 states and DC authorize EMTs to administer an opioid antagonist. Among the 40 US jurisdictions (39 states and DC) that define the EMR or a comparable first responder licensure level in state law, 37 states and DC authorize their EMRs to administer an opioid antagonist. Paramedics are authorized to administer opioid antagonists in all 50 states, DC, and PR. All 49 of the US jurisdictions (48 states and DC) that define the advanced emergency medical technician (AEMT) or a comparable intermediate EMS licensure level in state law authorize their AEMTs to administer an opioid antagonist. 49 out of 52 US jurisdictions (50 states, DC, and PR) authorize all existing levels of EMS licensure levels to administer an opioid antagonist. Expanding access to this medication can save lives, especially in communities that have limited

  1. Development of specific dopamine D-1 agonists and antagonists

    International Nuclear Information System (INIS)

    Sakolchai, S.

    1987-01-01

    To develop potentially selective dopamine D-1 agonists and to investigate on the structural requirement for D-1 activity, the derivatives of dibenzocycloheptadiene are synthesized and pharmacologically evaluated. The target compounds are 5-aminomethyl-10,11-dihydro-1,2-dihydroxy-5H-dibenzo[a,d]cycloheptene hydrobromide 10 and 9,10-dihydroxy-1,2,3,7,8,12b-hexahydrobenzo[1,2]cyclohepta[3,4,5d,e]isoquinoline hydrobromide 11. In a dopamine-sensitive rat retinal adenylate cyclase assay, a model for D-1 activity, compound 10 is essentially inert for both agonist and antagonist activity. In contrast, compound 11 is approximately equipotent to dopamine in activation of the D-1 receptor. Based on radioligand and binding data, IC 50 of compound 11 for displacement of 3 H-SCH 23390, a D-1 ligand, is about 7 fold less than that for displacement of 3 H-spiperone, a D-2 ligand. These data indicate that compound 11 is a potent selective dopamine D-1 agonist. This study provides a new structural class of dopamine D-1 acting agent: dihydroxy-benzocycloheptadiene analog which can serve as a lead compound for further drug development and as a probe for investigation on the nature of dopamine D-1 receptor

  2. Functional and structural changes of human erythrocyte catalase induced by cimetidine: proposed model of binding.

    Science.gov (United States)

    Yazdi, Fatemeh; Minai-Tehrani, Dariush; Jahngirvand, Mahboubeh; Almasirad, Ali; Mousavi, Zahra; Masoud, Masoudeh; Mollasalehi, Hamidreza

    2015-06-01

    In erythrocyte, catalase plays an important role to protect cells from hydrogen peroxide toxicity. Hydrogen peroxide is a byproduct compound which is produced during metabolic pathway of cells. Cimetidine, a histamine H2 receptor antagonist, is used for gastrointestinal tract diseases and prevents the extra release of gastric acid. In this study, the effect of cimetidine on the activity of human erythrocyte catalase was investigated. Erythrocytes were broken by hypotonic solution. The supernatant was used for catalase assay and kinetics study. Lineweaver-Burk plot was performed to determine the type of inhibition. The kinetics data revealed that cimetidine inhibited the catalase activity by mixed inhibition. The IC50 (1.54 μM) and Ki (0.45 μM) values of cimetidine determined that the drug was bound to the enzyme with high affinity. Circular dichroism and fluorescence measurement showed that the binding of cimetidine to the enzyme affected the content of secondary structure of the enzyme as well as its conformational changes. Docking studies were carried out to detect the site in which the drug was bound to the enzyme. Molecular modeling and energy calculation of the binding showed that the cyanoguanidine group of the drug connected to Asp59 via two hydrogen bonds, while the imidazole group of the drug interacted with Phe64 in the enzyme by a hydrophobic interaction. In conclusion, cimetidine could bind to human erythrocyte catalase, and its interaction caused functional and conformational changes in the enzyme.

  3. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel , Tracy M. (Vertex Pharm); (Leiden-MC); (USC); (BMS); (UCSD)

    2016-12-07

    CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

  4. (−) Arctigenin and (+) Pinoresinol Are Antagonists of the Human Thyroid Hormone Receptor β

    Science.gov (United States)

    2015-01-01

    Lignans are important biologically active dietary polyphenolic compounds. Consumption of foods that are rich in lignans is associated with positive health effects. Using modeling tools to probe the ligand-binding pockets of molecular receptors, we found that lignans have high docking affinity for the human thyroid hormone receptor β. Follow-up experimental results show that lignans (−) arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor β. The modeled complexes show key plausible interactions between the two ligands and important amino acid residues of the receptor. PMID:25383984

  5. Shared epitope-antagonistic ligands: a new therapeutic strategy in mice with erosive arthritis.

    Science.gov (United States)

    Ling, Song; Liu, Ying; Fu, Jiaqi; Colletta, Alessandro; Gilon, Chaim; Holoshitz, Joseph

    2015-05-01

    The mechanisms underlying bone damage in rheumatoid arthritis (RA) are incompletely understood. We recently identified the shared epitope (SE), an HLA-DRB1-coded 5-amino acid sequence motif carried by the majority of RA patients as a signal transduction ligand that interacts with cell surface calreticulin and accelerates osteoclast (OC)-mediated bone damage in collagen-induced arthritis (CIA). Given the role of the SE/calreticulin pathway in arthritis-associated bone damage, we sought to determine the therapeutic targetability of calreticulin. A library of backbone-cyclized peptidomimetic compounds, all carrying an identical core DKCLA sequence, was synthesized. The ability of these compounds to inhibit SE-activated signaling and OC differentiation was tested in vitro. The effect on disease severity and OC-mediated bone damage was studied by weekly intraperitoneal administration of the compounds to DBA/1 mice with CIA. Two members of the peptidomimetics library were found to have SE-antagonistic effects and antiosteoclast differentiation effects at picomolar concentrations in vitro. The lead mimetic compound, designated HS(4-4)c Trp, potently ameliorated arthritis and bone damage in vivo when administered in picogram doses to mice with CIA. Another mimetic analog, designated HS(3-4)c Trp, was found to lack activity, both in vitro and in vivo. The differential activity of the 2 analogs depended on minor differences in their respective ring sizes and correlated with distinctive geometry when computationally docked to the SE binding site on calreticulin. These findings identify calreticulin as a novel therapeutic target in erosive arthritis and provide sound rationale and early structure/activity relationships for future drug design. © 2015, American College of Rheumatology.

  6. Effects of organic ligands on fractionation of rare earth elements (REEs) in hydroponic plants: an application to the determination of binding capacities by humic acid for modeling.

    Science.gov (United States)

    Ding, ShiMing; Liang, Tao; Zhang, ChaoSheng; Yan, JunCai; Zhang, ZiLi

    2006-12-01

    Previous studies have revealed the fractionation processes of rare earth elements (REEs) in hydroponic plants, with a heavy REE (HREE, the elements from Gd to Lu) enrichment in leaves. In this study, effects on the HREE enrichment in soybean leaves with additions of carboxylic acids (acetate, malate, citrate, NTA, EDTA and DTPA) and two soil humic acids (HAs) were investigated. REE speciation in carboxylic acid and HA solutions was simulated using Visual MINTEQ and Model V, respectively. The results showed that the effects caused by carboxylic acids were strongly dependent on the differences between their binding strengths for light REEs (LREEs, the elements from La to Eu) and those for HREEs. A good correlation existed between these effects and the changes of free REE ions in solutions. This relationship was also observed for the HA treatments, provided that the intrinsic equilibrium constants of REEs for cation-proton exchange with HA (i.e., pK(MHA)) in Model V were estimated using a free-energy relationship with the stability constants for REE complexation with lactic acid. It is suggested that this set of pK(MHA) values is more suitable for use in Model V for the simulation of REE complexation with HA.

  7. Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat

    NARCIS (Netherlands)

    Cao, Y.; Shumsky, J. S.; Sabol, M. A.; Kushner, R. A.; Strittmatter, S.; Hamers, F. P. T.; Lee, D. H. S.; Rabacchi, S. A.; Murray, M.

    2008-01-01

    Objective. The myelin protein Nogo inhibits axon regeneration by binding to its receptor (NgR) on axons. Intrathecal delivery of an NgR antagonist (NEP1-40) promotes growth of injured corticospinal axons and recovery of motor function following a dorsal hemisection. The authors used a similar design

  8. Tritium labelling and characterization of the potent imidazoline I1 receptor antagonist [5,7-{sup 3}H] ({+-})-efaroxan at high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Egan, J.A.; Filer, C.N. E-mail: crist.filer@perkinelmer.com

    2003-06-01

    ({+-})-Efaroxan 1 is a selective antagonist at the imidazoline I1 receptor. [{sup 3}H] ({+-})-Efaroxan was required to explore its mechanism of action via receptor binding assay, and the radioligand was prepared by means of catalytic dehalogenation of a dibrominated precursor with tritium.

  9. Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists

    Czech Academy of Sciences Publication Activity Database

    Kwiatkowska, A.; Ptach, M.; Borovičková, Lenka; Slaninová, Jiřina; Lammek, B.; Prahl, A.

    2012-01-01

    Roč. 43, č. 2 (2012), s. 617-627 ISSN 0939-4451 Institutional research plan: CEZ:AV0Z40550506 Keywords : oxitocin antagonists * Atosiban * neurohypophyseal hormones analogues * arginine vasopressin (AVP) * antidiuretic hormone * binding affinity Subject RIV: CE - Biochemistry Impact factor: 3.914, year: 2012

  10. New selective and potent 5-HT1B/1D antagonists : Chemistry and pharmacological evaluation of N-piperazinylphenyl biphenylcarboxamides and biphenylsulfonamides

    NARCIS (Netherlands)

    Liao, Y; Bottcher, H; Harting, J; Greiner, H; van Amsterdam, C; Cremers, T; Sundell, S; Marz, J; Rautenberg, W; Wikstrom, H

    2000-01-01

    A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective 5-HT1B/1D antagonists were synthesized and evaluated pharmacologically. Their receptor binding profiles were comparable

  11. Effects of a novel bradykinin B1 receptor antagonist and angiotensin II receptor blockade on experimental myocardial infarction in rats.

    Directory of Open Access Journals (Sweden)

    Dongmei Wu

    Full Text Available The aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1 receptor antagonist after MI in rats.Sprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823 or AT1 receptor antagonist (irbesartan alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP; greater first derivative of left ventricular pressure (± dp/dt max, left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2 and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1 mRNA expression were not significantly affected by B1 receptor blockade.The present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI.

  12. Sexually antagonistic selection in human male homosexuality.

    Directory of Open Access Journals (Sweden)

    Andrea Camperio Ciani

    Full Text Available Several lines of evidence indicate the existence of genetic factors influencing male homosexuality and bisexuality. In spite of its relatively low frequency, the stable permanence in all human populations of this apparently detrimental trait constitutes a puzzling 'Darwinian paradox'. Furthermore, several studies have pointed out relevant asymmetries in the distribution of both male homosexuality and of female fecundity in the parental lines of homosexual vs. heterosexual males. A number of hypotheses have attempted to give an evolutionary explanation for the long-standing persistence of this trait, and for its asymmetric distribution in family lines; however a satisfactory understanding of the population genetics of male homosexuality is lacking at present. We perform a systematic mathematical analysis of the propagation and equilibrium of the putative genetic factors for male homosexuality in the population, based on the selection equation for one or two diallelic loci and Bayesian statistics for pedigree investigation. We show that only the two-locus genetic model with at least one locus on the X chromosome, and in which gene expression is sexually antagonistic (increasing female fitness but decreasing male fitness, accounts for all known empirical data. Our results help clarify the basic evolutionary dynamics of male homosexuality, establishing this as a clearly ascertained sexually antagonistic human trait.

  13. Hypocretin antagonists in insomnia treatment and beyond.

    Science.gov (United States)

    Ruoff, Chad; Cao, Michelle; Guilleminault, Christian

    2011-01-01

    Hypocretin neuropeptides have been shown to regulate transitions between wakefulness and sleep through stabilization of sleep promoting GABAergic and wake promoting cholinergic/monoaminergic neural pathways. Hypocretin also influences other physiologic processes such as metabolism, appetite, learning and memory, reward and addiction, and ventilatory drive. The discovery of hypocretin and its effect upon the sleep-wake cycle has led to the development of a new class of pharmacologic agents that antagonize the physiologic effects of hypocretin (i.e. hypocretin antagonists). Further investigation of these agents may lead to novel therapies for insomnia without the side-effect profile of currently available hypnotics (e.g. impaired cognition, confusional arousals, and motor balance difficulties). However, antagonizing a system that regulates the sleep-wake cycle while also influencing non-sleep physiologic processes may create an entirely different but equally concerning side-effect profile such as transient loss of muscle tone (i.e. cataplexy) and a dampened respiratory drive. In this review, we will discuss the discovery of hypocretin and its receptors, hypocretin and the sleep-wake cycle, hypocretin antagonists in the treatment of insomnia, and other implicated functions of the hypocretin system.

  14. Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

    Science.gov (United States)

    Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

    2009-01-01

    The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

  15. Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

    Science.gov (United States)

    Ha, Sookhee N; Hey, Pat J; Ransom, Rick W; Harrell, C Meacham; Murphy, Kathryn L; Chang, Ray; Chen, Tsing-Bau; Su, Dai-Shi; Markowitz, M Kristine; Bock, Mark G; Freidinger, Roger M; Hess, Fred J

    2005-05-27

    We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

  16. In vivo binding in rat brain and radiopharmaceutical preparation of radioiodinated HEAT, an alpha-1 adrenoceptor ligand

    International Nuclear Information System (INIS)

    Couch, M.W.; Greer, D.M.; Thonoor, C.M.; Williams, C.M.

    1988-01-01

    In vivo binding of [ 125 I]-2-[beta-(3-iodo-4-hydroxyphenyl)ethylaminomethyl tetralone) ([ 125 I]HEAT) to alpha-1 adrenoceptors in the rat brain was determined over 4 hr. Uptake in the thalamus and frontal cortex was approximately 0.1% injected dose per gram tissue. Thalamus/cerebellum ratios of 10:1 and frontal cortex/cerebellum ratios of 5:1 were found at 4 hr. Pretreatment with prazosin, an alpha-1 antagonist, completely inhibited the accumulation of [ 125 I]HEAT in thalamus and frontal cortex; yet uptake of radioactivity was not significantly affected by antagonists and agonists for other receptors classes (propranolol, beta-1; apomorphine, D-1; spiperone, D-2). Binding of [ 125 I]HEAT is saturable. At 4 hr, [ 125 I]HEAT or [ 123 I]HEAT was shown to be the only radioactive material in rat thalamus and frontal cortex. Iodine-123 HEAT and [ 125 I]HEAT were synthesized as radiopharmaceuticals within 3 hr in 99% radiochemical purity

  17. Identification of VDR Antagonists among Nuclear Receptor Ligands Using Virtual Screening

    Directory of Open Access Journals (Sweden)

    Kelly Teske

    2014-04-01

    Full Text Available Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR antagonists among nuclear receptor (NR ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available “Binding Database.” Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2D3 and 25(OH2D3. The first virtual screen identified 32 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 μM. The second screen identified 162 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ERα/β ligands (26%, TRα/β ligands (7%, and LxRα/β ligands (7%. The binding between VDR and ERα ligand H6036 as well as TRα/β ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.

  18. History of the 'geste antagoniste' sign in cervical dystonia.

    Science.gov (United States)

    Poisson, A; Krack, P; Thobois, S; Loiraud, C; Serra, G; Vial, C; Broussolle, E

    2012-08-01

    The geste antagoniste is a voluntary maneuver that temporarily reduces the severity of dystonic posture or movements. It is a classical feature of focal and particularly cervical dystonia. However, the precise historical aspects of geste antagoniste still remain obscure. The goals of this review were (1) to clarify the origin of the geste antagoniste sign; (2) to identify the factors that led to its diffusion in the international literature; (3) to follow the evolution of that term across the twentieth century. We used medical and neurological French, German and English literature of the late nineteenth and early twentieth centuries, and the PubMed database by entering the terms geste antagoniste, antagonistic gesture and sensory trick. The geste antagoniste sign is a legacy of the Paris Neurological School of the end of the nineteenth century. The term was introduced by Meige and Feindel in their 1902 book on tics, written in the vein of their master, Brissaud, who first described this sign in 1893. The almost immediate translations of this book by Giese into German and Kinnier Wilson into English contributed to the rapid spreading of the term geste antagoniste, which is still in use worldwide today. The term antagonistic gesture is the translation proposed by Kinnier Wilson, which also led to the use of the term geste antagonistique. The geste antagoniste sign has long been considered a solid argument for the psychogenic origins of dystonia until the 1980s when Marsden made strong arguments for its organic nature.

  19. Genetic and Dietary Determinants of Insulin-Like Growth Factor (IGF)-1 and IGF Binding Protein (BP)-3 Levels among Chinese Women

    Science.gov (United States)

    Li, Hui; McCullough, Lauren E.; Qi, Ya-na; Li, Jia-yuan; Zhang, Jing; Miller, Erline; Yang, Chun-xia; Smith, Jennifer S.

    2014-01-01

    Background Higher insulin-like growth factor (IGF)-1 and lower IGF binding protein (BP)-3 levels have been associated with higher commoncancer risk, including breast cancer. Dietary factors, genetic polymorphisms, and the combination of both may influence circulating IGF-1 and IGFBP-3 serum concentrations. Methods From September 2011 to July 2012, we collected demographic, reproductive and dietary data on 143 women (≥40 years). We genotyped IGF-1 rs1520220 and IGFBP-3 rs2854744 and measured circulating IGF-1 and IGFBP-3 levels in serum. Covariance analyses were used to estimate the associations of serum levels of IGF-1 and IGFBP-3, and the molar ratio of IGF-1to IGFBP-3 with IGF-1 rs1520220 and IGFBP-3 rs2854744 genotypes. We subsequently assessed the combined influence of genetics and diet (daily intake of protein, fat and soy isoflavones) on IGF-1 and IGFBP-3 levels. Results Among women aged less than 50 years, circulating IGF-1 serum levels were significantly lower for those with CC genotype for IGF-1 rs1520220 than levels for those with the GC or GG genotypes (in recessive model: P = 0.007).In gene-diet analyses among these women, we found carrying CC genotype for IGF-1 rs1520220 and high soy isoflavone intake tend to be associated with lower circulating IGF-1 levels synthetically (P = 0.002). Women with GG or GC genotypes for IGF-1 rs1520220 and with low intake of soy isoflavones had the highest levels of circulating IGF-1 (geometric mean [95% CI]: 195 [37, 1021] µg/L). Comparatively, women with both the CC genotype and high soy intake had the lowest levels of circulating IGF-1 (geometric mean [95% CI]: 120 [38,378] µg/L). Conclusions IGF-1 serum levels are significantly lower among women with the CC genotype for IGF-1-rs1520220. High soy isoflavone intake may interact with carrying CC genotype for IGF-1-rs1520220 to lower women's serum IGF-1 levels more. PMID:25285521

  20. New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues.

    Science.gov (United States)

    Bajusz, S; Csernus, V J; Janaky, T; Bokser, L; Fekete, M; Schally, A V

    1988-12-01

    Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.

  1. An assessment of the effects of serotonin 6 (5-HT6) receptor antagonists in rodent models of learning.

    Science.gov (United States)

    Lindner, Mark D; Hodges, Donald B; Hogan, John B; Orie, Anitra F; Corsa, Jason A; Barten, Donna M; Polson, Craig; Robertson, Barbara J; Guss, Valerie L; Gillman, Kevin W; Starrett, John E; Gribkoff, Valentin K

    2003-11-01

    Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

  2. Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Han, Gye Won; Abagyan, Ruben; Wu, Beili; Stevens, Raymond C.; Cherezov, Vadim; Kufareva, Irina; Handel, Tracy M. (USC); (Chinese Aca. Sci.); (UCSD)

    2017-06-01

    CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.

  3. Kappa-Opioid Antagonists for Psychiatric Disorders: From Bench to Clinical Trials.

    Science.gov (United States)

    Carlezon, William A; Krystal, Andrew D

    2016-10-01

    Kappa-opioid receptor (KOR) antagonists are currently being considered for the treatment of a variety of neuropsychiatric conditions, including depressive, anxiety, and substance abuse disorders. A general ability to mitigate the effects of stress, which can trigger or exacerbate these conditions, may explain their putative efficacy across such a broad array of conditions. The discovery of their potentially therapeutic effects evolved from preclinical research designed to characterize the molecular mechanisms by which experience causes neuroadaptations in the nucleus accumbens (NAc), a key element of brain reward circuitry. This research established that exposure to drugs of abuse or stress increases the activity of the transcription factor CREB (cAMP response element binding protein) in the NAc, which leads to elevated expression of the opioid peptide dynorphin that in turn causes core signs of depressive- and anxiety-related disorders. Disruption of KORs-the endogenous receptors for dynorphin-produces antidepressant- and anxiolytic-like actions in screening procedures that identify standard drugs of these classes, and reduces stress effects in tests used to study addiction and stress-related disorders. Although interest in this target is high, prototypical KOR antagonists have extraordinarily persistent pharmacodynamic effects that complicate clinical trials. The development of shorter acting KOR antagonists together with more rapid designs for clinical trials may soon provide insight on whether these drugs are efficacious as would be predicted by preclinical work. If successful, KOR antagonists would represent a unique example in psychiatry where the therapeutic mechanism of a drug class is understood before it is shown to be efficacious in humans. © 2016 Wiley Periodicals, Inc.

  4. The Attractiveness of Opposites: Agonists and Antagonists.

    LENUS (Irish Health Repository)

    O'Brien, Tony

    2015-02-02

    ABSTRACT Opioid-induced bowel dysfunction, of which constipation is the most common aspect, is a major limiting factor in the use of opioids for pain management. The availability of an oral, long-acting formulation of oxycodone and naloxone represents a highly significant development in pain management. The combination of an opioid analgesic with an opioid antagonist offers reliable pain control with a significant reduction in the burden of opioid-induced constipation. This report is adapted from paineurope 2014; Issue 3, ©Haymarket Medical Publications Ltd, and is presented with permission. paineurope is provided as a service to pain management by Mundipharma International, LTD and is distributed free of charge to healthcare professionals in Europe. Archival issues can be accessed via the website: http:\\/\\/www.paineurope.com at which European health professionals can register online to receive copies of the quarterly publication.

  5. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

    2017-07-31

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

  6. Competitive receptor binding radioassay for β-1 and β-2 adrenergic agents

    International Nuclear Information System (INIS)

    Hussain, M.N.; Culbreth, W.; Dalrymple, R.; Fung, C.; Ricks, C.

    1987-01-01

    A rapid and sensitive competitive receptor bonding assay for β-1 and β-2 adrenergic binding for adrenergic agents has been developed. The steps that are critical for the success of the assay are given in detail so that the assay can be set up in any routine laboratory with relative ease. The rationale behind the use of specific reagents is discussed. The assay requires microgram quantities of test compound, a radiolabeled specific β adrenergic antagonist [ 3 H]dihydroalprenolol (DHA), and turkey erythrocyte β-1 and rat erythrocyte β-2 receptor membranes. Serial dilutions of sample are incubated with appropriate receptor membranes and DHA for 1 hr at room temperature. After equilibrium is attained, the bound radioligand is separated by rapid filtration under vacuum through Whatman GF/B filters. The amount of bound DHA trapped on the filter is inversely proportional to the degree of β-1 and β-2 adrenergic binding of the sample. Separation of bound from free radioligand by filtration permits rapid determination of a large number of samples. This assay quantitates and differentiates β-1 and β-2 adrenergic binding of synthetic adrenergic agents

  7. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Directory of Open Access Journals (Sweden)

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  8. Antagonistic activity of marine sponges associated Actinobacteria

    Directory of Open Access Journals (Sweden)

    Selvakumar Dharmaraj

    2016-06-01

    Full Text Available Objective: To focus on the isolation and preliminary characterization of marine sponges associated Actinobacteria particularly Streptomyces species and also their antagonistic activities against bacterial and fungal pathogens. Methods: The sponges were collected from Kovalam and Vizhinjam port of south-west coast of Kerala, India. Isolation of strains was carried out from sponge extracts using international Streptomyces project media. For preliminary identification of the strains, morphological (mycelial colouration, soluble pigments, melanoid pigmentation, spore morphology, nutritional uptake (carbon utilisation, amonoacids influence, sodium chloride tolerance, physiological (pH, temperature and chemotaxonomical characterization were done. Antimicrobial studies were also carried out for the selected strains. Results: With the help of the spicule structures, the collected marine sponges were identified as Callyspongia diffusa, Mycale mytilorum, Tedania anhelans and Dysidea fragilis. Nearly 94 strains were primarily isolated from these sponges and further they were sub-cultured using international Streptomyces project media. The strains exhibited different mycelial colouration (aerial and substrate, soluble and melanoid pigmentations. The strains possessed three types of sporophore morphology namely rectus flexibilis, spiral and retinaculiaperti. Among the 94 isolates, seven exhibited antibacterial and antifungal activities with maximal zone of inhibition of 30 mm. The nutritional, physiological and chemotaxonomical characteristic study helped in the conventional identification of the seven strains and they all suggest that the strains to be grouped under the genus Streptomyces. Conclusions: The present study clearly helps in the preliminary identification of the isolates associated with marine sponges. Antagonistic activities prove the production of antimicrobial metabolites against the pathogens. Marine sponges associated Streptomyces are

  9. Alpha antagonists and intraoperative floppy iris syndrome: A spectrum

    Directory of Open Access Journals (Sweden)

    Sharif A Issa

    2008-07-01

    Full Text Available Sharif A Issa, Omar H Hadid, Oliver Baylis, Margaret DayanDepartment of Ophthalmology, Royal Victoria Infirmary, Newcastle upon Tyne, UKBackground: To determine occurrence of features of intraoperative floppy iris syndrome (IFIS during cataract surgery in patients taking systemic alpha-antagonists (AA.Methods: We prospectively studied patients on AA and who underwent phacoemulsification. The following were recorded: pupil diameter preoperatively, iris flaccidity, iris prolapse and peroperative miosis.Results: We studied 40 eyes of 31 subjects. Mean age was 78 years. Overall, 14 eyes (13 patients showed signs of IFIS: 9/13 (69% eyes of patients on tamsulosin, 1/18 (6% eyes in the doxazosin group, 2/2 prazosin patients, 1/4 eyes in the indoramin group, and 1/2 eyes in two patients on a combination of doxazosin and tamsulosin. Most cases (92% had only one or two signs of IFIS. Bilateral cataract surgery was undertaken in 9 patients but only one patient (on tamsulosin had features of IFIS in both eyes, while 4 patients (2 on tamsulosin and 2 on other AA showed signs of IFIS in one eye only, and 4 patients did not show IFIS in either eye.Conclusion: Most AA were associated with IFIS, but it tends to present as a spectrum of signs rather than full triad originally described. Tamsulosin was most likely to be associated with IFIS; however, its intake does not necessarily mean that IFIS will occur. For patients on AA, the behavior of the iris intraoperatively in one eye is a poor predictor of the other eye. Surgeons should anticipate the occurrence of IFIS in any patient on AA.Keywords: alpha blocker, alpha antagonist, cataract surgery, intraoperative floppy iris syndrome, tamsulosin.

  10. Vascular mechanism of action of endothelin-1: Effect of Ca2+ antagonists

    International Nuclear Information System (INIS)

    Chabrier, P.E.; Auguet, M.; Roubert, P.; Lonchampt, M.O.; Gillard, V.; Guillon, J.M.; Delaflotte, S.; Braquet, P.

    1989-01-01

    The vasoconstrictive properties of the endothelium-derived peptide, endothelin-1 (ET-1), were investigated on rat isolated aorta and on cultured rat aortic smooth muscle cells. In rat isolated aorta, endothelin-1 induced a slow and sustained contraction in a Ca2+-free medium; after calcium readmission, an additional sustained contraction was elicited. In vascular smooth muscle cells, endothelin-1 provoked a dose-dependent Ca2+ influx that was not inhibited by calcium entry blockers (nifedipine, D 600, or diltiazem). In these cells, [ 125 I]-endothelin-1 bound to a specific, saturable, and high affinity recognition site (Kd about 10(-9) M and Bmax = 52 +/- 2 fmol/10(6) cells). The binding was not reversible and not affected by calcium antagonists. These data do not support the hypothesis that endothelin-1 acts as an endogenous agonist of the voltage-dependent Ca2+ channels. The action of endothelin-1 can be separated into two components: one dependent on Ca2+ influx but insensitive to calcium antagonists and another independent of extracellular Ca2+. The irreversible binding of endothelin-1 may reflect an internalization of the ligand inside the cell membrane, leading to multiple contractile events

  11. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    Wolverton, C.

    2007-01-01

    Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

  12. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    Science.gov (United States)

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

    DEFF Research Database (Denmark)

    Tricoci, Pierluigi; Huang, Zhen; Held, Claes

    2012-01-01

    Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation.......Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation....

  14. Adverse cutaneous reactions induced by TNF-alpha antagonist therapy.

    Science.gov (United States)

    Borrás-Blasco, Joaquín; Navarro-Ruiz, Andrés; Borrás, Consuelo; Casterá, Elvira

    2009-11-01

    To review adverse cutaneous drug reactions induced by tumor necrosis factor alpha (TNF-alpha) antagonist therapy. A literature search was performed using PubMed (1996-March 2009), EMBASE, and selected MEDLINE Ovid bibliography searches. All language clinical trial data, case reports, letters, and review articles identified from the data sources were used. Since the introduction of TNF-alpha antagonist, the incidence of adverse cutaneous drug reactions has increased significantly. A wide range of different skin lesions might occur during TNF-alpha antagonist treatment. New onset or exacerbation of psoriasis has been reported in patients treated with TNF-alpha antagonists for a variety of rheumatologic conditions. TNF-alpha antagonist therapy has been associated with a lupus-like syndrome; most of these case reports occurred in patients receiving either etanercept or infliximab. Serious skin reactions such as erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis have been reported rarely with the use of TNF-alpha antagonists. As the use of TNF-alpha antagonists continues to increase, the diagnosis and management of cutaneous side effects will become an increasingly important challenge. In patients receiving TNF-alpha antagonist treatment, skin disease should be considered, and clinicians need to be aware of the adverse reactions of these drugs.

  15. Antagonistic and Bargaining Games in Optimal Marketing Decisions

    Science.gov (United States)

    Lipovetsky, S.

    2007-01-01

    Game theory approaches to find optimal marketing decisions are considered. Antagonistic games with and without complete information, and non-antagonistic games techniques are applied to paired comparison, ranking, or rating data for a firm and its competitors in the market. Mix strategy, equilibrium in bi-matrix games, bargaining models with…

  16. Vitamin K antagonist use and mortality in dialysis patients

    NARCIS (Netherlands)

    Voskamp, Pauline W.M.; Rookmaaker, Maarten B.; Verhaar, Marianne C.; Dekker, Friedo W.; Ocak, Gurbey

    2018-01-01

    Background. The risk-benefit ratio of vitamin K antagonists for different CHA2DS2-VASc scores in patients with end-stage renal disease treated with dialysis is unknown. The aim of this study was to investigate the association between vitamin K antagonist use and mortality for different CHA2DS2-VASc

  17. Interaction between Antagonist of Cannabinoid Receptor and Antagonist of Adrenergic Receptor on Anxiety in Male Rat

    Directory of Open Access Journals (Sweden)

    Alireza Komaki

    2014-07-01

    Full Text Available Introduction: Anxiety is among the most common and treatable mental disorders. Adrenergic and cannabinoid systems have an important role in the neurobiology of anxiety. The elevated plus-maze (EPM has broadly been used to investigate anxiolytic and anxiogenic compounds. The present study investigated the effects of intraperitoneal (IP injection of cannabinoid CB1 receptor antagonist (AM251 in the presence of alpha-1 adrenergic antagonist (Prazosin on rat behavior in the EPM. Methods: In this study, the data were obtained from male Wistar rat, which weighing 200- 250 g. Animal behavior in EPM were videotaped and saved in computer for 10 min after IP injection of saline, AM251 (0.3 mg/kg, Prazosin (0.3 mg/kg and AM251 + Prazosin, subsequently scored for conventional indices of anxiety. During the test period, the number of open and closed arms entries, the percentage of entries into the open arms of the EPM, and the spent time in open and closed arms were recorded. Diazepam was considered as a positive control drug with anxiolytic effect (0.3, 0.6, 1.2 mg/kg. Results: Diazepam increased the number of open arm entries and the percentage of spent time on the open arms. IP injection of AM251 before EPM trial decreased open arms exploration and open arm entry. Whereas, Prazosin increased open arms exploration and open arm entry. This study showed that both substances in simultaneous injection have conflicting effects on the responses of each of these two compounds in a single injection. Discussion: Injection of CB1 receptor antagonist may have an anxiogenic profile in rat, whereas adrenergic antagonist has an anxiolytic effect. Further investigations are essential for better understanding of anxiolytic and anxiogenic properties and neurobiological mechanisms of action and probable interactions of the two systems.

  18. Interaction between Antagonist of Cannabinoid Receptor and Antagonist of Adrenergic Receptor on Anxiety in Male Rat.

    Science.gov (United States)

    Komaki, Alireza; Abdollahzadeh, Fatemeh; Sarihi, Abdolrahman; Shahidi, Siamak; Salehi, Iraj

    2014-01-01

    Anxiety is among the most common and treatable mental disorders. Adrenergic and cannabinoid systems have an important role in the neurobiology of anxiety. The elevated plus-maze (EPM) has broadly been used to investigate anxiolytic and anxiogenic compounds. The present study investigated the effects of intraperitoneal (IP) injection of cannabinoid CB1 receptor antagonist (AM251) in the presence of alpha-1 adrenergic antagonist (Prazosin) on rat behavior in the EPM. In this study, the data were obtained from male Wistar rat, which weighing 200- 250 g. Animal behavior in EPM were videotaped and saved in computer for 10 min after IP injection of saline, AM251 (0.3 mg/kg), Prazosin (0.3 mg/kg) and AM251 + Prazosin, subsequently scored for conventional indices of anxiety. During the test period, the number of open and closed arms entries, the percentage of entries into the open arms of the EPM, and the spent time in open and closed arms were recorded. Diazepam was considered as a positive control drug with anxiolytic effect (0.3, 0.6, 1.2 mg/kg). Diazepam increased the number of open arm entries and the percentage of spent time on the open arms. IP injection of AM251 before EPM trial decreased open arms exploration and open arm entry. Whereas, Prazosin increased open arms exploration and open arm entry. This study showed that both substances in simultaneous injection have conflicting effects on the responses of each of these two compounds in a single injection. Injection of CB1 receptor antagonist may have an anxiogenic profile in rat, whereas adrenergic antagonist has an anxiolytic effect. Further investigations are essential for better understanding of anxiolytic and anxiogenic properties and neurobiological mechanisms of action and probable interactions of the two systems.

  19. Synthesis and evaluation of calcium channel antagonist activity of new 1, 4-dihydropyridines containing phenylamineimidazolyl substitute in guinea-pig ileal smooth muscle

    Directory of Open Access Journals (Sweden)

    A Fassihi

    2004-02-01

    Full Text Available Background: 1,4-dihydropyridines are a class of drugs which are used in the treatment of some cardiovascular disorders. The prototype, Nifedipine, does not have optimal pharmacokinetic and pharmacodynamic properties. Several new derivatives of 1, 4-dihydropyridine have been produced and pharmacologically evaluated in order to find drugs with better pharmacological properties. Among them, those with a substituted heteroaromatic ring in the C4 position of the 1, 4-dihydropyridine ring, instead of the phenyl ring in Nifedipine, are most considered. In this study, eight novel derivatives of this class with “2-methylthio-1-(phenylaminoimidazole-5-yl” in the C4, C3 and C5 positions were prepared and evaluated as calcium channel antagonist agents. Methods: To prepare these compounds, Hantzsch method for the synthesis of 1, 4-dihydropyridine derivatives was deployed. An aldehyde was reacted with appropriate acetoacetate ester and ammonium acetate. This aldehyde was prepared in three steps. Cumulative doses were applied to determine the relaxing effect of the compounds on the longitudinal smooth muscle of male albino guinea pigs. Results: Chemical structures of the compounds were characterized by 1H nuclear magnetic resonance, infrared and mass spectroscopy. The IC50 of each compound was graphically determined from the concentration-response curves. Conclusions: Two compounds were more active than Nifedipine. Both had lipophilic ester groups with low steric hindrance that met the merits of a better receptor binding of 1, 4-dihydropyridines. These derivatives have high potential for further study. Keywords: 1, 4-dihydropyridine, Calcium channel antagonist, Phenylamineimidazolyl, Cardiovascular disorder

  20. Stimulant effects of adenosine antagonists on operant behavior: differential actions of selective A2A and A1 antagonists

    Science.gov (United States)

    Randall, Patrick A.; Nunes, Eric J.; Janniere, Simone L.; Stopper, Colin M.; Farrar, Andrew M.; Sager, Thomas N.; Baqi, Younis; Hockemeyer, Jörg; Müller, Christa E.

    2012-01-01

    Rationale Adenosine A2A antagonists can reverse many of the behavioral effects of dopamine antagonists, including actions on instrumental behavior. However, little is known about the effects of selective adenosine antagonists on operant behavior when these drugs are administered alone. Objective The present studies were undertaken to investigate the potential for rate-dependent stimulant effects of both selective and nonselective adenosine antagonists. Methods Six drugs were tested: two nonselective adenosine antagonists (caffeine and theophylline), two adenosine A1 antagonists (DPCPX and CPT), and two adenosine A2A antagonists (istradefylline (KW6002) and MSX-3). Two schedules of reinforcement were employed; a fixed interval 240-s (FI-240 sec) schedule was used to generate low baseline rates of responding and a fixed ratio 20 (FR20) schedule generated high rates. Results Caffeine and theophylline produced rate-dependent effects on lever pressing, increasing responding on the FI-240 sec schedule but decreasing responding on the FR20 schedule. The A2A antagonists MSX-3 and istradefylline increased FI-240 sec lever pressing but did not suppress FR20 lever pressing in the dose range tested. In fact, there was a tendency for istradefylline to increase FR20 responding at a moderate dose. A1 antagonists failed to increase lever pressing rate, but DPCPX decreased FR20 responding at higher doses. Conclusions These results suggest that adenosine A2A antagonists enhance operant response rates, but A1 antagonists do not. The involvement of adenosine A2A receptors in regulating aspects of instrumental response output and behavioral activation may have implications for the treatment of effort-related psychiatric dysfunctions, such as psychomotor slowing and anergia in depression. PMID:21347642

  1. [11C]WAY-100635 PET imaging of 5-HT1A receptor binding in patients with temporal lobe epilepsy

    International Nuclear Information System (INIS)

    Sasai, Taeko; Matsuura, Masato; Itou, Shigeo; Suhara, Tetsuya; Yahata, Noriaki; Okubo, Yoshiro

    2006-01-01

    To understand the role of 5-HT in human temporal lobe epilepsy, here we measured 5-HT 1A receptor binding potential by positron emission tomography (PET) with [carbonyl- 11 C]WAY100635, a selective 5-HT 1A receptor antagonist, in patients with temporal lobe epilepsy and normal controls. Twelve patients with temporal lobe epilepsy and seventeen healthy controls participated in the study. For each subject, we conducted PET and magnetic resonance imaging (MRI), by which we measured the 5-HT 1A receptor binding potential, the R1-value, a relative indicator of cerebral blood flow in regions of interest, and the volume of gray matter. Patients with temporal lobe epilepsy showed significantly reduced 5-HT 1A receptor binding potential in the temporal lobe. The laterality of the reduction was coincided with the epileptogenic foci estimated by a scalp electroencephalography (EEG). In contrast, the R1-value and gray matter volume showed no difference between the patient and control groups. Our study revealed that 5-HT 1A receptor binding was reduced significantly at the epileptogenic foci. We suggest that PET imaging with [carbonyl- 11 C]WAY100635 is potentially a useful non-invasive method for determining the epileptogenic foci. (author)

  2. In vitro wear of four ceramic materials and human enamel on enamel antagonist.

    Science.gov (United States)

    Nakashima, Jun; Taira, Yohsuke; Sawase, Takashi

    2016-06-01

    The purpose of the present study was to evaluate the wear of four different ceramics and human enamel. The ceramics used were lithium disilicate glass (e.max Press), leucite-reinforced glass (GN-Ceram), yttria-stabilized zirconia (Aadva Zr), and feldspathic porcelain (Porcelain AAA). Hemispherical styli were fabricated with these ceramics and with tooth enamel. Flattened enamel was used for antagonistic specimens. After 100,000 wear cycles of a two-body wear test, the height and volume losses of the styli and enamel antagonists were determined. The mean and standard deviation for eight specimens were calculated and statistically analyzed using a non-parametric (Steel-Dwass) test (α = 0.05). GN-Ceram exhibited greater stylus height and volume losses than did Porcelain AAA. E.max Press, Porcelain AAA, and enamel styli showed no significant differences, and Aadva Zr exhibited the smallest stylus height and volume losses. The wear of the enamel antagonist was not significantly different among GN-Ceram, e.max Press, Porcelain AAA, and enamel styli. Aadva Zr resulted in significantly lower wear values of the enamel antagonist than did GN-Ceram, Porcelain AAA, and enamel styli. In conclusion, leucite-reinforced glass, lithium disilicate glass, and feldspathic porcelain showed wear values closer to those for human enamel than did yttria-stabilized zirconia. © 2016 Eur J Oral Sci.

  3. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2001-01-01

    Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified...

  4. Effect of long-term propranolol administration on specific binding of 3H-WB-4101 with rat mesenteric vascular membranes

    International Nuclear Information System (INIS)

    Ismailov, S.I.; Rozhanets, V.V.; Val'dman, A.V.

    1985-01-01

    The aim of this investigation was, first, to study the affinity of certain beta-adrenoblockers for specific binding sites of 3 H-WB-4101 (identifiable as alpha-adrenoreceptors) of brain membranes and, second, to study the characteristics of these same receptors in membranes of mesenteric vessels of rats during long-term administration of propranolol. Isotherms of specific binding, because of the limited quantity of vascular membranes, were determined by the use of three concentrations of 3 H-WB-4101: 0.1, 0.5, and 1.0 nM. It is shown that some beta-adrenoblockers have weak affinity for alpha-adrenoreceptors of brain synaptic membranes exhibited only when these compounds are present in relatively high concentrations. It is also shown that administration of propranolol for 15 days led to a significant decrease in affinity of the alpha-adrenorecptors for their specific antagonist WB-4101

  5. Study of the effect hydrogen binding in the solvation of alkaline earth cations with MeOH in nitromethane using 1 H NMR technique and determination of ionic solvation number

    CERN Document Server

    Alizadeh, N

    2001-01-01

    A proton NMR method for the study of the effect hydrogen binding and determination of solvation numbers of alkaline earth cations with methanol (MeOH) in in tromethane (NM) as diluent is described. The method is based on monitoring the resonance frequency of MeOH protons as a function of MeOH to metal ion mole ratio at constant metal ion concentration. the average solvation number of cation, n, at any MeOH/ metal ion mole ration was calculated from the NMR chemical shift-mole ration data and was plotted against the mole ration values. The solvation numbers of alkaline earth cations were obtained from the limiting values of the corresponding n, vs. mole ratio plots.

  6. Study of the effect hydrogen binding in the solvation of alkaline earth cations with MeOH in nitromethane using 1 H NMR technique and determination of ionic solvation number

    International Nuclear Information System (INIS)

    Alizadeh, N.

    2001-01-01

    A proton NMR method for the study of the effect hydrogen binding and determination of solvation numbers of alkaline earth cations with methanol (MeOH) in in tromethane (NM) as diluent is described. The method is based on monitoring the resonance frequency of MeOH protons as a function of MeOH to metal ion mole ratio at constant metal ion concentration. the average solvation number of cation, n, at any MeOH/ metal ion mole ration was calculated from the NMR chemical shift-mole ration data and was plotted against the mole ration values. The solvation numbers of alkaline earth cations were obtained from the limiting values of the corresponding n, vs. mole ratio plots

  7. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.

    2009-01-01

    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

  8. Evaluation of in vivo selective binding of [{sup 11}C]doxepin to histamine H{sub 1} receptors in five animal species

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, Kiichi E-mail: ishiwata@pet.tmig.or.jp; Kawamura, Kazunori; Wang Weifang; Tsukada, Hideo; Harada, Norihiro; Mochizuki, Hideki; Kimura, Yuichi; Ishii, Kenji; Iwata, Ren; Yanai, Kazuhiko

    2004-05-01

    The specific binding of [{sup 11}C]doxepin, which has been used as a radioligand for mapping histamine H{sub 1} receptors in human brain by positron emission tomography, was evaluated in five animal species. In mice the [{sup 11}C]doxepin uptake was reduced by treatment with cold doxepin and two H{sub 1} receptor antagonists, but not with H{sub 2}/H{sub 3} antagonists. The specific binding evaluated with treatment with (+)-chlorpheniramine (H{sub 1} antagonist) was in the range of 10-30% in mouse, rat, rabbit, and monkey, but was not detected in guinea pig.

  9. Determination of blood concentrations of main active compounds in Zi-Cao-Cheng-Qi decoction and their total plasma protein binding rates based on hollow fiber liquid phase microextraction coupled with high performance liquid chromatography.

    Science.gov (United States)

    Li, Miaomiao; Chen, Xuan; Hu, Shuang; Wang, Runqin; Peng, Xiaoli; Bai, Xiaohong

    2018-01-01

    Oil-in-salt hollow fiber liquid phase microextraction coupled with high performance liquid chromatography ultraviolet detection (HPLC-UV) was developed for determination of the blood concentrations of the main active compounds, hesperidin, honokiol, shikonin, magnolol, emodin and β,β'-dimethylacrylshikonin, after oral administration of Zi-Cao-Cheng-Qi decoction (ZCCQD) and their total plasma protein binding rates. In the procedure, a hollow fiber segment was immersed in organic solvent to fill the solvent in the fiber lumen and wall pore, and then the fiber was immersed into sodium chloride solution to cover a thin salt membrane on the fiber wall pore filling organic solvent. Various factors affecting the procedure, such as extraction solvent, sample phase pH, stirring rate, extraction time, NaCl concentration and fiber immersion time in the NaCl solution, were optimized. Under the optimum conditions, good linearities (r 2 ≥0.9905), low limits of detection (0.7-2.5ng/mL) or quantitation (1.2-12ng/mL), satisfactory precision (2.6%-12.8%) and accuracy (81.0%-114.2%) of this method, were observed. The results showed that, after oral administration of a 25g/kg dose, (1) the blood concentrations (at 0.5h) of hesperidin, honokiol, shikonin, magnolol, emodin and β,β'-dimethylacrylshikonin were 0.45, 0.40, 0.48, 0.74, 0.11 and 1.11μg/mL, respectively; (2) the total plasma protein binding rates of the six active compounds were 42.0% (hesperidin), 71.8% (honokiol), 64.6% (shikonin), 77.7% (magnolol), 75.3% (emodin) and 75.7% (β,β'-dimethylacrylshikonin), respectively. The proposed procedure coupled with HPLC shows obvious advantages, such as low solvent consumption, simple operation, high sensitivity and strong purifying and can be used for the determination of both the blood concentrations and total plasma protein binding rates of active compounds in traditional Chinese medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Current position of 5HT3 antagonists and the additional value of NK1 antagonists; a new class of antiemetics

    NARCIS (Netherlands)

    R. de Wit (Ronald)

    2003-01-01

    textabstractThe advent of the 5HT3 receptor antagonists (5HT3 antagonists) in the 1990s and the combination with dexamethasone has resulted in acute emesis protection in 70% of patients receiving highly emetogenic chemotherapy. Despite complete protection in the acute phase, however, 40% of patients

  11. Identification of an endogenous alpha-adrenergic receptor antagonist: studies on its possible role in endocrine and cardiovascular function

    International Nuclear Information System (INIS)

    Dunbar, J.C.; Wider, M.; House, F.; Campbell, R.

    1986-01-01

    The concept of α and β adrenergic receptors that are regulated by epinephrine or norepinephrine (NE) is well established. The reported receptor antagonists have been synthetic. A peptide extracted from the duodenal mucosa with α-2 antagonist properties has been identified. It specifically inhibits 3 H-yohimbine binding (α-2) but not 3 H dihydroalprenolol (β) binding in whole brain membranes. Partially purified preparations of the alpha receptor binding inhibitor (ABI) were tested for endocrine pancreatic and cardiovascular effects. When isolated islets were incubated in the presence of ABI with and without NE, ABI along did not alter insulin secretion but completely reversed the NE suppression of glucose stimulated insulin release. Glucagon secretion by these same islets was enhanced by ABI and augmented the stimulatory effect of NE. Intravenous (I.V.) infusion of ABI increased serum insulin in the presence of NE and decreased the serum glucose response to a glucose load. Infusion of ABI into the 4th ventricle, or I.V. resulted in a decrease (50-60%) in systolic and diastolic blood pressure as well as a decrease (10-20%) in heart rate. From these studies the authors conclude that a duodenal peptide with the capacity to inhibit α-2 agonist binding may play a role in endocrine and cardiovascular functions

  12. Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

    Science.gov (United States)

    Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

    2009-04-01

    Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5

  13. Combined Ligand/Structure-Based Virtual Screening and Molecular Dynamics Simulations of Steroidal Androgen Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Yuwei Wang

    2017-01-01

    Full Text Available The antiandrogens, such as bicalutamide, targeting the androgen receptor (AR, are the main endocrine therapies for prostate cancer (PCa. But as drug resistance to antiandrogens emerges in advanced PCa, there presents a high medical need for exploitation of novel AR antagonists. In this work, the relationships between the molecular structures and antiandrogenic activities of a series of 7α-substituted dihydrotestosterone derivatives were investigated. The proposed MLR model obtained high predictive ability. The thoroughly validated QSAR model was used to virtually screen new dihydrotestosterones derivatives taken from PubChem, resulting in the finding of novel compounds CID_70128824, CID_70127147, and CID_70126881, whose in silico bioactivities are much higher than the published best one, even higher than bicalutamide. In addition, molecular docking, molecular dynamics (MD simulations, and MM/GBSA have been employed to analyze and compare the binding modes between the novel compounds and AR. Through the analysis of the binding free energy and residue energy decomposition, we concluded that the newly discovered chemicals can in silico bind to AR with similar position and mechanism to the reported active compound and the van der Waals interaction is the main driving force during the binding process.

  14. Profiling the Interaction Mechanism of Quinoline/Quinazoline Derivatives as MCHR1 Antagonists: An in Silico Method

    Directory of Open Access Journals (Sweden)

    Mingwei Wu

    2014-09-01

    Full Text Available Melanin concentrating hormone receptor 1 (MCHR1, a crucial regulator of energy homeostasis involved in the control of feeding and energy metabolism, is a promising target for treatment of obesity. In the present work, the up-to-date largest set of 181 quinoline/quinazoline derivatives as MCHR1 antagonists was subjected to both ligand- and receptor-based three-dimensional quantitative structure–activity (3D-QSAR analysis applying comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA. The optimal predictable CoMSIA model exhibited significant validity with the cross-validated correlation coefficient (Q2 = 0.509, non-cross-validated correlation coefficient (R2ncv = 0.841 and the predicted correlation coefficient (R2pred = 0.745. In addition, docking studies and molecular dynamics (MD simulations were carried out for further elucidation of the binding modes of MCHR1 antagonists. MD simulations in both water and lipid bilayer systems were performed. We hope that the obtained models and information may help to provide an insight into the interaction mechanism of MCHR1 antagonists and facilitate the design and optimization of novel antagonists as anti-obesity agents.

  15. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Science.gov (United States)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  16. Upregulation of [125I] CGP42112 binding in the rat brainstem following nodose ganglionectomy

    International Nuclear Information System (INIS)

    Roulston, C.L.; Lawrence, A.J.; Jarrott, B.; Widdop, R.E.

    1999-01-01

    Full text: [ 125 I] CGP42112 is a specific ligand which has been used to demonstrate the presence of angiotensin AT 2 receptors in peripheral and brain tissue, although [ 125 I] CGP42112 also binds to a non-angiotensin II (Ang II) site. In the present study we have examined [ 125 I] CGP42112 binding in the brainstem following nodose ganglionectomy using autoradiography. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats underwent unilateral nodose ganglionectomy or sham operation (anaesthetised with methohexitone, 60mg/kg ip). Following a 14 day recovery period, animals were killed and slide mounted brainstem sections (14μm) were prepared and incubated in the presence of either [ 125 I] (Sar 1 Ile 8 )Ang II (0.5nM), [ 125 I] CGP42112 (0.3nM) or [ 3 H] PKI11195 (3nM, a marker for activated microglia). Following incubation, slides were apposed to film for 10 days. Specific binding was determined in adjacent sections by co-incubations with either the AT 1 receptor antagonist losartan, the AT 2 receptor antagonist PD 123319, Ang II or PKI11195 (all at 10μM). In the ganglionectomised groups, there were significant reductions in [ 125 I] (Sar 1 Ile 8 )Ang II binding on the denervated side of the nucleus of the solitary tract (NTS) by 30 ± 2 % (n=5, P 125 I] CGP42112 binding in the NTS revealed an AT 2 receptor component which was displaceable by PD 123319 and Ang II (∼60%), and a non-Ang II component (∼40%). Nodose ganglionectomy increased the density of [ 125 I] CGP42112 binding on the denervated side of the NTS by 55 ± 3 % (n=5, P 125 I] CGP42112 binding in the NTS of the ganglionectomised groups was comprised of a greater non-Ang II component than in the sham group, since only ∼30% was displaced by PD123319 and Ang II. [ 125 I] CGP42112 also revealed high binding density in the dorsal motor nucleus and the nucleus ambiguus on the denervated side in both SHR and WKY rats. This binding was absent in shams and was only ∼30-40% displaceable

  17. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  18. CysLT(1)R antagonists inhibit tumor growth in a xenograft model of colon cancer.

    Science.gov (United States)

    Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

    2013-01-01

    The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (Pcolon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.

  19. Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as revealed by X-ray crystallography, NMR and MD simulations.

    Directory of Open Access Journals (Sweden)

    Xuelu Huan

    Full Text Available The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD, which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity

  20. The cholinergic ligand binding material of axonal membranes

    International Nuclear Information System (INIS)

    Mautner, H.G.; Coronado, R.; Jumblatt, J.E.

    1986-01-01

    Choline acetyltransferase and acetylcholinesterase, the enzymes responsible for the synthesis and hydrolysis of ACh, are present in nerve fibers. In crustacean peripheral nerves, release of ACh from cut nerve fibers has been demonstrated. Previously closed membrane vesicles have been prepared from lobster walking leg nerve plasma membrane and saturable binding of cholinergic agonsist and antagonists to such membranes have been demonstrated. This paper studies this axonal cholinergic binding material, and elucidates its functions. The binding of tritium-nicotine to lobster nerve plasma membranes was antagonized by a series of cholinergic ligands as well as by a series of local anesthetics. This preparation was capable of binding I 125-alpha-bungarotoxin, a ligand widely believed to be a specific label for nicotinic ACh receptor. The labelling of 50 K petide band with tritium-MBTA following disulfide reduction is illustrated

  1. HIV-1 tropism testing and clinical management of CCR5 antagonists: Quebec review and recommendations.

    Science.gov (United States)

    Tremblay, Cécile; Hardy, Isabelle; Lalonde, Richard; Trottier, Benoit; Tsarevsky, Irina; Vézina, Louis-Philippe; Roger, Michel; Wainberg, Mark; Baril, Jean-Guy

    2013-01-01

    HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

  2. Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens

    OpenAIRE

    Singh, Tejinder P.; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K.

    2017-01-01

    Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were ...

  3. Antagonistic neural networks underlying differentiated leadership roles

    Science.gov (United States)

    Boyatzis, Richard E.; Rochford, Kylie; Jack, Anthony I.

    2014-01-01

    The emergence of two distinct leadership roles, the task leader and the socio-emotional leader, has been documented in the leadership literature since the 1950s. Recent research in neuroscience suggests that the division between task-oriented and socio-emotional-oriented roles derives from a fundamental feature of our neurobiology: an antagonistic relationship between two large-scale cortical networks – the task-positive network (TPN) and the default mode network (DMN). Neural activity in TPN tends to inhibit activity in the DMN, and vice versa. The TPN is important for problem solving, focusing of attention, making decisions, and control of action. The DMN plays a central role in emotional self-awareness, social cognition, and ethical decision making. It is also strongly linked to creativity and openness to new ideas. Because activation of the TPN tends to suppress activity in the DMN, an over-emphasis on task-oriented leadership may prove deleterious to social and emotional aspects of leadership. Similarly, an overemphasis on the DMN would result in difficulty focusing attention, making decisions, and solving known problems. In this paper, we will review major streams of theory and research on leadership roles in the context of recent findings from neuroscience and psychology. We conclude by suggesting that emerging research challenges the assumption that role differentiation is both natural and necessary, in particular when openness to new ideas, people, emotions, and ethical concerns are important to success. PMID:24624074

  4. Antagonistic Neural Networks Underlying Differentiated Leadership Roles

    Directory of Open Access Journals (Sweden)

    Richard Eleftherios Boyatzis

    2014-03-01

    Full Text Available The emergence of two distinct leadership roles, the task leader and the socio-emotional leader, has been documented in the leadership literature since the 1950’s. Recent research in neuroscience suggests that the division between task oriented and socio-emotional oriented roles derives from a fundamental feature of our neurobiology: an antagonistic relationship between two large-scale cortical networks -- the Task Positive Network (TPN and the Default Mode Network (DMN. Neural activity in TPN tends to inhibit activity in the DMN, and vice versa. The TPN is important for problem solving, focusing of attention, making decisions, and control of action. The DMN plays a central role in emotional self-awareness, social cognition, and ethical decision making. It is also strongly linked to creativity and openness to new ideas. Because activation of the TPN tends to suppress activity in the DMN, an over-emphasis on task oriented leadership may prove deleterious to social and emotional aspects of leadership. Similarly, an overemphasis on the DMN would result in difficulty focusing attention, making decisions and solving known problems. In this paper, we will review major streams of theory and research on leadership roles in the context of recent findings from neuroscience and psychology. We conclude by suggesting that emerging research challenges the assumption that role differentiation is both natural and necessary, in particular when openness to new ideas, people, emotions, and ethical concerns are important to success.

  5. Antagonistic neural networks underlying differentiated leadership roles.

    Science.gov (United States)

    Boyatzis, Richard E; Rochford, Kylie; Jack, Anthony I

    2014-01-01

    The emergence of two distinct leadership roles, the task leader and the socio-emotional leader, has been documented in the leadership literature since the 1950s. Recent research in neuroscience suggests that the division between task-oriented and socio-emotional-oriented roles derives from a fundamental feature of our neurobiology: an antagonistic relationship between two large-scale cortical networks - the task-positive network (TPN) and the default mode network (DMN). Neural activity in TPN tends to inhibit activity in the DMN, and vice versa. The TPN is important for problem solving, focusing of attention, making decisions, and control of action. The DMN plays a central role in emotional self-awareness, social cognition, and ethical decision making. It is also strongly linked to creativity and openness to new ideas. Because activation of the TPN tends to suppress activity in the DMN, an over-emphasis on task-oriented leadership may prove deleterious to social and emotional aspects of leadership. Similarly, an overemphasis on the DMN would result in difficulty focusing attention, making decisions, and solving known problems. In this paper, we will review major streams of theory and research on leadership roles in the context of recent findings from neuroscience and psychology. We conclude by suggesting that emerging research challenges the assumption that role differentiation is both natural and necessary, in particular when openness to new ideas, people, emotions, and ethical concerns are important to success.

  6. Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

    International Nuclear Information System (INIS)

    Pollack, A.E.; Wooten, G.F.

    1987-01-01

    The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

  7. Multiple Targeting Approaches on Histamine H3 Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Mohammad eKhanfar

    2016-05-01

    Full Text Available With the very recent market approval of pitolisant (Wakix®, the interest in clinical applications of novel multifunctional histamine H3 receptor antagonists has clearly increased. Since histamine H3 receptor antagonists in clinical development have been tested for a variety of different indications, the combination of pharmacological properties in one molecule for improved pharmacological effects and reduced unwanted side-effects is rationally based on the increasing knowledge on the complex neurotransmitter regulations. The polypharmacological approaches on histamine H3 receptor antagonists on different G-protein coupled receptors, transporters, enzymes as well as on NO-signaling mechanism are described, supported with some lead structures.

  8. Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Minuk, G.Y.; Bear, C.E.; Sarjeant, E.J.

    1987-01-01

    To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, [ 3 H]GABA, was added to sodium-free suspensions of Percoll-purified hepatocytes derived from collagenase-perfused rat livers under various experimental conditions and in the presence or absence of specific GABA receptor agonists (muscimol) and antagonists (bicuculline). The effects of GABA, muscimol, and bicuculline on hepatocyte resting membrane potentials were also determined. Specific binding of [ 3 H]GABA to hepatocytes was a consistent finding. GABA-hepatocyte interactions were reversible and temperature dependent. Muscimol and bicuculline inhibited binding in a dose-dependent manner (IC50, 30 nM and 50 microM, respectively), whereas strychnine (1.0-100 microM), a nonspecific central nervous system stimulant, had no appreciable effect. Both GABA and muscimol (100 microM) caused significant hyperpolarization of hepatocyte resting membrane potential (delta PD 5.4 +/- 3.1 and 22.2 +/- 16.2 mV, respectively, means +/- SD, P less than 0.0005). Bicuculline (100 microM) inhibited the effect of muscimol (P less than 0.05). The results of this study suggest that specific GABA receptor sites exist on the surface of isolated rat hepatocytes. The presence of such sites raises the possibility that, in addition to adrenergic and cholinergic innervation, hepatic function may be influenced by GABA-ergic neurotransmitter mechanisms

  9. Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors

    DEFF Research Database (Denmark)

    Kvist, Trine; Greenwood, Jeremy R; Hansen, Kasper B

    2013-01-01

    . In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid...... (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands....

  10. SAOS-2 osteosarcoma cells bind fibroblasts via ICAM-1 and this is increased by tumour necrosis factor-α.

    Directory of Open Access Journals (Sweden)

    Manu S David

    Full Text Available We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of "cellular sipping" changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001, and this was increased by h-GF pre-treatment with TNF-α (p<0.001. TNF-α stimulated binding was dose dependent and maximal at 1.16 nM (p<0.05 with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5 hr pre-treatment (p<0.001. h-GF retained maximal binding up to 6 hrs after TNF-α stimulation, but this was lost by 18 hrs (p<0.001. FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04. Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003, and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we

  11. Synergism between dexketoprofen and meloxicam in an orofacial formalin test was not modified by opioid antagonists.

    Science.gov (United States)

    Gonzalez, Claudia; Zegpi, Carlos; Noriega, Viviana; Prieto, Juan C; Miranda, Hugo F

    2011-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the management of acute and chronic pain. The role of the opioid system in the synergism between NSAIDs is not well characterized. Mice were injected with a 5% formalin solution (20 μl) into the upper right lip to perform an orofacial formalin test. The isobolographic method was used to determine the interaction between dexketoprofen, which is the (S)-(+) enantiomer of ketoprofen, and meloxicam co-administration. Additionally, the non-selective, opioid antagonist naltrexone, the selective δ opioid receptor (DOP) antagonist naltrindole and the selective κ opioid receptor (KOP) antagonist norbinaltorphimine were used to assess the opioid effects on this interaction. Intraperitoneal administration of dexketoprofen or meloxicam induced dose-dependent antinociception with different phase I and phase II potencies in the orofacial formalin test. Meloxicam displayed similar potencies (ED(50)) in phase I (7.20 mg/kg) and phase II (8.60 mg/kg). Dexketoprofen was more potent in phase I (19.96 mg/kg) than in phase II (50.90 mg/kg). The interactions between dexketoprofen and meloxicam were synergistic in both phases. This was determined based on the fixed ratios (1:1) of their ED(50) values, which were determined by isobolographic analysis. Furthermore, this antinociceptive activity does not seem to be modulated by opioid receptor blockers because they did not induce changes in the nature of this interaction. This finding may be relevant with regards to NSAID multi-modal analgesia where an opioid antagonist must be used.

  12. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  13. In vitro Evaluation of a Bombesin Antagonistic Analogue Conjugated with DOTA-Ala(SO3H)-Aminooctanoyl for Targeting of the Gastrin-releasing Peptide Receptor

    International Nuclear Information System (INIS)

    Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Lee, So Young; Choi, Sang Mu

    2014-01-01

    As Bombesin (BBS) binds with high affinity to GRPR, BBS derivatives have been labeled with various radionuclides such as 99 mTc, 111 In, 90 Y, 64 Cu, 177 Lu, 68 Ga, or 18 F and have proved to be successful candidates for peptide receptor radiotherapy (PRRT). In this study, we employed Ala(SO 3 H)-Aminooctanoyl as a linker of BBS antagonistic peptide sequence, Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH 2 , with DOTA to prepare radiolabeled candidates for GRPR targeting. A DOTA-conjugated BBS antagonistic analogue was synthesized and radiolabeled with 177 Lu, and in vitro characteristics on GRPR-overexpressing human prostate tumor cells were evaluated. In conclusion, a novel BBS antagonistic analogue, 177 Lu-DOTA-sBBNA, is a promising candidate for the targeting of GRPR-over-expressing tumors. Further investigations to evaluate its in vivo characteristics and therapeutic efficacy are needed

  14. Neuroprotective and memory-related actions of novel alpha-7 nicotinic agents with different mixed agonist/antagonist properties.

    Science.gov (United States)

    Meyer, E M; Tay, E T; Zoltewicz, J A; Meyers, C; King, M A; Papke, R L; De Fiebre, C M

    1998-03-01

    The goals of this study were to develop compounds that were selective and highly efficacious agonists at alpha-7 receptors, while varying in antagonist activity; and to test the hypothesis that these compounds had memory-related and neuroprotective actions associated with both agonist and antagonist alpha-7 receptor activities. Three compounds were identified; E,E-3-(cinnamylidene)anabaseine (3-CA), E,E-3-(2-methoxycinnamylidene) anabaseine (2-MeOCA) and E,E-3-(4-methoxycinnamylidene) anabaseine (4-MeOCA) each displaced [125I]alpha-bungarotoxin binding from rat brain membranes and activated rat alpha-7 receptors in a Xenopus oocyte expression system fully efficaciously. The potency series for binding and receptor activation was 2-MeOCA > 4-MeOCA = 3-CA and 2-MeOCA = 3-CA > 4-MeOCA, respectively. No compound significantly activated oocyte-expressed alpha-4beta-2 receptors. Although each cinnamylidene-anabaseine caused a long-term inhibition of alpha-7 receptors, as measured by ACh-application 5 min later, this inhibition ranged considerably, from less than 20% (3-CA) to 90% (2-MeOCA) at an identical concentration (10 microM). These compounds improved passive avoidance behavior in nucleus basalis lesioned rats, with 2-MeOCA most potent in this respect. In contrast, only 3-CA was neuroprotective against neurite loss during nerve growth factor deprivation in differentiated rat pheochromocytoma (PC12) cells. Choline, an efficacious alpha-7 agonist without antagonist activity, was also protective in this model. These results suggest that the neurite-protective action of alpha-7 receptor agonists may be more sensitive to potential long-term antagonist properties than acute behavioral actions are.

  15. A SELECTIVE ANTAGONIST OF MINERALOCORTICOID RECEPTOR EPLERENONE IN CARDIOLOGY PRACTICE

    Directory of Open Access Journals (Sweden)

    B. B. Gegenava

    2015-01-01

    Full Text Available The role of aldosterone in pathophysiological processes is considered. The effects of the selective antagonist of mineralocorticoid receptor eplerenone are analyzed. The advantages of eplerenone compared with spironolactone are discussed.

  16. Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375

    Science.gov (United States)

    Douglas, Stephen A; Behm, David J; Aiyar, Nambi V; Naselsky, Diane; Disa, Jyoti; Brooks, David P; Ohlstein, Eliot H; Gleason, John G; Sarau, Henry M; Foley, James J; Buckley, Peter T; Schmidt, Dulcie B; Wixted, William E; Widdowson, Katherine; Riley, Graham; Jin, Jian; Gallagher, Timothy F; Schmidt, Stanley J; Ridgers, Lance; Christmann, Lisa T; Keenan, Richard M; Knight, Steven D; Dhanak, Dashyant

    2005-01-01

    SB-706375 potently inhibited [125I]hU-II binding to both mammalian recombinant and ‘native' UT receptors (Ki 4.7±1.5 to 20.7±3.6 nM at rodent, feline and primate recombinant UT receptors and Ki 5.4±0.4 nM at the endogenous UT receptor in SJRH30 cells). Prior exposure to SB-706375 (1 μM, 30 min) did not alter [125I]hU-II binding affinity or density in recombinant cells (KD 3.1±0.4 vs 5.8±0.9 nM and Bmax 3.1±1.0 vs 2.8±0.8 pmol mg−1) consistent with a reversible mode of action. The novel, nonpeptidic radioligand [3H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (KD 2.6±0.4 nM, Bmax 0.86±0.12 pmol mg−1) in a manner that was inhibited by both U-II isopeptides and SB-706375 (Ki 4.6±1.4 to 17.6±5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. SB-706375 was a potent, competitive hU-II antagonist across species with pKb 7.29–8.00 in HEK293-UT receptor cells (inhibition of [Ca2+]i-mobilization) and pKb 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (Kapp∼20 nM). SB-706375 was a selective U-II antagonist with ⩾100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (Ki/IC50>1 μM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 μM). In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals. PMID:15852036

  17. Studies on Aryl-Substituted Phenylalanines: Synthesis, Activity, and Different Binding Modes at AMPA Receptors

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Frydenvang, Karla Andrea; Pickering, Darryl S

    2016-01-01

    , not previously seen for amino acid-based AMPA receptor antagonists, X-ray crystal structures of both eutomers in complex with the GluA2 ligand binding domain were solved. The cocrystal structures of (S)-37 and (R)-38 showed similar interactions of the amino acid parts but unexpected and different orientations...

  18. Similar serotonin-2A receptor binding in rats with different coping styles or levels of aggression

    DEFF Research Database (Denmark)

    Visser, Anniek Kd; Ettrup, Anders; Klein, Anders Bue

    2015-01-01

    is not an important molecular marker for coping style. Since neither an