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Sample records for high affinity influx

  1. ATP stimulates calcium influx in primary astrocyte cultures

    International Nuclear Information System (INIS)

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-01-01

    The effect of ATP and other purines on 45 Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45 Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45 Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45 Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced 45 Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

  2. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

    Science.gov (United States)

    Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

    2000-08-01

    The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

  3. Cytosine arabinoside influx and nucleoside transport sites in acute leukemia.

    Science.gov (United States)

    Wiley, J S; Jones, S P; Sawyer, W H; Paterson, A R

    1982-02-01

    Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10

  4. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  5. Ketamine inhibits 45Ca influx and catecholamine secretion by inhibiting 22Na influx in cultured bovine adrenal medullary cells

    International Nuclear Information System (INIS)

    Takara, Hiroshi; Wada, Akihiko; Arita, Masahide; Izumi, Futoshi; Sumikawa, Koji

    1986-01-01

    The effects of ketamine, an intravenous anesthetic, on 22 Na influx, 45 Ca influx and catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Ketamine inhibited carbachol-induced 45 Ca influx and catecholamine secretion in a concentration-dependent manner with a similar potency. Ketamine also reduced veratridine-induced 45 Ca influx and catecholamine secretion. The influx of 22 Na caused by carbachol or by veratridine was suppressed by ketamine with a concentration-inhibition curve similar to that of 45 Ca influx and catecholamine secretion. Inhibition by ketamine of the carbachol-induced influx of 22 Na, 45 Ca and secretion of catecholamines was not reversed by the increased concentrations of carbachol. These observations indicate that ketamine, at clinical concentrations, can inhibit nicotinic receptor-associated ionic channels and that the inhibition of Na influx via the receptor-associated ionic channels is responsible for the inhibition of carbachol-induced Ca influx and catecholamine secretion. (Auth.)

  6. Correlations between locked modes and impurity influxes

    Energy Technology Data Exchange (ETDEWEB)

    Fishpool, G M [Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking; Lawson, K D [UKAEA Culham Lab., Abingdon (United Kingdom)

    1994-07-01

    An analysis of pulses that were disturbed by medium Z impurity influxes (Cl, Cr, Fe and Ni) recorded during the 91/92 JET operations, has demonstrated that such influxes can result in MHD modes which subsequently ``lock``. A correlation is found between the power radiated by the influx and the time difference between the start of the influx and the beginning of the locked mode. The growth in the amplitude of the locked mode itself can lead to further impurity influxes. A correlation is noted between intense influxes (superior to 10 MW) and the mode ``unlocking``. (authors). 4 refs., 4 figs.

  7. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    Science.gov (United States)

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  8. Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A.

    Science.gov (United States)

    Wei, An-Chi; Liu, Ting; Cortassa, Sonia; Winslow, Raimond L; O'Rourke, Brian

    2011-07-01

    Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially

  9. Maintained LTP and Memory Are Lost by Zn2+ Influx into Dentate Granule Cells, but Not Ca2+ Influx.

    Science.gov (United States)

    Takeda, Atsushi; Tamano, Haruna; Hisatsune, Marie; Murakami, Taku; Nakada, Hiroyuki; Fujii, Hiroaki

    2018-02-01

    The idea that maintained LTP and memory are lost by either increase in intracellular Zn 2+ in dentate granule cells or increase in intracellular Ca 2+ was examined to clarify significance of the increases induced by excess synapse excitation. Both maintained LTP and space memory were impaired by injection of high K + into the dentate gyrus, but rescued by co-injection of CaEDTA, which blocked high K + -induced increase in intracellular Zn 2+ but not high K + -induced increase in intracellular Ca 2+ . High K + -induced disturbances of LTP and intracellular Zn 2+ are rescued by co-injection of 6-cyano-7-nitroquinoxakine-2,3-dione, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, but not by co-injection of blockers of NMDA receptors, metabotropic glutamate receptors, and voltage-dependent calcium channels. Furthermore, AMPA impaired maintained LTP and the impairment was also rescued by co-injection of CaEDTA, which blocked increase in intracellular Zn 2+ , but not increase in intracellular Ca 2+ . NMDA and glucocorticoid, which induced Zn 2+ release from the internal stores, did not impair maintained LTP. The present study indicates that increase in Zn 2+ influx into dentate granule cells through AMPA receptors loses maintained LTP and memory. Regulation of Zn 2+ influx into dentate granule cells is more critical for not only memory acquisition but also memory retention than that of Ca 2+ influx.

  10. High Ca2+ Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death.

    Science.gov (United States)

    Abdul-Muneer, P M; Long, Mathew; Conte, Adriano Andrea; Santhakumar, Vijayalakshmi; Pfister, Bryan J

    2017-08-01

    We investigated the hypothesis that high Ca 2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca 2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca 2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca 2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca 2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

  11. Radiation entropy influx as a measure of planetary dissipative processes

    International Nuclear Information System (INIS)

    Izakov, M.N.

    1989-01-01

    Dissipative processes including high flows of matter and energy occur at the planets. Radiation negentropy influx, resulting from difference of entropy fluxes of incoming solar and outgoing thermal radiation of the planet, is a measure of all these processes. Large share of radiation negentropy influx is spent in the vertical thermal fluxes which keep the planet temperature conditions. Next share of radiation negentropy consumption at the Earth is water evaporation. It's rest part is used for the dynamics, which is explained by the efficiency insignificant amount of heat engine, which generates movements in the atmosphere and ocean. Essentially higher share of radiation negentropy influx, than at the Earth, is spent at the Venus, where there are practically no water

  12. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  13. Toroidal asymmetries in divertor impurity influxes in NSTX

    Directory of Open Access Journals (Sweden)

    F. Scotti

    2017-08-01

    Full Text Available Toroidal asymmetries in divertor carbon and lithium influxes were observed in NSTX, due to toroidal differences in surface composition, tile leading edges, externally-applied three-dimensional (3D fields and toroidally-localized edge plasma modifications due to radio frequency heating. Understanding toroidal asymmetries in impurity influxes is critical for the evaluation of total impurity sources, often inferred from measurements with a limited toroidal coverage. The toroidally-asymmetric lithium deposition induced asymmetries in divertor lithium influxes. Enhanced impurity influxes at the leading edge of divertor tiles were the main cause of carbon toroidal asymmetries and were enhanced during edge localized modes. Externally-applied 3D fields led to strike point splitting and helical lobes observed in divertor impurity emission, but marginal changes to the toroidally-averaged impurity influxes. Power coupled to the scrape-off layer SOL plasma during radio frequency (RF heating of H-mode discharges enhanced impurity influxes along the non-axisymmetric divertor footprint of flux tubes connecting to plasma in front of the RF antenna.

  14. High-affinity hemoglobin and blood oxygen saturation in diving emperor penguins.

    Science.gov (United States)

    Meir, Jessica U; Ponganis, Paul J

    2009-10-01

    The emperor penguin (Aptenodytes forsteri) thrives in the Antarctic underwater environment, diving to depths greater than 500 m and for durations longer than 23 min. To examine mechanisms underlying the exceptional diving ability of this species and further describe blood oxygen (O2) transport and depletion while diving, we characterized the O2-hemoglobin (Hb) dissociation curve of the emperor penguin in whole blood. This allowed us to (1) investigate the biochemical adaptation of Hb in this species, and (2) address blood O2 depletion during diving, by applying the dissociation curve to previously collected partial pressure of O2 (PO2) profiles to estimate in vivo Hb saturation (SO2) changes during dives. This investigation revealed enhanced Hb-O2 affinity (P50=28 mmHg, pH 7.5) in the emperor penguin, similar to high-altitude birds and other penguin species. This allows for increased O2 at low blood PO2 levels during diving and more complete depletion of the respiratory O2 store. SO2 profiles during diving demonstrated that arterial SO2 levels are maintained near 100% throughout much of the dive, not decreasing significantly until the final ascent phase. End-of-dive venous SO2 values were widely distributed and optimization of the venous blood O2 store resulted from arterialization and near complete depletion of venous blood O2 during longer dives. The estimated contribution of the blood O2 store to diving metabolic rate was low and highly variable. This pattern is due, in part, to the influx of O2 from the lungs into the blood during diving, and variable rates of tissue O2 uptake.

  15. The influx of amino acids into the heart of the rat

    International Nuclear Information System (INIS)

    Banos, G.; Moorhouse, S.R.; Pratt, O.E.; Wilson, P.A.; Daniel, P.M.

    1978-01-01

    The influx of nineteen amino acids into the heart of the living rat was studied by a method specially devised for experiments under controlled conditions in vivo. When, in separate experiments, the concentration of each amino acid in turn was artificially raised in the circulation, the influx of that amino acid into the heart increased. The data indicate that at least ten of these amino acids enter the heart in vivo by means of saturable carrier-mediated transport systems. The transport rates conform, at least approximately, to Michaelis kinetics and the transport systems are clearly, in the case of many amino acids, active, i.e. energy-dependent. The amino acids which were studied had rates of influx into the heart which differed from each other over a range of more than 10 to 1, even when allowances were made for the differences in their concentration in the circulating blood. These differences in influx were not related to such factors as the molecular size of the individual amino acids. The amino acids which have a high influx into the heart are mainly those which are needed either to re-synthesize contractile protein or as oxidizable substrates. (author)

  16. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Predicting dietborne metal toxicity from metal influxes

    Science.gov (United States)

    Croteau, M.-N.; Luoma, S.N.

    2009-01-01

    Dietborne metal uptake prevails for many species in nature. However, the links between dietary metal exposure and toxicity are not well understood. Sources of uncertainty include the lack of suitable tracers to quantify exposure for metals such as copper, the difficulty to assess dietary processes such as food ingestion rate, and the complexity to link metal bioaccumulation and effects. We characterized dietborne copper, nickel, and cadmium influxes in a freshwater gastropod exposed to diatoms labeled with enriched stable metal isotopes. Metal influxes in Lymnaea stagnalis correlated linearly with dietborne metal concentrations over a range encompassing most environmental exposures. Dietary Cd and Ni uptake rate constants (kuf) were, respectively, 3.3 and 2.3 times higher than that for Cu. Detoxification rate constants (k detox) were similar among metals and appeared 100 times higher than efflux rate constants (ke). Extremely high Cu concentrations reduced feeding rates, causing the relationship between exposure and influx to deviate from linearity; i.e., Cu uptake rates leveled off between 1500 and 1800 nmol g-1 day-1. L. stagnalis rapidly takes up Cu, Cd, and Ni from food but detoxifies the accumulated metals, instead of reducing uptake or intensifying excretion. Above a threshold uptake rate, however, the detoxification capabilities of L. stagnalis are overwhelmed.

  18. Influx: A Tool and Framework for Reasoning under Uncertainty

    Science.gov (United States)

    2015-09-01

    document provides a high-level description of Influx1 from the reasoning perspective. The organisation of the document is given below. Section 2 presents a...exhibits behaviour similar to that of the proposed alternatives while maintaining mathematical simplicity and possessing highly-desirable

  19. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  20. High-throughput fragment screening by affinity LC-MS.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  1. TFTR L mode energy confinement related to deuterium influx

    International Nuclear Information System (INIS)

    Strachan, J.D.

    1999-01-01

    Tokamak energy confinement scaling in TFTR L mode and supershot regimes is discussed. The main result is that TFTR L mode plasmas fit the supershot scaling law for energy confinement. In both regimes, plasma transport coefficients increased with increased edge deuterium influx. The common L mode confinement scaling law on TFTR is also inversely proportional to the volume of wall material that is heated to a high temperature, possibly the temperature at which the deuterium sorbed in the material becomes detrapped and highly mobile. The deuterium influx is increased by: (a) increased beam power due to a deeper heated depth in the edge components and (b) decreased plasma current due to an increased wetted area as governed by the empirically observed dependence of the SOL width upon plasma current. (author). Letter-to-the-editor

  2. Calcium influx pathways in rat pancreatic ducts

    DEFF Research Database (Denmark)

    Hug, M J; Pahl, C; Novak, I

    1996-01-01

    A number of agonists increase intracellular Ca2+ activity, [Ca2+]i, in pancreatic ducts, but the influx/efflux pathways and intracellular Ca2+ stores in this epithelium are unknown. The aim of the present study was to characterise the Ca2+ influx pathways, especially their pH sensitivity, in nati...

  3. Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1987-01-01

    3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

  4. Structure of Greyhound hemoglobin: origin of high oxygen affinity.

    Science.gov (United States)

    Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

    2011-05-01

    This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

  5. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  6. Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

    Science.gov (United States)

    Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

    2001-12-01

    This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

  7. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    ] 3 H]cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 3 H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [ 3 H]cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  8. Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B.

    1987-01-01

    /sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

  9. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

    Directory of Open Access Journals (Sweden)

    Fabian König

    Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

  10. Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters.

    Science.gov (United States)

    Stefan, Christopher P; Zhang, Nannan; Sokabe, Takaaki; Rivetta, Alberto; Slayman, Clifford L; Montell, Craig; Cunningham, Kyle W

    2013-02-01

    In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca(2+) influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K(+) and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K(+) but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K(+) environments, suggesting they promote K(+) uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K(+) currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K(+) currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K(+) influx and that HACS may electrochemically respond to K(+) influx in much the same way as the homologous voltage-gated Ca(2+) channels in most animal cell types.

  11. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system

    International Nuclear Information System (INIS)

    Escandon, E.; Chao, M.V.

    1990-01-01

    In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with 125 I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system

  13. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  14. INMS measures an influx of molecules from Saturn's rings

    Science.gov (United States)

    Perry, M. E.

    2017-12-01

    In 1984, Connerney and Waite proposed water influx from Saturn's rings to explain the low electron densities measured during Pioneer and Voyager radio occultation experiments. Charge exchange with this minor species depleted the H+ ions and provided a faster path to electron recombination. With ice the primary constituent of the rings, water was the most likely in-falling molecule. During the Grand Finale orbits, Cassini's Ion and Neutral Mass Spectrometer (INMS) detected and quantified an influx from the rings. Unexpectedly, the primary influx molecules are CH4 and a heavier carbon-bearing species. Water was detected, but quantities were factors of ten lower than these other species. Distribution in both altitude and latitude are consistent with a ring influx. The concentration of the minor species in Saturn's atmosphere shows that they enter Saturn's atmosphere from the top. Both molecules have their highest concentrations at the highest altitudes, with concentrations >0.4% at 3,500 km altitude and only 0.02% at 2,700 km. Molecules from the rings deorbit to Saturn's atmosphere at altitudes near 4,000 km, consistent with the INMS measurements. The latitudinal dependence of the minor species indicates that their source is near the equatorial plane. At high altitudes, the minor species were observed primarily at zero latitude, where the 28u species was six times more concentrated than at 5° latitude. At lower altitudes, the peaking ratio was 1, indicating that the species had diffused and was fully mixed into Saturn's H2 atmosphere. The lighter molecule, CH4, diffuses more rapidly than the 28u species. INMS also detected both of these species during the earlier F-ring passes, finding that the neutrals were centered at the ring plane and extended 3,000 km (half width, half max) north and south.

  15. Measurement of calcium influx in tethered rings of rabbit aorta under tension

    International Nuclear Information System (INIS)

    Gleason, M.M.; Ratz, P.H.; Flaim, S.F.

    1985-01-01

    Calcium (Ca) influx in vascular smooth muscle is routinely measured in untethered preparations not under passive stretch, and Ca influx data are correlated with data for steady-state isometric tension obtained under parallel conditions from tethered preparations under passive stretch. The validity of this method was tested by simultaneous measurement of Ca influx and tension in tethered rings of rabbit thoracic aorta. Ca influx ( 45 Ca 3-min pulse) and tension were measured at 3 and 30 min after norepinephrine (NE) or KCl and under control (no agonist) conditions. Active tension was significantly altered by variations in passive tension. Ca influx was unaffected by passive tension under control, NE, or KCl conditions, and results were similar at 3 and 30 min. The results confirm the validity of correlating Ca influx data from untethered rings with steady-state contractile response data obtained from tethered rings under similar experimental conditions

  16. Characteristic of Extracellular Zn2+ Influx in the Middle-Aged Dentate Gyrus and Its Involvement in Attenuation of LTP.

    Science.gov (United States)

    Takeda, Atsushi; Koike, Yuta; Osaw, Misa; Tamano, Haruna

    2018-03-01

    An increased influx of extracellular Zn 2+ into neurons is a cause of cognitive decline. The influx of extracellular Zn 2+ into dentate granule cells was compared between young and middle-aged rats because of vulnerability of the dentate gyrus to aging. The influx of extracellular Zn 2+ into dentate granule cells was increased in middle-aged rats after injection of AMPA and high K + into the dentate gyrus, but not in young rats. Simultaneously, high K + -induced attenuation of LTP was observed in middle-aged rats, but not in young rats. The attenuation was rescued by co-injection of CaEDTA, an extracellular Zn 2+ chelator. Intracellular Zn 2+ in dentate granule cells was also increased in middle-aged slices with high K + , in which the increase in extracellular Zn 2+ was the same as young slices with high K + , suggesting that ability of extracellular Zn 2+ influx into dentate granule cells is greater in middle-aged rats. Furthermore, extracellular zinc concentration in the hippocampus was increased age-dependently. The present study suggests that the influx of extracellular Zn 2+ into dentate granule cells is more readily increased in middle-aged rats and that its increase is a cause of age-related attenuation of LTP in the dentate gyrus.

  17. Enhanced carbon influx into TFTR supershots

    International Nuclear Information System (INIS)

    Ramsey, A.T.; Bush, C.E.; Dylla, H.F.; Owens, D.K.; Pitcher, C.S.; Ulrickson, M.A.

    1991-01-01

    Under some conditions, a very large influx of carbon into TFTR occurs during neutral beam injection into low recycling plasmas (the supershot regime). These carbon ''blooms'' result in serious degradation of plasma parameters. The sources of this carbon have been identified as hot spots on the TFTR bumper limiter at or near the last closed flux surface. Two separate temperature thresholds have been identified. One threshold, at about 1650 deg. C, is consistent with radiation enhanced sublimation (RES). The other, at about 2300 deg. C, appears to be thermal sublimation of carbon from the limiter. The carbon influx can be quantitatively accounted for by taking laboratory values for RES rates, making reasonable assumptions about the extent of the blooming area and assuming unity carbon recycling at the limiter. Such high carbon recycling is expected, and it is shown that, in target plasmas at least, it is observed on TFTR. The sources of the carbon blooms are sites which have either loosely attached fragments of limiter material (caused by damage) or surfaces that are nearly perpendicular to the magnetic field lines. Such surfaces may have local power depositions two orders of magnitude higher than usual. The TFTR team modified the limiter during the opening of winter 1989-1990. The modifications greatly reduced the number and magnitude of the blooms, so that they are no longer a problem. (author). 27 refs, 9 figs

  18. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  19. Inhibiting the Ca2+ Influx Induced by Human CSF

    Directory of Open Access Journals (Sweden)

    Anna Drews

    2017-12-01

    Full Text Available One potential therapeutic strategy for Alzheimer’s disease (AD is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-β peptide (Aβ; and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.

  20. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

    International Nuclear Information System (INIS)

    Haber, R.S.; Loeb, J.N.

    1988-01-01

    The administration of thyroid hormone is known to result in an induction of the Na + -K + -adenosinetriphosphatase (Na + -K + -ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na + -K + -ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3',5-triiodo-L-thyronine (T 3 ) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na + -K + -ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K I s) for ouabain of ∼10 -7 and 10 -4 M, respectively. Measurement of the specific binding of [ 3 H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K d ) of slightly less than 10 -7 M, whose maximal binding capacity was increased by T 3 treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T 3 treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na + -K + -ATPase by ouabain-dependent phosphorylation from [ 32 P]orthophosphate confirmed that T 3 treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na + -K + -ATPase are subject to selective hormonal induction

  2. Calcium influx determines the muscular response to electrotransfer

    DEFF Research Database (Denmark)

    Møller, Pernille Højman; Brolin, Camilla; Gissel, Hanne

    2012-01-01

    expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA...... low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene...

  3. Aberrations in preliminary design of ITER divertor impurity influx monitor

    Energy Technology Data Exchange (ETDEWEB)

    Kitazawa, Sin-iti, E-mail: kitazawa.siniti@jaea.go.jp [Naka Fusion Institute, Japan Atomic Energy Agency, JAEA, Naka 311-0193 (Japan); Ogawa, Hiroaki [Naka Fusion Institute, Japan Atomic Energy Agency, JAEA, Naka 311-0193 (Japan); Katsunuma, Atsushi; Kitazawa, Daisuke [Core Technology Center, Nikon Corporation, Yokohama 244-8533 (Japan); Ohmori, Keisuke [Customized Products Business Unit, Nikon Corporation, Mito 310-0843 (Japan)

    2015-12-15

    Highlights: • Divertor impurity influx monitor for ITER (DIM) is procured by JADA. • DIM is designed to observe light from nuclear fusion plasma directly. • DIM is under preliminary design phase. • The spot diagrams were suppressed within the core of receiving fiber. • The aberration of DIM is suppressed in the preliminary design. - Abstract: Divertor impurity influx monitor for ITER (DIM) is a diagnostic system that observes light from nuclear fusion plasma directly. This system is affected by various aberrations because it observes light from the fan-array chord near the divertor in the ultraviolet–near infrared wavelength range. The aberrations should be suppressed to the extent possible to observe the light with very high spatial resolution. In the preliminary design of DIM, spot diagrams were suppressed within the core of the receiving fiber's cross section, and the resulting spatial resolutions satisfied the design requirements.

  4. Aberrations in preliminary design of ITER divertor impurity influx monitor

    International Nuclear Information System (INIS)

    Kitazawa, Sin-iti; Ogawa, Hiroaki; Katsunuma, Atsushi; Kitazawa, Daisuke; Ohmori, Keisuke

    2015-01-01

    Highlights: • Divertor impurity influx monitor for ITER (DIM) is procured by JADA. • DIM is designed to observe light from nuclear fusion plasma directly. • DIM is under preliminary design phase. • The spot diagrams were suppressed within the core of receiving fiber. • The aberration of DIM is suppressed in the preliminary design. - Abstract: Divertor impurity influx monitor for ITER (DIM) is a diagnostic system that observes light from nuclear fusion plasma directly. This system is affected by various aberrations because it observes light from the fan-array chord near the divertor in the ultraviolet–near infrared wavelength range. The aberrations should be suppressed to the extent possible to observe the light with very high spatial resolution. In the preliminary design of DIM, spot diagrams were suppressed within the core of the receiving fiber's cross section, and the resulting spatial resolutions satisfied the design requirements.

  5. High Performance Affinity Chromatography of Antithrombin III Based on Monodisperse Poly (glycidyl methacrylate) Beads

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse,non-porous,cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide,heparin was linked covalently to amino-PGMA-beads,which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII),good resolution and excellent mechanical properties and can be used under high flow rate.

  6. Excess influx of Zn(2+) into dentate granule cells affects object recognition memory via attenuated LTP.

    Science.gov (United States)

    Suzuki, Miki; Fujise, Yuki; Tsuchiya, Yuka; Tamano, Haruna; Takeda, Atsushi

    2015-08-01

    The influx of extracellular Zn(2+) into dentate granule cells is nonessential for dentate gyrus long-term potentiation (LTP) and the physiological significance of extracellular Zn(2+) dynamics is unknown in the dentate gyrus. Excess increase in extracellular Zn(2+) in the hippocampal CA1, which is induced with excitation of zincergic neurons, induces memory deficit via excess influx of Zn(2+) into CA1 pyramidal cells. In the present study, it was examined whether extracellular Zn(2+) induces object recognition memory deficit via excess influx of Zn(2+) into dentate granule cells. KCl (100 mM, 2 µl) was locally injected into the dentate gyrus. The increase in intracellular Zn(2+) in dentate granule cells induced with high K(+) was blocked by co-injection of CaEDTA and CNQX, an extracellular Zn(2+) chelator and an AMPA receptor antagonist, respectively, suggesting that high K(+) increases the influx of Zn(2+) into dentate granule cells via AMPA receptor activation. Dentate gyrus LTP induction was attenuated 1 h after KCl injection into the dentate gyrus and also attenuated when KCl was injected 5 min after the induction. Memory deficit was induced when training of object recognition test was performed 1 h after KCl injection into the dentate gyrus and also induced when KCl was injected 5 min after the training. High K(+)-induced impairments of LTP and memory were rescued by co-injection of CaEDTA. These results indicate that excess influx of Zn(2+) into dentate granule cells via AMPA receptor activation affects object recognition memory via attenuated LTP induction. Even in the dentate gyrus where is scarcely innervated by zincergic neurons, it is likely that extracellular Zn(2+) homeostasis is strictly regulated for cognition. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  8. Ca(2+) influx and neurotransmitter release at ribbon synapses.

    Science.gov (United States)

    Cho, Soyoun; von Gersdorff, Henrique

    2012-01-01

    Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

  10. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  11. Characterization of high-affinity (/sup 3/H)ouabain binding in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Hauger, R.; Luu, H.M.; Meyer, D.K.; Goodwin, F.K.; Paul, S.M.

    1985-06-01

    The characteristics of (/sup 3/H)ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. (/sup 3/H)Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific (/sup 3/H)ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for (/sup 3/H)ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal (/sup 3/H)ouabain binding was examined. Kainic acid lesions of the striatum reduced (/sup 3/H)ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the high-affinity (/sup 3/H)ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of (/sup 3/H)ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

  12. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  13. High-aluminum-affinity silica is a nanoparticle that seeds secondary aluminosilicate formation.

    Directory of Open Access Journals (Sweden)

    Ravin Jugdaohsingh

    Full Text Available Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7 we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP. Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m(2 g(-1 and it competes effectively with transferrin for Al(III binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III species binding monomeric silica to form early phase, non-toxic aluminosilicates.

  14. High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation

    Science.gov (United States)

    Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J.

    2013-01-01

    Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates. PMID:24349573

  15. Relationship between sodium influx and salt tolerance of nitrogen-fixing cyanobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Apte, S.K.; Reddy, B.R.; Thomas, J.

    1987-08-01

    The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na+ than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K+ (more than 25 mM) in the medium causes membrane depolarization and inhibits Na+ influx in both cyanobacteria (S.K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na+ influx accompanied by membrane depolarization. These experimental manipulations affecting Na+ uptake demonstrated a good negative correlation between Na+ influx and salt tolerance. All treatments which inhibited Na+ influx (such as alkaline pH, K+ above 25 mM, NO3-, and NH4+), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na+ influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria. (Refs. 27)

  16. Characterizing the glymphatic influx by utilizing intracisternal infusion of fluorescently conjugated cadaverine.

    Science.gov (United States)

    Zhang, Cui; Lin, Jun; Wei, Fang; Song, Jian; Chen, Wenyue; Shan, Lidong; Xue, Rong; Wang, Guoqing; Tao, Jin; Zhang, Guoxing; Xu, Guang-Yin; Wang, Linhui

    2018-05-15

    Accumulating evidence supports that cerebrospinal fluid (CSF) in the subarachnoid space (SAS) could reenter the brain parenchyma via the glymphatic influx. The present study was designed to characterize the detailed pathway of subarachnoid CSF influx by using a novel CSF tracer. Fluorescently conjugated cadaverine (A488-ca), for the first time, was employed to investigate CSF movement in the brain. Following intracisternal infusion of CSF tracers, mice brain was sliced and prepared for fluorescence imaging. Some brain sections were immunostained in order to observe tracer distribution and cellular uptake. A488-ca moved into the brain parenchyma rapidly, and the influx was time and region dependent. A488-ca entered the mice brain more readily and spread more widely than another commonly used CSF tracer-fluorescently conjugated ovalbumin (OA-45). Furthermore, A488-ca could enter the brain parenchyma either along the paravascular space or across the pial surface. Suppression of glymphatic transport by administration with acetazolamide strikingly reduced the influx of A488-ca. More importantly, relative to OA-45 largely remained in the extracellular space, A488-ca exhibited obvious cellular uptake by astrocytes surrounding the blood vessels and neurons in the cerebral cortex. Subarachnoid CSF could flow into the brain parenchyma via the glymphatic influx, in which the transcellular pathway was faithfully traced by intracisternal infusion with fluorescently conjugated cadaverine. These observations extend our comprehension on the glymphatic influx pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    International Nuclear Information System (INIS)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  18. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    Science.gov (United States)

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  19. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  20. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  1. Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

    Science.gov (United States)

    Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2010-02-05

    This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow

  2. A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

    Science.gov (United States)

    Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

    2003-01-01

    A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

  3. High blood oxygen affinity in the air-breathing swamp eel Monopterus albus.

    Science.gov (United States)

    Damsgaard, Christian; Findorf, Inge; Helbo, Signe; Kocagoz, Yigit; Buchanan, Rasmus; Huong, Do Thi Thanh; Weber, Roy E; Fago, Angela; Bayley, Mark; Wang, Tobias

    2014-12-01

    The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O₂ transport system to secure adequate O₂ delivery to the respiring tissues. We therefore investigated O₂ binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O₂ equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O₂ binding properties. The blood of M. albus has a high O₂ carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O₂ with an unusually high affinity (P₅₀=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O₂ affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue⁻¹, respectively). We suggest that the high Hct and high blood O₂ affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O₂ flux from the blood to the muscles. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

    Science.gov (United States)

    Fincham, D A; Ellory, J C; Young, J D

    1992-08-01

    In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target

  5. Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

    OpenAIRE

    Wiley, J. S.; Jones, S. P.; Sawyer, W. H.; Paterson, A. R. P.

    1982-01-01

    Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells...

  6. Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation.

    Science.gov (United States)

    Cherkaoui, A; Diene, S M; Renzoni, A; Emonet, S; Renzi, G; François, P; Schrenzel, J

    2017-02-01

    To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. All 46 imipenem-heteroresistant isolates (IMI hR ) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC 50 , 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMI hR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  7. Statistical removal of background signals from high-throughput 1H NMR line-broadening ligand-affinity screens

    International Nuclear Information System (INIS)

    Worley, Bradley; Sisco, Nicholas J.; Powers, Robert

    2015-01-01

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1 H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1 H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction

  8. Selective induction of high-ouabain-affinity isoform of Na sup + -K sup + -ATPase by thyroid hormone

    Energy Technology Data Exchange (ETDEWEB)

    Haber, R.S.; Loeb, J.N. (Columbia Univ., New York, NY (USA))

    1988-12-01

    The administration of thyroid hormone is known to result in an induction of the Na{sup +}-K{sup +}-adenosinetriphosphatase (Na{sup +}-K{sup +}-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na{sup +}-K{sup +}-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3{prime},5-triiodo-L-thyronine (T{sub 3}) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na{sup +}-K{sup +}-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K{sub I}s) for ouabain of {approximately}10{sup {minus}7} and 10{sup {minus}4} M, respectively. Measurement of the specific binding of ({sup 3}H)ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K{sub d}) of slightly less than 10{sup {minus}7}M, whose maximal binding capacity was increased by T{sub 3} treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T{sub 3} treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na{sup +}-K{sup +}-ATPase by ouabain-dependent phosphorylation from ({sup 32}P)orthophosphate confirmed that T{sub 3} treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na{sup +}-K{sup +}-ATPase are subject to selective hormonal induction.

  9. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  10. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  11. Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits. Molecular size as a determinant of plasma lipoprotein influx

    International Nuclear Information System (INIS)

    Stender, S.; Zilversmit, D.B.

    1981-01-01

    The arterial influx of esterified and free cholesterol from low density lipoproteins and very low density lipoproteins in 20 hypercholesterolemic rabbits was measured simultaneously by the use of lipoproteins labeled in vivo with [ 3 H]- and [ 14 C]-cholesterol. The simultaneous arterial influx of either [ 3 H]-leucine-labeled very low density lipoproteins, low density lipoproteins, high density lipoproteins, or plasma proteins was also measured in each rabbit. The arterial influx was calculated as intimal clearance, i.e., the influx of a given fraction divided by its plasma concentration. The intimal clearance of low density lipoprotein esterified cholesterol was equal to that for the apolipoproteins of that fraction, which is compatible with an arterial influx of intact low density lipoprotein molecules. The intimal clearance of very low density apolipoprotein or cholesteryl ester was less than that for low density lipoprotein, whereas high density lipoprotein and albumin clearances exceeded low density lipoprotein clearance by 1.5- to 3-fold. The intimal clearances of plasma proteins, high density, low density, and very low density lipoproteins decreased linearly with the logarithm of the macromolecular diameter. This indicates that the arterial influx of three plasma lipoprotein fractions and of plasma proteins proceeds by similar mechanisms. Apparently the relative intimal clearances of lipoproteins are more dependent on their size relative to pores or vesicular diameters at the plasma-artery interface than on specific interactions between lipoproteins and the arterial intimal surface

  12. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  13. Effective water influx control in gas reservoir development: Problems and countermeasures

    Directory of Open Access Journals (Sweden)

    Xi Feng

    2015-03-01

    Full Text Available Because of the diversity of geological characteristics and the complexity of percolation rules, many problems are found ineffective water influx control in gas reservoir development. The problems mainly focus on how to understand water influx rules, to establish appropriate countermeasures, and to ensure the effectiveness of technical measures. It is hard to obtain a complete applicable understanding through the isolated analysis of an individual gas reservoir due to many factors such as actual gas reservoir development phase, research work, pertinence and timeliness of measures, and so on. Over the past four decades, the exploration, practicing and tracking research have been conducted on water control in gas reservoir development in the Sichuan Basin, and a series of comprehensive water control technologies were developed integrating advanced concepts, successful experiences, specific theories and mature technologies. Though the development of most water-drive gas reservoirs was significantly improved, water control effects were quite different. Based on this background, from the perspective of the early-phase requirements of water influx control, the influencing factors of a water influx activity, the dynamic analysis method of water influx performance, the optimizing strategy of a water control, and the water control experience of typical gas reservoirs, this paper analyzed the key problems of water control, evaluated the influencing factors of water control effect, explored the practical water control strategies, and proposed that it should be inappropriate to apply the previous water control technological model to actual work but the pertinence should be improved according to actual circumstances. The research results in the paper provide technical reference for the optimization of water-invasion gas reservoir development.

  14. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  15. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    Energy Technology Data Exchange (ETDEWEB)

    Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  16. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    Orito, N; Umekage, S; Sakai, E; Tanaka, T; Kikuchi, Y; Sato, K; Kawauchi, S; Tanaka, H

    2012-01-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be K D = 2.25x10 -9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  17. Cryptococcal capsular glucuronoxylomannan reduces ischaemia-related neutrophil influx

    NARCIS (Netherlands)

    Ellerbroek, PM; Schoemaker, RG; van Veghel, R; Hoepelman, AIM; Coenjaerts, FEJ

    Background The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans interferes with the chemotaxis and transendothelial migration of neutrophils. Intravenous administration of purified GXM has been shown to reduce the influx of inflammatory cells in an animal model of

  18. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  19. Ethanol enhances GABA-induced 36Cl-influx in primary spinal cord cultured neurons

    International Nuclear Information System (INIS)

    Ticku, M.K.; Lowrimore, P.; Lehoullier, P.

    1986-01-01

    Ethanol has a pharmacological profile similar to other centrally acting drugs, which facilitate GABAergic transmission. GABA is known to produce its effects by increasing the conductance to Cl- ions. In this study, we have examined the effect of ethanol on GABA-induced 36Cl-influx in primary spinal cord cultured neurons. GABA produces a concentration-dependent, and saturable effect on 36Cl-influx in these neurons. Ethanol potentiates the effect of GABA on 36Cl-influx in these neurons. GABA (20 microM) increased the 36Cl-influx by 75% over the basal value, and in the presence of 50 mM ethanol, the observed increase was 142%. Eadie-Hoffstee analysis of the saturation curves indicated that ethanol decreases the Km value of GABA (10.6 microM to 4.2 microM), and also increases the Vmax. Besides potentiating the effect of GABA, ethanol also appears to have a direct effect in the absence of added GABA. These results suggest that ethanol enhances GABA-induced 36Cl-influx and indicate a role of GABAergic system in the actions of ethanol. These results also support the behavioral and electrophysiological studies, which have implicated GABA systems in the actions of ethanol. The potential mechanism(s) and the role of direct effect of ethanol is not clear at this time, but is currently being investigated

  20. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    International Nuclear Information System (INIS)

    Yashima, N.; Wada, A.; Izumi, F.

    1986-01-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of 45 Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of 45 Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of 45 Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the 45 Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the 45 Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of 45 Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of 45 Ca. Based on these findings, the authors suggest that inhibition of the 45 Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels

  1. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  2. Neutrophil elastase-induced elastin degradation mediates macrophage influx and lung injury in 60% O2-exposed neonatal rats.

    Science.gov (United States)

    Masood, Azhar; Yi, Man; Belcastro, Rosetta; Li, Jun; Lopez, Lianet; Kantores, Crystal; Jankov, Robert P; Tanswell, A Keith

    2015-07-01

    Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and α-elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to α-elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation. Copyright © 2015 the American Physiological Society.

  3. Affine and quasi-affine frames for rational dilations

    DEFF Research Database (Denmark)

    Bownik, Marcin; Lemvig, Jakob

    2011-01-01

    In this paper we extend the investigation of quasi-affine systems, which were originally introduced by Ron and Shen [J. Funct. Anal. 148 (1997), 408-447] for integer, expansive dilations, to the class of rational, expansive dilations. We show that an affine system is a frame if, and only if......, the corresponding family of quasi-affine systems are frames with uniform frame bounds. We also prove a similar equivalence result between pairs of dual affine frames and dual quasi-affine frames. Finally, we uncover some fundamental differences between the integer and rational settings by exhibiting an example...

  4. Photochemistry Saturn's Atmosphere. 2; Effects of an Influx of External Oxygen

    Science.gov (United States)

    Moses, Julianne I.; Lellouch, Emmanuel; Bezard, Bruno; Gladstone, G. Randall; Allen, Mark

    2000-01-01

    We use a one-dimensional diurnally averaged model of photochemistry and diffusion in Saturn's stratosphere to investigate the influence of extraplanetary debris on atmospheric chemistry. In particular, we consider the effects of an influx of oxygen from micrometeoroid ablation or from ring-particle diffusion; the contribution from cometary impacts, satellite debris, or ring vapor is deemed to be less important. The photochemical model results are compared directly with Infrared Space Observatory (ISO) observations to constrain the influx of extraplanetary oxygen to Saturn. From the ISO observations, we determine that the column densities of CO2 and H2O above 10 mbar in Saturn's atmosphere are (6.3 +/- 1) x 10(exp 14) and (1.4 +/- 0.4) x 10(exp 15)/ square cm, respectively; our models indicate that a globally averaged oxygen influx of (4+/-2) x 10(exp 6) O atoms /sq cm/s is required to explain these observations. Models with a locally enhanced influx of H20 operating over a small fraction of the projected area do not provide as good a fit to the ISO H2O observations. If volatile oxygen compounds comprise one-third to one-half of the exogenic source by mass, then Saturn is currently being bombarded with (3 +/- 2) x 10(exp -16) g/square cm/s of extraplanetary material. To reproduce the observed CO2/H2O ratio in Saturn's stratosphere, some of the exogenic oxygen must arrive in the form of a carbon-oxygen bonded species such as CO or CO2. An influx consistent with the composition of cometary ices fails to reproduce the high observed CO2/H2O ratio, suggesting that (i) the material has ices that are slightly more carbon-rich than is typical for comets, (ii) a contribution from an organic-rich component is required, or (iii) some of the hydrogen-oxygen bonded material is converted to carbon-oxygen bonded material without photochemistry (e.g., during the ablation process). We have also reanalyzed the 5-micron CO observations of Noll and Larson and determine that the CO

  5. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  6. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    International Nuclear Information System (INIS)

    Penefsky, H.S.

    1985-01-01

    Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

  7. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  8. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  9. Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

    Science.gov (United States)

    Whitehurst, Charles E; Annis, D Allen

    2008-07-01

    Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

  10. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Science.gov (United States)

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  11. Irreversible blockade of the high and low affinity (3H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

    International Nuclear Information System (INIS)

    Krizsan, D.; Varga, E.; Benyhe, S.; Szucs, M.; Borsodi, A.; Hosztafi, S.

    1991-01-01

    C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the ( 3 H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na + -index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of ( 3 H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested

  12. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Hemoglobin affinity in Andean rodents

    Directory of Open Access Journals (Sweden)

    HRVOJ OSTOJIC

    2002-01-01

    Full Text Available Blood hemoglobin oxygen affinity (P50 was measured in three Andean species and in the laboratory rat (control, all raised near sea level. Chinchilla lanigera (Molina, 1792 has an altitudinal habitat range from low Andean slopes up to 3000 m., while Chinchilla brevicaudata (Waterhouse, 1848 has an altitudinal range from 3000 to 5000 m. The laboratory type guinea pig, wild type guinea pig (Cavia porcellus, (Waterhouse, 1748, and laboratory rat (Rattus norvegicus were also raised at sea level. The Andean species had high hemoglobin oxygen affinities (low P50 compared with the rat. Chinchilla brevicaudata had a higher affinity than Chinchilla lanigera. The wild type guinea pig had a higher affinity than the laboratory type. As has been shown in other species, this is another example of an inverse correlation between the altitude level and the P50 values. This is the first hemoglobin oxygen affinity study in Chinchilla brevicaudata.

  14. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    International Nuclear Information System (INIS)

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P.

    1991-01-01

    The binding of the radiolabeled bombesin analogue [ 125 I-Tyr 4 ]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125 I-Tyr 4 ]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [ 125 I-Tyr 4 ]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125 I-Tyr 4 ]bombesin in an order of potencies as follows: [ 125 I-Tyr 4 ]bombesin > bombesin ≥ neuromedin C much-gt neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B

  15. Use of geochemical tracers for estimating groundwater influxes to the Big Sioux River, eastern South Dakota, USA

    Science.gov (United States)

    Neupane, Ram P.; Mehan, Sushant; Kumar, Sandeep

    2017-09-01

    Understanding the spatial distribution and variability of geochemical tracers is crucial for estimating groundwater influxes into a river and can contribute to better future water management strategies. Because of the much higher radon (222Rn) activities in groundwater compared to river water, 222Rn was used as the main tracer to estimate groundwater influxes to river discharge over a 323-km distance of the Big Sioux River, eastern South Dakota, USA; these influx estimates were compared to the estimates using Cl- concentrations. In the reaches overall, groundwater influxes using the 222Rn activity approach ranged between 0.3 and 6.4 m3/m/day (mean 1.8 m3/m/day) and the cumulative groundwater influx estimated during the study period was 3,982-146,594 m3/day (mean 40,568 m3/day), accounting for 0.2-41.9% (mean 12.5%) of the total river flow rate. The mean groundwater influx derived using the 222Rn activity approach was lower than that calculated based on Cl- concentration (35.6 m3/m/day) for most of the reaches. Based on the Cl- approach, groundwater accounted for 37.3% of the total river flow rate. The difference between the method estimates may be associated with minimal differences between groundwater and river Cl- concentrations. These assessments will provide a better understanding of estimates used for the allocation of water resources to sustain agricultural productivity in the basin. However, a more detailed sampling program is necessary for accurate influx estimation, and also to understand the influence of seasonal variation on groundwater influxes into the basin.

  16. High-throughput bioscreening system utilizing high-performance affinity magnetic carriers exhibiting minimal non-specific protein binding

    International Nuclear Information System (INIS)

    Hanyu, Naohiro; Nishio, Kosuke; Hatakeyama, Mamoru; Yasuno, Hiroshi; Tanaka, Toshiyuki; Tada, Masaru; Nakagawa, Takashi; Sandhu, Adarsh; Abe, Masanori; Handa, Hiroshi

    2009-01-01

    For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

  17. Thermal conductive heating in fractured bedrock: Screening calculations to assess the effect of groundwater influx

    Science.gov (United States)

    Baston, Daniel P.; Kueper, Bernard H.

    2009-02-01

    A two-dimensional semi-analytical heat transfer solution is developed and a parameter sensitivity analysis performed to determine the relative importance of rock material properties (density, thermal conductivity and heat capacity) and hydrogeological properties (hydraulic gradient, fracture aperture, fracture spacing) on the ability to heat fractured rock using thermal conductive heating (TCH). The solution is developed using a Green's function approach in which an integral equation is constructed for the temperature in the fracture. Subsurface temperature distributions are far more sensitive to hydrogeological properties than material properties. The bulk ground water influx ( q) can provide a good estimate of the extent of influx cooling when influx is low to moderate, allowing the prediction of temperatures during heating without specific knowledge of the aperture and spacing of fractures. Target temperatures may not be reached or may be significantly delayed when the groundwater influx is large.

  18. Observation of impurity accumulation and concurrent impurity influx in PBX

    International Nuclear Information System (INIS)

    Sesnic, S.S.; Fonck, R.J.; Ida, K.; Couture, P.; Kaita, R.; Kaye, S.; Kugel, H.; LeBlanc, B.; Okabayashi, M.; Paul, S.; Powell, E.T.; Reusch, M.; Takahashi, H.; Gammel, G.; Morris, W.

    1987-01-01

    Impurity studies in L- and H-mode discharges in PBX have shown that both types of discharges can evolve into either an impurity accumulative or nonaccumulative case. In a typical accumulative discharge, Z eff peaks in the center to values of about 5. The central metallic densities can be high, n met /n e ≅ 0.01, resulting in central radiated power densities in excess of 1 W/cm 3 , consistent with bolometric estimates. The radial profiles of metals obtained independently from the line radiation in the soft X-ray and the VUV regions are very peaked. Concurrent with the peaking, an increase in the impurity influx coming from the edge of the plasma is observed. At the beginning of the accumulation phase the inward particle flux for titanium has values of 6x10 10 and 10x10 10 particles/cm 2 s at minor radii of 6 and 17 cm. At the end of the accumulation phase, this particle flux is strongly increased to values of 3x10 12 and 1x10 12 particles/cm 2 s. This increased flux is mainly due to influx from the edge of the plasma and to a lesser extent due to increased convective transport. Using the measured particle flux, an estimate of the diffusion coefficient D and the convective velocity v is obtained. (orig.)

  19. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  20. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  1. Affinity imaging mass spectrometry (AIMS): high-throughput screening for specific small molecule interactions with frozen tissue sections.

    Science.gov (United States)

    Yoshimi, T; Kawabata, S; Taira, S; Okuno, A; Mikawa, R; Murayama, S; Tanaka, K; Takikawa, O

    2015-11-07

    A novel screening system, using affinity imaging mass spectrometry (AIMS), has been developed to identify protein aggregates or organ structures in unfixed human tissue. Frozen tissue sections are positioned on small (millimetre-scale) stainless steel chips and incubated with an extensive library of small molecules. Candidate molecules showing specific affinity for the tissue section are identified by imaging mass spectrometry (IMS). As an example application, we screened over a thousand compounds against Alzheimer's disease (AD) brain tissue and identified several compounds with high affinity for AD brain sections containing tau deposits compared to age-matched controls. It should also be possible to use AIMS to isolate chemical compounds with affinity for tissue structures or components that have been extensively modified by events such as oxidation, phosphorylation, acetylation, aggregation, racemization or truncation, for example, due to aging. It may also be applicable to biomarker screening programs.

  2. Molecular electron affinities

    International Nuclear Information System (INIS)

    Fukuda, E.K.

    1983-01-01

    Molecular electron affinities have historically been difficult quantities to measure accurately. These difficulties arise from differences in structure between the ion and neutral as well as the existence of excited negative ion states. To circumvent these problems, relative electron affinities were determined in this dissertation by studying equilibrium electron transfer reactions using a pulsed ion cyclotron resonance (ICR) spectrometer. Direct measurement of ion and neutral concentrations for reactions of the general type, A - + B = B - + A, allow calculation of the equilibrium constant and, therefore, the free energy change. The free energy difference is related to the difference in electron affinities between A and B. A relative electron affinity scale covering a range of about 45 kcal/mol was constructed with various substituted p-benzoquinones, nitrobenzenes, anhydrides, and benzophenones. To assign absolute electron affinities, various species with accurately known electron affinities are tied to the scale via ion-cyclotron double resonance bracketing techniques. After the relative scale is anchored to these species with well-known electron affinities, the scale is then used as a check on other electron affinity values as well as generating new electron affinity values. Many discrepancies were found between the electron affinities measured using the ICR technique and previous literature determinations

  3. High affinity, ligand specific uptake of complexed copper-67 by brain tissue incubated in vitro

    International Nuclear Information System (INIS)

    Barnea, A.; Hartter, D.E.

    1987-01-01

    Copper is an essential metal that is highly concentrated in the brain. The blood, the sole source of tissue Cu, contains 16-20 μM Cu, of which >95% is complexed to proteins and 2 was 10 times greater than that of CuAlbumin or Cu(II). Within the range of 0.2-150μM Cu, multiple uptake sites for CuHis were apparent. Increasing the molar ratio of His:Cu had a differential effect on Cu uptake: enhancing uptake at [Cu] 1 μM. Thus, using a His:Cu ratio of 1000, they observed a high affinity process exhibiting saturating and half saturating values of 5 μM and 1.5 μM Cu, respectively; using a His:Cu ratio of 2, they observed a low affinity process exhibiting saturating and half-saturating values of 100 μM and 40 μM Cu, respectively. Both processes required thermic but not metabolic energy, suggestive of facilitated diffusion. Considering the blood brain barrier for proteins, CuHis appears to be the major substrate for Cu uptake by neuronal tissue. They demonstrate the existence of a ligand specific, high affinity (apparent Km about 1.5 μM Cu) uptake process for CuHis in the brain, operative at the physiological concentration range of CuHis and histidine

  4. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  5. Mobile Technology Affinity in Renal Transplant Recipients.

    Science.gov (United States)

    Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

    Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Identification of high-affinity calmodulin-binding proteins in rat liver

    International Nuclear Information System (INIS)

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-01-01

    The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

  7. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  8. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    International Nuclear Information System (INIS)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M.

    1989-01-01

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125 I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity

  9. The utility of affine variables and affine coherent states

    International Nuclear Information System (INIS)

    Klauder, John R

    2012-01-01

    Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when appropriate. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’. (review)

  10. Selection of imprinted nanoparticles by affinity chromatography.

    Science.gov (United States)

    Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

    2009-04-15

    Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

  11. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby...... simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for Rgp...

  12. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  13. Trace element affinities in two high-Ge coals from China

    Energy Technology Data Exchange (ETDEWEB)

    Jing Li; Xinguo Zhuang; Xavier Querol [China University of Geosciences, Wuhan (China). Faculty of Earth Resources

    2011-01-15

    The Lincang (Yunnan Province, Southwest China) and Wulantuga (Inner Mongolia, Northeast China) coal deposits are known because of the high-Ge content. These coals have also a high concentration of a number of other elements. To determine the mode of occurrence of the enriched elements in both coals, six density fractions from {lt} 1.43 to {gt} 2.8 g/cm{sup 3} were obtained from two representative samples using heavy-liquids. A number of peculiar geochemical patterns characterize these high-Ge coals. Thus, the results of the chemical analysis of these density fractions showed that both coals (very distant and of a different geological age) are highly enriched (compared with the usual worldwide coal concentration ranges) in Ge, As, Sb, W, Be, and Tl. This may be due to similar geochemistry of hydrothermal fluids influencing the Earth Crust in these regions of China. Moreover, Wulantuga coal (Early Cretaceous subbituminous coal) is also enriched in Ca, Mg, and Na, and Lincang coal (Neogene subbituminous coal) in K, Rb, Nb, Mo, Sn, Cs, and U. A group of elements consisting of Ge, W, B, Nb, and Sb mostly occur with an organic affinity in both coals. Additionally, Be, U, and Mo (and partially Mn and Zn) in Lincang, and Na and Mg in Wulantuga occur also with a major organic affinity. Both coals have sulfide-arsenide mineral assemblages (Fe, S, As, Sn, and Pb, and in addition to Tl, Ta, and Cs in the Lincang coal). The occurrence of Al, P, Li, Sc, Ti, V, Cr, and Zr in both coals, and Ba in Lincang, are associated with the mineral assemblage of silico-aluminates and minor heavy minerals. Furthermore, P, Na, Li, Sc, Ti, Ga, Rb, Zr, Cr, Ba, Th, and LREE (La, Ce, Pr, Nd, and Gd) in Lincang are associated with mineral assemblages of phosphates and minor heavy minerals. The two later mineral assemblages are derived from the occurrence of detrital minerals. 34 refs., 7 figs., 3 tabs.

  14. A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues.

    Science.gov (United States)

    Diribe, C O; Warhurst, D C

    1985-09-01

    A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.

  15. Induction of nitrate transport in maize roots, and kinetics of influx, measured with nitrogen-13

    International Nuclear Information System (INIS)

    Hole, D.J.; Drew, M.C.; Emran, A.M.; Fares, Y.

    1990-01-01

    Unlike phosphate or potassium transport, uptake of nitrate by roots is induced, in part, by contact with the substrate ion. Plasmalemma influx of 13 N-labeled nitrate in maize roots was studied in relation to induction of the uptake system, and the influence of short-term N starvation. Maize (Zea mays) roots not previously exposed to nitrate had a constitutive transport system (state 1), but influx increased 250% during six hours of contact with 100 micromolar nitrate, by which time the transport mechanism appeared to be fully synthesized (state 2). A three-day period of N starvation prior to induction and measurement of nitrate influx resulted in a greater capacity to transport nitrate than in unstarved controls, but this was fully expressed only if roots were kept in contact with nitrate for the six hours needed for full induction (state 2E). A kinetic analysis indicated a 160% increase in maximum influx in N-starved, induced roots with a small decrease in K m . The inducible component to nitrate influx was induced only by contact with nitrate. Full expression of the nitrate inducible transport system was dependent upon mRNA synthesis. An inhibitor of cytoplasmic protein synthesis (cycloheximide) eliminated the formation of the transport system while inhibition by chloramphenicol of mitochondrial- or plastid-coded protein synthesis had no effect. Poisoning of membrane-bound proteins effectively disabled both the constitutive and induced transport systems

  16. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Calligaro, D.; Eldefrawi, M.

    1986-01-01

    Binding of [ 3 H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [ 3 H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95 0 C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [ 3 H]cocaine (15 nM) was inhibited by increasing concentrations of Na + ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [ 3 H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC 50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [ 3 H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC 50 values below μM concentrations

  17. Report: Affinity Chromatography.

    Science.gov (United States)

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  18. Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport.

    Science.gov (United States)

    Szczerba, Mark W; Britto, Dev T; Balkos, Konstantine D; Kronzucker, Herbert J

    2008-01-01

    Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

  19. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    Science.gov (United States)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  20. High-Affinity Methanotrophy Informed by Genome-Wide Analysis of Upland Soil Cluster Alpha (USCα) from Axel Heiberg Island, Canadian High Arctic

    Science.gov (United States)

    Rusley, C.; Onstott, T. C.; Lau, M.

    2017-12-01

    Methane (CH4) is a potent greenhouse gas whose proper budgeting is vital to climate predictions. Recent studies have identified upland Arctic mineral cryosols as consistent CH4 sinks, drawing CH4 from both the atmosphere and underlying anaerobic soil layers. Global atmospheric CH4 uptake is proposed to be mediated by high-affinity methanotrophs based on the detection of the marker gene pmoA (particulate methane monooxygenase beta subunit). However, a lack of pure cultures and scarcity of genomic information have hindered our understanding of their metabolic capabilities and versatility. Together with the missing genetic linkage between its pmoA and 16S ribosomal RNA (rRNA) gene, the factors that control the distribution and magnitude of high-affinity methanotrophy in the Arctic permafrost-affected region have remained elusive. Using 21 metagenomic datasets of surface soils obtained from long-term core incubation experiments,1 this bioinformatics study aimed to reconstruct the draft genome of the Upland Soil Cluster α-proteobacteria (USCα), the high-affinity methanotroph previously detected in the samples,2 and to determine its phylogeny and metabolic requirements. We obtained a genome bin containing the high-affinity form of the USCα-like pmoA gene. The 3.03 Mbp assembly is 91.6% complete with a unique set of single-copy marker genes. The 16S rRNA gene fragment of USCα belongs to the α-proteobacterial family Beijerinckiaceae. Genome annotation indicates possible formaldehyde oxidation via tetrahydromethanopterin-linked C1 transfer pathways, acetate utilization, carbon fixation via the Calvin-Benson-Bassham cycle, and glycogen production. Notably, the key enzymes for formaldehyde assimilation via the serine and ribulose monophosphate pathways are missing. The presence of genes encoding nitrate reductase and hemoglobin suggests adaptation to low O2 under water-logged conditions. Since USCα has versatile carbon metabolisms, it may not be an obligate methanotroph

  1. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  2. New methodology for aquifer influx status classification for single wells in a gas reservoir with aquifer support

    Directory of Open Access Journals (Sweden)

    Yong Li

    2016-10-01

    Full Text Available For gas reservoirs with strong bottom or edge aquifer support, the most important thing is avoiding aquifer breakthrough in a gas well. Water production in gas wells does not only result in processing problems in surface facilities, but it also explicitly reduces well productivity and reservoir recovery. There are a lot of studies on the prediction of water breakthrough time, but they are not completely practicable due to reservoir heterogeneity. This paper provides a new method together with three diagnostic curves to identify aquifer influx status for single gas wells; the aforementioned curves are based on well production and pressure data. The whole production period of a gas well can be classified into three periods based on the diagnostic curves: no aquifer influx period, early aquifer influx period, and middle-late aquifer influx period. This new method has been used for actual gas well analysis to accurately identify gas well aquifer influx status and the water breakthrough sequence of all wells in the same gas field. Additionally, the evaluation results are significantly beneficial for well production rate optimization and development of an effective gas field.

  3. Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields

    International Nuclear Information System (INIS)

    Stevenson, A.P.; Tobey, R.A.

    1985-01-01

    Potassium ion influx was measured by monitoring 42 KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+

  4. Structural insights into a high affinity nanobody:antigen complex by homology modelling.

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Characterization of high affinity [3H]triazolam binding in rat brain

    International Nuclear Information System (INIS)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-01-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on [ 3 H]TZ binding. Saturation studies showed a shift to lower affinity at 37 0 C (K/sub d/ = 0.25 +/- 0.01 nM at O 0 C; K/sub d/ = 1.46 +/- 0.03 nM at 37 0 C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0 0 C and 1001 +/- 43 fmoles/mg prot. at 37 0 C). Inhibition studies showed that [ 3 H]TZ binding displayed no GABA shift at 0 0 C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37 0 C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37 0 C. In Ro 15-1788/[ 3 H]TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on [ 3 H]TZ binding at both temperatures. In conclusion [ 3 H]TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists

  6. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    DEFF Research Database (Denmark)

    Jørgensen, Thomas R; vanKuyk, Patricia A; Poulsen, Bjarne R

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mst......-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays...

  7. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  8. Urban-Dome GHG Monitoring: Challenges and Perspectives from the INFLUX Project

    Science.gov (United States)

    Whetstone, J.; Shepson, P. B.; Davis, K. J.; Sweeney, C.; Gurney, K. R.; Miles, N. L.; Richardson, S.; Lauvaux, T.; Razlivanov, I.; Zhou, Y.; Song, Y.; Turnbull, J. C.; Karion, A.; Cambaliza, M. L.; Callahan, W.; Novakovskaia, E.; Crosson, E.; Rella, C.; Possolo, A.

    2012-04-01

    Quantification of carbon dynamics in urban areas using advanced and diverse observing systems enables the development of measurable, reportable, and verifiable (MRV) mitigation strategies as suggested in the Bali Action Plan, agreed upon at the 13th Conference of the Parties of the UNFCCC (COP 13, 2007). The National Institute of Standards and Technology (NIST), supports the Indianapolis Flux Experiment (INFLUX). INFLUX is focused on demonstrating the utility of dense, surface-based observing networks coupled with aircraft-based measurements, advanced atmospheric boundary layer observation and modeling to determine GHG emission source location and strength in urban areas. The ability to correctly model transport and mixing in the atmospheric boundary layer (ABL), responsible for carrying GHGs from their source to the point of measurement, is essential. The observing system design, using multiple instruments and observing methods, is intended to provide multi-scale measurements as a basis for mimicking the complex and evolving dynamics of a city. To better understand such a dynamic system, and incorporate this into models, reliable representations of horizontal and vertical transport, as well as ABL height, GHG mixing ratio measurements are planned for 11 tower locations, 2 are currently in operation with the remaining 9 planned for operational status in early to mid-2012. These observations are complimented by aircraft flights that measure mixing ratio as well as ABL parameters. Although measurements of ABL mixing heights and dynamics are presently only available intermittently, limiting efforts to evaluate ABL model performance and the uncertainties of GHG flux estimates, expansion of them is planned for the near future. INFLUX will significantly benefit from continuous, high resolution measurements of mixing depth, wind speed and direction, turbulence profiles in the boundary layer, as well as measurements of surface energy balance, momentum flux, and short and

  9. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  10. Influx mechanisms in the embryonic and adult rat choroid plexus

    DEFF Research Database (Denmark)

    Saunders, Norman R; Dziegielewska, Katarzyna M; Møllgård, Kjeld

    2015-01-01

    The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and a...

  11. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    Miles, L.A.; Plow, E.F.

    1986-01-01

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  12. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    OpenAIRE

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (?PO3 2??Zr4+?) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). Aft...

  13. Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines.

    Directory of Open Access Journals (Sweden)

    Brenda L Bloodgood

    2009-09-01

    Full Text Available Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.

  14. 14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

    Science.gov (United States)

    Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

    2017-11-05

    14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Influxed insects as Vectors for Campylobacter jejuni and Campylobacter coll in Danish Broiler Houses

    DEFF Research Database (Denmark)

    Hald, Birthe; Skovgård, Henrik; Pedersen, Karl

    2008-01-01

    ,816 flies captured from farm surroundings. Each individual fly was macerated, preenriched in Bolton broth for 24 h at 42 degrees C, streaked onto modified Campylobater blood-free selective agar and incubated under microaerobic conditions for 48 h at 42 degrees C. Second, the influx of insects to broiler...... houses was estimated by trapping of insects (n = 5,936) in ventilation vents. In total, 31 flies (28 of which were of the Muscidae family) caught in farm surroundings were Campylobacter spp.-positive (C. jejuni, n = 7; C. coli, n = 23; other Campylobacter spp., n = 1). Musca domestica (L) (house fly...... without other livestock, the prevalence was constantly below 1.0%. The average influx of insects per broiler rotation was estimated to be 30,728 +/- 2,443 SE (range 2,233 to 180,300), of which 21.4% were flies. The influx of insects correlated with the flow (m(3)/h) of ventilation air (P

  16. The Kinetics of Ouabain Inhibition and the Partition of Rubidium Influx in Human Red Blood Cells

    Science.gov (United States)

    Beauge, L. A.; Adragna, Norma

    1971-01-01

    In the development of ouabain inhibition of rubidium influx in human red blood cells a time lag can be detected which is a function of at least three variables: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70–80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10–20% of the total influx. PMID:5553102

  17. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  18. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides.

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-17

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K d =39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  19. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  20. Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

    Science.gov (United States)

    Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

    2018-01-01

    Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

  1. Hirota's solitons in the affine and the conformal affine Toda models

    International Nuclear Information System (INIS)

    Aratyn, H.; Constantinidis, C.P.; Ferreira, L.A.; Gomes, J.F.; Zimerman, A.H.

    1993-01-01

    We use Hirota's method formulated as a recursive scheme to construct a complete set of soliton solutions for the affine Toda field theory based on an arbitrary Lie algebra. Our solutions include a new class of solitons connected with two different types of degeneracies encountered in Hirota's perturbation approach. We also derive an universal mass formula for all Hirota's solutions to the affine Toda model valid for all underlying Lie groups. Embedding of the affine Toda model in the conformal affine Toda model plays a crucial role in this analysis. (orig.)

  2. Improving image segmentation by learning region affinities

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  3. The solutions of affine and conformal affine Toda field theory

    International Nuclear Information System (INIS)

    Papadopoulos, G.; Spence, B.

    1994-02-01

    We give new formulations of the solutions of the field equations of the affine Toda and conformal affine Toda theories on a cylinder and two-dimensional Minkowski space-time. These solutions are parameterised in terms of initial data and the resulting covariant phase spaces are diffeomorphic to the Hamiltonian ones. We derive the fundamental Poisson brackets of the parameters of the solutions and give the general static solutions for the affine theory. (authors). 10 refs

  4. Influence of self-affine roughness on the friction coefficient of rubber at high sliding velocity

    NARCIS (Netherlands)

    Palasantzas, G

    2004-01-01

    In this work we investigate the influence of self-affine roughness on the friction coefficient of a rubber body onto a solid surface at high speeds. The roughness is characterized by the rms amplitude w, the correlation length xi, and the roughness exponent H. It is shown that the friction

  5. Different endothelin receptor affinities in dog tissues

    International Nuclear Information System (INIS)

    Loeffler, B.M.L.; Loehrer, W.

    1991-01-01

    Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

  6. Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

    NARCIS (Netherlands)

    Bosdriesz, Evert; Wortel, Meike T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre Cortés, Pilar; Teusink, Bas

    2018-01-01

    Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear,

  7. Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

    NARCIS (Netherlands)

    Bosdriesz, Evert; Wortel, M.T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre, P.; Teusink, Bas

    2018-01-01

    Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains

  8. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  9. High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

    Science.gov (United States)

    Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

    1974-01-01

    Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

  10. Na+-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L. Delile

    Directory of Open Access Journals (Sweden)

    Lourdes Rubio

    2018-05-01

    Full Text Available Posidonia oceanica (L. Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−, which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+. Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+ increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

  11. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  12. Polish Perceptions on the Immigration Influx: a Critical Analysis

    Directory of Open Access Journals (Sweden)

    Kinga Hódor

    2017-02-01

    Full Text Available The article addresses the issue of Poles’ attitude to the problem of the influx of migrants to Poland in the context of the migration crisis, which Europe has to face today. The issues discussed in the present paper are aimed to illustrate the characteristic features specific to Poles’ attitudes in favor of or against the process of influx of migrants to the E.U. Member States or Poland. The analysis covers both positive and negative aspects of migration to Poland, which have been most often indicated by Poles with respects to migrants. On the one hand, they include fears with regard to national security, potential conflicts of cultural and religious background, fear of the alleged loss of jobs to migrants and their preying on the country’s social security system. All of the above result in anti-migration demonstrations and the language of hatred. On the other hand, positive aspects of the migration influx are believed to consist in cultural enrichment, benefits for the labor market resulting from the inflow of both qualified professionals and laborers with lower pay expectations in comparison to Polish workers and believing that migrants might be the chance of minimize the negative effects of the demographic crisis. The supporters of helping migrants also point out the issue of solidarity and sympathy for the victims and the fact that in the past it was the Poles who received support from other countries in Poland’s difficult moments. Thus, extending such help to others may prove to be beneficial in the future. The present paper is based on academic articles, internet sources and statistical data, which all reveal a division into two camps: supporters and opponents of receiving migrants in Poland, which prevents determining Poland’s definitive stance on this issue. All the aspects of the problem discussed in the paper are undoubtedly a basis for further analysis.

  13. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  14. A Ca2+ influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant

    International Nuclear Information System (INIS)

    Kerboeuf, D.; Cohen, J.

    1990-01-01

    A Paramecium possesses secretory organelles called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exocytosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 microM aminoethyl dextran as a vital secretagogue, we analyzed the movements of calcium (Ca 2+ ) during the discharge of trichocysts. We showed that (a) external Ca 2+ , at least at 3 X 10(-7) M, is necessary for AED to induce exocytosis; (b) a dramatic and transient influx of Ca 2+ as measured from 45 Ca uptake is induced by AED; (c) this influx is independent of the well-characterized voltage-operated Ca 2+ channels of the ciliary membranes since it persists in a mutant devoid of these channels; and (d) this influx is specifically abolished in one of the mutants unable to undergo exocytosis, nd12. We propose that the Ca 2+ influx induced by AED reflects an increase in membrane permeability through the opening of novel Ca 2+ channel or the activation of other Ca 2+ transport mechanism in the plasma membrane. The resulting rise in cytosolic Ca 2+ concentration would in turn induce membrane fusion. The mutation nd12 would affect a gene product involved in the control of plasma membrane permeability to Ca 2+ , specifically related to membrane fusion

  15. Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest

    Directory of Open Access Journals (Sweden)

    N.B. Iannucci

    2003-03-01

    Full Text Available High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively - was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes.

  16. Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension.

    Science.gov (United States)

    Fan, Kelong; Jiang, Bing; Guan, Zhe; He, Jiuyang; Yang, Dongling; Xie, Ni; Nie, Guohui; Xie, Can; Yan, Xiyun

    2018-04-10

    Nanobodies consist of a single domain variable fragment of a camelid heavy-chain antibody. Nanobodies have potential applications in biomedical fields because of their simple production procedures and low cost. Occasionally, nanobody clones of interest exhibit low affinities for their target antigens, which, together with their short half-life limit bioanalytical or therapeutic applications. Here, we developed a novel platform we named fenobody, in which a nanobody developed against H5N1 virus is displayed on the surface of ferritin in the form of a 24mer. We constructed a fenobody by substituting the fifth helix of ferritin with the nanobody. TEM analysis showed that nanobodies were displayed on the surface of ferritin in the form of 6 × 4 bundles, and that these clustered nanobodies are flexible for antigen binding in spatial structure. Comparing fenobodies with conventional nanobodies currently used revealed that the antigen binding apparent affinity of anti-H5N1 fenobody was dramatically increased (∼360-fold). Crucially, their half-life extension in a murine model was 10-fold longer than anti-H5N1 nanobody. In addition, we found that our fenobodies are highly expressed in Escherichia coli, and are both soluble and thermo-stable nanocages that self-assemble as 24-polymers. In conclusion, our results demonstrate that fenobodies have unique advantages over currently available systems for apparent affinity enhancement and half-life extension of nanobodies. Our fenobody system presents a suitable platform for various large-scale biotechnological processes and should greatly facilitate the application of nanobody technology in these areas.

  17. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    Science.gov (United States)

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  18. Lp-dual affine surface area

    Science.gov (United States)

    Wei, Wang; Binwu, He

    2008-12-01

    According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

  19. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  20. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

    Science.gov (United States)

    Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-21

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

  2. N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7  M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5  M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5  M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

  3. Role of H2O2 on the kinetics of low-affinity high-capacity Na+-dependent alanine transport in SHR proximal tubular epithelial cells

    International Nuclear Information System (INIS)

    Pinto, Vanda; Pinho, Maria Joao; Jose, Pedro A.; Soares-da-Silva, Patricio

    2010-01-01

    Research highlights: → H 2 O 2 in excess is required for the presence of a low-affinity high-capacity component for the Na + -dependent [ 14 C]-L-alanine uptake in SHR PTE cells only. → It is suggested that Na + binding in renal ASCT2 may be regulated by ROS in SHR PTE cells. -- Abstract: The presence of high and low sodium affinity states for the Na + -dependent [ 14 C]-L-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H 2 O 2 on the Na + -dependent [ 14 C]-L-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na + dependence of [ 14 C]-L-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na + removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H 2 O 2 levels in the extracellular medium significantly reduced Na + -K m and V max values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na + -dependent [ 14 C]-L-alanine uptake. After removal of apocynin from the culture medium, H 2 O 2 levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na + -K m and V max of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H 2 O 2 in excess is required for the presence of a low-affinity high-capacity component for the Na + -dependent [ 14 C]-L-alanine uptake in SHR PTE cells only. It is suggested that Na + binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.

  4. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  5. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells....

  6. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  7. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  8. Composition for limiting water influx into a well

    Energy Technology Data Exchange (ETDEWEB)

    Gazizov, A.Sh.; Budarina, L.A.; Kuznetsov, Ye.V.; Zhdanov, N.F.

    1982-01-01

    A composition is proposed for restricting water influx into a well. It contains acrylamide, ammonium persulfate, sodium hyposulfite, water and additive. It is distinguished by the fact that in order to improve water resistance of the copolymer formed in the bed and to preserve permeability of the bed for oil, it contains as an additive polymethacylic acid with the following ratio of components (% by weight): acrylamide 2.0-5.6; polymethacrylic acid 3.08.0; ammonium persulfate 0.020-0.072; sodium hyposulfite 0.018-0.068; water--the rest.

  9. The Indianapolis Flux Experiment (INFLUX: A test-bed for developing urban greenhouse gas emission measurements

    Directory of Open Access Journals (Sweden)

    Kenneth J. Davis

    2017-05-01

    Full Text Available The objective of the Indianapolis Flux Experiment (INFLUX is to develop, evaluate and improve methods for measuring greenhouse gas (GHG emissions from cities. INFLUX’s scientific objectives are to quantify CO2 and CH4 emission rates at 1 km2 resolution with a 10% or better accuracy and precision, to determine whole-city emissions with similar skill, and to achieve high (weekly or finer temporal resolution at both spatial resolutions. The experiment employs atmospheric GHG measurements from both towers and aircraft, atmospheric transport observations and models, and activity-based inventory products to quantify urban GHG emissions. Multiple, independent methods for estimating urban emissions are a central facet of our experimental design. INFLUX was initiated in 2010 and measurements and analyses are ongoing. To date we have quantified urban atmospheric GHG enhancements using aircraft and towers with measurements collected over multiple years, and have estimated whole-city CO2 and CH4 emissions using aircraft and tower GHG measurements, and inventory methods. Significant differences exist across methods; these differences have not yet been resolved; research to reduce uncertainties and reconcile these differences is underway. Sectorally- and spatially-resolved flux estimates, and detection of changes of fluxes over time, are also active research topics. Major challenges include developing methods for distinguishing anthropogenic from biogenic CO2 fluxes, improving our ability to interpret atmospheric GHG measurements close to urban GHG sources and across a broader range of atmospheric stability conditions, and quantifying uncertainties in inventory data products. INFLUX data and tools are intended to serve as an open resource and test bed for future investigations. Well-documented, public archival of data and methods is under development in support of this objective.

  10. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites...... as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile......, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases....

  11. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  12. 86Rb(K) influx and [3H]ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

    International Nuclear Information System (INIS)

    Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P.

    1989-01-01

    Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86 Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [ 3 H]ouabain binding by the human platelet. This 86 Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86 Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86 Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets

  13. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

    Directory of Open Access Journals (Sweden)

    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

  14. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    Science.gov (United States)

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain. PMID:8528085

  15. Continuous affine processes

    DEFF Research Database (Denmark)

    Buchardt, Kristian

    2016-01-01

    Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general transformati...

  16. Affinity in electrophoresis.

    Science.gov (United States)

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  17. Detection-Guided Fast Affine Projection Channel Estimator for Speech Applications

    Directory of Open Access Journals (Sweden)

    Yan Wu Jennifer

    2007-04-01

    Full Text Available In various adaptive estimation applications, such as acoustic echo cancellation within teleconferencing systems, the input signal is a highly correlated speech. This, in general, leads to extremely slow convergence of the NLMS adaptive FIR estimator. As a result, for such applications, the affine projection algorithm (APA or the low-complexity version, the fast affine projection (FAP algorithm, is commonly employed instead of the NLMS algorithm. In such applications, the signal propagation channel may have a relatively low-dimensional impulse response structure, that is, the number m of active or significant taps within the (discrete-time modelled channel impulse response is much less than the overall tap length n of the channel impulse response. For such cases, we investigate the inclusion of an active-parameter detection-guided concept within the fast affine projection FIR channel estimator. Simulation results indicate that the proposed detection-guided fast affine projection channel estimator has improved convergence speed and has lead to better steady-state performance than the standard fast affine projection channel estimator, especially in the important case of highly correlated speech input signals.

  18. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    Science.gov (United States)

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  19. The competitive advantage of a dual-transporter system.

    Science.gov (United States)

    Levy, Sagi; Kafri, Moshe; Carmi, Miri; Barkai, Naama

    2011-12-09

    Cells use transporters of different affinities to regulate nutrient influx. When nutrients are depleted, low-affinity transporters are replaced by high-affinity ones. High-affinity transporters are helpful when concentrations of nutrients are low, but the advantage of reducing their abundance when nutrients are abundant is less clear. When we eliminated such reduced production of the Saccharomyces cerevisiae high-affinity transporters for phosphate and zinc, the elapsed time from the initiation of the starvation program until the lack of nutrients limited growth was shortened, and recovery from starvation was delayed. The latter phenotype was rescued by constitutive activation of the starvation program. Dual-transporter systems appear to prolong preparation for starvation and to facilitate subsequent recovery, which may optimize sensing of nutrient depletion by integrating internal and external information about nutrient availability.

  20. Heterogeneous Cytoskeletal Force Distribution Delineates the Onset Ca2+ Influx Under Fluid Shear Stress in Astrocytes

    Directory of Open Access Journals (Sweden)

    Mohammad M. Maneshi

    2018-03-01

    Full Text Available Mechanical perturbations increase intracellular Ca2+ in cells, but the coupling of mechanical forces to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate defined fluid shear stress to cultured astrocytes, and simultaneously measured cytoskeletal forces using a force sensitive actinin optical sensor and intracellular Ca2+. Fluid shear generated non-uniform forces in actinin that critically depended on the stimulus rise time emphasizing the presence of viscoelasticity in the activating sequence. A short (ms shear pulse with fast rise time (2 ms produced an immediate increase in actinin tension at the upstream end of the cell with minimal changes at the downstream end. The onset of Ca2+ rise began at highly strained areas. In contrast to stimulus steps, slow ramp stimuli produced uniform forces throughout the cells and only a small Ca2+ response. The heterogeneity of force distribution is exaggerated in cells having fewer stress fibers and lower pre-tension in actinin. Disruption of cytoskeleton with cytochalasin-D (Cyt-D eliminated force gradients, and in those cells Ca2+ elevation started from the soma. Thus, Ca2+ influx with a mechanical stimulus depends on local stress within the cell and that is time dependent due to viscoelastic mechanics.

  1. The metric-affine gravitational theory as the gauge theory of the affine group

    International Nuclear Information System (INIS)

    Lord, E.A.

    1978-01-01

    The metric-affine gravitational theory is shown to be the gauge theory of the affine group, or equivalently, the gauge theory of the group GL(4,R) of tetrad deformations in a space-time with a locally Minkowskian metric. The identities of the metric-affine theory, and the relationship between them and those of general relativity and Sciama-Kibble theory, are derived. (Auth.)

  2. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  3. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

    International Nuclear Information System (INIS)

    Ogura, Kenji; Shiga, Takanori; Yokochi, Masashi; Yuzawa, Satoru; Burke, Terrence R.; Inagaki, Fuyuhiko

    2008-01-01

    The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity

  5. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  6. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  7. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  8. Influx of CO2 from Soil Incubated Organic Residues at Constant Temperature

    Directory of Open Access Journals (Sweden)

    Shoukat Ali Abro

    2016-06-01

    Full Text Available Temperature induced CO2 from genotypic residue substances is still less understood. Two types of organic residues (wheat- maize were incubated at a constant temperature (25°C to determine the rate and cumulative influx of CO2 in laboratory experiment for 40 days. Further, the effect of surface and incorporated crop residues with and without phosphorus addition was also studied. Results revealed that mixing of crop residues increased CO2-C evolution significantly & emission rare was 37% higher than that of control. At constant temperature, soil mixed residues, had higher emission rates CO2-C than the residues superimposed. There was linear correlation of CO2-C influxed for phosphorus levels and residue application ways with entire incubation at constant temperature. The mixing of organic residues to soil enhanced SOC levels and biomass of microbially bound N; however to little degree ammonium (NH4-N and nitrate NO3-N nitrogen were decreased.

  9. Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram

    DEFF Research Database (Denmark)

    Kumar, Vivek; Rahbek-Clemmensen, Troels; Billesbølle, Christian B

    2014-01-01

    Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demons...... demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells....

  10. A Generalized Affine Isoperimetric Inequality

    OpenAIRE

    Chen, Wenxiong; Howard, Ralph; Lutwak, Erwin; Yang, Deane; Zhang, Gaoyong

    2004-01-01

    A purely analytic proof is given for an inequality that has as a direct consequence the two most important affine isoperimetric inequalities of plane convex geometry: The Blaschke-Santalo inequality and the affine isoperimetric inequality of affine differential geometry.

  11. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  12. Compound immobilization and drug-affinity chromatography.

    Science.gov (United States)

    Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

    2012-01-01

    Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

  13. 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.

  14. Increased 22Na+-influx in lymphocytes from offspring of essential hypertensive patients

    DEFF Research Database (Denmark)

    Nielsen, J R; Pedersen, K E; Klitgaard, N A

    1989-01-01

    Lymphocytes were used as a cellular model for the in vitro measurements of 22Na+-influx during sodium pump inhibition by ouabain. The measurements were made using lymphocytes from young men at increased risk of developing essential hypertension in order to assess any changes and to analyse whether...

  15. Lp-mixed affine surface area

    Science.gov (United States)

    Wang, Weidong; Leng, Gangsong

    2007-11-01

    According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

  16. Content-Based High-Resolution Remote Sensing Image Retrieval via Unsupervised Feature Learning and Collaborative Affinity Metric Fusion

    Directory of Open Access Journals (Sweden)

    Yansheng Li

    2016-08-01

    Full Text Available With the urgent demand for automatic management of large numbers of high-resolution remote sensing images, content-based high-resolution remote sensing image retrieval (CB-HRRS-IR has attracted much research interest. Accordingly, this paper proposes a novel high-resolution remote sensing image retrieval approach via multiple feature representation and collaborative affinity metric fusion (IRMFRCAMF. In IRMFRCAMF, we design four unsupervised convolutional neural networks with different layers to generate four types of unsupervised features from the fine level to the coarse level. In addition to these four types of unsupervised features, we also implement four traditional feature descriptors, including local binary pattern (LBP, gray level co-occurrence (GLCM, maximal response 8 (MR8, and scale-invariant feature transform (SIFT. In order to fully incorporate the complementary information among multiple features of one image and the mutual information across auxiliary images in the image dataset, this paper advocates collaborative affinity metric fusion to measure the similarity between images. The performance evaluation of high-resolution remote sensing image retrieval is implemented on two public datasets, the UC Merced (UCM dataset and the Wuhan University (WH dataset. Large numbers of experiments show that our proposed IRMFRCAMF can significantly outperform the state-of-the-art approaches.

  17. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.

    1987-01-01

    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  18. Novel trends in affinity biosensors: current challenges and perspectives

    International Nuclear Information System (INIS)

    Arugula, Mary A; Simonian, Aleksandr

    2014-01-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives. (topical review)

  19. Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

    Directory of Open Access Journals (Sweden)

    Xiakun Chu

    2014-08-01

    Full Text Available Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less flexibility leads to weaker (stronger coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

  20. High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions

    NARCIS (Netherlands)

    Geuijen, K.P.M.; Schasfoort, R.B.; Wijffels, R.H.; Eppink, M.H.M.

    2014-01-01

    Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer

  1. The Lebanese–Syrian crisis: impact of influx of Syrian refugees to an already weak state

    Science.gov (United States)

    Cherri, Zeinab; Arcos González, Pedro; Castro Delgado, Rafael

    2016-01-01

    Background Lebanon, a small Middle Eastern country facing constant political and national unity challenges with a population of approximately 300,000 Palestinian and Iraqi refugees, has welcomed more than 1.2 million Office of the United Nations Commissioner for Refugees (UNHCR)-registered Syrian refugees since 2012. The Government of Lebanon considers individuals who crossed Lebanese–Syrian borders since 2011 as “displaced”, emphasizing its long-standing position that Lebanon is not a state for refugees, refusing to establish camps, and adopting a policy paper to reduce their numbers in October 2014. Humanitarian response to the Syrian influx to Lebanon has been constantly assembling with the UNHCR as the main acting body and the Lebanon Crisis Response Plan as the latest plan for 2016. Methods Review of secondary data from gray literature and reports focusing on the influx of Syrian refugees to Lebanon by visiting databases covering humanitarian response in complex emergencies. Limitations include obtaining majority of the data from gray literature and changing statistics due to the instability of the situation. Results The influx of Syrian refugees to Lebanon, an already weak and vulnerable state, has negatively impacted life in Lebanon on different levels including increasing demographics, regressing economy, exhausting social services, complicating politics, and decreasing security as well as worsened the life of displaced Syrians themselves. Conclusion Displaced Syrians and Lebanese people share aggravating hardships of a mutual and precarious crisis resulting from the Syrian influx to Lebanon. Although a lot of response has been initiated, both populations still lack much of their basic needs due to lack of funding and nonsustainable program initiatives. The two major recommendations for future interventions are to ensure continuous and effective monitoring and sustainability in order to alleviate current and future suffering in Lebanon. PMID:27471417

  2. The Lebanese-Syrian crisis: impact of influx of Syrian refugees to an already weak state.

    Science.gov (United States)

    Cherri, Zeinab; Arcos González, Pedro; Castro Delgado, Rafael

    2016-01-01

    Lebanon, a small Middle Eastern country facing constant political and national unity challenges with a population of approximately 300,000 Palestinian and Iraqi refugees, has welcomed more than 1.2 million Office of the United Nations Commissioner for Refugees (UNHCR)-registered Syrian refugees since 2012. The Government of Lebanon considers individuals who crossed Lebanese-Syrian borders since 2011 as "displaced", emphasizing its long-standing position that Lebanon is not a state for refugees, refusing to establish camps, and adopting a policy paper to reduce their numbers in October 2014. Humanitarian response to the Syrian influx to Lebanon has been constantly assembling with the UNHCR as the main acting body and the Lebanon Crisis Response Plan as the latest plan for 2016. Review of secondary data from gray literature and reports focusing on the influx of Syrian refugees to Lebanon by visiting databases covering humanitarian response in complex emergencies. Limitations include obtaining majority of the data from gray literature and changing statistics due to the instability of the situation. The influx of Syrian refugees to Lebanon, an already weak and vulnerable state, has negatively impacted life in Lebanon on different levels including increasing demographics, regressing economy, exhausting social services, complicating politics, and decreasing security as well as worsened the life of displaced Syrians themselves. Displaced Syrians and Lebanese people share aggravating hardships of a mutual and precarious crisis resulting from the Syrian influx to Lebanon. Although a lot of response has been initiated, both populations still lack much of their basic needs due to lack of funding and nonsustainable program initiatives. The two major recommendations for future interventions are to ensure continuous and effective monitoring and sustainability in order to alleviate current and future suffering in Lebanon.

  3. A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

    Science.gov (United States)

    Fujii, Rika; Schlom, Jeffrey; Hodge, James W

    2018-05-01

    OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

  4. High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

    Science.gov (United States)

    Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

    2015-01-01

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

  5. High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

    Science.gov (United States)

    Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

    2015-02-13

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

    Science.gov (United States)

    Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

    1989-07-01

    Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

  7. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screen...

  8. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...... charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from...

  9. sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))

    1989-08-01

    Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

  10. Mapping Affinities in Academic Organizations

    Directory of Open Access Journals (Sweden)

    Dario Rodighiero

    2018-02-01

    Full Text Available Scholarly affinities are one of the most fundamental hidden dynamics that drive scientific development. Some affinities are actual, and consequently can be measured through classical academic metrics such as co-authoring. Other affinities are potential, and therefore do not leave visible traces in information systems; for instance, some peers may share interests without actually knowing it. This article illustrates the development of a map of affinities for academic collectives, designed to be relevant to three audiences: the management, the scholars themselves, and the external public. Our case study involves the School of Architecture, Civil and Environmental Engineering of EPFL, hereinafter ENAC. The school consists of around 1,000 scholars, 70 laboratories, and 3 institutes. The actual affinities are modeled using the data available from the information systems reporting publications, teaching, and advising scholars, whereas the potential affinities are addressed through text mining of the publications. The major challenge for designing such a map is to represent the multi-dimensionality and multi-scale nature of the information. The affinities are not limited to the computation of heterogeneous sources of information; they also apply at different scales. The map, thus, shows local affinities inside a given laboratory, as well as global affinities among laboratories. This article presents a graphical grammar to represent affinities. Its effectiveness is illustrated by two actualizations of the design proposal: an interactive online system in which the map can be parameterized, and a large-scale carpet of 250 square meters. In both cases, we discuss how the materiality influences the representation of data, in particular the way key questions could be appropriately addressed considering the three target audiences: the insights gained by the management and their consequences in terms of governance, the understanding of the scholars’ own

  11. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  12. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    Science.gov (United States)

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  13. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  14. Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

    Science.gov (United States)

    Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

    1993-12-01

    A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

  15. Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

    Science.gov (United States)

    Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

    2006-06-01

    Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

  16. Fundamentals of affinity cell separations.

    Science.gov (United States)

    Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

    2018-03-01

    Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  18. High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

  19. [On the origin, course and influx-vessels of the V. basalis and the V. cerebri interna (author's transl)].

    Science.gov (United States)

    Lang, J; Köth, R; Reiss, G

    1981-01-01

    Origin, course and influx-vessels of the basal vein are investigated on 100 brains. An anterior formation of the basal vein (textbook) was found in 41%, a posterior formation in 34%. The different possibilities of drainage are examined procentually at the different types. Course and number of the different variations of the influx-vessels are taken into account: Vv. thalamostriata inferiores, gyri olfactorii, ventricularis inferior, peduncularis, cerebri interna, thalamostriata superioris, (terminalis), septi pellucidi anterior, septi pellucidi posterior, atrii medialis, atrii lateralis, nuclei caudati.

  20. Peptides in headlock ? a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    OpenAIRE

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ?-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once boun...

  1. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  2. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  3. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  4. Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

    Science.gov (United States)

    Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

    2017-05-13

    DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A High Affinity Adenosine Kinase from Anopheles gambiae

    Science.gov (United States)

    Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

  6. Affinity chromatography: A versatile technique for antibody purification.

    Science.gov (United States)

    Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

    2017-03-01

    Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

    Science.gov (United States)

    Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

    2014-02-01

    Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  8. Affine field theories

    International Nuclear Information System (INIS)

    Cadavid, A.C.

    1989-01-01

    The author constructs a non-Abelian field theory by gauging a Kac-Moody algebra, obtaining an infinite tower of interacting vector fields and associated ghosts, that obey slightly modified Feynman rules. She discusses the spontaneous symmetry breaking of such theory via the Higgs mechanism. If the Higgs particle lies in the Cartan subalgebra of the Kac-Moody algebra, the previously massless vectors acquire a mass spectrum that is linear in the Kac-Moody index and has additional fine structure depending on the associated Lie algebra. She proceeds to show that there is no obstacle in implementing the affine extension of supersymmetric Yang-Mills theories. The result is valid in four, six and ten space-time dimensions. Then the affine extension of supergravity is investigated. She discusses only the loop algebra since the affine extension of the super-Poincare algebra appears inconsistent. The construction of the affine supergravity theory is carried out by the group manifold method and leads to an action describing infinite towers of spin 2 and spin 3/2 fields that interact subject to the symmetries of the loop algebra. The equations of motion satisfy the usual consistency check. Finally, she postulates a theory in which both the vector and scalar fields lie in the loop algebra of SO(3). This theory has an expanded soliton sector, and corresponding to the original 't Hooft-Polyakov solitonic solutions she now finds an infinite family of exact, special solutions of the new equations. She also proposes a perturbation method for obtaining an arbitrary solution of those equations for each level of the affine index

  9. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

  10. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  11. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  12. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2011-02-15

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  13. Electron affinities: theoretical

    International Nuclear Information System (INIS)

    Kaufman, J.J.

    1976-01-01

    A brief description is given of the conceptual background and formalism of the various ab-initio and semi-ab-initio quantum computational techniques for calculating atomic and molecular electron affinities: Hartree--Fock--Roothaan SCF, configuration interaction (CI), multiconfiguration SCF (MC-SCF), Bethe--Goldstone, superposition of configurations (SOC), ab-initio effective core model potentials, Xα-MS, plus other less common methods. Illustrative and comparative examples of electron affinities calculated by these various methods are presented

  14. Transport of the alpha-amino-mono-carboxylic acid L-alanine by the beta-alanine carrier of the rabbit ileum

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Munck, B G

    1987-01-01

    The proposal that the beta-alanine carrier of the rabbit ileum is a high affinity carrier of the neutral amino acids was examined by means of measurements of influx across the brush border membrane of the intact epithelium using L-alanine as a representative of the neutral amino acids. Confirming...... the proposal, evidence was provided for mutual competitive inhibition between beta-alanine and L-alanine; and it was also demonstrated that a process contributes to the influx of L-alanine, which is characterized by a maximum rate of transport equal to that of beta-alanine and a Kt, which is equal to the Ki...... of L-alanine against the influx of beta-alanine. In the concentration range 0.01 to 0.125 mM the influx of L-alanine was found to be linearly related to the concentration indicating a significant unstirred layer influence on present and previous estimates of the Kt values for influx of amino acids...

  15. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    Science.gov (United States)

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-05

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

  16. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    International Nuclear Information System (INIS)

    Katz, D.M.; Kimelberg, H.K.

    1985-01-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [ 3 H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [ 3 H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [ 3 H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [ 3 H]-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of [ 3 H]-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate [ 3 H]-5-HT up to 44-fold at an external [ 3 H]-5-HT concentration of 10(-7) M. Inhibition of [ 3 H]-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of [ 3 H]-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on [ 3 H]-5-HT uptake

  17. Transformations Based on Continuous Piecewise-Affine Velocity Fields

    DEFF Research Database (Denmark)

    Freifeld, Oren; Hauberg, Søren; Batmanghelich, Kayhan

    2017-01-01

    We propose novel finite-dimensional spaces of well-behaved transformations. The latter are obtained by (fast and highly-accurate) integration of continuous piecewise-affine velocity fields. The proposed method is simple yet highly expressive, effortlessly handles optional constraints (e.g., volum...

  18. Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high-affinity choline uptake

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, R.H.; Boegman, R.J.; Quirion, R.; Riopelle, R.J.; Ludwin, S.K.

    1987-08-01

    A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific (/sup 3/H)hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.

  19. The Lebanese–Syrian crisis: impact of influx of Syrian refugees to an already weak state

    Directory of Open Access Journals (Sweden)

    Cherri Z

    2016-07-01

    Full Text Available Zeinab Cherri, Pedro Arcos González, Rafael Castro Delgado Unit for Research in Emergency and Disaster, Department of Medicine, University of Oviedo, Oviedo, Asturias, Spain Background: Lebanon, a small Middle Eastern country facing constant political and national unity challenges with a population of approximately 300,000 Palestinian and Iraqi refugees, has welcomed more than 1.2 million Office of the United Nations Commissioner for Refugees (UNHCR-registered Syrian refugees since 2012. The Government of Lebanon considers individuals who crossed Lebanese–Syrian borders since 2011 as “displaced”, emphasizing its long-standing position that Lebanon is not a state for refugees, refusing to establish camps, and adopting a policy paper to reduce their numbers in October 2014. Humanitarian response to the Syrian influx to Lebanon has been constantly assembling with the UNHCR as the main acting body and the Lebanon Crisis Response Plan as the latest plan for 2016. Methods: Review of secondary data from gray literature and reports focusing on the influx of Syrian refugees to Lebanon by visiting databases covering humanitarian response in complex emergencies. Limitations include obtaining majority of the data from gray literature and changing statistics due to the instability of the situation. Results: The influx of Syrian refugees to Lebanon, an already weak and vulnerable state, has negatively impacted life in Lebanon on different levels including increasing demographics, regressing economy, exhausting social services, complicating politics, and decreasing security as well as worsened the life of displaced Syrians themselves. Conclusion: Displaced Syrians and Lebanese people share aggravating hardships of a mutual and precarious crisis resulting from the Syrian influx to Lebanon. Although a lot of response has been initiated, both populations still lack much of their basic needs due to lack of funding and nonsustainable program initiatives

  20. Comparison of high affinity binding of 3H-proadifen and 3H-(-)-cocaine t rat liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1995-01-01

    The characteristics of the binding of 3 H-proadifen to rat liver membranes were studied and compared to those of 3 H-cocaine. It was found that 3 H-proadifen was bound reversibly with high affinity (K D =1.8±0.5 nM) and large capacity (B max =2010±340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3 H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 3 H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 μM CdCl 2 in the incubation buffer it was possible to differentiate between two 3 H-cocaine binding sites with K d values of 1.6 and 7.7 nM and B max values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3 H-proadifen and 3 H-cocaine inhibited the binding of 3 H-proadifen (IC 50 =10 nM) and proadifen that of 3 H-cocaine (IC 50 =1 nM). There was a high correlation coefficient (r r =0.972; P 50 =100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl 2 , ZnCl 2 and CuCl 2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd 2+ on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15μM. The similarity of the characteristics of the binding of these ligands with that of 3 H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.)

  1. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  2. Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

    International Nuclear Information System (INIS)

    Kutty, R.K.; Singh, Y.; Santostasi, G.; Krishna, G.

    1989-01-01

    Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 pM at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [14C]adenine nucleotides from hepatocytes prelabeled with [14C]-adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (less than 0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (greater than 1ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death

  3. Affine-Invariant Geometric Constraints-Based High Accuracy Simultaneous Localization and Mapping

    Directory of Open Access Journals (Sweden)

    Gangchen Hua

    2017-01-01

    Full Text Available In this study we describe a new appearance-based loop-closure detection method for online incremental simultaneous localization and mapping (SLAM using affine-invariant-based geometric constraints. Unlike other pure bag-of-words-based approaches, our proposed method uses geometric constraints as a supplement to improve accuracy. By establishing an affine-invariant hypothesis, the proposed method excludes incorrect visual words and calculates the dispersion of correctly matched visual words to improve the accuracy of the likelihood calculation. In addition, camera’s intrinsic parameters and distortion coefficients are adequate for this method. 3D measuring is not necessary. We use the mechanism of Long-Term Memory and Working Memory (WM to manage the memory. Only a limited size of the WM is used for loop-closure detection; therefore the proposed method is suitable for large-scale real-time SLAM. We tested our method using the CityCenter and Lip6Indoor datasets. Our proposed method results can effectively correct the typical false-positive localization of previous methods, thus gaining better recall ratios and better precision.

  4. Isolation and characterization of high affinity aptamers against DNA polymerase iota.

    Science.gov (United States)

    Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

    2012-02-01

    Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

  5. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  6. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  7. Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications.

    Science.gov (United States)

    Hage, David S

    2017-06-01

    The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically related binding agent, are 2 methods that can be used to study these interactions. This review presents various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. © 2016 American Association for Clinical Chemistry.

  8. The role of antibody affinity and titre in immunity to Schistosoma mansoni following vaccination with highly irradiated cercariae

    International Nuclear Information System (INIS)

    Vignali, D.A.A.; Devey, M.E.; Bickle, Q.D.; Taylor, M.G.

    1990-01-01

    Sera from rabbits and rats vaccinated with highly irradiated cercariae of Schistosoma mansoni (VRabS, VRatS) were found to be of substantially higher affinity than sera from CBA mice vaccinated four times (4 x CVMS), single sex sera (SSS) or chronic infection sera (CIS). In contrast, immunoprecipitation studies demonstrated that sera from vaccinated LA mice (LVMS) recognized 125 I-labelled schistosomular surface antigens more intensely than sera from vaccinated HA mice (HVMS). However, peritoneal macrophages from HA and LA mice in the presence of HVMS, LVMS or 4 x CVMS, and naive macrophages activated in vitro with interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) mediated comparable levels of schistosomula killing in vitro. The experiments described here provide evidence that the titre of antibody rather than its affinity may be a more critical factor in the development of optimal immunity to S. mansoni. (author)

  9. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed...... displayed lower affinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH2 and (5-30)NH2 . Agonism was only observed for GIP(1-30)NH2 with an Emax on 100% of GIP(1-42) and GIP(2-30)NH2 (Emax 20%). GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and right-shifts of the GIP(1-42)-response......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

  10. Covalent labeling of the beta-adrenergic ligand-binding site with para-(bromoacetamidyl)benzylcarazolol. A highly potent beta-adrenergic affinity label

    International Nuclear Information System (INIS)

    Dickinson, K.E.; Heald, S.L.; Jeffs, P.W.; Lefkowitz, R.J.; Caron, M.G.

    1985-01-01

    Para-(Bromoacetamidyl)benzylcarazolol (pBABC) was synthesized and found to be an extremely potent affinity label for beta-adrenergic receptors. Its interaction with mammalian (rabbit and hamster lung) and nonmammalian (turkey and frog erythrocyte) beta-adrenergic receptors was similar, displaying EC 50 values of 400-900 pM for inhibiting 125 I-cyanopindolol binding to these receptors. pBABC reduced the number of beta-adrenergic receptors in frog erythrocyte membranes, without any change in the affinity of the remaining sites for [ 125 I]iodocyanopindolol. pBABC has been radioiodinated. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this affinity probe specifically labeled the beta-adrenergic peptide of a purified preparation of hamster lung, with high efficiency (approximately 40%) and with a pharmacological specificity characteristic of an interaction at the beta 2-adrenergic receptor ligand-binding site. Comparison of the proteolyzed products derived from purified receptor labeled with [ 125 I]pBABC and with the photoaffinity agent [ 125 I]p-azidobenzylcarazolol suggested that covalent labeling of the beta-adrenergic receptor by these probes occurs at similar domains of the beta-adrenergic receptor

  11. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko

    2014-04-01

    Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

  12. Involvement of plasma membrane calcium influx in bacterial induction of the K+/H+ and hypersensitive responses in tobacco

    International Nuclear Information System (INIS)

    Atkinson, M.M.; Keppler, L.D.; Orlandi, E.W.; Baker, C.J.; Mischke, C.F.

    1990-01-01

    An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K + /H + response characterized by specific plasma membrane K + efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca 2+ influx of 0.02 to 0.06 micromole per gram per hour as determined from 45 Ca 2+ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K + /H + response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca 2+ influx was prevented by EGTA and calcium channel blockers such as La 3+ , Co 2+ , and Cd 2+ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K + /H + response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increase plasmalemma Ca 2+ influx is required for the K + /H + and hypersensitive responses in tobacco

  13. In Vitro and In Vivo Evaluations of A High Affinity and Specificity Photoacoustic Nanoparticle Targeting to Cancer

    DEFF Research Database (Denmark)

    Ma, Lixin; Xu, Hang; Lee, Li Ean

    oxide (SIO) nanoparticle as a potent cancer cell selective PA contrast agent, with a high binding affinity and selectivity to the gastrin releasing peptide receptor (GRPR) which is overexpressed in many human cancers including prostate cancer, breast cancer and small cell lung cancer etc.......Photoacoustic (PA) imaging uses a short-pulsed laser to create ultrasound signals for the high resolution acoustic imaging. The development of targeting PA contrast agents offers a unique opportunity to improve the early detection of cancer cells. This work aims to develop a silica coated iron...

  14. Impaired leukocyte influx in cervix of postterm women not responding to prostaglandin priming

    Directory of Open Access Journals (Sweden)

    Masironi Britt

    2008-09-01

    Full Text Available Abstract Background Prolonged pregnancies are associated with increased rate of maternal and fetal complications. Post term women could be divided into at least two subgroups, one where parturition is possible to induce by prostaglandins and one where it is not. Our aim was to study parameters in cervical biopsies in women with spontaneous delivery at term (controls and compare to those that are successfully induced post term (responders, and those that are not induced (non-responders, by local prostaglandin treatment. Methods Stromal parameters examined in this study were the accumulation of leukocytes (CD45, CD68, mRNAs and/or proteins for the extracellular matrix degrading enzymes (matrix metalloproteinase (MMP-2, MMP-8 and MMP-9, their inhibitors (tissue inhibitor of MMP (TIMP-1 and TIMP-2, interleukin-8 (IL-8, the platelet activating factor-receptor (PAF-R, syndecan-1 and estrogen binding receptors (estrogen receptor (ERα, ERβ and G-coupled protein receptor (GPR 30 as well as the proliferation marker Ki-67. Results The influx of leukocytes as assessed by CD45 was strongest in the responders, thereafter in the controls and significantly lower in the non-responders. IL-8, PAF-R and MMP-9, all predominantly expressed in leukocytes, showed significantly reduced immunostaining in the group of non-responders, while ERα and GPR30 were more abundant in the non-responders, as compared to the controls. Conclusion The impaired leukocyte influx, as reflected by the reduced number of CD45 positive cells as well as decreased immunostaining of IL-8, PAF-R and MMP-9 in the non-responders, could be one explanation of the failed ripening of the cervix in post term women. If the decreased leukocyte influx is a primary explanation to absent ripening or secondary, as a result of other factors, is yet to be established.

  15. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB 1 Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 2 Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 2 Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB 2 Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  16. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

  17. Pharmacologic study of calcium influx pathways in rabbit aortic smooth muscle

    International Nuclear Information System (INIS)

    Lukeman, D.S.

    1987-01-01

    Functional characteristics and pharmacologic domains of receptor-operated and potential-sensitive calcium (Ca 2+ ) channels (ROCs and PSCs, respectively) were derived via measurements of 45 Ca 2+ influx (M/sup Ca/) during activation by the neurotransmitters norepinephrine (NE), histamine (HS), and serotonin (5-HT) and by elevated extracellular potassium (K + ) in the individual or combined presence of organic Ca 2+ channel antagonists (CAts), calmodulin antagonists (Calm-ants), lanthanum (La 3+ ), and agents that increase intracellular levels of cyclic AMP

  18. 2017 Guralp Affinity Digitizer Evaluation.

    Energy Technology Data Exchange (ETDEWEB)

    Merchant, Bion J.

    2018-03-01

    Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  19. Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives

    NARCIS (Netherlands)

    Rios, Pablo; Carter, Tom S; Mooibroek, Tiddo J; Crump, Matthew P; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T; Boons, Geert-Jan|info:eu-repo/dai/nl/088245489; Davis, Anthony P

    2016-01-01

    The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside

  20. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.; Catterall, W.A.

    1982-01-01

    Specific binding of 3 H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors

  1. Two-parameter quantum affine algebra Ur,s(sln-circumflex), Drinfeld realization and quantum affine Lyndon basis

    International Nuclear Information System (INIS)

    Hu Naihong; Rosso, M.; Zhang Honglian

    2006-12-01

    We further find the defining structure of a two-parameter quantum affine algebra U r,s (sl n -circumflex) (n > 2) in the sense of Benkart-Witherspoon [BW1] after the work of [BGH1], [HS] and [BH], which turns out to be a Drinfeld double. Of more importance for the 'affine' cases is that we work out the compatible two-parameter version of the Drinfeld realization as a quantum affinization of U r,s (sl n ) and establish the Drinfeld isomorphism Theorem in the two-parameter setting via developing a new remarkable combinatorial approach - quantum 'affine' Lyndon basis with an explicit valid algorithm, based on the Drinfeld realization. (author)

  2. Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor

    OpenAIRE

    Smith, J R; Subbarao, K; Franc, D T; Haribabu, B; Rosenbaum, J T

    2004-01-01

    Aim: To investigate the role of arachidonic acid derived chemotactic factor, LTB4, in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB4 receptor.

  3. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific

  4. IA-2 autoantibody affinity in children at risk for type 1 diabetes.

    Science.gov (United States)

    Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

    2012-12-01

    Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

    Science.gov (United States)

    Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

    2018-04-25

    Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  6. Differential potassium influx influences growth of two cotton varieties in hydroponics

    International Nuclear Information System (INIS)

    Ali, L.; Maqsood, M.A.; Kanwal, S.; Aziz, T.

    2010-01-01

    Potassium uptake rate of two cotton (Gossypium hirsutum L.) varieties viz., NIBGE-2 and MNH-786 was investigated in nutrient solution culture having deficient K at the rate 0.3 mM and deficient K+ Na at the rate 0.3 +2.7 mM. Depletion of K from solution was monitored over a period of 24 h at regular time intervals after 0, 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h to estimate K uptake kinetics of the roots i.e. maximum influx, I/sub max/ and the Michaelis-Menten constant, Km. NIBGE-2 had about 2-fold higher (2.0 mg g rdw-1 hr-1) I/sub max/ value for K uptake rate at deficient K+Na than that (1.207 mg g rdw-1 hr-1) for MNH-786. Higher, Michaelis-Menten constant, Km (12.82 ppm) for K uptake rate was observed in both cultivars NIBGE-2 and MNH-786 at deficient K+Na than that at deficient K. Main effects of treatments and varieties had significant (p< 0.05) effect on shoot dry matter, root dry matter, total dry matter and leaf area per plant. Maximum K influx in NIBGE-2 at deficient K and deficient K +Na was attributed to enhanced growth response as compared to that in MNH-786. (author)

  7. Effect of selective blockade of oxygen consumption, glucose transport, and Ca2+ influx on thyroxine action in human mononuclear cells

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E

    1990-01-01

    The effect of selective blockade of cellular glucose transporters, Ca2+ influx, and mitochondrial oxygen consumption on thyroxine (T4)-stimulated oxygen consumption and glucose uptake was examined in human mononuclear blood cells. Blockade of glucose transporters by cytochalasin B (1 x 10(-5) mol....../L) and of Ca2+ influx by alprenolol (1 x 10(-5) mol/L) and verapamil (4 x 10(-4) mol/L) inhibited T4-activated glucose uptaken and reduced T4-stimulated oxygen consumption by 20%. Uncoupling of mitochondrial oxygen consumption by azide (1 x 10(-3) mol/L) inhibited T4-stimulated oxygen consumption, but had...... no effect on glucose uptake. We conclude that T4-stimulated glucose uptake in human mononuclear blood cells is dependent on intact glucose transporters and Ca2+ influx, but not on mitochondrial oxygen consumption. However, oxygen consumption is, in part, dependent on intact glucose uptake....

  8. Representations of affine Hecke algebras

    CERN Document Server

    Xi, Nanhua

    1994-01-01

    Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

  9. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    International Nuclear Information System (INIS)

    Asermely, K.E.; O'Neill, J.J.

    1986-01-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37 0 C in buffers containing tritium choline (5 μM 0.124 μC/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10 -5 M compared with 50% inhibition on brain synaptosomes. At 5 x 10 -4 M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K + -ion (25 mM) were unsuccessful; tissue 3 H-choline uptake appeared to oscillate over a 30 min period

  10. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    Energy Technology Data Exchange (ETDEWEB)

    Asermely, K.E.; O' Neill, J.J.

    1986-03-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37/sup 0/C in buffers containing tritium choline (5 ..mu..M 0.124 ..mu..C/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10/sup -5/ M compared with 50% inhibition on brain synaptosomes. At 5 x 10/sup -4/ M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K/sup +/-ion (25 mM) were unsuccessful; tissue /sup 3/H-choline uptake appeared to oscillate over a 30 min period.

  11. The role of antibody affinity and titre in immunity to Schistosoma mansoni following vaccination with highly irradiated cercariae

    Energy Technology Data Exchange (ETDEWEB)

    Vignali, D.A.A.; Devey, M.E.; Bickle, Q.D.; Taylor, M.G. (London School of Hygiene and Tropical Medicine (UK))

    1990-02-01

    Sera from rabbits and rats vaccinated with highly irradiated cercariae of Schistosoma mansoni (VRabS, VRatS) were found to be of substantially higher affinity than sera from CBA mice vaccinated four times (4 x CVMS), single sex sera (SSS) or chronic infection sera (CIS). In contrast, immunoprecipitation studies demonstrated that sera from vaccinated LA mice (LVMS) recognized {sup 125}I-labelled schistosomular surface antigens more intensely than sera from vaccinated HA mice (HVMS). However, peritoneal macrophages from HA and LA mice in the presence of HVMS, LVMS or 4 x CVMS, and naive macrophages activated in vitro with interferon-gamma (IFN-{gamma})/lipopolysaccharide (LPS) mediated comparable levels of schistosomula killing in vitro. The experiments described here provide evidence that the titre of antibody rather than its affinity may be a more critical factor in the development of optimal immunity to S. mansoni. (author).

  12. Thermal and mass implications of magmatic evolution in the Lassen volcanic region, California, and minimum constraints on basalt influx to the lower crust

    Science.gov (United States)

    Guffanti, M.; Clynne, M.A.; Muffler, L.J.P.

    1996-01-01

    We have analyzed the heat and mass demands of a petrologic model of basaltdriven magmatic evolution in which variously fractionated mafic magmas mix with silicic partial melts of the lower crust. We have formulated steady state heat budgets for two volcanically distinct areas in the Lassen region: the large, late Quaternary, intermediate to silicic Lassen volcanic center and the nearby, coeval, less evolved Caribou volcanic field. At Caribou volcanic field, heat provided by cooling and fractional crystallization of 52 km3 of basalt is more than sufficient to produce 10 km3 of rhyolitic melt by partial melting of lower crust. Net heat added by basalt intrusion at Caribou volcanic field is equivalent to an increase in lower crustal heat flow of ???7 mW m-2, indicating that the field is not a major crustal thermal anomaly. Addition of cumulates from fractionation is offset by removal of erupted partial melts. A minimum basalt influx of 0.3 km3 (km2 Ma)-1 is needed to supply Caribou volcanic field. Our methodology does not fully account for an influx of basalt that remains in the crust as derivative intrusives. On the basis of comparison to deep heat flow, the input of basalt could be ???3 to 7 times the amount we calculate. At Lassen volcanic center, at least 203 km3 of mantle-derived basalt is needed to produce 141 km3 of partial melt and drive the volcanic system. Partial melting mobilizes lower crustal material, augmenting the magmatic volume available for eruption at Lassen volcanic center; thus the erupted volume of 215 km3 exceeds the calculated basalt input of 203 km3. The minimum basalt input of 1.6 km3 (km2 Ma)-1 is >5 times the minimum influx to the Caribou volcanic field. Basalt influx high enough to sustain considerable partial melting, coupled with locally high extension rate, is a crucial factor in development of Lassen volcanic center; in contrast. Caribou volcanic field has failed to develop into a large silicic center primarily because basalt supply

  13. Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    International Nuclear Information System (INIS)

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-01-01

    Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

  14. Chronic Ca2+ influx through voltage-dependent Ca2+ channels enhance delayed rectifier K+ currents via activating Src family tyrosine kinase in rat hippocampal neurons.

    Science.gov (United States)

    Yang, Yoon-Sil; Jeon, Sang-Chan; Kim, Dong-Kwan; Eun, Su-Yong; Jung, Sung-Cherl

    2017-03-01

    Excessive influx and the subsequent rapid cytosolic elevation of Ca 2+ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca 2+ level in normal as well as pathological conditions. Delayed rectifier K + channels (I DR channels) play a role to suppress membrane excitability by inducing K + outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca 2+ -mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of I DR channels to hyperexcitable conditions induced by high Ca 2+ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca 2+ -treatment significantly increased the amplitude of I DR without changes of gating kinetics. Nimodipine but not APV blocked Ca 2+ -induced I DR enhancement, confirming that the change of I DR might be targeted by Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I DR enhancement was not affected by either Ca 2+ -induced Ca 2+ release (CICR) or small conductance Ca 2+ -activated K + channels (SK channels). Furthermore, PP2 but not H89 completely abolished I DR enhancement under high Ca 2+ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca 2+ -mediated I DR enhancement. Thus, SFKs may be sensitive to excessive Ca 2+ influx through VDCCs and enhance I DR to activate a neuroprotective mechanism against Ca 2+ -mediated hyperexcitability in neurons.

  15. High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidicacid

    DEFF Research Database (Denmark)

    Khandelia, Himanshu

    2008-01-01

    , comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of XPA ) 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 ( 80 × 106. An MD simulation on the siramesine:PA interaction...

  16. Antisymmetric tensor generalizations of affine vector fields.

    Science.gov (United States)

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  17. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    Science.gov (United States)

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  18. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  19. Manifolds with integrable affine shape operator

    Directory of Open Access Journals (Sweden)

    Daniel A. Joaquín

    2005-05-01

    Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

  20. Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches.

    Science.gov (United States)

    Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis; Ricci, Francesco

    2013-12-23

    Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

  1. Constraints on the affinity term for modeling long-term glass dissolution rates

    International Nuclear Information System (INIS)

    Bourcier, W.L.; Carroll, S.A.; Phillips, B.L.

    1993-11-01

    Predictions of long-term glass dissolution rates are highly dependent on the form of the affinity term in the rate expression. Analysis of the quantitative effect of saturation state on glass dissolution rate for CSG glass (a simple analog of SRL-165 glass), shows that a simple (1-Q/K) affinity term does not match experimental results. Our data at 100 degree C show that the data is better fit by an affinity term having the form (1 - (Q/K) 1 /σ) where σ = 10

  2. Ca2+ influx and efflux in animal cells in the presence of panax notoginseng extracts: investigated by using 45Ca as a radioactive tracer

    International Nuclear Information System (INIS)

    Yang Yuanyou; Liu Ning; Mo Shangwu; Liao Jiali; Xu Falun

    2010-01-01

    In this paper, the influence of extracts of Panax notoginseng on Ca 2+ influx and efflux in isolated rat visceral organs was investigated by using 45 Ca as a radioactive tracer. The results indicated that both extracts, the total flavonoids and total saponins of Panax notoginseng had significant influence on Ca 2+ influx and efflux in the isolated rat aorta, heart, and kidney, in those organs it could markedly block 45 Ca entering into cell and could facilitate efflux of intracellular Ca 2+ . Compared with the total flavonoids, total saponins had stronger role in the regulation of Ca 2+ influx and efflux. Also, regulation effects of Ca 2+ influx and efflux of the total saponins were compared with positive drug Verapamil, or even better. This implies that the total flavonoids and total saponins of Panax notoginseng have calcium antagonistic effect, and both may be the active ingredients in Panax notoginseng for coronary heart disease treatment. (authors)

  3. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  4. Affine LIBOR Models with Multiple Curves

    DEFF Research Database (Denmark)

    Grbac, Zorana; Papapantoleon, Antonis; Schoenmakers, John

    2015-01-01

    are specified following the methodology of the affine LIBOR models and are driven by the wide and flexible class of affine processes. The affine property is preserved under forward measures, which allows us to derive Fourier pricing formulas for caps, swaptions, and basis swaptions. A model specification...... with dependent LIBOR rates is developed that allows for an efficient and accurate calibration to a system of caplet prices....

  5. Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

    Science.gov (United States)

    Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R.; Botana, Luis M.

    2013-01-01

    Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. PMID:24379088

  6. Surface plasmon resonance biosensor method for palytoxin detection based on Na+,K+-ATPase affinity.

    Science.gov (United States)

    Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R; Botana, Luis M

    2013-12-27

    Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (Kobs). From the representation of Kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (K(D)) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is K(D) = 6.38 × 10-7 ± 6.67 × 10-8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.

  7. Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

    Directory of Open Access Journals (Sweden)

    Amparo Alfonso

    2013-12-01

    Full Text Available Palytoxin (PLTX, produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs. From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.

  8. A Novel Fast and Robust Binary Affine Invariant Descriptor for Image Matching

    Directory of Open Access Journals (Sweden)

    Xiujie Qu

    2014-01-01

    Full Text Available As the current binary descriptors have disadvantages of high computational complexity, no affine invariance, and the high false matching rate with viewpoint changes, a new binary affine invariant descriptor, called BAND, is proposed. Different from other descriptors, BAND has an irregular pattern, which is based on local affine invariant region surrounding a feature point, and it has five orientations, which are obtained by LBP effectively. Ultimately, a 256 bits binary string is computed by simple random sampling pattern. Experimental results demonstrate that BAND has a good matching result in the conditions of rotating, image zooming, noising, lighting, and small-scale perspective transformation. It has better matching performance compared with current mainstream descriptors, while it costs less time.

  9. Affinity Programs and the Real Estate Brokerage Industry

    OpenAIRE

    G Stacy Sirmans; David A. Macpherson

    2001-01-01

    This study surveys active real estate brokers obtaining information on involvement in affinity programs and referral/relocation networks. Some results regarding affinity involvement are: (a) 13% of respondents reported affinity affilliations, 75% reported no affiliations, and 12% indicated plans to become involved within the next year; (b) about half having affinity affiliations were involved with 2-4 groups; (c) affinity relationships were most often with membership organizations, corporatio...

  10. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  11. Reconstitution of high affinity α2 adrenergic agonist binding by fusion with a pertussis toxin substrate

    International Nuclear Information System (INIS)

    Kim, M.H.; Neubig, R.R.

    1986-01-01

    High affinity α 2 adrenergic agonist binding is thought to occur via a coupling of the α 2 receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 μM phenoxybenzamine to block α 2 receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the α 2 agonist [ 3 H]UK 14,304 (UK) and the antagonist [ 3 H] yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain α 2 receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance [ 3 H] UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity α 2 agonist binding

  12. Development of 99mTc labelled somatostatin analogues with high affinity for somatostatin receptors

    International Nuclear Information System (INIS)

    Maina, T.; Nock, B.; Nicolopoulou, A.; Tsipra, C.; Poppe, M.; Chiotellis, E.

    2001-01-01

    A recent development in oncology involves the use of metabolically stabilized peptide hormone analogues labelled with metallic radionuclides for the diagnosis or therapy of malignant disease. This approach was successfully applied for the first time in the visualization of somatostatin positive tumours and their metastases with 111 In DTPA-octreotide. In an effort to obtain a 99m Tc somatostatin receptor affine radioligand we describe herein the synthesis, radiochemistry and preliminary biological evaluation of two novel 99m Tc labelled somatostatin analogues, N 4 -TOC and N 4 -RC-160. In these compounds a tetraamine bifunctional unit was covalently attached to the N-terminal (D)Phe 1 of the peptide chain using Boc-protection strategies. The peptide conjugates were purified by high performance liquid chromatography (HPLC) and characterized by UV/Vis and ES-MS spectroscopies. As revealed by HPLC, 99m Tc labelling was quantitative under mild conditions, leading to a single 99m Tc species in high specific activities. Affinity of 99m Tc N 4 -TOC for the somatostatin receptor, as determined by in vitro binding assays in rat brain cortex membranes, was found unaffected by the presence of the bulky metal chelate. The binding properties of 99m Tc N 4 -RC-160 could not be determined by this assay due to an extremely high non-specific binding of this radioligand, and will be shortly investigated by other methods. Tissue distribution in healthy mice revealed that 99m Tc N 4 -TOC is clearing mainly through the kidneys and the urinary tract whereas 99m Tc N 4 -RC-160 shows a high accumulation in the liver as a result of its lipophilicity. Analysis of urine samples by HPLC showed that 99m Tc N 4 -TOC is excreted integer from the body of mice, while 99m Tc N 4 -RC-160 is totally transformed to an unidentified hydrophilic metabolite in vivo. The location of this metabolism is currently investigated. In vivo blocking experiments using animals pre-treated with 50 μg octreotide

  13. Experimental and theoretical binding affinity between polyvinylpolypyrrolidone and selected phenolic compounds from food matrices.

    Science.gov (United States)

    Durán-Lara, Esteban F; López-Cortés, Xaviera A; Castro, Ricardo I; Avila-Salas, Fabián; González-Nilo, Fernando D; Laurie, V Felipe; Santos, Leonardo S

    2015-02-01

    Polyvinylpolypyrrolidone (PVPP) is a fining agent, widely used in winemaking and brewing, whose mode of action in removing phenolic compounds has not been fully characterised. The aim of this study was to evaluate the experimental and theoretical binding affinity of PVPP towards six phenolic compounds representing different types of phenolic species. The interaction between PVPP and phenolics was evaluated in model solutions, where hydroxyl groups, hydrophobic bonding and steric hindrance were characterised. The results of the study indicated that PVPP exhibits high affinity for quercetin and catechin, moderate affinity for epicatechin, gallic acid and lower affinity for 4-methylcatechol and caffeic acid. The affinity has a direct correlation with the hydroxylation degree of each compound. The results show that the affinity of PVPP towards phenols is related with frontier orbitals. This work demonstrates a direct correlation between the experimental affinity and the interaction energy calculations obtained through computational chemistry methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Using Affinity Diagrams to Evaluate Interactive Prototypes

    DEFF Research Database (Denmark)

    Lucero, Andrés

    2015-01-01

    our particular use of affinity diagramming in prototype evaluations. We reflect on a decade’s experience using affinity diagramming across a number of projects, both in industry and academia. Our affinity diagramming process in interaction design has been tailored and consists of four stages: creating...

  15. Fundamental and practical studies on high-performance liquid affinity chromatography of biopolymers with novel stationary phases

    Energy Technology Data Exchange (ETDEWEB)

    Bacolod, M.D.

    1992-01-01

    Rigid microparticulate stationary phases having surface-bound metal chelating functions were developed and evaluated in high performance metal chelate affinity chromatography of proteins. Silica- and polystyrene-divinylbenzene-based metal chelate sorbents were produced in wide pore and in non-porous type of column packings. A major effort has been placed on development of non-porous highly crosslinked polystyrene-divinylbenzene (PSDVB). These PSDVB microparticles were produced by a two-step swelling polymerization, and exhibited excellent mechanical strength over a wide range of flow-rates and composition used in high performance liquid chromatography (HPLC). Simple and reproducible hydrophilic coatings were developed for the surface modification of hydrophobic PSDVB supports. A tetradentate metal chelating ligand, ethylenediamine-N, N[prime]-diacetic acid (EDDA), was covalently bound to the surface of the various supports. Sorbents having iminodiacetic acid (IDA) metal chelating functions were also evaluated. The hydrophilic character and surface coverage of various stationary phases were assessed chromatographically. Studies concerning the effects of eluent pH as well as the nature and concentration of salts on retention and selectivity with different metal chelate stationary phases having various immobilized metal ions were carried out. Elution schemes were developed for rapid separation of proteins in metal chelate affinity chromatography. EDDA stationary phases in metal forms can be viewed as complementary to IDA stationary phases since they afforded different selectivity and retentivity toward proteins. Hydrophilic PSDVB could be functionalized with IDA or EDDA metal chelating ligands or lectins. The non-porous metal chelate stationary phases afforded rapid separation of proteins by the development of multiple gradient systems, which permitted higher column peak capacity, enabling the separation of a greater number of proteins in a single chromatographic run.

  16. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    Science.gov (United States)

    Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

    2007-06-01

    Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  17. Limitation of the influx of formation water into oil wells. Ogranichenie pritoka plastovykh vod v neftyanye skvazhiny

    Energy Technology Data Exchange (ETDEWEB)

    Bulgakov, R.T.; Gazizov, A.Sh.; Gabdullin, R.G.; Yusupov, I.G.

    1976-01-01

    The problems of limiting the influx of water into oil wells are examined. On the basis of studies, systemization, and generalization of the reasons for the premature flooding of wells, the improvement of strata by polymer-cement solutions with consolidating liquid phases is considered. A detailed description is given of the technology and results of cementing well using solutions based on plugging cement and water-soluble phenol-formaldehyde resins of the TSD-9 type. Results are reported on the study of the properties of selective water-insulating substances based on acrylamide monomers and hydrolyzed polyacrylonitriles. Industrial testing of these materials is generalized. An economic evaluation is made of the efficiency of measures undertaken to prevent water influx into oil wells.

  18. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  19. Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

    Science.gov (United States)

    Holdgate, Geoff A; Anderson, Malcolm; Edfeldt, Fredrik; Geschwindner, Stefan

    2010-10-01

    Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

  20. Single-step affinity purification for fungal proteomics.

    Science.gov (United States)

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  1. Analysis of the plasma impurity influx from alkali-metal coatings for fusion-reactor applications

    International Nuclear Information System (INIS)

    DeWald, A.B.; Davidson, J.N.; Krauss, A.R.; Gruen, D.M.

    1982-01-01

    Recently, it has been proposed that alkali-metal covered surfaces be applied to magnetic fusion devices as a means of controlling plasma impurity contamination and shielding the substrate from erosion. Monolayer films of alkali metals have been shown to sputter primarily as ions under particle bombardment. Thus, it is thought that a sheath potential and/or magnetic fields encountered by a sputtered ion will return the ion to the surface without entering the plasma. In this paper, we investigate the net wall impurity influx associated with coatings which exhibit substantial secondary ion emission as compared to those which sputter only as neutral atoms. Included in the analysis are sputtered substrate atoms. These are sometimes found to be a significant fraction of the total sputtering yield for low-Z alkali monolayers and affect the overall performance of such coatings. Estimates of the impurity influx made in the neighborhood of a sheath potential show that secondary-ion emitting coatings are effective as a means of inhibiting plasma impurity contamination and wall erosion

  2. The production of high affinity monoclonal antibodies to human growth hormone

    International Nuclear Information System (INIS)

    Stuart, M.C.; Walichnowski, C.M.; Hussain, S.; Underwood, P.A.; Harman, D.F.; Rathjen, D.A.; Sturmer, S.R. von

    1983-01-01

    The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4x10 10 l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay. (Auth.)

  3. A pharmacological profile of the high-affinity GluK5 kainate receptor

    DEFF Research Database (Denmark)

    Møllerud, Stine; Kastrup, Jette Sandholm Jensen; Pickering, Darryl S

    2016-01-01

    -hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2 S,4 R)−4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar...

  4. Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate ssupply

    Directory of Open Access Journals (Sweden)

    Rui Hu

    2016-10-01

    Full Text Available The large nitrate transporter 1/peptide transporter family (NPF has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa root-specific expressed member OsNPF7.2 has not been characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, and affects rice growth under high nitrate supply. The expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterogenous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.

  5. Plasma-polymerized films providing selective affinity to the polarity of vaporized organic solvents

    International Nuclear Information System (INIS)

    Akimoto, Takuo; Ikeshita, Yusuke; Terashima, Ryo; Karube, Isao

    2009-01-01

    Plasma-polymerized films (PPFs) were fabricated as recognition membranes for a vapor-sensing device, and their affinity to vaporized organic solvents was evaluated with surface plasmon resonance. The affinity we intended to create is the selective sorption of the vaporized organic solvents depending on their polarity. For this purpose, acetonitrile, ethylenediamine (EDA), styrene, hexamethyldisiloxane (HMDSO), and hexamethyldisilazane were used to fabricate PPFs. Vaporized methanol, ethanol, and 1-propanol were used as high-polar solvents to be analyzed. Hexane, toluene, and p-xylene were used as low-polar solvents. As a result, the HMDSO-PPF with 97.3 o of contact angle was found to provide affinity to the low-polar solvents. In contrast, the EDA-PPF with 7.1 o of contact angle provided affinity to the high-polar solvents. Observations of the surface morphology of the HMDSO- and EDA-PPFs with a scanning electron microscope revealed that they are composed of nano-scale islands.

  6. Locked nucleic acid (LNA): High affinity targeting of RNA for diagnostics and therapeutics

    DEFF Research Database (Denmark)

    Kauppinen, S.; Vester, Birte; Wengel, Jesper

    2005-01-01

    Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. This conformational restriction results in unprecedented hybridization affinity towards complementary single stranded RN...

  7. Connections between quantized affine algebras and superalgebras

    International Nuclear Information System (INIS)

    Zhang, R.B.

    1992-08-01

    Every affine superalgebra with a symmetrizable Cartan matrix is closely related to an ordinary affine algebra with the same Cartan matrix. It is shown that the quantum supergroup associated with the former is essentially isomorphic to the quantum group associated with the latter in an appropriate class of representations. At the classical level, each integrable irreducible highest weight representation of the affine superalgebra has a corresponding irreducible representation of the affine algebra, which has the same weight space decomposition. (author). 5 refs, 3 tabs

  8. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  9. Insulin binding and stimulation of hexose and amino acid transport by normal and receptor-defective human fibroblasts

    International Nuclear Information System (INIS)

    Longo, N.; Nagata, N.; Danner, D.; Priest, J.; Elsas, L.

    1986-01-01

    The authors analyzed insulin receptors in cells cultured from a sibship of related parents who had two offspring with severe insulin resistance (Leprechaunism). 124 I-Insulin (1 ng/ml) binding to skin fibroblasts from the proband, mother, and father was 9, 60 and 62% of control cells, respectively, at equilibrium, Non-linear regression analysis, utilizing a two receptors model, of curvilinear Scatchard plots indicated a reduced number of high-affinity binding sites in both parents. Influx of L-Proline (System A), L-Serine (ASC) and L-Leucine (L) was similar in control and mutant cells. Similarly, during the depletion of intracellular amino acid pools, there was a release from transinhibition for System A and a decrease of transstimulation of Systems ASC and L in both cell lines. Surprisingly, insulin augmented, normally, A system influx with an ED 50 = 70 ng/ml at 24 0 C and 7 ng/ml at 37 0 C. By contrast insulin failed to simulated 3-0-methyl-D-glucose influx into the proband's cells, while normal cells were stimulated 30% with an ED 50 of 6 ng/ml. These results indicate that defective high-affinity insulin binding is inherited as an autosomal recessive trait; that general membrane functions are intact; that insulin regulates A system amino acid and hexose transport by two different mechanisms; and, that the latter mechanism is impaired by this family's receptor mutation

  10. TeBG- and CBG-bound steroid hormones in rabbits are available for influx into uterus in vivo

    International Nuclear Information System (INIS)

    Chaudhuri, G.; Steingold, K.A.; Pardridge, W.M.; Judd, H.L.

    1988-01-01

    The metabolic clearance rate (MCR) of gonadal or adrenal steroid hormones in rabbits often does not bear the expected inverse relationship with hormone binding to testosterone-binding globulin (TeBG) or corticosteroid-binding globulin (CBG). This suggests TeBG or CBG may not impede steroid hormone delivery to tissues. The effects of rabbit plasma proteins on the influxes of 3 H-labeled steroids from the circulation into the rabbit uterus were measured in vivo using a tissue sampling single-injection technique. In the absence of plasma proteins, estradiol (E 2 ) and testosterone (T) were freely diffusible through the uterine microvasculature (i.e., extraction >80%). The extractions of dihydrostestosterone (DHT) and corticosterone (B) ranged from 60 to 72%, while that of cortisol (F) was reduced at 40%. Rabbit serum exerted no inhibition of the influxes of the steroids tested. The influxes of T and B greatly exceeded the rates that would be expected if only the free and albumin-bound fractions estimated in vitro were diffusible in vivo. However, the extraction of [ 3 H]corticosteroid-binding globulin or bovine [ 3 H]albumin were low, consistent with little, if any, extravascular uptake of the plasma proteins. The results indicate both albumin-bound and globulin-bound steroid hormone are available for transport into the uterus in the rabbit in vivo without significant exodus of the plasma protein, per se

  11. Deep-sea spherules from Pacific clay - Mass distribution and influx rate. [extraterrestrial origins from optical and electron microscopy

    Science.gov (United States)

    Murrell, M. T.; Davis, P. A., Jr.; Nishiizumi, K.; Millard, H. T., Jr.

    1980-01-01

    From 411 kg of Pacific clay, 22 mg of stony spherules and 50 mg of iron spherules larger than 150 microns were concentrated. The extraterrestrial origin of these particles was evaluated with the aid of optical and electron microscopy and atomic absorption elemental analysis. An expression for the integral number of stony particles from this sediment in the mass range 20-300 micrograms was derived. The world-wide influx rate of stony particles in the mass range which survive atmospheric heating and ocean sediment storage is calculated to be 90 tons/yr. The relative contributions of ablation debris vs fused interplanetary dust to the influx of stony spherules is discussed, but no conclusions could be made.

  12. Screening for Natural Inhibitors of Topoisomerases I from Rhamnus davurica by Affinity Ultrafiltration and High-Performance Liquid Chromatography–Mass Spectrometry

    Science.gov (United States)

    Chen, Guilin; Guo, Mingquan

    2017-01-01

    Topoisomerase I (Topo I) catalyzes topological interconversion of duplex DNA during DNA replication and transcription, and has been deemed as important antineoplastic targets. In this study, the fraction R.d-60 from ethyl acetate extracts of Rhamnus davurica showed higher inhibitory rates against SGC-7901 and HT-29 compared with the R.d-30 fraction in vitro. However, the specific active components of R.d-60 fraction remain elusive. To this end, a method based on bio-affinity ultrafiltration and high performance liquid chromatography/electrospray mass spectrometry (HPLC- ESI-MS/MS) was developed to rapidly screen and identify the Topo I inhibitors in this fraction. The enrichment factors (EFs) were calculated to evaluate the binding affinities between the bioactive constituents and Topo I. As a result, eight ligands were identified and six of which with higher EFs showed more potential antitumor activity. Furthermore, antiproliferative assays in vitro (IC50 values) with two representative candidates (apigenin, quercetin) against SGC-7901, HT-29 and Hep G2 cells were conducted and further validated. Finally, the structure-activity relationships revealed that flavones contain a C2-C3 double bond of C ring exhibited higher bio-affinities to Topo I than those without it. This integrated method combining Topo I ultrafiltration with HPLC-MS/MS proved to be very efficient in rapid screening and identification of potential Topo I inhibitors from the complex extracts of medicinal plants, and could be further explored as a valuable high-throughput screening platform in the early drug discovery stage. PMID:28919906

  13. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  14. Affine Fullerene C60 in a GS-Quasigroup

    Directory of Open Access Journals (Sweden)

    Vladimir Volenec

    2014-01-01

    Full Text Available It will be shown that the affine fullerene C60, which is defined as an affine image of buckminsterfullerene C60, can be obtained only by means of the golden section. The concept of the affine fullerene C60 will be constructed in a general GS-quasigroup using the statements about the relationships between affine regular pentagons and affine regular hexagons. The geometrical interpretation of all discovered relations in a general GS-quasigroup will be given in the GS-quasigroup C(1/2(1+5.

  15. [Kinetic properties of the fructose influx across the brush border of the rat jejunum. Effects of a diet rich in fructose].

    Science.gov (United States)

    Crouzoulon, G

    1978-10-01

    The unidirectional influx (i.e. initial rate of uptake) of D-fructose across the brush border of rat jejunum is a saturable function of concentration, with a Kt of 125 mM, which implicates a carrier mechanism. This mechanism appears to be very specific for fructose in view of the lack of influx inhibition observed in the presence of large concentrations of the sugars or polyols, D-glucose, D-galactose, D-mannose, D-xylose, L-sorbose, D-tagatose, sorbitol or mannitol. D-Fructose uptake is inhibited by incubation, preceded by a 30-min preincubation in the same inhibitory conditions, in the absence of Na, or in the presence of metabolic poisons, NaF, 2,4-dinitrophenol, monoiodoacetate. Phloridzin (10-3 M), with or without preincubation, has no effect on uptake. D-Fructose influx is stimulated by fructose feeding, mainly because the augmentation of the number of active sites of transfer: Jmax is increased two-fold, Kt is more weakly affected.

  16. High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions

    DEFF Research Database (Denmark)

    Plenge, Per; Wiborg, Ove

    2005-01-01

    of presumed importance. Binding of S-citalopram, both to the high-affinity-binding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two...

  17. Affinity Spaces and 21st Century Learning

    Science.gov (United States)

    Gee, James Paul

    2017-01-01

    This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

  18. Enhanced carbon influx into TFTR supershots

    International Nuclear Information System (INIS)

    Ramsey, A.T.; Bush, C.E.; Dylla, H.F.; Owens, D.K.; Pitcher, C.S.; Ulrickson, M.

    1990-12-01

    Under some conditions, a very large influx of carbon into TFTR occurs during beam injection into low recycling plasmas (the Supershot regime). These carbon ''blooms'' result in serious degradation of plasma parameters. The sources of this carbon have been identified as hot spots on the TFTR bumper limiter at or near the last closed flux surface. Two separate temperature thresholds have been identified. One, at about 1650 degree C, is consistent with radiation enhanced sublimation. The other, at about 2300 degree C, appears to be thermal sublimation of carbon from the limiter. To account for the increased density caused by the blooms, near unity recycling of the carbon at the limiter by physical sputtering is required; this effect is expected from laboratory measurements, and we believe we are seeing it on TFTR. The sources of the carbon blooms are sites which have either loosely attached fragments of limiter material (caused by damage) or surfaces nearly perpendicular to the magnetic field lines. Such surfaces may have local power depositions two orders of magnitude higher than usual. The TFTR team modified the limiter during the opening of Winter 1989--90. The modifications greatly reduced the number and magnitude of the blooms, so that they are no longer a problem

  19. New immunogenic form for vasopressin: production of high-affinity antiserum and RIA for plasmatic AVP

    International Nuclear Information System (INIS)

    Rougon-Rappuzi, G.; Delaage, M.A.; Conte-Devolx, B.; Millet, Y.

    1977-01-01

    A highly sensitive and specific radioimmunoassay (RIA) for arginine-vasopressin (AVP) was developped and applied to the measurement of AVP in human plasma. High-affinity antivasopressin antibodies with limited association constant heterogeneity have been induced by immunizing rabbits with Lysine-vasopressine (LVP) coupled to a human immunoglobulin (IgA). Replacing air drying of acetone-petroleum ether extracts by lyophilisation increased significantly the yields of AVP. Equilibrium dialysis was used for separating bound and free antigen, thus reducing the total time required for the assay to 48 hours. Only 1 ml of plasma was required for routine determinations due to a sensitivity threshold better than 0.5 pg/ml. Plasma AVP levels of normal subjects and of patients with inappropriate ADH secretion (SIADH) were determined during different hydratation states and following nicotin of ethanol infusions. (orig.) [de

  20. On the structure of self-affine convex bodies

    Energy Technology Data Exchange (ETDEWEB)

    Voynov, A S [M. V. Lomonosov Moscow State University, Faculty of Mechanics and Mathematics, Moscow (Russian Federation)

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  1. PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

    Science.gov (United States)

    Wang, Han; Sieburth, Derek

    2013-01-01

    Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels. PMID:24086161

  2. PKA controls calcium influx into motor neurons during a rhythmic behavior.

    Directory of Open Access Journals (Sweden)

    Han Wang

    Full Text Available Cyclic adenosine monophosphate (cAMP has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

  3. Affinity membranes for hormone removal from aqueous solutions

    NARCIS (Netherlands)

    Urmenyi, A.M.; Poot, Andreas A.; Wessling, Matthias; Mulder, M.H.V.

    2005-01-01

    A novel affinity membrane was prepared by covalent binding of antibodies (against 17--estradiol) to a micro-porous poly(ethylene vinyl alcohol) (EVAL) membrane, taking benefit from the high surface area of EVAL membranes and the large number of reactive groups available for further surface

  4. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  5. DOTA-derivatives of octreotide dicarba-analogues with high affinity for somatostatin sst2,5 receptors

    Science.gov (United States)

    Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M.; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

    2017-02-01

    In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

  6. Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec-7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion.

    Science.gov (United States)

    Prescher, Horst; Frank, Martin; Gütgemann, Stephan; Kuhfeldt, Elena; Schweizer, Astrid; Nitschke, Lars; Watzl, Carsten; Brossmer, Reinhard

    2017-02-09

    Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar K d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

  7. Polynomials associated with equilibria of affine Toda-Sutherland systems

    International Nuclear Information System (INIS)

    Odake, S; Sasaki, R

    2004-01-01

    An affine Toda-Sutherland system is a quasi-exactly solvable multi-particle dynamics based on an affine simple root system. It is a 'cross' between two well-known integrable multi-particle dynamics, an affine Toda molecule (exponential potential, periodic nearest-neighbour interaction) and a Sutherland system (inverse sine-square interaction). Polynomials describing the equilibrium positions of affine Toda-Sutherland systems are determined for all affine simple root systems

  8. Duals of Affine Grassmann Codes and Their Relatives

    DEFF Research Database (Denmark)

    Beelen, P.; Ghorpade, S. R.; Hoholdt, T.

    2012-01-01

    Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine...... Grassmann code of any level and compute its minimum distance. Further, we ameliorate the results by Beelen concerning the automorphism group of affine Grassmann codes. Finally, we prove that affine Grassmann codes and their duals have the property that they are linear codes generated by their minimum......-weight codewords. This provides a clean analogue of a corresponding result for generalized Reed-Muller codes....

  9. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. A Novel Vertex Affinity for Community Detection

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  11. Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity

    International Nuclear Information System (INIS)

    Breillat, Christelle; Thoumine, Olivier; Choquet, Daniel

    2007-01-01

    In order to better probe SynCAM function in neurons, we produced a fusion protein between the extracellular domain of SynCAM1 and the constant fragment of human IgG (SynCAM-Fc). Whether in soluble form or immobilized on latex microspheres, the chimera bound specifically to the surface of hippocampal neurons and recruited endogenous SynCAM molecules. SynCAM-Fc was also used in combination with Quantum Dots to follow the mobility of transfected SynCAM receptors at the neuronal surface. Both immobile and highly mobile SynCAM were found. Thus, SynCAM-Fc behaves as a high affinity ligand that can be used to study the function of SynCAM at the neuronal membrane

  12. Complementary three-dimensional quantitative structure-activity relationship modeling of binding affinity and functional potency

    DEFF Research Database (Denmark)

    Tosco, Paolo; Ahring, Philip K; Dyhring, Tino

    2009-01-01

    Complementary 3D-QSAR modeling of binding affinity and functional potency is proposed as a tool to pinpoint the molecular features of the ligands, and the corresponding amino acids in the receptor, responsible for high affinity binding vs those driving agonist behavior and receptor activation. Th...

  13. On affine non-negative matrix factorization

    DEFF Research Database (Denmark)

    Laurberg, Hans; Hansen, Lars Kai

    2007-01-01

    We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate id...

  14. Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

    Science.gov (United States)

    Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

    2012-01-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

  15. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  16. Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Justesen, Sune Frederik Lamdahl; Lamberth, Kasper

    2009-01-01

    , better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput...... the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range...... screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009: 173-180)...

  17. An analysis of the plasma impurity influx from alkali-metal coatings for fusion reactor applications

    International Nuclear Information System (INIS)

    DeWald, A.B.; Davidson, J.N.; Krauss, A.R.; Gruen, D.M.

    1982-01-01

    Recently, it has been proposed that alkali-metal covered surfaces be applied to magnetic fusion devices as a means of controlling plasma impurity contamination and shielding the substrate from erosion. Monolayer films of alkali metals have been shown to sputter primarily as ions under particle bombardment. Thus, it is thought that a sheath potential and/or magnetic fields encountered by a sputtered ion will return the ion to the surface without entering the plasma. In this paper, we investigate the net wall impurity influx associated with coatings which exhibit substantial secondary ion emission compared with those which sputter only as neutral atoms. Included in the analysis are sputtered substrate atoms. These are sometimes found to be a significant fraction of the total sputtering yield for low-Z alkali monolayers and affect the overall performance of such coatings. Estimates of the impurity influx made in the neighborhood of a sheath potential show that secondary-ion emitting coatings are effective as a means of inhibiting plasma impurity contamination and wall erosion. (orig.)

  18. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  19. Heavy sediment influx during early Holocene: Inference from clay mineral studies in a core from the western Bay of Bengal

    Digital Repository Service at National Institute of Oceanography (India)

    Reddy, N.P.C.; Rao, K.M.

    is attributed to heavy sediment influx du r- ing Mid Termination (MT) (12,500 ? 10,000 years BP), due to i n creased precipitation and run - off resulting from high inte n sity monsoonal regime. C LAY minerals are a powerful source for the interpret a... to address to the prov e nance of the sediments in a sediment core from the western Bengal Fan. In this paper, we report clay mi n eralogy and provide an explanation for the heavy sed i ment deposition du r ing the Holocene. A sediment core of 650...

  20. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    Science.gov (United States)

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

  1. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  2. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    Science.gov (United States)

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  3. Contractions of affine spherical varieties

    International Nuclear Information System (INIS)

    Arzhantsev, I V

    1999-01-01

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL 2 -varieties are considered

  4. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K+ uptake and Na+ transport in yeast

    International Nuclear Information System (INIS)

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-01-01

    Highlights: → The AtCCX5 protein coding a putative cation calcium exchanger was characterized. → AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. → AtCCX5 protein did not show the same transport properties as the CAXs. → AtCCX5 protein involves in mediating high-affinity K + uptake in yeast. → AtCCX5 protein also involves in Na + transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K + , Na + , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , Co 2+ , Cd 2+ , Mn 2+ , Ba 2+ , Ni 2+ , Zn 2+ , and Li + ) were analyzed. AtCCX5 expression was found to affect the response to K + and Na + in yeast. The AtCCX5 transformant also showed a little better growth to Zn 2+ . The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K + (0.5 mM), and also suppressed its Na + sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K + uptake and was also involved in Na + transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K + uptake and Na + transport in yeast.

  5. Amine-functionalized PVA-co-PE nanofibrous membrane as affinity membrane with high adsorption capacity for bilirubin.

    Science.gov (United States)

    Wang, Wenwen; Zhang, Hao; Zhang, Zhifeng; Luo, Mengying; Wang, Yuedan; Liu, Qiongzhen; Chen, Yuanli; Li, Mufang; Wang, Dong

    2017-02-01

    In this study, poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane was activated by sodium hydroxide and cyanuric chloride, and then the activated membranes were functionalized by 1,3-propanediamine, hexamethylenediamine and diethylenetriamine to be affinity membranes for bilirubin removal, respectively. The chemical structures and morphologies of membranes were investigated by SEM, FTIR and XPS. And the adsorption ability of different amine-functionalized nanofibrous membranes for bilirubin was characterized. Furthermore, the effects of temperature, initial concentration of bilirubin, NaCl concentration and BSA concentration on the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane were studied. Results indicated that the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane could reach 85mg/g membrane when the initial bilirubin concentration was 200mg/L while the adsorption capacity could be increased to 110mg/g membrane if the initial bilirubin concentration was more than 400mg/L. The dynamic adsorption of diethylenetriamine-functionalized nanofibrous membrane showed that the ligands of amine groups on the membrane surface could be used as far as possible by recirculating the plasma with certain flow rates. Therefore, the diethylenetriamine-functionalized PVA-co-PE nanofibrous membrane possessed high adsorption capacity for bilirubin and it can be candidate as affinity membrane for bilirubin removal. Copyright © 2016. Published by Elsevier B.V.

  6. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are ...... of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke....

  7. hPEPT1 Affinity and Translocation of Selected Gln-Sar and Glu-Sar Dipeptide Derivatives

    DEFF Research Database (Denmark)

    Eriksson, A. H.; Elm, Peter L.; Begtrup, Mikael

    2005-01-01

    using 14C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides l-Gln(N,N-dimethyl)-Sar and l-Gln(N-piperidinyl)-Sar were...... been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size...... translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must...

  8. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    Science.gov (United States)

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals.

    Science.gov (United States)

    Li, Li; Chen, Hongpu; Lv, Xiaolan; Wang, Min; Jiang, Xizhi; Jiang, Yifei; Wang, Heye; Zhao, Yongfu; Xia, Liru

    2018-03-01

    Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 μg·kg -1 , respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81-86% for deoxynivalenol, 89-117% for zearalenone, 79-86% for T-2 toxin, and 78-83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstract Paper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone.

  10. Providing affinity

    DEFF Research Database (Denmark)

    Guglielmi, Michel; Johannesen, Hl

    2004-01-01

    , Essex, Hertfordshire, Norfolk and Suffolk. Research found that there was a lack of identity or sense of belonging and nothing anchoring people to the region as a whole. Common affinity is somehow forced to the people of East England and thereby we came to the conclusion that a single landmark...... and potential situations but also virtual events that calls for an undeterminated process of resolution. This process is activated by the user who co-produces the actualisation as an answer to a virtual reality that we defined at the first place. The potential situations or the possible it is a fantomatic real....... The possible is like the real. It is determinated and it only lakes existence. While the possible is already made, the virtual is like a problematic which needs to be resolved and actualized. Our installations are based on high tech interactivity where we use sensors and remote communication to offer a sense...

  11. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

    Science.gov (United States)

    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

    2014-01-01

    Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

  12. Modeling DNA affinity landscape through two-round support vector regression with weighted degree kernels

    KAUST Repository

    Wang, Xiaolei

    2014-12-12

    Background: A quantitative understanding of interactions between transcription factors (TFs) and their DNA binding sites is key to the rational design of gene regulatory networks. Recent advances in high-throughput technologies have enabled high-resolution measurements of protein-DNA binding affinity. Importantly, such experiments revealed the complex nature of TF-DNA interactions, whereby the effects of nucleotide changes on the binding affinity were observed to be context dependent. A systematic method to give high-quality estimates of such complex affinity landscapes is, thus, essential to the control of gene expression and the advance of synthetic biology. Results: Here, we propose a two-round prediction method that is based on support vector regression (SVR) with weighted degree (WD) kernels. In the first round, a WD kernel with shifts and mismatches is used with SVR to detect the importance of subsequences with different lengths at different positions. The subsequences identified as important in the first round are then fed into a second WD kernel to fit the experimentally measured affinities. To our knowledge, this is the first attempt to increase the accuracy of the affinity prediction by applying two rounds of string kernels and by identifying a small number of crucial k-mers. The proposed method was tested by predicting the binding affinity landscape of Gcn4p in Saccharomyces cerevisiae using datasets from HiTS-FLIP. Our method explicitly identified important subsequences and showed significant performance improvements when compared with other state-of-the-art methods. Based on the identified important subsequences, we discovered two surprisingly stable 10-mers and one sensitive 10-mer which were not reported before. Further test on four other TFs in S. cerevisiae demonstrated the generality of our method. Conclusion: We proposed in this paper a two-round method to quantitatively model the DNA binding affinity landscape. Since the ability to modify

  13. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

    Directory of Open Access Journals (Sweden)

    Ivo M B Francischetti

    2010-02-01

    Full Text Available Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  14. Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

    Science.gov (United States)

    Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

    2017-06-20

    Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

  15. Extreme disorder in an ultrahigh-affinity protein complex

    DEFF Research Database (Denmark)

    Borgia, Alessandro; Borgia, Madeleine B; Bugge, Katrine

    2018-01-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions...... with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring...... or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex...

  16. Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

    Science.gov (United States)

    McQuaid, Jeffrey B.; Kustka, Adam B.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Karas, Bogumil J.; Zheng, Hong; Kindeberg, Theodor; Andersson, Andreas J.; Barbeau, Katherine A.; Allen, Andrew E.

    2018-03-01

    In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

  17. Global affine differential geometry of hypersurfaces

    CERN Document Server

    Li, An-Min; Zhao, Guosong; Hu, Zejun

    2015-01-01

    This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

  18. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  19. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T. [Samsung Medical Center, Seoul (Korea, Republic of)

    2005-07-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2{alpha} of 5.2 min and late T1/2{beta} of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8{+-}0.5 ml/min, T1/2 of 4.1{+-}0.4 hr, and volume of distribution of 69.7{+-}4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors.

  20. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    International Nuclear Information System (INIS)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T.

    2005-01-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2α of 5.2 min and late T1/2β of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8±0.5 ml/min, T1/2 of 4.1±0.4 hr, and volume of distribution of 69.7±4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors

  1. Enhanced binding affinity, remarkable selectivity, and high capacity of CO 2 by dual functionalization of a rht-type metal-organic framework

    KAUST Repository

    Li, Baiyan

    2011-12-23

    Open and friendly: The smallest member of the rht-type metal-organic frameworks (MOFs, see picture) constructed by a hexacarboxylate ligand with a nitrogen-rich imino triazine backbone shows a significantly enhanced gas binding affinity relative to all other isoreticular rht-type MOFs. The high adsorption capacity and remarkable selectivity of CO 2 are attributed to the high density of open metal and Lewis basic sites in the framework. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

    Science.gov (United States)

    Fawaz, Maria V; Brooks, Allen F; Rodnick, Melissa E; Carpenter, Garrett M; Shao, Xia; Desmond, Timothy J; Sherman, Phillip; Quesada, Carole A; Hockley, Brian G; Kilbourn, Michael R; Albin, Roger L; Frey, Kirk A; Scott, Peter J H

    2014-08-20

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

  3. Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

    Science.gov (United States)

    Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

    1993-03-10

    Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

  4. New unitary affine-Virasoro constructions

    International Nuclear Information System (INIS)

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M.; Yamron, J.P.

    1990-01-01

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g

  5. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  6. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    Science.gov (United States)

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  7. Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.; Kimmich, G.A.

    1982-01-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 μg/ml cholera toxin (CT) at 37 0 C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na + influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na + entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na + influx suggest that the reactivation of the Na + transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP

  8. Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

    OpenAIRE

    Stewart, Rachel C.; Bansal, Prashant N.; Entezari, Vahid; Lusic, Hrvoje; Nazarian, Rosalynn M.; Snyder, Brian D.; Grinstaff, Mark W.

    2013-01-01

    The high affinity of a cationic iodinated contrast agent for cartilage provides better tissue visualization, easier segmentation, higher contrast-to-noise ratios, and longer usable imaging windows and requires a lower dose of injected contrast agent compared with an anionic contrast agent.

  9. Extracellular Zn2+ Influx into Nigral Dopaminergic Neurons Plays a Key Role for Pathogenesis of 6-Hydroxydopamine-Induced Parkinson's Disease in Rats.

    Science.gov (United States)

    Tamano, Haruna; Nishio, Ryusuke; Morioka, Hiroki; Takeda, Atsushi

    2018-04-29

    Parkinson's disease (PD) is a progressive neurological disease characterized by a selective loss of nigrostriatal dopaminergic neurons. The exact cause of the neuronal loss remains unclear. Here, we report a unique mechanism of nigrostriatal dopaminergic neurodegeneration, in which extracellular Zn 2+ influx plays a key role for PD pathogenesis induced with 6-hydroxydopamine (6-OHDA) in rats. 6-OHDA rapidly increased intracellular Zn 2+ only in the substantia nigra pars compacta (SNpc) of brain slices and this increase was blocked in the presence of CaEDTA, an extracellular Zn 2+ chelator, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, indicating that 6-OHDA rapidly increases extracellular Zn 2+ influx via AMPA receptor activation in the SNpc. Extracellular Zn 2+ concentration was decreased under in vivo SNpc perfusion with 6-OHDA and this decrease was blocked by co-perfusion with CNQX, supporting 6-OHDA-induced Zn 2+ influx via AMPA receptor activation in the SNpc. Interestingly, both 6-OHDA-induced loss of nigrostriatal dopaminergic neurons and turning behavior to apomorphine were ameliorated by co-injection of intracellular Zn 2+ chelators, i.e., ZnAF-2DA and N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Co-injection of TPEN into the SNpc blocked 6-OHDA-induced increase in intracellular Zn 2+ but not in intracellular Ca 2+ . These results suggest that the rapid influx of extracellular Zn 2+ into dopaminergic neurons via AMPA receptor activation in the SNpc induces nigrostriatal dopaminergic neurodegeneration, resulting in 6-OHDA-induced PD in rats.

  10. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

    Science.gov (United States)

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  11. Affine stochastic mortality

    NARCIS (Netherlands)

    Schrager, D.F.

    2006-01-01

    We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing

  12. Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs

    International Nuclear Information System (INIS)

    Ong, Hooi Tin; Loo, Joachim S. C.; Boey, Freddy Y. C.; Russell, Stephen J.; Ma Jan; Peng, Kah-Whye

    2008-01-01

    Hydroxyapatite is biocompatible and used in various biomedical applications. Here, we generated hydroxyapatite nanoparticles (HNPs) of various sizes (40-200 nm) and demonstrated that they can be stably loaded with drugs or radioisotopes by exploiting the high-affinity HA-(poly)phosphonate interaction. Clinically available phosphonates, clodronate, and Tc-99m-methylene-diphosphonate (Tc-99m-MDP), were efficiently loaded onto HNPs within 15 min. Biodistribution of radiolabeled HNP-MDP-Tc99m in mice was monitored non-invasively using microSPECT-CT. Imaging and dosimetry studies indicated that the HNPs, regardless of size, were quickly taken up by Kupffer cells in the liver after systemic administration into mice. Clodronate loaded onto HNPs remained biologically active and were able to result in selective depletion of Kupffer cells. This method of drug or isotope loading on HA is fast and easy as it eliminates the need for additional surface modifications of the nanoparticles

  13. Rank Two Affine Manifolds in Genus 3

    OpenAIRE

    Aulicino, David; Nguyen, Duc-Manh

    2016-01-01

    We complete the classification of rank two affine manifolds in the moduli space of translation surfaces in genus three. Combined with a recent result of Mirzakhani and Wright, this completes the classification of higher rank affine manifolds in genus three.

  14. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  15. Comparative cation dependency of sugar transport by crustacean hepatopancreas and intestine

    Directory of Open Access Journals (Sweden)

    Ada Duka

    2014-06-01

    Full Text Available Glucose is transported in crustacean hepatopancreas and intestine by Na+-dependent co-transport, while Na+-dependent D-fructose influx has only been described for the hepatopancreas. It is still unclear if the two sugars are independently transported by two distinct cation-dependent co-transporter carrier systems. In this study, lobster (Homarus americanus hepatopancreas brush border membrane vesicles (BBMV were used to characterize, in detail, the cation-dependency of both D-[3H]-glucose and D-[3H]-fructose influxes, while in vitro perfused intestines were employed to determine the nature of cation-dependent sugar transport across this organ. Over the sodium concentration range of 0–100 mM, both [3H]-glucose and [3H]-fructose influxes (0.1 mM; 1 min uptakes by hepatopancreatic BBMV were hyperbolic functions of [Na+]. [3H]-glucose and [3H]-fructose influxes by hepatopancreatic BBMV over a potassium concentration range of 15–100 mM were hyperbolic functions of [K+]. Both sugars displayed significant (p<0.01 Na+/K+-dependent and cation-independent uptake processes. Transepithelial 25 µM [3H]-glucose and [3H]-fructose fluxes across lobster intestine over luminal sodium and potassium concentration ranges of 0–50 mM and 5–100 mM, respectively, were hyperbolic functions of luminal [Na+] and [K+]. As with hepatopancreatic sugar transport, transepithelial intestinal sugar transport exhibited both significant (p<0.01 Na+/K+-dependent and cation-independent processes. Results suggest that both D-glucose and D-fructose are transported by a single SGLT-type carrier in each organ with sodium being the “preferred”, high affinity, cation for both sugars in the hepatopancreas, and potassium being the “preferred”, high affinity, cation for both sugars in the intestine.

  16. Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

    Science.gov (United States)

    Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

    2018-02-23

    Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Better well control through safe drilling margin identification, influx analysis and direct bottom hole pressure control method for deep water

    Energy Technology Data Exchange (ETDEWEB)

    Veeningen, Daan [National Oilwell Varco IntelliServ (NOV), Houston, TX (United States)

    2012-07-01

    Currently, well control events are almost exclusively detected by using surface measurements. Measuring a volume increase in the 'closed loop' mud circulation system; a standpipe pressure decrease; or changes in a variety of drilling parameters provide indicators of a kick. Especially in deep water, where the riser comprises a substantial section of the well bore, early kick detection is paramount for limiting the severity of a well bore influx and improve the ability to regain well control. While downhole data is presently available from downhole tools nearby the bit, available data rates are sparse as mud pulse telemetry bandwidth is limited and well bore measurements compete with transmission of other subsurface data. Further, data transfer is one-directional, latency is significant and conditions along the string are unknown. High-bandwidth downhole data transmission system, via a wired or networked drill string system, has the unique capability to acquire real-time pressure and temperature measurement at a number of locations along the drill string. This system provides high-resolution downhole data available at very high speed, eliminating latency and restrictions that typically limit the availability of downhole data. The paper describes well control opportunities for deep water operations through the use of downhole data independent from surface measurements. First, the networked drill string provides efficient ways to identify pore pressure, fracture gradient, and true mud weight that comprise the safe drilling margin. Second, the independent measurement capability provides early kick detection and improved ability to analyze an influx even with a heterogeneous mud column through distributed along-string annular pressure measurements. Third, a methodology is proposed for a direct measurement method using downhole real-time pressure for maintaining constant bottom hole pressure during well kills in deep water. (author)

  18. Food and value motivation: Linking consumer affinities to different types of food products.

    Science.gov (United States)

    de Boer, Joop; Schösler, Hanna

    2016-08-01

    This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Dynamics of Open Systems with Affine Maps

    International Nuclear Information System (INIS)

    Zhang Da-Jian; Liu Chong-Long; Tong Dian-Min

    2015-01-01

    Many quantum systems of interest are initially correlated with their environments and the reduced dynamics of open systems are an interesting while challenging topic. Affine maps, as an extension of completely positive maps, are a useful tool to describe the reduced dynamics of open systems with initial correlations. However, it is unclear what kind of initial state shares an affine map. In this study, we give a sufficient condition of initial states, in which the reduced dynamics can always be described by an affine map. Our result shows that if the initial states of the combined system constitute a convex set, and if the correspondence between the initial states of the open system and those of the combined system, defined by taking the partial trace, is a bijection, then the reduced dynamics of the open system can be described by an affine map. (paper)

  20. USING MICROSCALE THERMOPHORESIS TO EASILY MEASURE BINDING AFFINITY

    Directory of Open Access Journals (Sweden)

    Dennis Breitsprecher*

    2018-03-01

    Full Text Available While it’s very common for biologists and chemists to test whether or not two molecules interact with each other, it’s much more useful to gather information on the nature of that interaction. How strong is it? How long will it last? What does that mean for its biological function? One way to answer these questions is to study affinity. Binding affinity is defined as the strength of the binding interaction between a single biomolecule to its binding partner, or ligand, and it can be quantifiably measured, providing information on whether or not molecules are interacting, as well as assigning a value to the affinity. When measuring binding affinity, there are several parameters to look at, but the dissociation constant (Kd, which defines the likelihood that an interaction between two molecules will break, is a very common measurement. The smaller the dissociation constant, the more tightly bound the ligand is, and the higher the affinity is between the two molecules.

  1. Single-Step Affinity Purification for Fungal Proteomics ▿ †

    OpenAIRE

    Liu, Hui-Lin; Osmani, Aysha H.; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B.; De Souza, Colin P.; Osmani, Stephen A.

    2010-01-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  2. Blood O2 affinity of a large polar elasmobranch, the Greenland shark Somniosus microcephalus

    DEFF Research Database (Denmark)

    Herbert, N.A.; Skov, Peter Vilhelm; Tirsgaard, B.

    2017-01-01

    The Greenland shark (Somniosus microcephalus. Bloch & Schneider 1801) is a polar elasmobranch that is hypothesised to possess a unique metabolic physiology due to its extreme large size, the cold waters it inhabits and its slow swimming lifestyle. Our results therefore provide the first insight...... into the metabolic physiology of this unique shark, with a focus on blood O2 affinity. An evaluation of blood O2 affinity at 2 °C using tonometry revealed a P50 of 11.7 mmHg at a PCO2 of 2.25 mmHg and a Bohr effect (binding sensitivity of blood to pH, ϕ = Δlog P50/ΔpH) of −0.26. A comparative evaluation of blood O2...... affinity across elasmobranch fishes suggests that S. microcephalus has a high blood O2 affinity (i.e., low P50) and a small Bohr effect but these are common traits in sluggish elasmobranch fishes, with little evidence for any relationship of blood O2 affinity to the low metabolic rates, low environmental...

  3. The Structure of Affine Buildings

    CERN Document Server

    Weiss, Richard M

    2009-01-01

    In The Structure of Affine Buildings, Richard Weiss gives a detailed presentation of the complete proof of the classification of Bruhat-Tits buildings first completed by Jacques Tits in 1986. The book includes numerous results about automorphisms, completions, and residues of these buildings. It also includes tables correlating the results in the locally finite case with the results of Tits's classification of absolutely simple algebraic groups defined over a local field. A companion to Weiss's The Structure of Spherical Buildings, The Structure of Affine Buildings is organized around the clas

  4. The dynamics of metric-affine gravity

    International Nuclear Information System (INIS)

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-01-01

    Highlights: → The role and the dynamics of the connection in metric-affine theories is explored. → The most general second order action does not lead to a dynamical connection. → Including higher order invariants excites new degrees of freedom in the connection. → f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy

  5. 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

  6. Spectral affinity in protein networks.

    Science.gov (United States)

    Voevodski, Konstantin; Teng, Shang-Hua; Xia, Yu

    2009-11-29

    Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to quickly find nodes closest to a queried vertex in any protein

  7. Genetically based low oxygen affinities of felid hemoglobins: lack of biochemical adaptation to high-altitude hypoxia in the snow leopard

    Science.gov (United States)

    Janecka, Jan E.; Nielsen, Simone S. E.; Andersen, Sidsel D.; Hoffmann, Federico G.; Weber, Roy E.; Anderson, Trevor; Storz, Jay F.; Fago, Angela

    2015-01-01

    ABSTRACT Genetically based modifications of hemoglobin (Hb) function that increase blood–O2 affinity are hallmarks of hypoxia adaptation in vertebrates. Among mammals, felid Hbs are unusual in that they have low intrinsic O2 affinities and reduced sensitivities to the allosteric cofactor 2,3-diphosphoglycerate (DPG). This combination of features compromises the acclimatization capacity of blood–O2 affinity and has led to the hypothesis that felids have a restricted physiological niche breadth relative to other mammals. In seeming defiance of this conjecture, the snow leopard (Panthera uncia) has an extraordinarily broad elevational distribution and occurs at elevations above 6000 m in the Himalayas. Here, we characterized structural and functional variation of big cat Hbs and investigated molecular mechanisms of Hb adaptation and allosteric regulation that may contribute to the extreme hypoxia tolerance of the snow leopard. Experiments revealed that purified Hbs from snow leopard and African lion exhibited equally low O2 affinities and DPG sensitivities. Both properties are primarily attributable to a single amino acid substitution, β2His→Phe, which occurred in the common ancestor of Felidae. Given the low O2 affinity and reduced regulatory capacity of feline Hbs, the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the O2-transport pathway. PMID:26246610

  8. High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

    Science.gov (United States)

    2014-01-01

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [18F]lansoprazole, [11C]N-methyl lansoprazole, and [18F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [18F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials. PMID:24896980

  9. Multiprocessor Real-Time Scheduling with Hierarchical Processor Affinities

    OpenAIRE

    Bonifaci , Vincenzo; Brandenburg , Björn; D'Angelo , Gianlorenzo; Marchetti-Spaccamela , Alberto

    2016-01-01

    International audience; Many multiprocessor real-time operating systems offer the possibility to restrict the migrations of any task to a specified subset of processors by setting affinity masks. A notion of " strong arbitrary processor affinity scheduling " (strong APA scheduling) has been proposed; this notion avoids schedulability losses due to overly simple implementations of processor affinities. Due to potential overheads, strong APA has not been implemented so far in a real-time operat...

  10. Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

    Science.gov (United States)

    Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

    2012-04-01

    Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the

  11. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  12. Simulated electron affinity tuning in metal-insulator-metal (MIM) diodes

    Science.gov (United States)

    Mistry, Kissan; Yavuz, Mustafa; Musselman, Kevin P.

    2017-05-01

    Metal-insulator-metal diodes for rectification applications must exhibit high asymmetry, nonlinearity, and responsivity. Traditional methods of improving these figures of merit have consisted of increasing insulator thickness, adding multiple insulator layers, and utilizing a variety of metal contact combinations. However, these methods have come with the price of increasing the diode resistance and ultimately limiting the operating frequency to well below the terahertz regime. In this work, an Airy Function Transfer Matrix simulation method was used to observe the effect of tuning the electron affinity of the insulator as a technique to decrease the diode resistance. It was shown that a small increase in electron affinity can result in a resistance decrease in upwards of five orders of magnitude, corresponding to an increase in operating frequency on the same order. Electron affinity tuning has a minimal effect on the diode figures of merit, where asymmetry improves or remains unaffected and slight decreases in nonlinearity and responsivity are likely to be greatly outweighed by the improved operating frequency of the diode.

  13. Influence of a chinese crude drug on Ca2+ influx and efflux in rat visceral organs:Investigation and evaluation by 45Ca

    International Nuclear Information System (INIS)

    Yang Yuanyou; Liu Ning; Mo Zhengji; Xie Jianping; Liao Jiali; Mo Shangwu

    2006-01-01

    The influences of a Chinese crude drug, Herba Epimedii (HE), on Ca 2+ influx and efflux in the isolated rat aorta and some visceral organs were evaluated by using 45 Ca as a radioactive tracer. Additionally, its protective effect on myocardial ischemia was investigated in live animals. The results indicated that HE has significant influence on Ca 2+ influx and efflux in the isolated rat aorta, heart, and kidney, in that it can markedly block 45 Ca entering into cell and can facilitate efflux of intracellular Ca 2+ . However, among the three kinds of extracts from HE, the alkali extracts have the most obvious effect on calcium channels in visceral organs. Even if the alkali extracts are diluted by water for 10 times, the material still has a rather strong inhibition effect on calcium channels. Fortunately, the three kinds of extracts have favorable protective effect on myocardial ischemia induced by drugs or by the ligation of the coronary artery. This is consistent with the results about the Ca 2+ influx and efflux obtained by isotope tracer technique, and implies that the Chinese crude drug has attractive potential for the treatment of heart, cerebrovascular and other diseases

  14. Synthesis, biodistribution and in vitro evaluation of brain permeable high affinity type 2 cannabinoid receptor agonists [11C]MA2 and [18F]MA3

    Directory of Open Access Journals (Sweden)

    Muneer Ahamed

    2016-09-01

    Full Text Available Abstract The type 2 cannabinoid receptor (CB2 is a member of the endocannabinoid system and is known for its important role in (neuroinflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2 and a fluorine-18 ([18F]MA3 labeled analogue of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM. MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analogue 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  15. Affine fractal functions as bases of continuous funtions | Navascues ...

    African Journals Online (AJOL)

    The objective of the present paper is the study of affine transformations of the plane, which provide self-affine curves as attractors. The properties of these curves depend decisively of the coefficients of the system of affinities involved. The corresponding functions are continuous on a compact interval. If the scale factors are ...

  16. Antibody Affinity Maturation in Fishes—Our Current Understanding

    Directory of Open Access Journals (Sweden)

    Brad G. Magor

    2015-07-01

    Full Text Available It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig mutator enzyme activation-induced cytidine deaminase (AID. We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

  17. High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

    Science.gov (United States)

    Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

    2015-01-01

    Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

  18. Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice

    Energy Technology Data Exchange (ETDEWEB)

    Inoguchi, Noriko; Mizuno, Nobuhiro; Baba, Seiki; Kumasaka, Takashi; Natarajan, Chandrasekhar; Storz, Jay F.; Moriyama, Hideaki; Permyakov, Eugene A.

    2017-03-31

    Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

  19. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucoc...

  20. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  1. Synthesis, in vitro validation and in vivo pharmacokinetics of [{sup 125}I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    John, Christy S; Gulden, Mary E; Vilner, Bertold J; Bowen, Wayne D

    1996-08-01

    N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[{sup 125}I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[{sup 125}I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 {+-} 0.40, 32.6 {+-} 2.75, and 48.1 {+-} 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[{sup 125}I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki 100-200 nM) affinity for the sites labeled by 4-[{sup 125}I]PEMP in these cells. Biodistribution studies of 4-[{sup 125}I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[{sup 125}I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[{sup 125}I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

  2. Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

    Science.gov (United States)

    Lorkin, P A; Stephens, A D; Beard, M E; Wrigley, P F; Adams, L; Lehmann, H

    1975-01-01

    A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident. PMID:124

  3. Thermokinetic model of borosilicate glass dissolution: contextual affinity

    International Nuclear Information System (INIS)

    Advocat, T.; Vernaz, E.; Crovisier, J.L.; Fritz, B.

    1989-01-01

    Short and long-term geochemical interactions of R7T7 nuclear glass with water at 100 0 C were simulated with the DISSOL thermokinetic computer code. Both the dissolved glass quantity and the resulting water composition, saturation states and mineral quantities produced were calculated as a function of time. The rate equation used in the simulation was first proposed by Aagaard and Helgeson. It simulates a gradually diminishing dissolution rate as the reaction affinity diminishes. The best agreement with 1-year experimental data was obtained with a reaction affinity calculated from silica activity (Grambow's hypothesis) rather than taking into account the activity of all the glass components as proposed by Jantzen and Plodinec. The concept of residual affinity was introduced by Grambow to express the fact that the glass dissolution rate does not cease. We prefer to replace the term residual affinity by contextual affinity, which expresses the influence on the dissolution rate of three factors: the solution chemistry, the metastability of SiO 2 (m), and the possible precipitation of certain aluminosilicates such as zeolites. 19 refs

  4. Affinity Strings: Enterprise Data for Resource Recommendations

    Directory of Open Access Journals (Sweden)

    Shane Nackerud

    2008-12-01

    Full Text Available The University of Minnesota Libraries have created a MyLibrary portal, with databases and e-journals targeted to users, based on their affiliations. The University's enterprise authentication system provides an "affinity string", now used to personalize the MyLibrary portal. This affinity string automates discovery of a user's relationship to the University--describing a user's academic department and degree program or position at the University. Affinity strings also provide the Libraries with an anonymized view of resource usage, allowing data collection that respects users' privacy and lays the groundwork for automated recommendation of relevant resources based on the practices and habits of their peers.

  5. Affine coherent states and Toeplitz operators

    Science.gov (United States)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  6. 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

  7. Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

    Science.gov (United States)

    Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

    2016-03-01

    Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Affinity between information retrieval system and search topic

    International Nuclear Information System (INIS)

    Ebinuma, Yukio

    1979-01-01

    Ten search profiles are tested on the INIS system at the Japan Atomic Energy Research Institute. The results are plotted on recall-precision chart ranging from 100% recall to 100% precision. The curves are not purely systems-dependent nor search-dependent, and are determined substantially by the ''affinity'' between the system and the search topic. The curves are named ''Affinity curves of search topics with information retrieval systems'', and hence retrieval affinity factors are derived. They are obtained not only for individual search topics but also for averages in the system. By such a quantitative examination, the difference of affinity among search topics in a given system, that of the same search topic among various systems, and that of systems to the same group of search topics can be compared reasonably. (author)

  9. Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.

    Science.gov (United States)

    Wright, Traver J; Davis, Randall W

    2015-07-01

    Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40 to 4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melon-headed whale, which had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives. © 2015. Published by The Company of Biologists Ltd.

  10. Affine group formulation of the Standard Model coupled to gravity

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Ching-Yi, E-mail: l2897107@mail.ncku.edu.tw [Department of Physics, National Cheng Kung University, Taiwan (China); Ita, Eyo, E-mail: ita@usna.edu [Department of Physics, US Naval Academy, Annapolis, MD (United States); Soo, Chopin, E-mail: cpsoo@mail.ncku.edu.tw [Department of Physics, National Cheng Kung University, Taiwan (China)

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  11. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    International Nuclear Information System (INIS)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [ 99m Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise

  12. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August E-mail: schubiger@psi.ch

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [{sup 99m}Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  13. Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells

    Czech Academy of Sciences Publication Activity Database

    Laňková, Martina; Smith, R. S.; Pešek, Bedřich; Kubeš, Martin; Zažímalová, Eva; Petrášek, Jan; Hoyerová, Klára

    2010-01-01

    Roč. 61, č. 13 (2010), s. 3589-3598 ISSN 0022-0957 R&D Projects: GA AV ČR KJB600380702; GA MŠk(CZ) LC06034 Grant - others:_(CZ) CZ.2.16/3.1.00/21159 Institutional research plan: CEZ:AV0Z50380511 Keywords : Auxin efflux carrier * auxin influx carrier * auxin transport Subject RIV: EF - Botanics Impact factor: 4.818, year: 2010

  14. Sequence2Vec: A novel embedding approach for modeling transcription factor binding affinity landscape

    KAUST Repository

    Dai, Hanjun

    2017-07-26

    Motivation: An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Results: Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model (HMM) which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these HMMs into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA data sets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods.

  15. Live Imaging of Calcium Dynamics during Axon Degeneration Reveals Two Functionally Distinct Phases of Calcium Influx

    Science.gov (United States)

    Yamagishi, Yuya; Tessier-Lavigne, Marc

    2015-01-01

    Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program. SIGNIFICANCE STATEMENT Axon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled

  16. Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

    Science.gov (United States)

    Li, Qianjin; Liu, Zhen

    2015-01-01

    Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

  17. Single-cell measurement of red blood cell oxygen affinity.

    Science.gov (United States)

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

    2015-08-11

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

  18. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  19. Affine.m—Mathematica package for computations in representation theory of finite-dimensional and affine Lie algebras

    Science.gov (United States)

    Nazarov, Anton

    2012-11-01

    In this paper we present Affine.m-a program for computations in representation theory of finite-dimensional and affine Lie algebras and describe implemented algorithms. The algorithms are based on the properties of weights and Weyl symmetry. Computation of weight multiplicities in irreducible and Verma modules, branching of representations and tensor product decomposition are the most important problems for us. These problems have numerous applications in physics and we provide some examples of these applications. The program is implemented in the popular computer algebra system Mathematica and works with finite-dimensional and affine Lie algebras. Catalogue identifier: AENA_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENB_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, UK Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 24 844 No. of bytes in distributed program, including test data, etc.: 1 045 908 Distribution format: tar.gz Programming language: Mathematica. Computer: i386-i686, x86_64. Operating system: Linux, Windows, Mac OS, Solaris. RAM: 5-500 Mb Classification: 4.2, 5. Nature of problem: Representation theory of finite-dimensional Lie algebras has many applications in different branches of physics, including elementary particle physics, molecular physics, nuclear physics. Representations of affine Lie algebras appear in string theories and two-dimensional conformal field theory used for the description of critical phenomena in two-dimensional systems. Also Lie symmetries play a major role in a study of quantum integrable systems. Solution method: We work with weights and roots of finite-dimensional and affine Lie algebras and use Weyl symmetry extensively. Central problems which are the computations of weight multiplicities, branching and fusion coefficients are solved using one general recurrent

  20. Extreme disorder in an ultrahigh-affinity protein complex

    Science.gov (United States)

    Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

    2018-03-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

  1. Spectral affinity in protein networks

    Directory of Open Access Journals (Sweden)

    Teng Shang-Hua

    2009-11-01

    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to

  2. Relationship Between Accumulation and Influx of Pollutants in Highway Ponds

    DEFF Research Database (Denmark)

    Bentzen, Thomas Ruby; Larsen, Torben; Rasmussen, Michael R.

    The paper discusses the long term mass balance of pollutants in highway ponds. The accumulations of five polycyclic aromatic hydrocarbons (PAHs) and six heavy metals have been measured in eight Danish detention ponds, which receive runoff from highways only. For each pollutant the accumulation has...... been compared to the long-term influx, estimated from short-term measurements of concentrations in highway runoff. The results show that a large proportion of the incoming heavy metals in short-term runoff events has accumulated in the ponds. This is not the case for the toxic organic compounds....... The results also show that the accumulation rates for the heavy metals depend significantly on the relative pond area (pond area divided by catchment area). The conclusion is that the mass balances of heavy metals and PAHs in highway ponds can be estimated with acceptable accuracy from a combination of short...

  3. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  4. Thermokinetic model of borosilicate glass dissolution: Contextual affinity

    International Nuclear Information System (INIS)

    Advocat, T.; Vernaz, E.; Crovisier, J.L.; Fritz, B.

    1990-01-01

    Short and long-term geochemical interactions of R7T7 nuclear glass with water at 100C were simulated with the DISSOL thermokinetic computer code. Both the dissolved glass quantity and the resulting water composition, saturation states and mineral quantities produced were calculated as a function of time. The rate equation used in the simulation was first proposed by Aagaard and Hegelson: v = k + · S · a( H + ) -n · (1 - e -(A/RT) ). It simulates a gradually diminishing dissolution rate as the reaction affinity diminishes. The best agreement with 1-year experimental data was obtained with a reaction affinity calculated from silica activity (Grambow's hypothesis) rather than taking into account the activity of all the glass components as proposed by Jantzen and Plodinec. The concept of residual affinity was introduced by Grambow to express the fact that the glass dissolution rate does not cease. The authors prefer to replace the term residual affinity by contextual affinity, which expresses the influence on the dissolution rate of three factors: the solution chemistry, the metastability of SiO 2 (m), and the possible precipitation of certain aluminosilicates such as zeolites

  5. A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

    Directory of Open Access Journals (Sweden)

    Lykke Pedersen

    2014-01-01

    Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

  6. Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

    2017-01-01

    -affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

  7. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    Science.gov (United States)

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  8. 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.

  9. Sodium-22 influx into erythrocytes from diabetic hypertensive patients on maintenance hemodialysis

    International Nuclear Information System (INIS)

    Gambhir, K.K.; Mathews, J.; Parui, R.; Cruz, I.A.; Hosten, A.O.; Dillard, M.G.

    1990-01-01

    We have studied the percentage of 22Na+ uptake in cell suspensions; 0.4 to 2.0 x 10(9) erythrocytes/mL from diabetic uremic patients with secondary hypertension and from normal subjects. Suspensions from diabetic uremic patients with secondary hypertension 0.42 +/- 0.06 to 2.05 +/- 0.28; normal subjects showed a percentage uptake of 22Na+ of 0.27 +/- 0.05 to 1.28 +/- 0.22. The uptake of 22Na+ in 2.0 x 10(9) cells/mL was 60% more (P less than .05) in diabetic uremic patients than in the controls. These studies indicate that 22Na+ influx determinations may be used to distinguish secondary hypertensive patients from normal subjects

  10. Estrone-1-sulphate (E1S) has impact on the kinetics parameters of transporter mediated taurine and glutamate influx in Caco-2 cells

    DEFF Research Database (Denmark)

    Steffansen, Bente; El-Sayed, F

    Previously, we have suggested estrone-1-sulfate (E1S) to be intercalated into the phospholipid membrane 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC). The overall hypothesis of the present study was that E1S intercalation in the cell membrane of Caco-2 cells may changes the functionality...... of membrane transporters. The aim was therefore to investigate if addition of E1S to the growth medium of Caco-2 cells before but not during the influx study, change the kinetic parameters of transporter-mediated influx of taurine and glutamate by respective TAUT and EAAT transporters. The results show that 4...

  11. Calculation of protein-ligand binding affinities.

    Science.gov (United States)

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  12. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  13. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  14. ASIFT: An Algorithm for Fully Affine Invariant Comparison

    Directory of Open Access Journals (Sweden)

    Guoshen Yu

    2011-02-01

    Full Text Available If a physical object has a smooth or piecewise smooth boundary, its images obtained by cameras in varying positions undergo smooth apparent deformations. These deformations are locally well approximated by affine transforms of the image plane. In consequence the solid object recognition problem has often been led back to the computation of affine invariant image local features. The similarity invariance (invariance to translation, rotation, and zoom is dealt with rigorously by the SIFT method The method illustrated and demonstrated in this work, Affine-SIFT (ASIFT, simulates a set of sample views of the initial images, obtainable by varying the two camera axis orientation parameters, namely the latitude and the longitude angles, which are not treated by the SIFT method. Then it applies the SIFT method itself to all images thus generated. Thus, ASIFT covers effectively all six parameters of the affine transform.

  15. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  16. Applications of Affine and Weyl geometry

    CERN Document Server

    García-Río, Eduardo; Nikcevic, Stana

    2013-01-01

    Pseudo-Riemannian geometry is, to a large extent, the study of the Levi-Civita connection, which is the unique torsion-free connection compatible with the metric structure. There are, however, other affine connections which arise in different contexts, such as conformal geometry, contact structures, Weyl structures, and almost Hermitian geometry. In this book, we reverse this point of view and instead associate an auxiliary pseudo-Riemannian structure of neutral signature to certain affine connections and use this correspondence to study both geometries. We examine Walker structures, Riemannia

  17. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because

  18. Theoretical determination of proton affinity differences in zeolites

    NARCIS (Netherlands)

    Kramer, G.J.; Santen, van R.A.

    1993-01-01

    An important factor in zeolite catalysis is the proton affinity, i.e., the energy required to remove a proton from the zeolite lattice. Differences in proton affinity are expected to influence the catalytic activity of acid sites, making the catalytically active sites inhomogeneous (within one

  19. Pseudo-affinity chromatography of rumen microbial cellulase on ...

    African Journals Online (AJOL)

    Pseudo-affinity chromatography of rumen microbial cellulase on Sepharose- Cibacron Blue F3GA. ... African Journal of Biotechnology ... Pseudo affinity adsorption of bioproducts on Sepharose-cibacron blue F3-GA was subjected to rumen microbial enzyme evaluation through batch binding and column chromatography of ...

  20. Volatility Components, Affine Restrictions and Non-Normal Innovations

    DEFF Research Database (Denmark)

    Christoffersen, Peter; Jacobs, Kris; Dorian, Christian

    Recent work by Engle and Lee (1999) shows that allowing for long-run and short-run components greatly enhances a GARCH model's ability fit daily equity return dynamics. Using the risk-neutralization in Duan (1995), we assess the option valuation performance of the Engle-Lee model and compare...... models to four conditionally non-normal versions. As in Hsieh and Ritchken (2005), we find that non-affine models dominate affine models both in terms of fitting return and in terms of option valuation. For the affine models we find strong evidence in favor of the component structure for both returns...

  1. Realization of Robertson-Walker spacetimes as affine hypersurfaces

    International Nuclear Information System (INIS)

    Chen Bangyen

    2007-01-01

    Due to the growing interest in embeddings of spacetimes in higher dimensional spaces, we consider a special type of embedding. We prove that Robertson-Walker spacetimes can be embedded as centroaffine hypersurfaces and graph hypersurfaces in some affine spaces in such a way that the induced relative metrics are exactly the Lorentzian metrics on the Robertson-Walker spacetimes. Such realizations allow us to view Robertson-Walker spacetimes and their submanifolds as affine submanifolds in a natural way. Consequently, our realizations make it possible to apply the tools of affine differential geometry to study Robertson-Walker spacetimes and their submanifolds

  2. Overcoming HERG affinity in the discovery of the CCR5 antagonist maraviroc.

    Science.gov (United States)

    Price, David A; Armour, Duncan; de Groot, Marcel; Leishman, Derek; Napier, Carolyn; Perros, Manos; Stammen, Blanda L; Wood, Anthony

    2006-09-01

    The discovery of maraviroc 17 is described with particular reference to the generation of high selectivity over affinity for the HERG potassium channel. This was achieved through the use of a high throughput binding assay for the HERG channel that is known to show an excellent correlation with functional effects.

  3. Methyl Cation Affinities of Neutral and Anionic Maingroup-Element Hydrides: Trends Across the Periodic Table and Correlation with Proton Affinities

    NARCIS (Netherlands)

    Mulder, R. Joshua; Guerra, Celia Fonseca; Bickelhaupt, F. Matthias

    2010-01-01

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and

  4. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

    Science.gov (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J

    2015-11-01

    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  5. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    International Nuclear Information System (INIS)

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-01-01

    Highlights: → Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. → Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca 2+ permeant SACs. → The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. → Stretch-induced ET-1 production depends on a calcium influx. → SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch ( 2+ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  6. Cyanated diazatetracene diimides with ultrahigh electron affinity for n-channel field effect transistors

    KAUST Repository

    Ye, Qun

    2013-03-15

    Several diazatetracene diimides with high electron affinity (up to 4.66 eV!) were prepared and well characterized. The LUMO energy level of these electron-deficient molecules was found to be closely related to their material stability. Compound 7 with ultrahigh electron affinity suffered from reduction and hydrolysis in the presence of silica gel or water. The stable compounds 3 and 6 showed n-channel FET behavior with an average electron mobility of 0.002 and 0.005 cm2 V-1 s-1, respectively, using a solution processing method. © 2013 American Chemical Society.

  7. Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

    Science.gov (United States)

    Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

    2001-10-30

    The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

  8. Generalized Warburg impedance on realistic self-affine fractals ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals.

  9. Piecewise affine control for fast unmanned ground vehicles

    OpenAIRE

    Benine Neto , André; Grand , Christophe

    2012-01-01

    International audience; Unmanned ground vehicles (UGV) may experience skidding when moving at high speeds, and therefore have its safety jeopardized. For this reason the nonlinear dynamics of lateral tire forces must be taken into account into the design of steering controllers for autonomous vehicles. This paper presents the design of a state feedback piecewise affine controller applied to an UGV to coordinate the steering and torque distribution inputs in order to reduce vehicle skidding on...

  10. Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

    2013-07-01

    In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

  11. Automated Kick Control Procedure for an Influx in Managed Pressure Drilling Operations

    Directory of Open Access Journals (Sweden)

    Jing Zhou

    2016-01-01

    Full Text Available Within drilling of oil and gas wells, the Managed Pressure Drilling (MPD method with active control of wellbore pressure during drilling has partly evolved from conventional well control procedures. However, for MPD operations the instrumentation is typically more extensive compared to conventional drilling. Despite this, any influx of formation fluids (commonly known as a kick during MPD operations is typically handled by conventional well control methods, at least if the kick is estimated to be larger than a threshold value. Conventional well control procedures rely on manual control of the blow out preventer, pumps, and choke valves and do not capitalize on the benefits from the instrumentation level associated with MPD. This paper investigates two alternative well control procedures specially adapted to backpressure MPD: the dynamic shut-in (DSI procedure and the automatic kick control (AKC procedure. Both methods capitalize on improvements in Pressure While Drilling (PWD technology. A commercially available PWD tool buffers high-resolution pressure measurements, which can be used in an automated well control procedure. By using backpressure MPD, the choke valve opening is tuned automatically using a feedback-feedforward control method. The two procedures are evaluated using a high fidelity well flow model and cases from a North Sea drilling operation are simulated. The results show that using AKC procedure reduces the time needed to establish control of the well compared to DSI procedure. It also indicates that the AKC procedure reduces the total kick size compared to the DSI procedure, and thereby reduces the risk of lost circulation.

  12. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    International Nuclear Information System (INIS)

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

    2011-01-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  13. Low-complexity piecewise-affine virtual sensors: theory and design

    Science.gov (United States)

    Rubagotti, Matteo; Poggi, Tomaso; Oliveri, Alberto; Pascucci, Carlo Alberto; Bemporad, Alberto; Storace, Marco

    2014-03-01

    This paper is focused on the theoretical development and the hardware implementation of low-complexity piecewise-affine direct virtual sensors for the estimation of unmeasured variables of interest of nonlinear systems. The direct virtual sensor is designed directly from measured inputs and outputs of the system and does not require a dynamical model. The proposed approach allows one to design estimators which mitigate the effect of the so-called 'curse of dimensionality' of simplicial piecewise-affine functions, and can be therefore applied to relatively high-order systems, enjoying convergence and optimality properties. An automatic toolchain is also presented to generate the VHDL code describing the digital circuit implementing the virtual sensor, starting from the set of measured input and output data. The proposed methodology is applied to generate an FPGA implementation of the virtual sensor for the estimation of vehicle lateral velocity, using a hardware-in-the-loop setting.

  14. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  15. Generalized Warburg impedance on realistic self-affine fractals

    Indian Academy of Sciences (India)

    We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals. The information about the ...

  16. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

    2017-06-15

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC{sub 50} value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  17. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    International Nuclear Information System (INIS)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin; Yu, Ruijin; Lai, Weihua; Xiong, Yonghua

    2017-01-01

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC_5_0 value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  18. Affine pairings on ARM

    NARCIS (Netherlands)

    Acar, T.; Lauter, K.; Naehrig, M.; Shumow, D.

    2011-01-01

    Pairings on elliptic curves are being used in an increasing number of cryptographic applications on many different devices and platforms, but few performance numbers for cryptographic pairings have been reported on embedded and mobile devices. In this paper we give performance numbers for affine and

  19. New polymer-supported ion-complexing agents: Design, preparation and metal ion affinities of immobilized ligands

    International Nuclear Information System (INIS)

    Alexandratos, Spiro D.

    2007-01-01

    Polymer-supported reagents are comprised of crosslinked polymer networks that have been modified with ligands capable of selective metal ion complexation. Applications of these polymers are in environmental remediation, ion chromatography, sensor technology, and hydrometallurgy. Bifunctional polymers with diphosphonate/sulfonate ligands have a high selectivity for actinide ions. The distribution coefficient for the uranyl ion from 1 M nitric acid is 70,000, compared to 900 for the monophosphonate/sulfonate polymer and 200 for the sulfonic acid ion-exchange resin. A bifunctional trihexyl/triethylammonium polymer has a high affinity and selectivity for pertechnetate and perchlorate anions from groundwater. In one example, its distribution coefficient for perchlorate ions in the presence of competing anions is 3,300,000, compared to 203,180 for a commercially available anion-exchange resin. Polystyrene modified with N-methyl-D-glucamine ligands is capable of selectively complexing arsenate from groundwater. It complexes 99% of the arsenate present in a solution of 100 mg/L arsenate with 560 mg/L sulfate ions. Its selectivity is retained even in the presence of 400 mg/L phosphate. There is no affinity for arsenate above pH 9, allowing for the polymer to be regenerated with moderate alkali solution. In studies aimed at developing a Hg(II)-selective resin, simple amine resins were found to have a high Hg(II) affinity and that affinity is dependent upon the solution pH and the counterion

  20. Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

    Science.gov (United States)

    Chen, Guilin; Huang, Bill X; Guo, Mingquan

    2018-05-21

    Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases. To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value. The ultrafiltration mass spectrometry (UF-LC/MS) is a powerful bio-analytical method by combining affinity ultrafiltration and liquid chromatography-mass spectrometry (LC/MS), which could rapidly screen and identify small active molecules that bind to biological targets of interest at the same time. Compared with other analytical methods, affinity UF-LC/MS has the characteristics of fast, sensitive and high throughput, and is especially suitable for the complicated extracts of medicinal plants. In this review, the basic principle, characteristics and some most recent challenges in UF-LC/MS have been demonstrated. Meanwhile, the progress and applications of affinity UF-LC/MS in the discovery of the active components from natural medicinal plants and the interactions between small molecules and biological target proteins are also briefly summarised. In addition, the future directions for UF-LC/MS are also prospected. Affinity UF-LC/MS is a powerful tool in studies on the interactions between small active molecules and biological protein targets, especially in the high-throughput screening of active components from the natural medicinal plants. Copyright © 2018 John Wiley & Sons, Ltd.

  1. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    Science.gov (United States)

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  2. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

  3. Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

    Science.gov (United States)

    Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

    2015-12-07

    The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

  4. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  5. ODE/IM correspondence and modified affine Toda field equations

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Katsushi; Locke, Christopher

    2014-08-15

    We study the two-dimensional affine Toda field equations for affine Lie algebra g{sup ^} modified by a conformal transformation and the associated linear equations. In the conformal limit, the associated linear problem reduces to a (pseudo-)differential equation. For classical affine Lie algebra g{sup ^}, we obtain a (pseudo-)differential equation corresponding to the Bethe equations for the Langlands dual of the Lie algebra g, which were found by Dorey et al. in study of the ODE/IM correspondence.

  6. A highly selective dispersive liquid-liquid microextraction approach based on the unique fluorous affinity for the extraction and detection of per- and polyfluoroalkyl substances coupled with high performance liquid chromatography tandem-mass spectrometry.

    Science.gov (United States)

    Wang, Juan; Shi, Yali; Cai, Yaqi

    2018-04-06

    In the present study, a highly selective fluorous affinity-based dispersive liquid-liquid microextraction (DLLME) technique was developed for the extraction and analysis of per- and polyfluoroalkyl substances (PFASs) followed by high performance liquid chromatography tandem-mass spectrometry. Perfluoro-tert-butanol with multiple C-F bonds was chosen as the extraction solvent, which was injected into the aqueous samples with a dispersive solvent (acetonitrile) in a 120:800 (μL, v/v) mixture for PFASs enrichment. The fluorous affinity-based extraction mechanism was confirmed by the significantly higher extraction recoveries for PFASs containing multiple fluorine atoms than those for compounds with fewer or no fluorine atoms. The extraction recoveries of medium and long-chain PFASs (CF 2  > 5) exceeded 70%, except perfluoroheptanoic acid, while those of short-chain PFASs were lower than 50%, implying that the proposed DLLME may not be suitable for their extraction due to weak fluorous affinity. This highly fluoroselective DLLME technique can greatly decrease the matrix effect that occurs in mass spectrometry detection when applied to the analysis of urine samples. Under the optimum conditions, the relative recoveries of PFASs with CF 2  > 5 ranged from 80.6-121.4% for tap water, river water and urine samples spiked with concentrations of 10, 50 and 100 ng/L. The method limits of quantification for PFASs in water and urine samples were in the range of 0.6-8.7 ng/L. Furthermore, comparable concentrations of PFASs were obtained via DLLME and solid-phase extraction, confirming that the developed DLLME technique is a promising method for the extraction of PFASs in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. In vitro evolution and affinity-maturation with Coliphage qβ display.

    Directory of Open Access Journals (Sweden)

    Claudia Skamel

    Full Text Available The Escherichia coli bacteriophage, Qβ (Coliphage Qβ, offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV. DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

  8. Characterization of self-affinity in the global regime

    Science.gov (United States)

    Neimark, Alexander V.

    1994-11-01

    Methods for characterization of self-affine surfaces and measurements of their roughness exponents H are developed. It is shown that for smoothed surfaces, which underwent particular coarse graining or averaging of the small-scale fluctuations, the excess surface area Sex and the mean square root radius of curvature ac are related by two distinct asymptotic power laws if ac is well below or well above a certain crossover scale acr. In the local regime of self-affinity, when acSex~(ac/acr)-(1-H). In the global regime of self-affinity, when ac>>acr, Sex~(ac/acr)-2(1-H)/(2-H). The former scaling relationship is consistent with the well known definition of local fractal dimensions dloc=dtop+1-H. The latter scaling relationship offers alternatives for characterization of self-affinity over large scales by means of excess dimensions defined as dex=dtop+2(1-H)/(2-H) and can be used for determination of roughness exponents from the measurements provided in the global regime. The thermodynamic method of fractal analysis, proposed earlier for self-similar surfaces (A.V. Neimark, Pis'ma Zh. Eksp. Teor. Fiz. 51, 535 (1990) [JETP Lett. 51, 607 (1990)]; Physica A 191, 258 (1992)), is extended for self-affine surfaces for determination of fractal dimensions and roughness exponents from adsorption and capillary experimental data.

  9. 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.

  10. A multi-method approach for the study of lanthanum speciation in coastal and estuarine sediments

    Digital Repository Service at National Institute of Oceanography (India)

    Chakraborty, P.; Babu, P.V.R.; Sarma, V.V.

    ) in different fields (agricultural field, water treatment technology, nuclear industry, glass industry and medicine manufacturing industry) (Guo, 1987; Zhang et al. 1988; Eisenbud and Paschoa, 1989; Tribe et al. 1990; Buckingham et al. 1995) is expected... through the displacement or replacement of calcium in different cell functions and also its high affinity for phosphate groups of biological macromolecules. La 3+ ion usually prevents the influx of Ca 2+ ion and competes for available binding sites...

  11. Photolabeling of tonoplast from sugar beet cell suspensions by [3H]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar Na+/H+ antiport

    International Nuclear Information System (INIS)

    Barkla, B.J.; Charuk, J.H.M.; Blumwald, E.; Cragoe, E.J. Jr.

    1990-01-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na + /H + antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na + /H + exchange in a competitive manner with a K i of 2.5 and 5.9 micromolar for ΔpH-dependent 22 Na + influx in tonoplast vesicles and Na + -dependent H + efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [ 3 H]MIA to tonoplast membranes revealed a high affinity binding component with a K d of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na + /H + antiport. Photolabeling of the tonoplast with [ 3 H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog

  12. Photolabeling of tonoplast from sugar beet cell suspensions by [3H]-MIA, an inhibitor of the vacuolar Na+/H+ antiport

    International Nuclear Information System (INIS)

    Barkla, B.J.; Blumwald, E.

    1990-01-01

    A radiolabeled amiloride analog, [ 3 H]-MIA, was used for equilibrium binding studies and photolabeling of purified tonoplast vesicles. Scatchard analysis revealed a high affinity binding component with a K 4 of 1.4 μM which is closely related to constants of inhibition obtained for Na + -dependent H + efflux (5.9 μM) and pH-dependent 22 Na + influx (2.5 μM). This suggests that the high affinity component represents a class of sites associated with the Na + /H + antiport. Photolabeling of tonoplast with [ 3 H]-MIA in the presence of amiloride revealed the presence of two classes of receptors with distinct affinities for MIA, possibly representing the Na + /H + antiport and the Na + channel. In order to identify these receptors, amiloride analogues specific for the Na + /H + antiport or the Na + channel are being used to protect differentially against labeling of tonoplast proteins by photo-irradiation of [ 3 H]-MIA

  13. Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

    Science.gov (United States)

    Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

    1990-07-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

  14. Effect of dietary protein level on nitrogen utilization and ruminal influx of endogenous urea nitrogen in growing animals

    International Nuclear Information System (INIS)

    Bunting, L.D.

    1987-01-01

    Three experiments were conducted to evaluate the impact of ruminal influx of blood urea nitrogen (BUN) on intestinal protein supply and nitrogen (N) metabolism in growing animals at both excess and growth-limiting protein intake. In Experiment 1, wether lambs were given diets, either high or low in protein, containing 25% cottonseed hulls and 75% corn-soybean meal hourly in 24 equal portions. Single injections of 14 C- and 15 N-urea, and 15 N-ammonium sulfate (AS) were made into the BUN and ruminal ammonia N (RAN) pools, respectively, to measure rate of flux through, and transfer of N between these and the bacterial N pool. In Experiment 2, beef calves were given HP and LP diets containing 30% cottonseed hulls and 70% corn-soybean meal every 4 h in 6 equal portions. Single injections of 15 N-urea and 15 N-AS were made into the BUN and RAN pools, respectively, to measure rate of flux through, and transfer of N between these and the bacterial N pool. Abomasal N flow was 24% greater than intake in LP and 29% less than intake in HP. An inverse relationship may exist between level of N intake and rate of influx of BUN into the rumen. In Experiment 3 ruminal fluid samples were obtained. With HP, BUN-derived 15 N-ammonia appeared to rapidly equilibrate with RAN in the primary digesta mass. In contrast, with LP, there appeared to be an enrichment gradient for both RAN and bacterial N, declining from the rumen wall toward the center of the digesta mass, suggesting that bacteria at or near the rumen wall may preferentially utilized some BUN-derived ammonia N entering through the rumen wall

  15. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    Science.gov (United States)

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values.

  16. DAYA ANTIBAKTERI EKSTRAK ETANOL DAUN SENGGANI (Melastoma affine D. Don

    Directory of Open Access Journals (Sweden)

    Ika Trisharyanti Dian Kusumowati

    2014-08-01

    Full Text Available Melastoma affine D. Don had some activities such as anthelmintic, antibacteria, antiinfiammation, antifungal, and antitumor. The aims of this research was determine antibacteria activity of ethanolic extract of Melastoma affine D. Don. The antimicrobial activity was tested by solid dilution method to get Minimum Inhibition Concentration (MIC. The compounds in Melastoma affine D. Don was analyzed by tube test method and Thin Layer Chromatography (TLC with chloroform : methanol : formic acid (8,5:1,5:0,5 as mobile phase and silica gel GF254 as stationary phase. The result showed ethanolic extract of Melastoma affine D. Don contains alkaloid, polyphenol, fiavonoid, saponin, and essential oil. The MIC of Senggani against Staphylococcus aureus was 2% and 3% against Escherichia coli and the extract could not inhibit Staphylococcus aureus and Escherichia coli multiresistant until concentration 7% extract ethanol. Keywords: Melastoma affine D. Don, Staphylococcus aureus, Escherichia coli

  17. Computation of piecewise affine terminal cost functions for model predictive control

    NARCIS (Netherlands)

    Brunner, F.D.; Lazar, M.; Allgöwer, F.; Fränzle, Martin; Lygeros, John

    2014-01-01

    This paper proposes a method for the construction of piecewise affine terminal cost functions for model predictive control (MPC). The terminal cost function is constructed on a predefined partition by solving a linear program for a given piecewise affine system, a stabilizing piecewise affine

  18. Earth's influx of different populations of sporadic meteoroids from photographic and television data

    International Nuclear Information System (INIS)

    Ceplecha, Z.

    1988-01-01

    Precise photographic and television double- and multi-station data on 3624 sporadic meteors in the mass range from 2 x 10 -5 grams to 2 x 10 7 grams form the basis of this paper. The applied classification criteria and procedures are defined and described. A survey of 7 different populations of sporadic meteoroids known so far is presented. The total numbers and masses of meteoroids as a function of mass are given for individual groups and for all sporadic meteors. The absolute calibration of the influx to the Earth was carried out by comparison with the results of Halliday et al. (1984). The comparison with the visual and cratering data revealed good agreement in the narrow ''visual'' interval of masses, and disagreement in the extrapolated parts of the visual and cratering flux curves. The slope of the cumulative number curve for the meteorite-dropping fireballs (type I) with masses larger than 1 kg was found as -0.69 in perfect agreement with the results of Halliday et al. (1984). The final mass scale derived in this paper is situated between the scale of McCrosky and the scale of Halliday. The relative significance of the different groups of meteoroids changes with the mass quite dramatically. The total influx of sporadic meteoroids in the mass interval of 12 orders from 2 x 10 7 to 2 x 10 -5 grams resulted in 5 x 10 9 grams per year for the entire Earth's surface. Most of this mass comes in the form of larger meteoroids. Bulk densities and ablation coefficient are presented for the individual meteor groups depending on different ablation models of several authors and some extreme concepts of this problem are discussed. (author). 3 figs., 6 tabs., 38 refs

  19. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  20. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  1. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report ...

  2. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    Science.gov (United States)

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-05

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

  3. On Affine Fusion and the Phase Model

    Directory of Open Access Journals (Sweden)

    Mark A. Walton

    2012-11-01

    Full Text Available A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n Wess-Zumino-Novikov-Witten (WZNW conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  4. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a

  5. Single cell wound generates electric current circuit and cell membrane potential variations that requires calcium influx.

    Science.gov (United States)

    Luxardi, Guillaume; Reid, Brian; Maillard, Pauline; Zhao, Min

    2014-07-24

    Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

  6. Specificity and affinity quantification of protein-protein interactions.

    Science.gov (United States)

    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  7. Exploring Girls' Science Affinities Through an Informal Science Education Program

    Science.gov (United States)

    Todd, Brandy; Zvoch, Keith

    2017-10-01

    This study examines science interests, efficacy, attitudes, and identity—referred to as affinities, in the context of an informal science outreach program for girls. A mixed methods design was used to explore girls' science affinities before, during, and after participation in a cohort-based summer science camp. Multivariate analysis of survey data revealed that girls' science affinities varied as a function of the joint relationship between family background and number of years in the program, with girls from more affluent families predicted to increase affinities over time and girls from lower income families to experience initial gains in affinities that diminish over time. Qualitative examination of girls' perspectives on gender and science efficacy, attitudes toward science, and elements of science identities revealed a complex interplay of gendered stereotypes of science and girls' personal desires to prove themselves knowledgeable and competent scientists. Implications for the best practice in fostering science engagement and identities in middle school-aged girls are discussed.

  8. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  9. Responding to a Refugee Influx: Lessons from Lebanon

    Directory of Open Access Journals (Sweden)

    Ninette Kelley

    2017-02-01

    Full Text Available Between 2011 and 2015, Lebanon received over one million Syrian refugees. There is no country in the world that has taken in as many refugees in proportion to its size: by 2015, one in four of its residents was a refugee from Syria. Already beset, prior to the Syrian crisis, by political divisions, insecure borders, severely strained infrastructure, and over-stretched public services, the mass influx of refugees further taxed the country. That Lebanon withstood what is often characterized as an existential threat is primarily due to the remarkable resilience of the Lebanese people. It is also due to the unprecedented levels of humanitarian funding that the international community provided to support refugees and the communities that hosted them. UN, international, and national partners scaled up more than a hundred-fold to meet ever-burgeoning needs and creatively endeavored to meet challenges on the ground. And while the refugee response was not perfect, and funding fell well below needs, thousands of lives were saved, protection was extended, essential services were provided, and efforts were made to improve through education the future prospects of the close to half-a-million refugee children residing in Lebanon. This paper examines what worked well and where the refugee response stumbled, focusing on areas where improved efforts in planning, delivery, coordination, innovation, funding, and partnerships can enhance future emergency responses.

  10. Efficiency of nitrate uptake in spinach : impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

    NARCIS (Netherlands)

    Ter Steege, MW; Stulen, [No Value; Wiersema, PK; Posthumus, F; Vaalburg, W

    1999-01-01

    Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with N-13- and N-15-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration.

  11. Affinity-tuning leukocyte integrin for development of safe therapeutics

    Science.gov (United States)

    Park, Spencer

    Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

  12. Affine pairings on ARM

    NARCIS (Netherlands)

    Acar, T.; Lauter, K.; Naehrig, M.; Shumow, D.; Abdalla, M.; Lange, T.

    2013-01-01

    We report on relative performance numbers for affine and projective pairings on a dual-core Cortex A9 ARM processor. Using a fast inversion in the base field and doing inversion in extension fields by using the norm map to reduce to inversions in smaller fields, we find a very low ratio of

  13. Affine planes, ternary rings, and examples of non-Desarguesian planes

    OpenAIRE

    Ivanov, Nikolai V.

    2016-01-01

    The paper is devoted to a detailed self-contained exposition of a part of the theory of affine planes leading to a construction of affine (or, equivalently, projective) planes not satisfying the Desarques axiom. It is intended to complement the introductory expositions of the theory of affine and projective planes. A novelty of our exposition is a new notation for the ternary operation in a ternary ring, much more suggestive than the standard one.

  14. Fermionic construction of vertex operators for twisted affine algebras

    International Nuclear Information System (INIS)

    Frappat, L.; Sorba, P.; Sciarrino, A.

    1988-03-01

    We construct vertex operator representations of the twisted affine algebras in terms of fermionic (or parafermionic in some cases) elementary fields. The folding method applied to the extended Dynkin diagrams of the affine algebras allows us to determine explicitly these fermionic fields as vertex operators

  15. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  16. Excited state electron affinity calculations for aluminum

    Science.gov (United States)

    Hussein, Adnan Yousif

    2017-08-01

    Excited states of negative aluminum ion are reviewed, and calculations of electron affinities of the states (3s^23p^2)^1D and (3s3p^3){^5}{S}° relative to the (3s^23p)^2P° and (3s3p^2)^4P respectively of the neutral aluminum atom are reported in the framework of nonrelativistic configuration interaction (CI) method. A priori selected CI (SCI) with truncation energy error (Bunge in J Chem Phys 125:014107, 2006) and CI by parts (Bunge and Carbó-Dorca in J Chem Phys 125:014108, 2006) are used to approximate the valence nonrelativistic energy. Systematic studies of convergence of electron affinity with respect to the CI excitation level are reported. The calculated value of the electron affinity for ^1D state is 78.675(3) meV. Detailed Calculations on the ^5S°c state reveals that is 1216.8166(3) meV below the ^4P state.

  17. k-Schur functions and affine Schubert calculus

    CERN Document Server

    Lam, Thomas; Morse, Jennifer; Schilling, Anne; Shimozono, Mark; Zabrocki, Mike

    2014-01-01

    This book gives an introduction to the very active field of combinatorics of affine Schubert calculus, explains the current state of the art, and states the current open problems. Affine Schubert calculus lies at the crossroads of combinatorics, geometry, and representation theory. Its modern development is motivated by two seemingly unrelated directions. One is the introduction of k-Schur functions in the study of Macdonald polynomial positivity, a mostly combinatorial branch of symmetric function theory. The other direction is the study of the Schubert bases of the (co)homology of the affine Grassmannian, an algebro-topological formulation of a problem in enumerative geometry. This is the first introductory text on this subject. It contains many examples in Sage, a free open source general purpose mathematical software system, to entice the reader to investigate the open problems. This book is written for advanced undergraduate and graduate students, as well as researchers, who want to become familiar with ...

  18. Binding Affinity of a Highly Sensitive Au/Ag/Au/Chitosan-Graphene Oxide Sensor Based on Direct Detection of Pb2+ and Hg2+ Ions

    Directory of Open Access Journals (Sweden)

    Nur Hasiba Kamaruddin

    2017-10-01

    Full Text Available The study of binding affinity is essential in surface plasmon resonance (SPR sensing because it allows researchers to quantify the affinity between the analyte and immobilised ligands of an SPR sensor. In this study, we demonstrate the derivation of the binding affinity constant, K, for Pb2+ and Hg2+ ions according to their SPR response using a gold/silver/gold/chitosan–graphene oxide (Au/Ag/Au/CS–GO sensor for the concentration range of 0.1–5 ppm. The higher affinity of Pb2+ to binding with the CS–GO sensor explains the outstanding sensitivity of 2.05 °ppm−1 against 1.66 °ppm−1 of Hg2+. The maximum signal-to-noise ratio (SNR upon detection of Pb2+ is 1.53, and exceeds the suggested logical criterion of an SNR. The Au/Ag/Au/CS–GO SPR sensor also exhibits excellent repeatability in Pb2+ due to the strong bond between its functional groups and this cation. The adsorption data of Pb2+ and Hg2+ on the CS–GO sensor fits well with the Langmuir isotherm model where the affinity constant, K, of Pb2+ and Hg2+ ions is computed. The affinity of Pb2+ ions to the Au/Ag/Au/CS–GO sensor is significantly higher than that of Hg2+ based on the value of K, 7 × 105 M−1 and 4 × 105 M−1, respectively. The higher shift in SPR angles due to Pb2+ and Hg2+ compared to Cr3+, Cu2+ and Zn2+ ions also reveals the greater affinity of the CS–GO SPR sensor to them, thus supporting the rationale for obtaining K for these two heavy metals. This study provides a better understanding on the sensing performance of such sensors in detecting heavy metal ions.

  19. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    Science.gov (United States)

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Local fibroblast proliferation but not influx is responsible for synovial hyperplasia in a murine model of rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Matsuo, Yusuke; Mizoguchi, Fumitaka; Saito, Tetsuya [Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519 (Japan); Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST) Program, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 (Japan); Kawahata, Kimito [Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519 (Japan); Ueha, Satoshi; Matsushima, Kouji [Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST) Program, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 (Japan); Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 (Japan); Inagaki, Yutaka [Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST) Program, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 (Japan); Center for Matrix Biology and Medicine, Graduate School of Medicine and the Institute of Medical Sciences, Tokai University, 143 Shimo-kasuya, Isehara, Kanagawa, 259-1193 (Japan); Miyasaka, Nobuyuki [Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519 (Japan); Kohsaka, Hitoshi, E-mail: kohsaka.rheu@tmd.ac.jp [Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519 (Japan); Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST) Program, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 (Japan)

    2016-02-12

    Synovial fibroblasts play crucial roles in inflammation and joint destruction in rheumatoid arthritis (RA). How they accumulate in the RA joints remains unclear. This study was conducted to discern whether cellular influx from the outside of the joints and local proliferation are responsible for synovial fibroblast accumulation in an animal model of RA. We found that synovial fibroblasts were identified as GFP+ cells using collagen type I alpha 2 (Col1a2)-GFP transgenic reporter mice. Then, bone marrow transplantation and parabiosis techniques were utilized to study the cellular influx. Irradiated wild-type mice were transplanted with bone marrow from Col1a2-GFP mice. Col1a2-GFP and wild-type mice were conjoined for parabiosis. The transplanted mice and the parabionts were subjected to collagen antibody-induced arthritis (CAIA). We found no GFP+ cells in the hyperplastic synovial tissues from the transplanted mice with CAIA and from the wild-type parabionts with CAIA. Furthermore, normal and CAIA synovial tissues from Col1a2-GFP mice and from fluorescent ubiquitination-based cell cycle indicator (Fucci) transgenic mice, in which cells in S/G{sub 2}/M phases of the cell cycle express Azami-Green, were studied for Ki67, a cellular proliferation marker, and vimentin, a fibroblast marker, expression. The percentages of Ki67+/GFP+ and Azami-Green+/vimentin+ cells in the CAIA synovial tissues were higher than those in the untreated synovial tissues (34% vs. 0.40% and 19% vs. 0.26%, respectively). These findings indicate that local fibroblast proliferation but not cellular influx is responsible for the synovial hyperplasia in CAIA. Suppression of proliferation of the local synovial fibroblasts should be a promising treatment for RA. - Highlights: • We studied how synovial fibroblasts accumulate in joints in a murine model of RA. • Bone marrow-derived cells did not accumulate in arthritic joints. • Synovial fibroblasts did not accumulate in arthritic joints via