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Sample records for high-affinity desensitized state

  1. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

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    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

  2. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across...... that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation...

  3. Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second high-affinity open state.

    Science.gov (United States)

    Gingrich, Kevin J; Wagner, Larry E

    2016-06-01

    Local anesthetics (LAs) block resting, open, and inactivated states of voltage-gated Na(+) channels where inactivated states are thought to bind with highest affinity. However, reports of fast-onset block occurring over milliseconds hint at high-affinity block of open channels. Movement of voltage-sensor domain IV-segment 4 (DIVS4) has been associated with high affinity LA block termed voltage-sensor block (VSB) that also leads to a second open state. These observations point to a second high-affinity open state that may underlie fast-onset block. To test for this state, we analyzed the modulation of Na(+) currents by lidocaine and its quaternary derivative (QX222) from heterologously expressed (Xenopus laevis oocytes) rat skeletal muscle μ1 NaV1.4 (rSkM1) with β1 (WT-β1), and a mutant form (IFM-QQQ mutation in the III-IV interdomain, QQQ) lacking fast inactivation, in combination with Markov kinetic gating models. 100 μM lidocaine induced fast-onset (τonset≈2 ms), long-lived (τrecovery≈120 ms) block of WT-β1 macroscopic currents. Lidocaine blocked single-channel and macroscopic QQQ currents in agreement with our previously described mechanism of dual, open-channel block (DOB mechanism). A DOB kinetic model reproduced lidocaine effects on QQQ currents. The DOB model was extended to include trapping fast-inactivation and activation gates, and a second open state (OS2); the latter arising from DIVS4 translocation that precedes inactivation and exhibits high-affinity, lidocaine binding (apparent Kd=25 μM) that accords with VSB (DOB-S2VSB mechanism). The DOB-S2VSB kinetic model predicted fast-onset block of WT-β1. The findings support the involvement of a second, high-affinity, open state in lidocaine modulation of Na(+) channels.

  4. Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

    Science.gov (United States)

    Perrin, Mark J; Kuchel, Philip W; Campbell, Terence J; Vandenberg, Jamie I

    2008-11-01

    Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

  5. The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFβ1.

    Science.gov (United States)

    van Caam, A; Blaney Davidson, E; Garcia de Vinuesa, A; van Geffen, E; van den Berg, W; Goumans, M-J; ten Dijke, P; van der Kraan, P

    2015-06-01

    In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFβ1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFβ1 signaling was also studied. Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFβ1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFβ/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFβ1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFβ/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFβ1. This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  6. DESENSITIZATION PROPERTIES OF P2X3 RECEPTORS SHAPING PAIN SIGNALLING

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    Rashid eGiniatullin

    2013-12-01

    Full Text Available ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called ‘high affinity desensitization’. We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called ‘left flipper’ converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given such their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain.

  7. Between innovation and tradition: the paradoxical relationship between eye movement desensitization and reprocessing and altered states of consciousness.

    Science.gov (United States)

    Stein, Debra; Rousseau, Cécile; Lacroix, Louise

    2004-03-01

    Eye movement desensitization and reprocessing (EMDR) is a relatively new form of psychotherapy to emerge in the West. Using both a case analysis and literature review we situate EMDR within the use of altered states of consciousness (ASCs) in psychological healing practices across times and cultures. We discuss EMDR's unique predicament as a therapy that draws upon techniques common to most therapeutic ASCs, while at the same time distancing itself from this tradition through its pseudoscientific language and technologic aesthetic. Our conclusion attempts to shed light on this paradox and raise questions for further study.

  8. Dyes with high affinity for polylactide

    Institute of Scientific and Technical Information of China (English)

    Liang He; Shu Fen Zhang; Bing Tao Tang; Li Li Wang; Jin Zong Yang

    2007-01-01

    Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes.The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.

  9. Effectiveness of oxaliplatin desensitization protocols.

    Science.gov (United States)

    Cortijo-Cascajares, Susana; Nacle-López, Inmaculada; García-Escobar, Ignacio; Aguilella-Vizcaíno, María José; Herreros-de-Tejada, Alberto; Cortés-Funes Castro, Hernán; Calleja-Hernández, Miguel-Ángel

    2013-03-01

    Hypersensitivity reaction (HSR) to antineoplastic drugs can force doctors to stop treatment and seek other alternatives. These alternatives may be less effective, not as well tolerated and/or more expensive. Another option is to use desensitization protocols that induce a temporary state of tolerance by gradually administering small quantities of the antineoplastic drug until the therapeutic dosage is reached. The aim of this study is to assess the effectiveness of oxaliplatin desensitization protocols. A retrospective observational study was carried out between January 2006 and May 2011. The inclusion criteria were patients undergoing chemotherapy treatment with oxaliplatin who had developed an HSR to the drug and who were candidates for continuing the treatment using a desensitization protocol. The patients' clinical records were reviewed and variables were gathered relating to the patient, the treatment, the HSR, and the desensitization protocol administered. The data were analysed using version 18.0 of the statistics program SPSS. A total of 53 desensitization protocols were administered to 21 patients. In 89 % of these cases, no new reactions occurred while the drug was being administered. New reactions of mild severity only occurred in 11 % of cases, and none of these reactions were severe enough for treatment to be stopped. All patients were able to complete the desensitization protocol. This study confirms that oxaliplatin desensitization protocols are safe and effective and allow patients to continue with the treatment that initially caused an HSR.

  10. Desensitization to antibiotics in children.

    Science.gov (United States)

    Cernadas, Josefina R

    2013-02-01

    Drug hypersensitivity reactions can occur to almost all drugs and antibiotics are among the most common cause for this kind of reactions. Drug hypersensitivity may affect any organ or system, and manifestations range widely in clinical severity from mild pruritus to anaphylaxis. In most cases, the suspected drug is avoided in the future. In case of infection, there is usually a safe antibiotic alternative. Nonetheless, in some cases, no alternative treatment exists for optimal therapy. Under these circumstances, desensitization may be performed. Drug desensitization is defined as the induction of a temporary state of tolerance to a drug which can only be maintained by continuous administration of the medication responsible for the hypersensitivity reaction. Desensitization is mainly performed in IgE-mediated reactions. Increasing doses of the implicated drug are administered over a short period of time, until the therapeutic dose is achieved and tolerated. Very few studies confined to children are found in literature. Most of them are case reports. In general, the proposed desensitization schemes are similar to those used in adults differing only in the final dose administered. The purpose of this study is to review desensitization to antibiotics in children presenting and discussing three clinical practical cases of desensitization in this age group. © 2012 John Wiley & Sons A/S.

  11. Medication Desensitization: Characterization of Outcomes and Risk Factors for Reactions.

    Science.gov (United States)

    Murray, Taryn S; Rice, Todd W; Wheeler, Arthur P; Phillips, Elizabeth J; Dworski, Ryszard T; Stollings, Joanna L

    2016-03-01

    Although its mechanisms are poorly understood, desensitization has been used to induce a temporary state of immune unresponsiveness in patients who have IgE-, non-IgE-, or pharmacologically mediated reactions when a drug has no alternatives. The purpose of this study was to characterize the outcomes and identify risk factors for reactions during drug desensitization. A retrospective review of electronic medical records of adult patients undergoing drug desensitization from January 1, 2011, to December 31, 2013, was conducted in 2 intensive care units at a tertiary medical center. We used multivariate analysis to determine if specified risk factors were associated with reacting during the desensitization. Reactions were classified according to the pretest probability prior to desensitization, and then, reactions during desensitization were classified based on the occurrence of cutaneous reactions as follows: successful with no reaction, mild reaction, moderate reaction, or failed. Failure could result from any systemic allergic or cutaneous reaction resulting in procedure termination. The desensitizations were also assessed to determine if the patient required de-escalation secondary to a reaction. A total of 88 desensitizations were performed in 69 patients. Desensitization was completed with no cutaneous reaction in 85% of patients. No baseline characteristic, medication class (P = 0.46), or indication for desensitization (P = 0.59) was associated with having a reaction. Reported histories of urticaria (P desensitization. However, neither history of urticaria nor labored breathing was independently associated with having a reaction in multivariate analysis (OR = 0.979, 95% CI = 0.325-2.952, P = 0.970, and OR = 1.626, 95% CI = 0.536-4.931, P = 0.739, respectively). Drug desensitization is safe for patients who have no alternative for therapy. Reported allergy histories of urticaria and labored breathing are both associated with having a reaction during the

  12. Desensitization with oxaliplatin in patients intolerant of carboplatin desensitization.

    Science.gov (United States)

    Rose, Peter G; Metz, Carol; Link, Nicolas

    2014-11-01

    The tolerance and efficacy of oxaliplatin desensitization in patients who were intolerant of carboplatin desensitization were determined. We retrospectively reviewed the Gynecologic Oncology patients who received carboplatin or oxaliplatin from December 2007 until August 2014. The number of treatments and number of patients of carboplatin standard infusions, carboplatin desensitizations, and oxaliplatin desensitizations were determined. Carboplatin infusions (2294) were administered to 281 patients. Twenty-eight (10%) of these patients developed carboplatin hypersensitivity and were treated with 205 carboplatin desensitizations. Nine (29%) patients were subsequently treated with 61 oxaliplatin desensitizations due to intolerance of carboplatin desensitization. Nine of the 10 patients tolerated this infusion well. Four of 9 evaluable patients had an objective response, 2 complete and 2 partial. Oxaliplatin desensitization seems well tolerated and effective in most patients who are intolerant of carboplatin desensitization.

  13. Platinum hypersensitivity and desensitization

    National Research Council Canada - National Science Library

    Miyamoto, Shingo; Okada, Rika; Ando, Kazumichi

    2015-01-01

    .... Desensitization, which gradually increases the concentration of the anticancer drug considered to be the antigen until the target dosage, has been reported as being particularly effective, with a success rate of 80-100...

  14. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    Science.gov (United States)

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  15. High affinity ligands for 'diazepam-insensitive' benzodiazepine receptors.

    Science.gov (United States)

    Wong, G; Skolnick, P

    1992-01-14

    Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their 'diazepam-sensitive' (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the 'diazepam-insensitive' (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (Ki approximately 2.6-20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio approximately 1; other substances had ratios ranging from 13 to greater than 1000. Ligands with high to moderate affinities at DI were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as 'GABA neutral' (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.

  16. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

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    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. (Univ. of Maryland, Baltimore (USA))

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  17. Kinetics of desensitization and recovery from desensitization for human a4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

    Institute of Scientific and Technical Information of China (English)

    Kewei D YU; Qiang LIU; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human a4p2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Methods: Whole-cell patch clamp recordings were performed to evaluate a4p2-nAChR currents.Results: Application of 0.1 nmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ~70 and ~700 ms, for the onset of a4(32-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of a4p2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs'1' from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human a4p2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings'21 using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse.Conclusion: These findings indicate the existence of a4p2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.

  18. Shock desensitizing of solid explosive

    Energy Technology Data Exchange (ETDEWEB)

    Davis, William C [Los Alamos National Laboratory

    2010-01-01

    Solid explosive can be desensitized by a shock wave too weak to initiate it promptly, and desensitized explosive does not react although its chemical composition is almost unchanged. A strong second shock does not cause reaction until it overtakes the first shock. The first shock, if it is strong enough, accelerates very slowly at first, and then more rapidly as detonation approaches. These facts suggest that there are two competing reactions. One is the usual explosive goes to products with the release of energy, and the other is explosive goes to dead explosive with no chemical change and no energy release. The first reaction rate is very sensitive to the local state, and the second is only weakly so. At low pressure very little energy is released and the change to dead explosive dominates. At high pressure, quite the other way, most of the explosive goes to products. Numerous experiments in both the initiation and the full detonation regimes are discussed and compared in testing these ideas.

  19. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  20. Platinum hypersensitivity and desensitization.

    Science.gov (United States)

    Miyamoto, Shingo; Okada, Rika; Ando, Kazumichi

    2015-09-01

    Platinum agents are drugs used for various types of cancer. With increased frequency of administration of platinum agents, hypersensitivity reactions appear more frequently, occurring in over 25% of cases from the seventh cycle or second line onward. It then becomes difficult to conduct treatment using these agents. Various approaches have been investigated to address hypersensitivity reactions to platinum agents. Desensitization, which gradually increases the concentration of the anticancer drug considered to be the antigen until the target dosage, has been reported as being particularly effective, with a success rate of 80-100%. The aims of this paper are to present the current findings regarding hypersensitivity reactions to platinum agents and to discuss attempts of using desensitization against hypersensitivity reactions worldwide. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. The high-affinity immunoglobulin E receptor as pharmacological target.

    Science.gov (United States)

    Blank, Ulrich; Charles, Nicolas; Benhamou, Marc

    2016-05-05

    The high-affinity receptor for immunoglobulin E is expressed mainly on mast cells and basophils, but also on neutrophils, eosinophils, platelets, monocytes, Langerhans and dendritic cells, airway smooth muscle cells and some nerve cells. Its main function is, upon its engagement by IgE and specific antigen, to trigger a powerful defense against invading pathogens and a rapid neutralization of dangerous toxic substances introduced in the body. This powerful response could be wielded against tumors. But, when control over this receptor is lost, its unchecked activation can induce an array of diseases, some of which can lead to death. In this review we will summarize the pharmacological approaches and strategies that are currently used, or under study, to harness or wield activation of this receptor for therapeutic purposes.

  2. 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.

  3. A high-affinity molybdate transporter in eukaryotes.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  4. Development and characterization of high affinity leptins and leptin antagonists.

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-02-11

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

  5. Development and Characterization of High Affinity Leptins and Leptin Antagonists*

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-01-01

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin. PMID:21119198

  6. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  7. Study on Property of Desensitized Explosive Film

    Institute of Scientific and Technical Information of China (English)

    李国新; 王晓丽; 焦清介; 刘淑珍

    2004-01-01

    The mechanical sensitivity, the critical thickness of detonation wave propagation and detonation velocity of desensitized PETN film were studied by experiments. The relationship between the mass of desensitizer paraffin wax and the friction sensitivity of desensitized PETN film was tested. According to the microstructure of film, the function of desensitizer was explained. It was proved that the explosive film could make explosive element micromation and kept its inherence properties by the result of testing the propagating critical dimension of the desensitized PETN film detonation wave. The explosive velocity of confined desensitized PETN film was tested by the multiplex optical fibre.

  8. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  9. Children's exposure to violent video games and desensitization to violence.

    Science.gov (United States)

    Funk, Jeanne B

    2005-07-01

    Desensitization to violence is cited frequently as being an outcome of exposure to media violence and a condition that contributes to increased aggression. This article initiates the development of a conceptual model for describing possible relationships among violent video games, brain function, and desensitization by using empathy and attitudes toward violence as proxy measures of desensitization. More work is needed to understand how specific game content may affect brain activity, how brain development may be affected by heavy play at young ages, and how personality and lifestyle variables may moderate game influence. Given the current state of knowledge, recommendations are made for clinicians to help parents monitor and limit exposure to violent video games and encourage critical thinking about media violence.

  10. Clinical applications of drug desensitization in the Asia-Pacific region.

    Science.gov (United States)

    Thong, Bernard Yu-Hor

    2011-04-01

    Drug desensitization is the induction, within hours to days, of a temporary state of tolerance to a drug which the patient has developed a hypersensitivity reaction to. It may be used for IgE and non-IgE mediated allergic reactions, and certain non-allergic reactions. The indication for desensitization is where no alternative medications are available for the treatment of that condition, and where the benefits of desensitization outweigh the risks. Desensitization is a therapeutic modality for drug allergy (similar to allergen specific immunotherapy for allergic rhinitis and insect venom anaphylaxis). In contrast, the drug provocation test is a diagnostic modality used to confirm or refute the diagnosis of drug allergy. This review discusses the clinical applications of desensitization for the treatment of common infectious, metabolic and cardiovascular diseases, and oncological conditions in the Asia-Pacific region.

  11. Desensitization of metastable intermolecular composites

    Science.gov (United States)

    Busse, James R [South Fork, CO; Dye, Robert C [Los Alamos, NM; Foley, Timothy J [Los Alamos, NM; Higa, Kelvin T [Ridgecrest, CA; Jorgensen, Betty S [Jemez Springs, NM; Sanders, Victor E [White Rock, NM; Son, Steven F [Los Alamos, NM

    2011-04-26

    A method to substantially desensitize a metastable intermolecular composite material to electrostatic discharge and friction comprising mixing the composite material with an organic diluent and removing enough organic diluent from the mixture to form a mixture with a substantially putty-like consistency, as well as a concomitant method of recovering the metastable intermolecular composite material.

  12. Desensitization to rituximab in a multidisciplinary setting.

    Science.gov (United States)

    Amorós-Reboredo, Patrícia; Sánchez-López, Jaime; Bastida-Fernández, Carla; do Pazo-Oubiña, Fernando; Borràs-Maixenchs, Núria; Giné, Eva; Valero, Antonio; Creus-Baró, Natàlia

    2015-10-01

    The need to offer first-line therapy to the increasing number of patients who have suffered an hypersensitivity reaction has stimulated the use of rapid desensitization protocols. To present our experience working as a multidisciplinary team using a rituximab rapid desensitization scheme. Patient demographics, allergic reaction, skin tests to rituximab, number of desensitizations, reactions during the desensitization protocol and actions taken, number of administered and completed cycles, were retrospectively collected in patients who received at least one desensitization to rituximab. Number of desensitizations successfully managed. Between 2012 and June 2013 five patients received a total of 19 desensitizations to rituximab using a 12 step rapid desensitization protocol. All patients received the scheduled chemotherapeutic cycles as inpatients, with no delay in administration dates. Three patients presented a hypersensitivity reaction during the first desensitization and in one patient the event occurred again during the second treatment cycle. All reactions occurred in the last step, when the infusion rate reached the maximum speed. The developed protocol for rapid desensitization was successful in five patients receiving rituximab. Patients could receive the full intended dose.

  13. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    Science.gov (United States)

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  14. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    Science.gov (United States)

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  15. Desensitization mechanism in prokaryotic ligand-gated ion channel.

    Science.gov (United States)

    Velisetty, Phanindra; Chakrapani, Sudha

    2012-05-25

    Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

  16. Drug desensitization in the management of hypersensitivity reactions to monoclonal antibodies and chemotherapy.

    Science.gov (United States)

    Mezzano, Veronica; Giavina-Bianchi, Pedro; Picard, Matthieu; Caiado, Joana; Castells, Mariana

    2014-04-01

    Hypersensitivity reactions to monoclonal antibodies and chemotherapy, which may vary in severity from mild to life-threatening, can lead to their discontinuation and replacement by alternative agents that are often less effective, more toxic, and/or more expensive. Drug desensitization has emerged as the best treatment modality capable of allowing re-introduction of the hypersensitivity reaction-inducing medication in highly sensitized patients in need of first line therapies. In recent years, the availability of new anti-neoplastic drugs and therapeutic monoclonal antibodies has increased, as has the potential for hypersensitivity reactions. Development of desensitization protocols for these new medications requires a careful assessment of the potential risks and benefits. The purposes of this review are to provide an overview of the presentation of hypersensitivity reactions amenable to desensitization and to increase awareness of the indications for and outcomes of desensitization protocols. Rapid drug desensitization has proven to be a safe and effective way of administering first line therapy to patients with hypersensitivity reactions, providing an extremely powerful treatment modality for patients for whom alternative drugs are deemed unacceptable. Rapid drug desensitization protocols should be administered only by highly trained allergists and nurses who have experience in determining which reactions are amenable to desensitization, and can identify high risk patients and provide them with appropriate care. Efforts should be made to increase awareness of the remarkable safety and efficacy of rapid drug desensitization among non-allergists, especially in the fields of oncology and rheumatology, so as to favor its universal application. Development of desensitization units to provide state-of-the-art care is possible only through coordinated teamwork.

  17. Pore mutations of the Escherichia coli MscS channel affect desensitization but not ionic preference.

    Science.gov (United States)

    Edwards, Michelle D; Bartlett, Wendy; Booth, Ian R

    2008-04-15

    Mechanosensitive channels rescue bacterial cells from a fate of lysis when they transfer from a high- to low-osmolarity environment. Of three Escherichia coli mechanosensitive proteins studied to date, only MscS-Ec demonstrates a small anionic preference and a desensitized, nonconducting state under sustained pressure. Little is known about the mechanisms generating these distinctive properties. Eliminating the sole positive charge in the MscS-Ec pore region (Arg(88)) did not alter anionic preference. Adding positive charges at either end of the pore did not augment anionic preference, and placing negative charges within the pore did not diminish it. Thus, pore charges do not control this characteristic. However, from this analysis we identified mutations in the hinge region of the MscS-Ec pore helix (at Gly(113)) that profoundly affected ability of the channel to desensitize. Substitution with nonpolar (Ala, Pro) or polar (Asp, Arg, Ser) residues inhibited transition to the desensitized state. Interestingly, Gly(113) replaced with Met did not impede desensitization. Thus, although Gly is not specifically required at position 113, MscS desensitization is strongly influenced by the residue situated here. Mutations at residues further into the pore also regulated desensitization. Transition to this unique mechanosensitive channel state is discussed in terms of existing data.

  18. Guided Mastery and Performance Desensitization Treatments.

    Science.gov (United States)

    Williams, S. Lloyd; And Others

    1985-01-01

    Compared desensitization and self-efficacy models of phobia treatment in height phobics. Self-efficacy treatment proved to be significantly more effective than desensitization treatment in restoring subjects' behavioral functioning, in raising their perceptions of self-efficacy, and in reducing their anticipated anxiety and thoughts of danger.…

  19. High-affinity olfactory receptor for the death-associated odor cadaverine

    OpenAIRE

    2013-01-01

    Cadaverine and putrescine, two diamines emanating from decaying flesh, are strongly repulsive odors to humans but serve as innate attractive or social cues in other species. Here we show that zebrafish, a vertebrate model system, exhibit powerful and innate avoidance behavior to both diamines, and identify a high-affinity olfactory receptor for cadaverine.

  20. Isolation and cloning of the gene encoding high affinity phosphate transporter in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High affinity phosphate transporter plays an important role in plant adapting to low phosphorus. Isolation of genes coding this kind of protein has attracted worldwide scholars to accomplish. We aimed to isolate the gene and transfer it to target plants for breeding.

  1. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; Ruiter, de M.V.; Cornelissen, J.J.L.M.; Jonkheijm, P.

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  2. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue;

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects...

  3. 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 radiol

  4. N-Oxide analogs of WAY-100635 : new high affinity 5-HT1A receptor antagonists

    NARCIS (Netherlands)

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

    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 radiol

  5. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; de Ruiter, Mark Vincent; Cornelissen, Jeroen Johannes Lambertus Maria; Jonkheijm, Pascal

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  6. High affinity, bioavailable 3-amino-1,4-benzodiazepine-based gamma-secretase inhibitors.

    Science.gov (United States)

    Owens, Andrew P; Nadin, Alan; Talbot, Adam C; Clarke, Earl E; Harrison, Timothy; Lewis, Huw D; Reilly, Michael; Wrigley, Jonathan D J; Castro, José L

    2003-11-17

    In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.

  7. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    Science.gov (United States)

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  8. The dual aptamer approach: rational design of a high-affinity FAD aptamer.

    Science.gov (United States)

    Merkle, T; Holder, I T; Hartig, J S

    2016-01-14

    A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP- and FMN-aptamers. Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.

  9. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K...

  10. Rapid desensitization for hypersensitivity reactions to medications.

    Science.gov (United States)

    Castells, Mariana

    2009-08-01

    Drug desensitization is the induction of temporary clinical unresponsiveness to drug antigens to which patients have presented severe hypersensitivity reactions. It is typically achieved by gradual reintroduction of small doses of drug antigens at fixed time intervals, and it is aimed at providing increased safety and protection from side effects, including anaphylaxis. Delivery of full therapeutic doses is achieved during desensitization, allowing patients to receive firstline chemotherapy, antibiotics, or monoclonal antibodies, as well as other drugs such as insulin, aspirin, and iron. Desensitizations are high-risk interventions. Inhibition of cellular activation mechanisms occurs during drug desensitization, allowing for the protective clinical outcomes and lack of side effects in the majority of cases, but the cellular and molecular inhibitory mechanisms are incompletely understood. The indication for desensitization protocols can only be done by trained allergists and immunologists and should be implemented as standard of care because of their high success rates and outcomes-demonstrated safety profile.

  11. Aspirin Allergy Desensitization in Cerebrovascular Disease

    Science.gov (United States)

    Zuckerman, Scott L; Seder, David B; Tsujiura, Crystiana; Cushing, Deborah; Gallup, Holly; Mocco, J; Hanel, Richard A; Ecker, Robert D

    2014-01-01

    Summary Aspirin (ASA) is the mainstay of treatment in cerebrovascular and systemic vascular disease. ASA hypersensitivity can pose a challenge to achieving optimum medical management prior to and after neurointerventional treatment. Desensitization to ASA is well described in the allergy and cardiovascular literature, but there are no similar discussions specific to neurointervention. The purpose of our study was to describe our experience with ASA hypersensitivity management and review the relevant literature. Two cases of patients with symptomatic cerebrovascular disease requiring neurointervention who were successfully desensitized to their ASA hypersensitivity prior to treatment are described. The subsequent literature is reviewed. Several ASA desensitization protocols exist and have been proven to successfully treat ASA hypersensitivity and allow for ASA therapy to be safely initiated. We describe several previously published protocols. ASA desensitization is a safe and simple way to manage ASA hypersensitivity. We provide comprehensive management guidelines for the neurointerventionalist engaging in ASA desensitization. PMID:24556294

  12. Cholesterol modulates open probability and desensitization of NMDA receptors

    Science.gov (United States)

    Korinek, Miloslav; Vyklicky, Vojtech; Borovska, Jirina; Lichnerova, Katarina; Kaniakova, Martina; Krausova, Barbora; Krusek, Jan; Balik, Ales; Smejkalova, Tereza; Horak, Martin; Vyklicky, Ladislav

    2015-01-01

    NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid–NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-β-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-β-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs. Key points NMDA receptors (NMDARs) are tetrameric cation channels permeable to calcium; they mediate excitatory synaptic transmission in the CNS and their excessive activation can lead to

  13. Dentin tubule occluding ability of dentin desensitizers.

    Science.gov (United States)

    Han, Linlin; Okiji, Takashi

    2015-04-01

    To compare the dentin tubule-occluding ability of fluoroaluminocalciumsilicate-based (Nanoseal), calcium phosphate-based (Teethmate Desensitizer), resin-containing oxalate (MS Coat ONE) and diamine silver fluoride (Saforide) dentin desensitizers using artificially demineralized bovine dentin. Simulated hypersensitive dentin was created using cervical dentin sections derived from bovine incisors using phosphoric acid etching followed by polishing with a paste containing hydroxyapatite. The test desensitizers were applied in one, two, or three cycles, where each cycle involved desensitizer application, brushing, and immersion in artificial saliva (n= 5 each). The dentin surfaces were examined with scanning electron microscopy, and the dentin tubule occlusion rate was calculated. The elemental composition of the deposits was analyzed with electron probe microanalysis. Data were analyzed by one-way ANOVA and the Tukey honestly significant different test. Marked deposit formation was observed on the specimens treated with Nanoseal or Teethmate Desensitizer, and tags were detected in the specimens' dentin tubules. These findings became more prominent as the number of application cycles increased. The major elemental components of the tags were Ca, F, and Al (Nanoseal) and Ca and P (Teethmate Desensitizer). The tubule occlusion rates of MS Coat ONE and Saforide were significantly lower than those of Nanoseal and Teethmate Desensitizer (P< 0.05).

  14. Antibiotic Desensitization Therapy in Secondary Syphilis and Listeria Infection: Case Reports and Review of Desensitization Therapy

    Science.gov (United States)

    Magpantay, Gil; Madar, Cristian S; Hsue, Gunther; Belnap, Conrad

    2011-01-01

    Two adult cases, one of secondary syphilis and one of Listeria monocytogenes bacteremia, in which antibiotic desensitization therapy was utilized to assist treatment of active infection in the face of severe penicillin allergy. Clinical considerations are discussed that led to the decision to employ a formal desensitization procedure. Antibiotic desensitization protocols can facilitate optimal and safe antibiotic therapy in the appropriate clinical setting. PMID:22187514

  15. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  16. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  17. 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.

  18. A High-Affinity Metal-Binding Peptide From Escherichia Coli Hypb

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.C.Chan; Cao, L.; Dias, A.V.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.

  19. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

    Science.gov (United States)

    Maute, Roy L; Gordon, Sydney R; Mayer, Aaron T; McCracken, Melissa N; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M; Manglik, Aashish; Kruse, Andrew C; Gambhir, Sanjiv S; Weissman, Irving L; Ring, Aaron M

    2015-11-24

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

  20. 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.

  1. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; YASUHARA, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

  2. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    Science.gov (United States)

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  3. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-01

    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 (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 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/sup 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/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 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.

  4. 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.

  5. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

  6. 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.

  7. [3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

    Science.gov (United States)

    Hoo, K; Legutko, B; Rizkalla, G; Deverill, M; Hawes, C R; Ellis, G J; Stensbol, T B; Krogsgaard-Larsen, P; Skolnick, P; Bleakman, D

    1999-12-01

    The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

  8. 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.

  9. Characterization of a genetically reconstituted high-affinity system for serotonin transport

    Energy Technology Data Exchange (ETDEWEB)

    Chang, A.S.S.; Lam, D.M.K. (Baylor College of Medicine, Woodlands, TX (USA) Baylor College of Medicine, Houston, TX (USA)); Frnka, J.V.; Chen, D. (Baylor College of Medicine, Woodlands, TX (USA))

    1989-12-01

    By transfecting mouse fibroblast L-M cells with human genomic DNA, the authors have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-({sup 3}H)HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na{sup +} and temperature dependence; (iv) kinetic saturability; and (v) high affinity for 5-HT. This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, they postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neutrotransmitter-uptake systems as well as substrate for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.

  10. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    Science.gov (United States)

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  11. 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...

  12. Hyperventilation in a motion sickness desensitization program

    NARCIS (Netherlands)

    Mert, A.; Bles, W.; Nooij, S.A.E.

    2007-01-01

    Introduction: In motion sickness desensitization programs, the motion sickness provocative stimulus is often a forward bending of the trunk on a rotating chair, inducing Coriolis effects. Since respiratory relaxation techniques are applied successfully in these courses, we investigated whether these

  13. Pain ameliorating effect of eye movement desensitization.

    Science.gov (United States)

    Hekmat, H; Groth, S; Rogers, D

    1994-06-01

    This study explores the efficacy of eye movement desensitization and reprocessing (EMD/R) in the management of acute pain induced by hand exposures to ice water. Thirty participants were randomly assigned to one of the following interventions: (a) eye movement desensitization and reprocessing, (b) eye movement desensitization with music (EMD/M), and (c) control. The EMD/R participants focused on negative experiences associated with exposure to ice water, generated positive self-talk, and diverted their attention away from pain by focusing on a rapidly moving light on a monitor. The EMD with music group received eye movement desensitization coupled with preferred music. Repeated measures univariate and multivariate analysis of covariance was used to analyze the data. Results indicated that both procedures alleviated participants' pain to a similar degree and significantly more than the control, P < 0.05.

  14. Systematic Desensitization: A Technique Worth Trying

    Science.gov (United States)

    Bugg, Charles A.

    1972-01-01

    The author relates his experiences in using a modified form of systematic desensitization in a public school setting with counselees whose success and development are hampered by test anxiety and fear of public speaking. (Author)

  15. 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.

  16. Carboplatin hypersensitivity: evaluation and successful desensitization protocol.

    Science.gov (United States)

    Bruchim, Ilan; Goldberg, Arnon; Fishman, Ami; Confino-Cohen, Ronit

    2014-01-01

    Carboplatin-induced immediate hypersensitivity reactions are relatively common among patients with gynecological malignancies. Once this occurs, the patient might be at risk for future carboplatin-induced reactions. This study evaluated the efficacy of allergic consultation, carboplatin skin testing and desensitization as a single intervention strategy in this population. Patients with a well-documented immediate reaction to carboplatin were offered allergy consultation, carboplatin skin testing and a desensitization plan in a single visit between scheduled chemotherapy sessions. Fifty-five patients with an immediate reaction were evaluated. After allergist assessment, 44 (89%) of 49 patients skin tested had a positive result. A total of 207 carboplatin desensitization cycles were administered to 49 women. Among them, 10 patients had a mild immediate hypersensitivity reaction during desensitization. Five patients subsequently tolerated carboplatin administered in the prolonged desensitization protocol. In the data presented, we propose a strategy that is both cost effective and very convenient for the patient. The diagnostic procedure, including allergist consultation and skin test, can be completed in less than 2 h. In most cases where carboplatin is indispensable, desensitization can be administered without overnight hospitalization.

  17. Monomeric TonB and the Ton box are required for the Formation of a High-Affinity Transporter-TonB Complex†

    Science.gov (United States)

    Freed, Daniel M.; Lukasik, Stephen M.; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S.

    2013-01-01

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In the present study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB to the outer membrane transporters BtuB, FecA and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high-affinity (tens of nM) to both BtuB and FhuA; however, no high-affinity binding was observed to FecA. In BtuB, the high affinity binding of TonB(33–239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33–239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33–239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When bound to the outer membrane transporter, DEER shows that the TonB(33–239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle. PMID:23517233

  18. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    Science.gov (United States)

    Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S

    2013-04-16

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

  19. In Vivo Desensitization: An Alternative to Systematic Desensitization with College Students

    Science.gov (United States)

    Atkinson, Donald R.

    1973-01-01

    It is the thesis of this article that college counselors would make more economical and efficacious use of their time and would greatly reduce the risk of turning off'' students if they employed in vivo desensitization rather than systematic desensitization when dealing with anxious clients. (Author)

  20. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI)

    OpenAIRE

    1992-01-01

    It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast c...

  1. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  2. 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.

  3. 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.

  4. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

    Science.gov (United States)

    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

  5. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  6. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

    )/sup 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 (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 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 (/sup 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 (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  7. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    Science.gov (United States)

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  8. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  9. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae.

    Science.gov (United States)

    Aouida, Mustapha; Leduc, Anick; Poulin, Richard; Ramotar, Dindial

    2005-06-24

    Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

  10. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    Science.gov (United States)

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.

  11. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  12. [Experience of rapid drug desensitization therapy in the treatment of mycobacterial disease].

    Science.gov (United States)

    Sasaki, Yuka; Kurashima, Atsuyuki; Morimoto, Kozo; Okumura, Masao; Watanabe, Masato; Yoshiyama, Takashi; Ogata, Hideo; Gotoh, Hajime; Kudoh, Shoji; Suzuki, Hiroaki

    2014-11-01

    Drugs for tuberculosis and non-tuberculosis mycobacterial diseases are limited. In particular, no new drugs for non-tuberculosis mycobacterial disease have been developed in recent years. Antimycobacterial drugs have many adverse reactions, for which drug desensitization therapy has been used. Rapid drug desensitization (RDD) therapy, including antituberculosis drugs and clarithromycin, has been implemented in many regions in Europe and the United States. We investigated the validity of RDD therapy in Japan. We report our experience with RDD therapy in 13 patients who developed severe drug allergy to antimycobacterial treatment. The desensitization protocol reported by Holland and Cernandas was adapted. The underlying diseases were 7 cases of pulmonary Mycobacterium avium complex disease and 6 cases of pulmonary tuberculosis. Isoniazid was readministered in 2 (100%) of 2 patients; rifampicin, in 8 (67.7%) of 12 patients; ethambutol, in 4 (67.7%) of 6 patients; and clarithromycin, in 2 (100%) of 2 patients. In Japan, the desensitization therapy recommended by the Treatment Committee of the Japanese Society for Tuberculosis have been implemented generally. We think RDD therapy is effective and safe as the other desensitization therapy. We will continue to investigate the efficiency of RDD therapy in patients who had discontinued antimycobacterial treatment because of the drug allergic reaction.

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

    OpenAIRE

    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-affin...

  14. Playing violent video games and desensitization to violence.

    Science.gov (United States)

    Brockmyer, Jeanne Funk

    2015-01-01

    This article examines current research linking exposure to violent video games and desensitization to violence. Data from questionnaire, behavioral, and psychophysiologic research are reviewed to determine if exposure to violent video games is a risk factor for desensitization to violence. Real-world implications of desensitization are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Profilin desensitization: A case series.

    Science.gov (United States)

    Nucera, Eleonora; Aruanno, Arianna; Rizzi, Angela; Pecora, Valentina; Patriarca, Giampiero; Buonomo, Alessandro; Mezzacappa, Simona; Schiavino, Domenico

    2016-09-01

    The role of profilin as an allergen has long been questioned. The capacity of profilin to induce respiratory symptoms has recently been demonstrated; moreover, over 50% of patients sensitized to profilin experienced symptoms after the ingestion of plant-derived foods, suggesting that profilin should be considered as a clinically relevant food allergen.We describe the cases of seven allergic patients with oral allergy syndrome and other adverse reactions after eating plant-derived food, that have been undergone to profilin desensitization treatment.The protocol started with a drop of profilin solution (50 µg/mL) diluted 1:10(18) in water until the highest dose of 10 drops of undiluted solution three times a week. At the end of the treatment we observed a decreased mean diameter of profilin wheal in skin prick test (SPT) in five of the seven participants and in profilin specific IgE values in six patients that repeated the test. Regarding basophil activation test (BAT) and the detection of IgG4, we do not have significant results because the tests have to be repeated in some patients. Regarding the double-blind placebo-controlled challenges, after about 10 months of induction phase all the patients showed tolerance to several foods that they previously did not tolerate.Moreover, the immunotherapy with profilin has proved to be safe because no serious adverse events have been reported in our patients.In summary, the results of this exploratory study of sublingual immunotherapy (SLIT) for profilin allergy show that it can be a promising therapeutic option that could modify the clinical reactivity of the patients to the intake of plant-derived food. © The Author(s) 2015.

  16. Desensitization of human adipose tissue to adrenaline stimulation studied by microdialysis

    DEFF Research Database (Denmark)

    Stallknecht, Bente; Bülow, J; Frandsen, E

    1997-01-01

    1. Desensitization of fat cell lipolysis to catecholamine exposure has been studied extensively in vitro but only to a small extent in human adipose tissue in vivo. 2. We measured interstitial glycerol concentrations by microdialysis in subcutaneous, abdominal adipose tissue in healthy humans......M, respectively) or a low, a high and a low concentration (2.5, 4.6 and 2.6 nM, respectively) in order to examine both desensitization and the dose dependency of adipose tissue lipolysis to adrenaline. 3. Adipose tissue lipolysis was calculated and was found to vary directly with arterial adrenaline concentration...... in adipose tissue blood flow in response to adrenaline was also reduced by prior adrenaline exposure, but no consistent desensitization could be demonstrated for whole-body energy expenditure, blood pressure and heart rate. 5. In the basal state, arterial plasma and interstitial adrenaline concentrations did...

  17. Desensitizing nano powders to electrostatic discharge ignition

    Energy Technology Data Exchange (ETDEWEB)

    Steelman, Ryan [Texas Tech Univ., Lubbock, TX (United States). Dept. of Mechanical Engineering; Clark, Billy [Texas Tech Univ., Lubbock, TX (United States). Dept. of Mechanical Engineering; Pantoya, Michelle L. [Texas Tech Univ., Lubbock, TX (United States). Dept. of Mechanical Engineering; Heaps, Ronald J. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Daniels, Michael A. [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2015-08-01

    Electrostatic discharge (ESD) is a main cause for ignition in powder media ranging from grain silos to fireworks. Nanoscale particles are orders of magnitude more ESD ignition sensitive than their micron scale counterparts. This study shows that at least 13 vol. % carbon nanotubes (CNT) added to nano-aluminum and nano-copper oxide particles (nAl + CuO) eliminates ESD ignition sensitivity. The CNT act as a conduit for electric energy and directs electric charge through the powder to desensitize the reactive mixture to ignition. For nanoparticles, the required CNT concentration for desensitizing ESD ignition acts as a diluent to quench energy propagation.

  18. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    Science.gov (United States)

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

  19. Receptor downregulation and desensitization enhance the information processing ability of signalling receptors

    Directory of Open Access Journals (Sweden)

    Resat Haluk

    2007-11-01

    Full Text Available Abstract Background In addition to initiating signaling events, the activation of cell surface receptors also triggers regulatory processes that restrict the duration of signaling. Acute attenuation of signaling can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation or via ligand-induced receptor desensitization. These phenomena have traditionally been viewed in the context of adaptation wherein the receptor system enters a refractory state in the presence of sustained ligand stimuli and thereby prevents the cell from over-responding to the ligand. Here we use the epidermal growth factor receptor (EGFR and G-protein coupled receptors (GPCR as model systems to respectively examine the effects of downregulation and desensitization on the ability of signaling receptors to decode time-varying ligand stimuli. Results Using a mathematical model, we show that downregulation and desensitization mechanisms can lead to tight and efficient input-output coupling thereby ensuring synchronous processing of ligand inputs. Frequency response analysis indicates that upstream elements of the EGFR and GPCR networks behave like low-pass filters with the system being able to faithfully transduce inputs below a critical frequency. Receptor downregulation and desensitization increase the filter bandwidth thereby enabling the receptor systems to decode inputs in a wider frequency range. Further, system-theoretic analysis reveals that the receptor systems are analogous to classical mechanical over-damped systems. This analogy enables us to metaphorically describe downregulation and desensitization as phenomena that make the systems more resilient in responding to ligand perturbations thereby improving the stability of the system resting state. Conclusion Our findings suggest that in addition to serving as mechanisms for adaptation, receptor downregulation and desensitization can play a critical role in temporal information

  20. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.

    Science.gov (United States)

    Norholm, Morten H H; Nour-Eldin, Hussam H; Brodersen, Peter; Mundy, John; Halkier, Barbara A

    2006-04-17

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

  1. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis.

    Science.gov (United States)

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-08-31

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E(+)) and acquire a high-affinity conformation with an 'open' headpiece (H(+)). The canonical switchblade model of integrin activation proposes that the E(+) conformation precedes H(+), and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E(-)H(+) conformation. E(-)H(+) β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.

  2. Selection of high affine peptide ligands for detection of Clostridium Tyrobutyricum spores.

    Science.gov (United States)

    Lavilla, María; De Luis, Ruth; Pérez, María Dolores; Calvo, Miguel; Sánchez, Lourdes

    2009-11-01

    Clostridium tyrobutyricum is the main agent responsible for "late blowing" in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN); however, it is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.

  3. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.

    Science.gov (United States)

    Altschuler, Sarah E; Lewis, Karen A; Wuttke, Deborah S

    2013-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  4. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    Directory of Open Access Journals (Sweden)

    Sarah E. Altschuler

    2013-09-01

    Full Text Available The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  5. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil.

    Science.gov (United States)

    Tomatsu, Hajime; Takano, Junpei; Takahashi, Hideki; Watanabe-Takahashi, Akiko; Shibagaki, Nakako; Fujiwara, Toru

    2007-11-20

    Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the K(m) value of MOT1 for molybdate is approximately 20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

  6. Experimental conditions can obscure the second high-affinity site in LeuT.

    Science.gov (United States)

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  7. Cytisine derivatives as high affinity nAChR ligands: synthesis and comparative molecular field analysis.

    Science.gov (United States)

    Nicolotti, O; Canu Boido, C; Sparatore, F; Carotti, A

    2002-06-01

    A number of new N-substituted cytisine derivatives were prepared and tested, along with similar compounds already described by us and others, as high affinity neuronal acetylcholine receptor ligands. Structure-affinity relationships were discussed in the light of our recently proposed pharmacophore model for nicotinic receptor agonists. The most significant physicochemical interactions modulating the receptor-ligand binding were detected at the three dimensional (3D) level by means of comparative molecular field analysis (CoMFA). The best predictive PLS model was a single-field steric model showing good statistical figures: n = 17, Q2 = 0.717, s(ev) = 0.566, r2 = 0.942, s = 0.275.

  8. A linker peptide with high affinity towards silica-containing materials.

    Science.gov (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

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

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    B 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....

  10. A complex water network contributes to high-affinity binding in an antibody–antigen interface

    Directory of Open Access Journals (Sweden)

    S.F. Marino

    2016-03-01

    Full Text Available This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment, J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA. The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: “Potent anti-tumor response by targeting B cell maturation antigen (BCMA in a mouse model of multiple myeloma”, Mol. Oncol. 9 (7 (2015 pp. 1348–58.

  11. Expression of a Hybrid Human Superoxide Dismutase with a High Affinity for Heparin

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A designed heparin-affinity of human Cu, Zn-SOD is described. The natural leader peptide of P.leiognathi Cu, Zn-SOD and a heparin-binding peptide containing a stretch of 7 Arg were fused to the N-terminal and the C-terminal of human Cu, Zn-SOD respectively. The resulted hybrid enzyme had not only a normal SOD activity but also a high affinity for heparin eluted on the heparin-Sepharose column at 0.4 mol/L NaCl. Some properties, such as the optimum pH, the thermostability and the half-life in the circulation of rats, were also analyzed.

  12. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    DEFF Research Database (Denmark)

    Rao, C N; Castronovo, V; Schmitt, M C;

    1989-01-01

    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...... al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse......, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues...

  13. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 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 (/sup 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 (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 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 (/sup 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 (/sup 3/H)-5-HT uptake.

  15. The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors.

    Science.gov (United States)

    Nezami, Azin; Freire, Ernesto

    2002-12-04

    The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.

  16. 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.

  17. 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.

  18. Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging.

    Science.gov (United States)

    Dhar, Alok; Feiss, Michael

    2005-03-18

    DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.

  19. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    Science.gov (United States)

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  20. A Review of Eye Movement Desensitization and Reprocessing (EMDR): Research Findings and Implications for Counsellors.

    Science.gov (United States)

    MacCluskie, Kathryn C.

    1998-01-01

    States that within the last six years a new therapeutic technique for the treatment of posttraumatic stress disorder, Eye Movement Desensitization and Reprocessing (EMDR), has emerged. Examines the strengths and weaknesses of published studies concerning EMDR, describes the nature of the debate about the efficacy of EMDR, and reviews implications…

  1. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

    Science.gov (United States)

    Miethke, Marcus; Monteferrante, Carmine G; Marahiel, Mohamed A; van Dijl, Jan Maarten

    2013-10-01

    Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Intravenous desensitization to beta-lactam antibiotics.

    Science.gov (United States)

    Borish, L; Tamir, R; Rosenwasser, L J

    1987-09-01

    Patients allergic to penicillin (PCN) often require treatment with beta-lactam antibiotics for life-threatening bacterial infections. In this article, we review our experience with rapid intravenous desensitization for patients who gave a history of PCN allergy and who had hypersensitivity demonstrated by skin tests. Skin testing was performed with both prick and intradermal techniques and with the recommended antibiotic as well as PCN G, penicilloyl polylysine, and a minor determinant mixture. Patients were transferred to the intensive care unit, and desensitization was performed with a buret technique that required minimal preparation and was easily applied to any antibiotic. Fifteen desensitizations in 12 patients were associated with no immediate reactions. One patient developed a delayed reaction consisting of a pruritic rash and angioedema. A second patient developed a more serious delayed serum sickness-like illness with fever, rash, eosinophilia, abnormal liver function tests, and urinary abnormalities. These reactions did not necessitate stopping the antibiotic, although the latter patient required corticosteroids to suppress his symptoms. Rapid intravenous desensitization is a rapid, safe, and effective technique for patients demonstrating hypersensitivity to beta-lactam antibiotics who require therapy with these medications.

  3. Desensitization and recovery of metastable intermolecular composites

    Science.gov (United States)

    Busse, James R [South Fork, CO; Dye, Robert C [Los Alamos, NM; Foley, Timothy J [Los Alamos, NM; Higa, Kelvin T [Ridgecrest, CA; Jorgensen, Betty S [Jemez Springs, NM; Sanders, Victor E [White Rock, NM; Son, Steven F [Los Alamos, NM

    2010-09-07

    A method to substantially desensitize a metastable intermolecular composite material to electrostatic discharge and friction comprising mixing the composite material with an organic diluent and removing enough organic diluent from the mixture to form a mixture with a substantially putty-like consistency, as well as a concomitant method of recovering the metastable intermolecular composite material.

  4. Clinical practice - Drug desensitization in children

    NARCIS (Netherlands)

    de Groot, H.; Mulder, W.M.C.

    2010-01-01

    Immediate type allergic reactions to medication are potentially life threatening and can hamper drug therapy of several medical conditions. Exact incidence and prevalence data for these reactions in children are lacking. If no alternative drug treatment is available, a desensitization procedure may

  5. Clinical practice - Drug desensitization in children

    NARCIS (Netherlands)

    H. de Groot; W.M.C. Mulder

    2010-01-01

    Immediate type allergic reactions to medication are potentially life threatening and can hamper drug therapy of several medical conditions. Exact incidence and prevalence data for these reactions in children are lacking. If no alternative drug treatment is available, a desensitization procedure may

  6. Investigation into the Coating and Desensitization Effect on HNIW of Paraffin Wax/Stearic Acid Composite System

    Science.gov (United States)

    Wang, Dong-Xu; Chen, Shu-Sen; Jin, Shao-Hua; Shu, Qing-Hai; Jiang, Zhen-Ming; Shang, Feng-Qin; Li, Jin-Xin

    2016-01-01

    2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (HNIW) was bonded by fluorine rubber and then desensitized by paraffin wax (PW), stearic acid (SA), and a PW/SA composite system using an aqueous suspension method. The coating and desensitization effects of the composite systems on HNIW and the influence of the addition of SA on the mechanical properties of the coated HNIW samples were studied. In addition, the PW/SA composite solution was simulated using a molecular dynamics method, and the relationship between the desensitization effect on HNIW and the properties of the composite solution was investigated. The results showed that the PW/SA composite system, of which the desensitization effect on HNIW was between those of the two desensitizers, could effectively coat HNIW and that the composite solution had the most stable and well-distributed state when using benzene as solvent with the mass ratio of PW/SA equal to 7/3 or 3/7, thus resulting in the best desensitization effect on HNIW. Moreover, the addition of stearic acid was successful in enhancing the mechanical properties of the coated HNIW samples.

  7. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Ton, Hoai T; Smart, Amanda E; Aguilar, Brittany L; Olson, Thao T; Kellar, Kenneth J; Ahern, Gerard P

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

  8. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

  9. Dentin desensitizing agents: SEM and X-ray microanalysis assessment.

    Science.gov (United States)

    Jain, P; Vargas, M A; Denehy, G E; Boyer, D B

    1997-02-01

    To evaluate the effect of four proprietary dentin desensitizing agents on dentin tubular occlusion, chemical composition changes on the dentin surface, and the effect of saliva and toothbrushing on these agents. Fifty dentin discs, obtained from 50 freshly extracted human premolar and molar teeth were used in this study. These were divided into five groups of 10 discs each. Five discs from each group were treated with the desensitizing agents, viewed under the SEM and subjected to energy dispersive X-ray analysis. The other five discs were treated with the desensitizing agents, immersed in artificial saliva, subjected to simulated toothbrushing equivalent to 3 weeks of normal brushing and viewed under the SEM. The agents studied were Sensodyne Dentin Desensitizer, Therma-Trol Desensitizer Gel, Gluma Desensitizer and All-Bond DS. The Kruskal-Wallis test followed by the Wilcoxon signed-rank test showed that Sensodyne Dentin Desensitizer exhibited the greatest amount of tubular occlusion among the unbrushed samples, followed by Therma-Trol Desensitizer Gel, Gluma Desensitizer and All-Bond DS (P Sensodyne Dentin Desensitizer treated samples.

  10. 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

  11. 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

    2013-10-24

    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 [(3)H]-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 [(3)H]-1 binding shows high specificity to the high-affinity GHB binding sites.

  12. 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....... 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....

  13. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

    Science.gov (United States)

    Reis, C R; van der Sloot, A M; Natoni, A; Szegezdi, E; Setroikromo, R; Meijer, M; Sjollema, K; Stricher, F; Cool, R H; Samali, A; Serrano, L; Quax, W J

    2010-10-21

    The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

  14. 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

  15. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  16. Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

    Science.gov (United States)

    Bennmann, Dorit; Kannicht, Christoph; Fisseau, Claudine; Jacobs, Kathleen; Navarette-Santos, Alexander; Hofmann, Britt; Horstkorte, Rüdiger

    2015-09-01

    AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 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{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 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 ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  18. 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.

  19. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

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    Kaval Kaur

    Full Text Available Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE. Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  20. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    Science.gov (United States)

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

  1. Peptide array-based characterization and design of ZnO-high affinity peptides.

    Science.gov (United States)

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  2. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin.

    Science.gov (United States)

    Shulman, Ziv; Shinder, Vera; Klein, Eugenia; Grabovsky, Valentin; Yeger, Orna; Geron, Erez; Montresor, Alessio; Bolomini-Vittori, Matteo; Feigelson, Sara W; Kirchhausen, Tomas; Laudanna, Carlo; Shakhar, Guy; Alon, Ronen

    2009-03-20

    Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

  3. High-affinity DNA base analogs as supramolecular, nanoscale promoters of macroscopic adhesion.

    Science.gov (United States)

    Anderson, Cyrus A; Jones, Amanda R; Briggs, Ellen M; Novitsky, Eric J; Kuykendall, Darrell W; Sottos, Nancy R; Zimmerman, Steven C

    2013-05-15

    Adhesion phenomena are essential to many biological processes and to synthetic adhesives and manufactured coatings and composites. Supramolecular interactions are often implicated in various adhesion mechanisms. Recently, supramolecular building blocks, such as synthetic DNA base-pair mimics, have drawn attention in the context of molecular recognition, self-assembly, and supramolecular polymers. These reversible, hydrogen-bonding interactions have been studied extensively for their adhesive capabilities at the nano- and microscale, however, much less is known about their utility for practical adhesion in macroscopic systems. Herein, we report the preparation and evaluation of supramolecular coupling agents based on high-affinity, high-fidelity quadruple hydrogen-bonding units (e.g., DAN·DeUG, Kassoc = 10(8) M(-1) in chloroform). Macroscopic adhesion between polystyrene films and glass surfaces modified with 2,7-diamidonaphthyridine (DAN) and ureido-7-deazaguanine (DeUG) units was evaluated by mechanical testing. Structure-property relationships indicate that the designed supramolecular interaction at the nanoscale plays a key role in the observed macroscopic adhesive response. Experiments probing reversible adhesion or self-healing properties of bulk samples indicate that significant recovery of initial strength can be realized after failure but that the designed noncovalent interaction does not lead to healing during the process of adhesion loss.

  4. Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

    Directory of Open Access Journals (Sweden)

    Amanda E Siglin

    Full Text Available Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC and the dynactin p150(Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

  5. High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

    Science.gov (United States)

    Vernhes, Emeline; Renouard, Madalena; Gilquin, Bernard; Cuniasse, Philippe; Durand, Dominique; England, Patrick; Hoos, Sylviane; Huet, Alexis; Conway, James F.; Glukhov, Anatoly; Ksenzenko, Vladimir; Jacquet, Eric; Nhiri, Naïma; Zinn-Justin, Sophie; Boulanger, Pascale

    2017-01-01

    Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments. PMID:28165000

  6. [The high-affinity IgE receptor: lessons from structural analysis].

    Science.gov (United States)

    Blank, Ulrich; Jouvin, Marie-Hélène; Guérin-Marchand, Claudine; Kinet, Jean-Pierre

    2003-01-01

    The high affinity receptor for IgE, FcERI, is at the core of the allergic reaction. This receptor is expressed mainly on mast cells and basophils. Interaction of an allergen with its specific IgE bound to FcERI triggers cell activation, which induces the release of numerous mediators that are responsible for allergic manifestations. The recent increase in the prevalence of allergic diseases in developed countries has resulted in renewed efforts towards the development of new drugs. One of these is a humanised antibody directed against the IgE ligand. This antibody recognises specifically free but not FcERI-bound IgE thus preventing ligand binding and subsequent cell activation. This antibody has shown some efficacy in clinical trials involving patients with asthma and allergic rhinitis. The recent elucidation of the tridimensional structure of the complex between IgE and FcERI provides unexpected information regarding the mechanism of assembly of the complex, which now can be used to design small chemical compounds capable of specifically inhibiting this interaction.

  7. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    Energy Technology Data Exchange (ETDEWEB)

    Raza, H.; Chung, W.L.; Mukhtar, H. (Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH (USA))

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  8. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    2013-01-01

    Full Text Available Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB with high affinity (KD values from 2.55 to 36.27 nM. RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p. administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  9. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    Science.gov (United States)

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  10. Sequential administration of the high affinity CXCR4 antagonist BKT140 promotes megakaryopoiesis and platelet production.

    Science.gov (United States)

    Abraham, Michal; Weiss, Ido D; Wald, Hanna; Wald, Ori; Nagler, Arnon; Beider, Katia; Eizenberg, Orly; Peled, Amnon

    2013-10-01

    Platelets are the terminal differentiation product of megakaryocytes (MKs). Cytokines, such as thrombopoietin (TPO), are known to influence different steps in MK development; however, the complex differentiation and platelet localization processes are not fully understood. MKs express the receptor CXCR4 and have been shown to migrate in response to CXCL12 and to increase their platelet production. In this study, we studied the role of CXCR4 in platelet production with the high affinity CXCR4 antagonist, BKT140. Single and sequential administration of BKT140 significantly increased the number of MKs and haematopoietic progenitors (HPCs) within the bone marrow (BM). Increased megakaryopoiesis was associated with increased platelet production. Single and sequential administration of BKT140 also increased the number of HPCs in the blood. In a model of 5-fluorouracil-induced thrombocytopenia, BKT140 significantly reduced the severity and duration of thrombocytopenia and cytopenia when administered before and after chemotherapy. Our results demonstrated that the CXCR4 antagonist, BKT140, mediated unique beneficial effects by stimulating megakaryopoiesis and platelet production. These results provide evidence for the possible therapeutic use of BKT140 for modulating platelet numbers in thrombocytopenic conditions. © 2013 John Wiley & Sons Ltd.

  11. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  12. A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine.

    Science.gov (United States)

    Bray, Lauriane; Moulédous, Lionel; Tafani, Jean A M; Germanier, Maryse; Zajac, Jean-Marie

    2014-05-15

    A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.

  13. High affinity germinal center B cells are actively selected into the plasma cell compartment.

    Science.gov (United States)

    Phan, Tri Giang; Paus, Didrik; Chan, Tyani D; Turner, Marian L; Nutt, Stephen L; Basten, Antony; Brink, Robert

    2006-10-30

    A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.

  14. 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.

  15. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

    Directory of Open Access Journals (Sweden)

    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

  16. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    Science.gov (United States)

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  17. PREPARATION AND CHARACTERISTICS OF ANIONIC POLYACRYLAMIDES CONTAINING DIRECT DYE WITH A HIGH AFFINITY FOR CELLULOSE

    Directory of Open Access Journals (Sweden)

    Shingo Yokota

    2009-05-01

    Full Text Available Direct dye with a high affinity for cellulose substrate was utilized as a cellulose anchor to promote retention of paper strengthening additives under various conditions associated with the wet end of a paper machine. Direct Red 28 (DR was covalently linked to anionic polyacrylamide (A-PAM via a condensation reaction using water-soluble carbodiimide. The DR-conjugated A-PAM (DR-A-PAM demonstrated good retention efficiency, resulting in strength enhancement of handsheets. Anionic trash showed no interference with the performance of DR-A-PAM in the wet end, while the additive performance was sensitive to calcium ions. Surface plasmon resonance analysis gave useful information on the cellulose-anchoring ability of DR-A-PAM. Dye molecules were irreversibly adsorbed onto the cellulose substrate under aqueous conditions, while A-PAM possessed no significant affinity for cellulose. These results suggest that anionic DR moieties in DR-A-PAM molecules served as a cellulose-anchor, possibly due to multiple CH-π interaction between hydrophobic face of cellulose substrate and π-conjugated system of dye molecules. Such a unique interaction of direct dye and cellulose provides a new insight into the wet end system, and does not depend on conventional electrostatic attraction.

  18. Benson's Relaxation Effect in Comparing to Systematic Desensitization on Anxiety of Female Nurses: A Randomized Clinical Trial.

    Science.gov (United States)

    Sajadi, Mahbobeh; Goudarzi, Khatereh; Khosravi, Sharareh; Farmahini-Farahani, Molod; Mohammadbeig, Abolfazl

    2017-01-01

    Nursing staffs expose to a high level of anxiety. This study aimed to compare the effect of Benson's relaxation and systematic desensitization methods for decreasing the anxiety score of nurses. In a randomized clinical trial, 72 female nurses were assigned randomly to three different groups. Benson's relaxation and systematic desensitization were used as intervention beside control group. After intervention, the Spielberger state-trait anxiety inventory was used for measuring the anxiety score. Analysis of variance (ANOVA), Tukey test, and paired t-test were applied for comparing three group scores. The ANOVA test showed that a significant difference among three groups regarding scores of posttrait and poststate anxiety (P < 0.05). The Tukey test showed that both Benson's relaxation and systematic desensitization methods were effective in decreasing of the anxiety score of nurses. Moreover, the mean change in trait and state anxiety scores at Benson's relaxation and systematic desensitization groups was more than control group, respectively, and was statistically significant. Both Benson's relaxation and systematic desensitization methods are effective in improvement of the state and trait dimensions of anxiety. However, these methods could be applied in stressful situation among medical staffs of students.

  19. Using humor in systematic desensitization to reduce fear.

    Science.gov (United States)

    Ventis, W L; Higbee, G; Murdock, S A

    2001-04-01

    Effectiveness of systematic desensitization for fear reduction, using humorous hierarchy scenes without relaxation, was tested. Participants were 40 students highly fearful of spiders. Using a 24-item behavioral approach test with an American tarantula, participants were matched on fear level and randomly assigned to 1 of 3 treatment groups: (a) systematic desensitization, (b) humor desensitization, and (c) untreated controls. Each participant was seen for 6 sessions, including pretest and posttest. Analyses of covariance of posttest scores revealed that the 2 treatment groups showed greater reduction in fear than the controls on 3 measures but did not differ from each other. Therefore, humor in systematic desensitization reduced fear as effectively as more traditional desensitization. This finding may have therapeutic applications; however, it may also be applicable in advertising to desensitize fear of a dangerous product, such as cigarettes.

  20. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K

    2014-01-01

    released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

  1. Desensitization protocol for rituximab-induced serum sickness.

    Science.gov (United States)

    Fajt, Merritt L; Petrov, Andrej A

    2014-01-01

    Rituximab, a chimeric anti-CD20 monoclonal antibody, is used to treat rheumatologic and hematologic diseases. Serum sickness, a Type III delayed hypersensitivity reaction, has been reported with rituximab treatment. Traditionally, drug desensitization has been used to treat Type I IgE-mediated hypersensitivity reactions. We report the first case of successful drug desensitization to rituximab in a patient with medication-induced serum sickness. In our case, a 37-year-old woman with Sjogren's syndrome and papillary thyroid carcinoma developed serum sickness 72 hours following rituximab infusion for gastric mucosal associated lymphoma tissue (MALT). Her MALT progressed after stopping rituximab. She underwent a rapid 12-step intravenous rituximab desensitization without recurrence of serum sickness. Following the completion of 4 rituximab desensitizations, she had gastric MALT remission. She received 25 maintenance rituximab doses using this desensitization protocol quarterly without complications. This is the first report documenting rituximab desensitization for the treatment of delayed drug reactions like serum sickness.

  2. Desensitization: Overcoming the Immunologic Barriers to Transplantation

    Directory of Open Access Journals (Sweden)

    Supreet Sethi

    2017-01-01

    Full Text Available HLA (Human Leucocyte Antigen sensitization is a significant barrier to successful kidney transplantation. It often translates into difficult crossmatch before transplant and increased risk of acute and chronic antibody mediated rejection after transplant. Over the last decade, several immunomodulatory therapies have emerged allowing for increased access to kidney transplantation for the immunologically disadvantaged group of HLA sensitized end stage kidney disease patients. These include IgG inactivating agents, anti-cytokine antibodies, costimulatory molecule blockers, complement inhibitors, and agents targeting plasma cells. In this review, we discuss currently available agents for desensitization and provide a brief analysis of data on novel biologics, which will likely improve desensitization outcomes, and have potential implications in treatment of antibody mediated rejection.

  3. Desensitization: Overcoming the Immunologic Barriers to Transplantation

    Science.gov (United States)

    Choi, Jua; Vo, Ashley; Peng, Alice; Jordan, Stanley C.

    2017-01-01

    HLA (Human Leucocyte Antigen) sensitization is a significant barrier to successful kidney transplantation. It often translates into difficult crossmatch before transplant and increased risk of acute and chronic antibody mediated rejection after transplant. Over the last decade, several immunomodulatory therapies have emerged allowing for increased access to kidney transplantation for the immunologically disadvantaged group of HLA sensitized end stage kidney disease patients. These include IgG inactivating agents, anti-cytokine antibodies, costimulatory molecule blockers, complement inhibitors, and agents targeting plasma cells. In this review, we discuss currently available agents for desensitization and provide a brief analysis of data on novel biologics, which will likely improve desensitization outcomes, and have potential implications in treatment of antibody mediated rejection. PMID:28127571

  4. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  5. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Science.gov (United States)

    Stockner, Thomas; Montgomery, Therese R; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F; Freissmuth, Michael; Sitte, Harald H

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  6. Evaluation of Antibacterial Effectiveness of Desensitizers against Oral Bacteria

    OpenAIRE

    Duran, Ismet; Sengun, Abdulkadir; Hadimli, Hasan Huseyin; Ulker, Mustafa

    2008-01-01

    Objectives Desensitizers contribute to better clinical results by reducing the rate of cervical dentin sensitivity. However, information on their antibacterial effect is limited. This study examined the antibacterial activities of a triclosan containing (Seal & Protect), a benzalconium containing desensitizer (Micro Prime), a fluoride containing prophilaxy paste (Sultan Desensitizer), two fluoride containing varnishes (Cavity Shealth and Ultra EZ), and a dentin bonding primer (All Bond). Meth...

  7. Nursing Care of Patients Undergoing Chemotherapy Desensitization: Part II.

    Science.gov (United States)

    Jakel, Patricia; Carsten, Cynthia; Carino, Arvie; Braskett, Melinda

    2016-04-01

    Chemotherapy desensitization protocols are safe, but labor-intensive, processes that allow patients with cancer to receive medications even if they initially experienced severe hypersensitivity reactions. Part I of this column discussed the pathophysiology of hypersensitivity reactions and described the development of desensitization protocols in oncology settings. Part II incorporates the experiences of an academic medical center and provides a practical guide for the nursing care of patients undergoing chemotherapy desensitization.
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  8. Cutaneous toxicity from epidermal growth factor receptor inhibitors: would a subcutaneous desensitization be helpful? Case report.

    Science.gov (United States)

    D'Alessio, Andrea; Cecchini, Sara; Di Mauro, Daniela; Geroli, Luca; Villa, Simonetta; Quadri, Antonello; Resta, Davide; Fortugno, Carmelo

    2016-11-11

    Cetuximab and panitumumab are monoclonal antibody inhibitors that bind the epidermal growth factor receptor (EGFR) currently used in the treatment of metastatic colorectal cancer. The main adverse event related to EGFR inhibitors (EGFR-Is) is cutaneous toxicity, which can cause dosage reduction and interruption of treatment. State-of-the-art management of skin toxicity associated with EGFR-Is therapy involves the topical administration of corticosteroids and oral antibiotics, but is not completely effective in the management of toxicity. Subcutaneous desensitization with increasing concentrations of monoclonal antibodies can induce a tolerance to drug administration and reduce cutaneous adverse effects. To our knowledge, this is the first case in which a reduction or a disappearance of skin toxicity caused by EGFR-Is through subcutaneous desensitization has been achieved. We present cases of 2 Caucasian patients with adenocarcinoma of the colon treated with EGFR-Is who developed severe cutaneous toxicity. A 73-year-old man presented grade 4 skin toxicity of the face and grade 3 skin toxicity of the trunk during treatment with cetuximab. A 68-year-old woman developed G2 rash on the face after the first administration of cetuximab. These patients underwent subcutaneous desensitization with increasing concentrations of EGFR-Is. After this procedure, patients restarted therapy at the optimal dosage with reduction or disappearance of skin toxicity. These cases suggest that by giving rising doses of antibody it is possible to obtain desensitization able to prevent severe cutaneous adverse events in patients treated with EGFR-Is.

  9. Desensitization in delayed drug hypersensitivity reactions -- an EAACI position paper of the Drug Allergy Interest Group.

    Science.gov (United States)

    Scherer, K; Brockow, K; Aberer, W; Gooi, J H C; Demoly, P; Romano, A; Schnyder, B; Whitaker, P; Cernadas, J S R; Bircher, A J

    2013-07-01

    Drug hypersensitivity may deprive patients of drug therapy, and occasionally no effective alternative treatment is available. Successful desensitization has been well documented in delayed drug hypersensitivity reactions. In certain situations, such as sulfonamide hypersensitivity in HIV-positive patients or hypersensitivity to antibiotics in patients with cystic fibrosis, published success rates reach 80%, and this procedure appears helpful for the patient management. A state of clinical tolerance may be achieved by the administration of increasing doses of the previously offending drug. However, in most cases, a pre-existent sensitization has not been proven by positive skin tests. Successful re-administration may have occurred in nonsensitized patients. A better understanding of the underlying mechanisms of desensitization is needed. Currently, desensitization in delayed hypersensitivity reactions is restricted to mild, uncomplicated exanthems and fixed drug eruptions. The published success rates vary depending on clinical manifestations, drugs, and applied protocols. Slower protocols tend to be more effective than rush protocols; however, underreporting of unsuccessful procedures is very probable. The decision to desensitize a patient must always be made on an individual basis, balancing risks and benefits. This paper reviews the literature and presents the expert experience of the Drug Hypersensitivity Interest Group of the European Academy of Allergy and Clinical Immunology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  11. 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...

  12. 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...

  13. 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)prop...

  14. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  15. Structure-affinity properties of a high-affinity ligand of FKBP12 studied by molecular simulations of a binding intermediate.

    Directory of Open Access Journals (Sweden)

    Lilian Olivieri

    Full Text Available With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant [Formula: see text] of this intermediate should be decreased, resulting in an increase of the affinity constant [Formula: see text]. The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

  16. Desensitized Optimal Filtering and Sensor Fusion Tool Kit Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Research on desensitized optimal filtering techniques and a navigation and sensor fusion tool kit using advanced filtering techniques is proposed. Research focuses...

  17. Prospective Iterative Trial of Proteasome Inhibitor‐Based Desensitization

    National Research Council Canada - National Science Library

    Woodle, E. S; Shields, A. R; Ejaz, N. S; Sadaka, B; Girnita, A; Walsh, R. C; Alloway, R. R; Brailey, P; Cardi, M. A; Abu Jawdeh, B. G; Roy‐Chaudhury, P; Govil, A; Mogilishetty, G

    2015-01-01

    A prospective, iterative trial of five proteasome inhibitor (bortezomib)‐based desensitization regimens demonstrates that these regimens can provide significant reductions in HLA antibodies with substantial durability...

  18. High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

    Science.gov (United States)

    Goldmacher, Victor S; Audette, Charlene A; Guan, Yinghua; Sidhom, Eriene-Heidi; Shah, Jagesh V; Whiteman, Kathleen R; Kovtun, Yelena V

    2015-01-01

    The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

  19. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    Science.gov (United States)

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging.

  20. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    Science.gov (United States)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

    High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.

  1. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    Energy Technology Data Exchange (ETDEWEB)

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James (NIH); (Kansas); (HWMRI)

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

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

    Science.gov (United States)

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

    2016-10-05

    Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [(3)H]-kainate in receptor binding assays (Kd=6.9nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128kDa) and deglycosylated (111kDa) protein, which was identical to the band pattern of native rat brain GluK5. A pharmacological profile of the high-affinity kainate receptor GluK5 is described which is distinct from the profiles of other kainate receptors (GluK1-3). The 27 tested ligands generally show a preferential affinity to GluK1 over GluK5, the exceptions being: dihydrokainate, (S)-5-fluorowillardiine, (S)-glutamate and quisqualate, where the affinity is similar at GluK1 and GluK5. In contrast, quisqualate shows 40-fold higher affinity at GluK5 over GluK3 whereas (S)-1-(2'-amino-2'-caboxyethyl)thienol[3,4-d]pyrimidin-2,4-dione (NF1231), (RS)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2S,4R)-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 disorder), selective GluK5 ligands could have therapeutic potential. The distinct pharmacological profile of GluK5 suggests that it would be possible to design ligands with selectivity towards GluK5.

  3. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    Directory of Open Access Journals (Sweden)

    Klaus B Nissen

    Full Text Available PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  4. In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

    Science.gov (United States)

    Elshafey, Reda; Siaj, Mohamed; Zourob, Mohammed

    2014-09-16

    Contamination of freshwater with cyanotoxin cylindrospermopsin (CYN) represents a significant global concern for public health. The sensitive detection of CYN is necessary to effectively manage and control the treatment of water resources. Here we report a novel, highly sensitive label-free aptasensor for CYN analysis, using aptamers as specific receptors. We have selected the DNA aptamers from a diverse random library using the in vitro screening SELEX approach. The aptamers exhibited high affinity for CYN with Kd of nanomolar range. One aptamer exhibited conformational change upon CYN recognition (CD analysis) and was used to fabricate the label-free impedimetric aptasensor for CYN. A self-assembled monolayer from a disulfide-derivatized aptamer was formed on a gold electrode to fabricate the aptasensor. Upon CYN capturing to the aptasensor surface, a marked drop in the electron transfer resistance was obtained, which was used as the principle of detection of CYN. This resulted from the aptamer's conformational change induced by CYN recognition. The present aptasensor could detect CYN with the limit of detection as low as 100 pM and a wide linear range of 0.1 to 80 nM. When mounted on the gold surface, the aptamer exhibited a lower dissociation constant for CYN than that observed in the fluorescence assay, implying that the anchoring of the aptamer on the Au surface improved its affinity to CYN. Moreover, the aptasensor showed high specificity toward other coexistent cyanobacterial toxins of microcystin-LR and Anatoxin-a. Further biosensor designs will be generated using those aptamers for simple and sensitive CYN monitoring.

  5. The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids.

    Science.gov (United States)

    Lyakhova, Tatyana A; Knight, Jefferson D

    2014-09-01

    Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

  6. Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9.

    Science.gov (United States)

    Heikkilä, Outi; Susi, Petri; Stanway, Glyn; Hyypiä, Timo

    2009-01-01

    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

  7. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    Science.gov (United States)

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-01-01

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 Å cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the “off-target” effect of a small molecule is mediated by an MAI mechanism. PMID:20194791

  8. High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons.

    Science.gov (United States)

    Cosgrove, Kathleen E; Meriney, Stephen D; Barrionuevo, Germán

    2011-12-01

    Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 μM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 μM produced no further decrease in MF EPSC amplitude compared with 20 μM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 μM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40 Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs.

  9. In silico analysis of high affinity potassium transporter (HKT) isoforms in different plants.

    Science.gov (United States)

    Zamani Babgohari, Mahbobeh; Ebrahimie, Esmaeil; Niazi, Ali

    2014-01-01

    High affinity potassium transporters (HKTs) are located in the plasma membrane of the vessels and have significant influence on salt tolerance in some plants. They exclude Na(+) from the parenchyma cells to reduce Na(+) concentration. Despite many studies, the underlying regulatory mechanisms and the exact functions of HKTs within different genomic backgrounds are relatively unknown. In this study, various bioinformatics techniques, including promoter analysis, identification of HKT-surrounding genes, and construction of gene networks, were applied to investigate the HKT regulatory mechanism. Promoter analysis showed that rice HKTs carry ABA response elements. Additionally, jasmonic acid response elements were detected on promoter region of TmHKT1;5. In silico synteny highlighted several unknown and new loci near rice, Arabidopsis thaliana and Physcomitrella patent HKTs, which may play a significant role in salt stress tolerance in concert with HKTs. Gene network prediction unravelled that crosstalk between jasmonate and ethylene reduces AtHKT1;1 expression. Furthermore, antiporter and transferase proteins were found in AtHKT1;1 gene network. Interestingly, regulatory elements on the promoter region of HKT in wild genotype (TmHKT1;5) were more frequent and variable than the ones in cultivated wheat (TaHKT1;5) which provides the possibility of rapid response and better understanding of environmental conditions for wild genotype. Detecting ABA and jasmonic acid response elements on promoter regions of HKTs provide valuable clues on underlying regulatory mechanisms of HKTs. In silico synteny and pathway discovery indicated several candidates which act in concert with HKTs in stress condition. We highlighted different arrangement of regulatory elements on promoter region of wild wheat (TmHKT1;5) compared to bread wheat (TaHKT1;5) in this study.

  10. Taking Advantage: High Affinity B cells in the Germinal Center Have Lower Death Rates, But Similar Rates of Division Compared to Low Affinity Cells1

    OpenAIRE

    2009-01-01

    B lymphocytes producing high affinity antibodies (Abs) are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high affinity B cells are selected during the immune response has never been elucidated. Though it has been shown that high affinity cells directly outcompete low affinity cells in the germinal center (GC)2, whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity c...

  11. Structures of the ultra-high-affinity protein–protein complexes of pyocins S2 and AP41 and their cognate immunity proteins from pseudomonas aeruginosa

    OpenAIRE

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna; Lowe, Edward; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander; Cogdell, Richard; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despit...

  12. Desensitization to Oxcarbazepine: Long-Term Efficacy and Tolerability

    Science.gov (United States)

    Lee, Jiwon; Park, Eu Gene; Lee, Munhyang

    2017-01-01

    Background and Purpose Antiepileptic drug (AED)-associated cutaneous adverse drug reactions can lead to the discontinuation of medications. The aim of this study was to determine the long-term efficacy and safety of performing desensitization to oxcarbazepine. Methods This study involved 20 patients who exhibited cutaneous adverse drug reactions associated with oxcarbazepine use between July 2009 and March 2016 at Samsung Medical Center. All of the participants had to discontinue oxcarbazepine despite presenting initially positive responses. Human leukocyte antigen genotyping was performed to detect the genetic predisposition to Stevens-Johnson syndrome. The desensitization to oxcarbazepine was performed with a starting dosage of 0.1 mg/day. Efficacy was evaluated by comparing the frequency of seizures before and at 1 and 3 years after desensitization. Adverse events occurring during desensitization and the retention rate after desensitization were also investigated. Results Nineteen patients (95%) safely completed the desensitization protocol. One withdrew owing to emotional problems that appeared to be associated with oxcarbazepine. The follow-up period was 4.6±1.2 years (mean±SD), and oxcarbazepine was maintained for more than 3 years after desensitization in 15 patients (83.3%). The response rates were 84.2% and 77.8% at 1 and 3 years after desensitization, respectively. Eight patients remained seizure-free for 3 years, and two discontinued all AEDs. Transient adverse reactions such as mild rash and itching were reported by five patients during desensitization. Conclusions This study has demonstrated the long-term efficacy and safety of desensitization to oxcarbazepine in patients exhibiting cutaneous adverse drug reactions. This favorable outcome should encourage the implementation of desensitization in patients presenting with hypersensitivity to oxcarbazepine as an alternative strategy in clinical practice. PMID:27730770

  13. Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

    Directory of Open Access Journals (Sweden)

    Mirick Gary

    2009-04-01

    Full Text Available Abstract Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL (DvLPBaPPP2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a

  14. Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism.

    Science.gov (United States)

    Smith, Kayla J; Murray, Iain A; Tanos, Rachel; Tellew, John; Boitano, Anthony E; Bisson, William H; Kolluri, Siva K; Cooke, Michael P; Perdew, Gary H

    2011-07-01

    The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a

  15. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  16. Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.

    Science.gov (United States)

    Bortolus, Marco; Bisaglia, Marco; Zoleo, Alfonso; Fittipaldi, Maria; Benfatto, Maurizio; Bubacco, Luigi; Maniero, Anna Lisa

    2010-12-29

    Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

  17. Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

    Science.gov (United States)

    Luo, Dan; Chen, Lei; Yu, Baoping

    2017-06-17

    The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. We report for the first time that CHT1 may be involved

  18. Galvanic Skin Response and Reported Anxiety During Systematic Desensitization

    Science.gov (United States)

    Hyman, Edward T.; Gale, Elliot N.

    1973-01-01

    The purpose of the present study was to investigate the GSR during systematic desensitization. Three groups of females each were preselected for high snake fear. Outcome measures indicated that the desensitization group reduced phobic behavior most, followed by the relaxation group, and then the exposure groups. (Author)

  19. In Vitro Desensitization of Human Skin Mast Cells

    Science.gov (United States)

    Zhao, Wei; Gomez, Gregorio; Macey, Matthew; Kepley, Christopher L.

    2013-01-01

    Desensitization is a clinical procedure whereby incremental doses of a drug are administered over several hours to a sensitive patient until a therapeutic dose and clinical tolerance are achieved. Clinical tolerance may occur in part by attenuating the mast cell response. In the present study, primary human skin mast cells were used to establish and characterize an in vitro model of desensitization. Mast cells in culture were armed with allergen-specific (4-hydroxy-3-nitro-phenylacety and Der p2) and non-specific IgE antibodies, and then desensitized by incremental exposures to 4-hydroxy-3-nitrophenylacety-BSA. This desensitization procedure abrogated the subsequent degranulation response to the desensitizing allergen, to an unrelated allergen, and to IgG anti-FcεRI, but not to C5a, substance P, compound 48/80, and calcium ionophore. Desensitized cells regained their FcεRI-dependent degranulation capability by 24–48 h after free allergen had been removed. Therefore, sensitized human skin mast cells are reversibly desensitized in vitro by exposure to incremental doses of that allergen, which also cross-desensitizes them to an unrelated allergen. PMID:22009002

  20. Desensitization to Mycofenolate Mofetil: a novel 12 step protocol.

    Science.gov (United States)

    Smith, M; Gonzalez-Estrada, A; Fernandez, J; Subramanian, A

    2016-07-01

    The use of MMF has become standard practice in many solid organ transplant recipients due its efficacy and favorable risk profile compared to other immunosuppressants. There has been a single case report of successful MMF desensitization. However, this protocol did not follow current Drug practice parameters. We report a successful desensitization to MMF in a double heart-kidney transplant recipient.

  1. Rapid desensitization with autologous sweat in cholinergic urticaria.

    Science.gov (United States)

    Kozaru, Takeshi; Fukunaga, Atsushi; Taguchi, Kumiko; Ogura, Kanako; Nagano, Tohru; Oka, Masahiro; Horikawa, Tatsuya; Nishigori, Chikako

    2011-09-01

    The majority of patients with cholinergic urticaria presents with strong hypersensitivity to autologous sweat. Patients with severe cholinergic urticaria are frequently resistant to H(1) antagonists which are used in conventional therapies for various types of urticaria. It has been reported that desensitization using partially purified sweat antigen was effective in a patient with cholinergic urticaria. The aim of this study is to determine the usefulness of rapid desensitization with autologous sweat in severe cholinergic urticaria, because rapid desensitization has proven to be a quick and effective immunotherapy for allergies to various allergens. Six patients with severe cholinergic urticaria who are resistant to H(1) antagonists and have sweat hypersensitivity were enrolled in a rapid desensitization protocol. In all six patients, the responses for skin tests with autologous sweat were attenuated after rapid desensitization with autologous sweat. Two of the three cholinergic urticaria patients showed reduced histamine release with autologous sweat after the rapid desensitization with autologous sweat. Further, the rapid desensitization and subsequent maintenance treatment reduced the symptoms in five of the six patients. This study provides evidence that rapid desensitization with autologous sweat is beneficial for treating cholinergic urticaria patients resistant to conventional therapy who have sweat hypersensitivity.

  2. Hypnosis Versus Systematic Desensitization in the Treatment of Test Anxiety

    Science.gov (United States)

    Melnick, Joseph; Russell, Ronald W.

    1976-01-01

    This study compared the effectiveness of systematic desensitization and the directed experience hypnotic technique in reducing self-reported test anxiety and increasing the academic performance of test-anxious undergraduates (N=36). The results are discussed as evidence for systematic desensitization as the more effective treatment in reducing…

  3. Desensitization in patients with beta-lactam drug allergy.

    Science.gov (United States)

    Yusin, J S; Klaustermeyer, W; Simmons, C W; Baum, M

    2013-01-01

    Patients with a history of beta-lactam antibiotic allergy are often admitted to the hospital with severe or life-threatening infections requiring beta-lactam antibiotics. Strict avoidance of beta lactams to such patients may prevent them from getting adequate coverage and can lead to an increase in the use of alternative antibiotics, which can predispose to antibiotic resistance. Past studies revealed a lower incidence of pen allergy then patients' histories suggest. Fortunately today, there are three options for patients presenting with a history of beta-lactam allergy. Penicillin skin testing, beta-lactam challenge or beta-lactam desensitization. Recently Pre Pen has been FDA re-approved and when combined with Pen G is a valid way to determine if patients are able to tolerate beta-lactam antibiotic. When these agents are not available one must decide about desensitization or challenge. When a patient has a positive penicillin skin test, desensitization or beta-lactam avoidance are the only options. This paper reviews the safety of beta-lactam desensitization. To perform a chart review on patients desensitised with beta lactam to determine if desensitizations can be performed safely without minimal complications. A retrospective chart review was performed on allergy and immunology inpatient consultations for beta-lactam desensitization between September 2003 and August 2006 at the Cedars-Sinai Medical Centre in Los Angeles. Patient data and outcomes of desensitization were analysed. A total of 13 intravenous desensitizations were performed on 12 patients. The patients consisted of eight females and four males with an average age of 65 years. Age range was 36-92 years old. All 13 intravenous desensitizations were completed without complications. No patient required a slower rate of desensitization or discontinuance of the desensitization. Patients were able to tolerate the initial therapeutic dose of their beta-lactam antibiotic and were then able to complete full

  4. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

  5. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    Science.gov (United States)

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  6. 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......, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high...... density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using (3)H-HOCPCA we analyzed the GHB binding protein profile during mouse...

  7. Bladder sensory desensitization decreases urinary urgency

    Directory of Open Access Journals (Sweden)

    Avelino António

    2007-06-01

    Full Text Available Abstract Background Bladder desensitization has been investigated as an alternative treatment for refractory detrusor overactivity. Most open and controlled clinical trials conducted with intravesical RTX showed that desensitization delays the appearance of involuntary detrusor contractions during bladder filling and decreases the number of episodes of urgency incontinence. Urgency is being recognised as the fundamental symptom of overactive bladder (OAB, a symptomatic complex which recent epidemiological studies have shown to affect more than 10% of the Western population. As anti-muscarinic drugs, the first line treatment for OAB, are far from being able to fully control urgency, the opportunity to test other therapeutic approaches is created. The present work was, therefore, designed as an exploratory investigation to evaluate the effect of bladder desensitization on urinary urgency. Methods Twenty-three OAB patients with refractory urgency entered, after given informed consent, a 30 days run-in period in which medications influencing the bladder function were interrupted. At the end of this period patients filled a seven-day voiding chart where they scored, using a 0–4 scale, the bladder sensations felt before each voiding. Then, patients were instilled with 100 ml of 10% ethanol in saline (vehicle solution and 30 days later a second seven-day voiding chart was collected. Finally, patients were instilled with 100 ml of 50 nM RTX in 10% ethanol in saline. At 1 and 3 months additional voiding charts were collected. At the end of the vehicle and 3 months period patients were asked to give their subjective impression about the outcome of the treatment and about the willingness to repeat the previous instillation. Results At the end of the run-in period the mean number of episodes of urgency per week was 71 ± 12 (mean ± SEM. After vehicle instillation, the mean number of episodes of urgency was 56 ± 11, but only 4 patients (17% considered

  8. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    Science.gov (United States)

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-01-28

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  9. Immunotherapy Expands and Maintains the Function of High-Affinity Tumor-Infiltrating CD8 T Cells In Situ.

    Science.gov (United States)

    Moran, Amy E; Polesso, Fanny; Weinberg, Andrew D

    2016-09-15

    Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.

  10. α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

    DEFF Research Database (Denmark)

    Absalom, N.; Karim, N.; Eghorn, L.F.;

    2012-01-01

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinit...... and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism....

  11. Soluble T Cell Receptor Vβ Domains Engineered for High-Affinity Binding to Staphylococcal or Streptococcal Superantigens

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    2014-01-01

    Full Text Available Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs, so-called because they stimulate a large fraction of an individual’s T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR on a T cell and a class II product of the major histocompatibility complex (MHC on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1 or S. pyogenes (SpeA and SpeC have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  12. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    Science.gov (United States)

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  13. Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.

    Directory of Open Access Journals (Sweden)

    Huaying Zhao

    Full Text Available Sedimentation velocity analytical ultracentrifugation (SV is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.

  14. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.

  15. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    Science.gov (United States)

    Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

    2015-12-01

    During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

  16. Desensitization for Peanut Allergies in Children

    Science.gov (United States)

    Jhamnani, Rekha D.

    2016-01-01

    Opinion Statement Immunotherapy for peanut allergy has been an exploding topic of study within the last few years. Sublingual, epicutaneous, and oral immunotherapy are being investigated and show promise in the treatment of peanut allergy. Oral immunotherapy has shown the most clinical benefit; however, sublingual and epicutaneous immunotherapy appear to have the most favorable safety profiles. Most studies to date suggest that only a minority of subjects achieve sustained unresponsiveness to peanut after discontinuation of immunotherapy. Recent efforts have been focused on identifying adjunct therapies, such as omalizumab, that may assist patients in achieving peanut desensitization more quickly and with greater success. Several underlying immunologic mechanisms, including a switch from IgE to IgG4 production and induction of T regulatory cells, have been studied although more research is needed to identify reliable biomarkers. This article will describe the immunotherapy approaches that are being investigated to induce peanut desensitization, and highlight the benefits and risks of these therapies that need to be considered before they are ready for routine clinical practice. PMID:28042528

  17. Discovery of high affinity ligands for β2-adrenergic receptor through pharmacophore-based high-throughput virtual screening and docking.

    Science.gov (United States)

    Yakar, Ruya; Akten, Ebru Demet

    2014-09-01

    Novel high affinity compounds for human β2-adrenergic receptor (β2-AR) were searched among the clean drug-like subset of ZINC database consisting of 9,928,465 molecules that satisfy the Lipinski's rule of five. The screening protocol consisted of a high-throughput pharmacophore screening followed by an extensive amount of docking and rescoring. The pharmacophore model was composed of key features shared by all five inactive states of β2-AR in complex with inverse agonists and antagonists. To test the discriminatory power of the pharmacophore model, a small-scale screening was initially performed on a database consisting of 117 compounds of which 53 antagonists were taken as active inhibitors and 64 agonists as inactive inhibitors. Accordingly, 7.3% of the ZINC database subset (729,413 compounds) satisfied the pharmacophore requirements, along with 44 antagonists and 17 agonists. Afterwards, all these hit compounds were docked to the inactive apo form of the receptor using various docking and scoring protocols. Following each docking experiment, the best pose was further evaluated based on the existence of key residues for antagonist binding in its vicinity. After final evaluations based on the human intestinal absorption (HIA) and the blood brain barrier (BBB) penetration properties, 62 hit compounds have been clustered based on their structural similarity and as a result four scaffolds were revealed. Two of these scaffolds were also observed in three high affinity compounds with experimentally known Ki values. Moreover, novel chemical compounds with distinct structures have been determined as potential β2-AR drug candidates.

  18. Biological effects of exogenous adenosine 5 prime -triphosphate on cultured mammalian cells: Evidence for a receptor mechanism and its regulation by desensitization

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, F.A.

    1989-01-01

    Exogenous adenosine 5{prime}-triphosphate (ATP) mobilized intracellular calcium in human carcinoma A43l cells and in Swiss 3T3 and 3T6 mouse fibroblasts by increasing inositol trisphosphate similar to well down growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bradykinin (BK), serum). Calcium mobilization was examined by video imaging of fura-2 fluorescence is single cells, following the radioactive isotope {sup 45}Ca, and monitoring the decrease in fluorescence of cells loaded with chlortetracycline. Uridine 5{prime}-triphosphate, but not other nucleotides, mimicked ATP. Single-cell analysis revealed synchronous responses in 10 sec to ATP, BK or serum, while PDGF (3T3) and EGF (A431) produced slower signals with significant cell-to-cell variation. PDGF desensitized 3T3 cells to ATP and BK added 100 sec later but ATP or BK did not desensitized to PDGF. Homologous desensitization was seen with all agonists. Heterologous desensitization was also observed in A431 cells where ATP desensitized to serum, but serum did not to ATP. ATP-stimulated calcium entry was detected after 10 sec in A431 cells, but not in Swiss 3T6 cells. Entry started before significant efflux had occurred and did not fit the capacitance model of Putney. A 2-3 hr ATP pretreatment produced a homologous desensitization state that required 20 hr to disappear, probably due to down-regulation of the putative ATP receptors.

  19. MACV/Radio integrated navigation for Mars powered descent via robust desensitized central difference Kalman filter

    Science.gov (United States)

    Lou, Taishan; Liu, Jie; Jin, Pan; Wang, Yan

    2017-01-01

    An innovative integrated navigation scheme based on MCAV/Radio measurement information during Mars powered descent phase, and a robust desensitized central difference Kalman filter (DCDKF) for systems with uncertain parameters or biases are proposed to improve the navigation descent accuracy. Based on the altitude and velocity information of the Miniature Coherent Altimeter and Velocimeter (MCAV), the radio-range information is added into the integrated navigation system to correct the horizontal position error of the vehicle during the Mars powered descent phase. Based the central difference transform, the sensitivity propagation of the state estimate errors in the DCDKF is described, and a designed desensitized cost function is minimized to obtain the gain matrix of the DCDKF. The performances of the innovative navigation scheme and the proposed DCDKF are all demonstrated by two Monte Carlo simulations with the Inertial Measurement Unit biases during the Mars power descent phase.

  20. μ-Opioid receptor desensitization: homologous or heterologous?

    Science.gov (United States)

    Llorente, Javier; Lowe, Janet D; Sanderson, Helen S; Tsisanova, Elena; Kelly, Eamonn; Henderson, Graeme; Bailey, Chris P

    2012-12-01

    There is considerable controversy over whether μ-opioid receptor (MOPr) desensitization is homologous or heterologous and over the mechanisms underlying such desensitization. In different cell types MOPr desensitization has been reported to involve receptor phosphorylation by various kinases, including G-protein-coupled receptor kinases (GRKs), second messenger and other kinases as well as perturbation of the MOPr effector pathway by GRK sequestration of G protein βγ subunits or ion channel modulation. Here we report that in brainstem locus coeruleus (LC) neurons prepared from relatively mature rats (5-8 weeks old) rapid MOPr desensitization induced by the high-efficacy opioid peptides methionine enkephalin and DAMGO was homologous and not heterologous to α(2)-adrenoceptors and somatostatin SST(2) receptors. Given that these receptors all couple through G proteins to the same set of G-protein inwardly rectifying (GIRK) channels it is unlikely therefore that in mature neurons MOPr desensitization involves G protein βγ subunit sequestration or ion channel modulation. In contrast, in slices from immature animals (less than postnatal day 20), MOPr desensitization was observed to be heterologous and could be downstream of the receptor. Heterologous MOPr desensitization was not dependent on protein kinase C or c-Jun N-terminal kinase activity, but the change from heterologous to homologous desensitization with age was correlated with a decrease in the expression levels of GRK2 in the LC and other brain regions. The observation that the mechanisms underlying MOPr desensitization change with neuronal development is important when extrapolating to the mature brain results obtained from experiments on expression systems, cell lines and immature neuronal preparations.

  1. Immediate Desensitization in Teeth Affected by Amelogenesis Imperfecta.

    Science.gov (United States)

    Moreira, Rudá França; Figueiredo, Rossana Gomes; Oliveira, Henrique Eduardo; Fonseca, Ana Christina Lamosa da; Miranda, Mauro Sayão de

    2016-01-01

    The aim of this paper was to describe a clinical case of immediate dental desensitization using a self-etch adhesive system in an adolescent patient diagnosed with amelogenesis imperfecta (AI). AI was associated with severe tooth sensitivity, treated by the application of a universal adhesive system for desensitization of the teeth affected by AI. Reduction of tooth sensitivity was assessed using a visual analog scale during all reevaluations. The technique was effective for reducing tooth sensitivity. It was concluded that the adhesive system for tooth desensitization had an immediate effect and maintained its effectiveness during a 12-month follow-up period.

  2. Outpatient desensitization in selected patients with platinum hypersensitivity reactions.

    Science.gov (United States)

    O'Malley, David M; Vetter, Monica Hagan; Cohn, David E; Khan, Ambar; Hays, John L

    2017-06-01

    Platinum-based chemotherapies are a standard treatment for both initial and recurrent gynecologic cancers. Given this widespread use, it is important to be aware of the features of platinum hypersensitivity reactions and the subsequent treatment of these reactions. There is also increasing interest in the development of desensitization protocols to allow patients with a history of platinum hypersensitivity to receive further platinum based therapy. In this review, we describe the management of platinum hypersensitivity reactions and the desensitization protocols utilized at our institution. We also describe the clinical categorizations utilized to triage patients to appropriate desensitization protocols. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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

  4. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    Science.gov (United States)

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  5. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

    Science.gov (United States)

    Stagg, Amy R.; Fleming, Judith C.; Baker, Meghan A.; Sakamoto, Massayuki; Cohen, Nadine; Neufeld, Ellis J.

    1999-01-01

    We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400–550 nM; Vmax 11 pmol/min/106 cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death. J. Clin. Invest. 103:723–729 (1999). PMID:10074490

  6. 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.

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

    Science.gov (United States)

    Klein, A B; Bay, T; Villumsen, I S; Falk-Petersen, C B; Marek, A; Frølund, B; Clausen, R P; Hansen, H D; Knudsen, G M; Wellendorph, P

    2016-11-01

    GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB 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, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using (3)H-HOCPCA we analyzed the GHB binding protein profile during mouse 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 the commonly used radioligand (3)H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

  8. 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.

  9. (/sup 3/H)pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex

    Energy Technology Data Exchange (ETDEWEB)

    Watson, M.; Roeske, W.R.; Yamamura, H.I.

    1982-11-01

    The specific binding of (/sup 3/H)pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of (/sup 3/H)pirenzepine in the cerebral cortex as defined by displacement with atropine sulfate (1..mu..M) was of high affinity (K/sub d/ = 4-10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the muscarinic receptor. In contrast, few (/sup 3/H)pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.

  10. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    OpenAIRE

    Liu, F T; Albrandt, K; Robertson, M W

    1988-01-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, w...

  11. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin.

    Science.gov (United States)

    Ryan, Ali J; Chung, Chun-Wa; Curry, Stephen

    2011-04-18

    Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which--in drugs sites 1 and 2 on the protein--are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  12. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    Directory of Open Access Journals (Sweden)

    Chung Chun-wa

    2011-04-01

    Full Text Available Abstract Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA. It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  13. (TH)205-501, a non-catechol dopaminergic agonist, labels selectively and with high affinity dopamine D2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Closse, A.; Frick, W.; Markstein, R.; Maurer, R.; Nordmann, R.

    1985-01-01

    (TH)205-501, a non dopaminergic agonist, is presented as a ligand with high affinity (Ksub(D) approx= 1 nM) and high selectivity for dopamine receptors. pKsubi values of dopaminergic agonists derived from competition isotherms in the (TH)205-501 binding assay correlate very well with their potency in the acetylcholine release assay, which is controlled by dopamine D2 receptors. There is however no correlation with their potency stimulating aldenylate cyclase, a process controlled by dopamine D1 receptors. Thus (TH)205-501 is the first agonist ligand selective for dopamine D2 receptors. (Author).

  14. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    Energy Technology Data Exchange (ETDEWEB)

    Steingroewer, Juliane [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)]. E-mail: juliane.steingroewer@tu-dresden.de; Bley, Thomas [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany); Bergemann, Christian [Chemicell GmbH, D-10823, Berlin (Germany); Boschke, Elke [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)

    2007-04-15

    Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

  15. Contribution of Pretesting to Several Measures of Semantic Desensitization Effectiveness

    Science.gov (United States)

    Israel, Allen C.; And Others

    1977-01-01

    Snake- or spider-phobic subjects (N=32) were randomly assigned to one of four groups. Subjects receiving semantic desensitization therapy showed less posttest anxiety on the semantic differential than control subjects regardless of testing condition. (Author)

  16. Desensitized Optimal Filtering and Sensor Fusion Tool Kit Project

    Data.gov (United States)

    National Aeronautics and Space Administration — It is proposed to develop desensitized optimal filtering techniques and to implement these algorithms in a navigation and sensor fusion tool kit. These proposed...

  17. A Group Desensitization Approach to Public Speaking Anxiety

    Science.gov (United States)

    Akin, Clifford; Kunzman, Glen G.

    1974-01-01

    Program evaluation shows that a mixed behavioral format emphasizing practice situations produces results similar to traditionally operant or desensitization formats, with less attrition in attendance. Statistically significant changes on fear inventory scores is the measurable outcome of the program. (Author)

  18. [Adaptive desensitization for acetylsalicylic acid hypersensitivity: A success story?].

    Science.gov (United States)

    Mühlmeier, G; Hausch, R; Maier, H

    2015-10-01

    Adaptive desensitization still remains the only causative therapy for acetylsalicylic acid (ASA) hypersensitivity and is carried out nearly worldwide. To date there are hardly any data available on disease development under current desensitization therapy and longitudinal data in particular are missing. Out of a large collective of patients with proven hypersensitivity to ASA, 194 patients with initiated desensitization treatment were observed for periods up to 5 years (average 32 months). Patients with immediate reactions to systemic challenge tests revealed a response rate of 77% after 12 months of therapy. In this period 12% reached complete remission, 38% showed a clear reduction in symptoms, 32% reached partial remission, 13% remained unchanged and 5% suffered from disease progression. Adaptive desensitization therapy for hypersensitivity to ASA has been shown to be an effective causative therapy and chronic hyperplastic sinusitis as well as bronchial asthma could be improved. For the determination of maintenance dosages and required time periods more data are needed.

  19. Nursing Care of Patients Undergoing Chemotherapy Desensitization: Part I.

    Science.gov (United States)

    Jakel, Patricia; Carsten, Cynthia; Braskett, Melinda; Carino, Arvie

    2016-02-01

    Hypersensitivity reactions to chemotherapeutic agents can cause the discontinuation of first-line therapies. Chemotherapy desensitization is a safe, but labor-intensive, process to administer these important medications. A desensitization protocol can enable a patient to receive the entire target dose of a medication, even if the patient has a history of severe infusion reactions. In this article, the authors explain the pathophysiology of hypersensitivity reactions and describe the recent development of desensitization protocols in oncology. In part II of this article, which will appear in the April 2016 issue of the Clinical Journal of Oncology Nursing, the authors will give a detailed account of how a desensitization protocol is performed at an academic medical center.
.

  20. Receptor desensitization by neurotransmitters in membranes: are neurotransmitters the endogenous anesthetics?

    Science.gov (United States)

    Cantor, Robert S

    2003-10-21

    A mechanism of anesthesia is proposed that addresses one of the most troubling peculiarities of general anesthesia: the remarkably small variability of sensitivity within the human population and across a broad range of animal phyla. It is hypothesized that in addition to the rapid, saturable binding of a neurotransmitter to its receptor that results in activation, the neurotransmitter also acts indirectly on the receptor by diffusing into the postsynaptic membrane and changing its physical properties, causing a shift in receptor conformational equilibrium (desensitization). Unlike binding, this slower indirect mechanism is nonspecific: each neurotransmitter will, in principle, affect all receptors in the membrane. For proteins modeled as having only resting and active conformational states, time-dependent ion currents are predicted that exhibit many characteristics of desensitization for both inhibitory and excitatory channels. If receptors have been engineered to regulate the time course of ion currents by this mechanism, then (a) mutations that significantly alter receptor sensitivity to this effect would be lethal and (b) by design, excitatory receptors would be inhibited, but inhibitory receptors activated, so that their effects are not counterproductive. The wide range of exogenous molecules that affect the physical properties of membranes as do neurotransmitters, but that do not bind to receptors, would thus inhibit excitatory channels and activate inhibitory channels, i.e., they would act as anesthesics. The endogenous anesthetics would thus be the neurotransmitters, the survival advantage conferred by their proper membrane-mediated desensitization of receptors explaining the selection pressure for anesthesic sensitivity.

  1. Immediate Desensitization in Teeth Affected by Amelogenesis Imperfecta

    OpenAIRE

    Moreira,Rudá França; Figueiredo,Rossana Gomes; Oliveira,Henrique Eduardo de; Fonseca,Ana Christina Lamosa da; Miranda,Mauro Sayão de

    2016-01-01

    Abstract The aim of this paper was to describe a clinical case of immediate dental desensitization using a self-etch adhesive system in an adolescent patient diagnosed with amelogenesis imperfecta (AI). AI was associated with severe tooth sensitivity, treated by the application of a universal adhesive system for desensitization of the teeth affected by AI. Reduction of tooth sensitivity was assessed using a visual analog scale during all reevaluations. The technique was effective for reducing...

  2. Desensitization for solid organ and hematopoietic stem cell transplantation.

    Science.gov (United States)

    Zachary, Andrea A; Leffell, Mary S

    2014-03-01

    Desensitization protocols are being used worldwide to enable kidney transplantation across immunologic barriers, i.e. antibody to donor HLA or ABO antigens, which were once thought to be absolute contraindications to transplantation. Desensitization protocols are also being applied to permit transplantation of HLA mismatched hematopoietic stem cells to patients with antibody to donor HLA, to enhance the opportunity for transplantation of non-renal organs, and to treat antibody-mediated rejection. Although desensitization for organ transplantation carries an increased risk of antibody-mediated rejection, ultimately these transplants extend and enhance the quality of life for solid organ recipients, and desensitization that permits transplantation of hematopoietic stem cells is life saving for patients with limited donor options. Complex patient factors and variability in treatment protocols have made it difficult to identify, precisely, the mechanisms underlying the downregulation of donor-specific antibodies. The mechanisms underlying desensitization may differ among the various protocols in use, although there are likely to be some common features. However, it is likely that desensitization achieves a sort of immune detente by first reducing the immunologic barrier and then by creating an environment in which an autoregulatory process restricts the immune response to the allograft. © 2014 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

  3. Linezolid desensitization for a patient with multiple medication hypersensitivity reactions.

    Science.gov (United States)

    Bagwell, Autumn D; Stollings, Joanna L; White, Katie D; Fadugba, Olajumoke O; Choi, Jane J

    2013-01-01

    To describe a case in which a linezolid desensitization protocol was successfully used for a polymicrobial surgical wound infection in a patient with multiple drug hypersensitivity reactions. A 24-year-old woman with vocal cord dysfunction requiring tracheostomy was admitted for a surgical wound infection following a tracheostomy fistula closure procedure. The patient reported multiple antibiotic allergies including penicillins (rash), sulfonamides (rash), vancomycin (anaphylaxis), azithromycin (rash), cephalosporins (anaphylaxis), levofloxacin (unspecified), clindamycin (unspecified), and carbapenems (unspecified). Gram stain of the purulent wound drainage demonstrated mixed gram-negative and gram-positive flora, and bacterial cultures were overgrown with Proteus mirabilis, which precluded identification of other pathogens. Following failed test doses of linezolid, tigecycline, and daptomycin, all of which resulted in hypersensitivity reactions, a 16-step linezolid desensitization protocol was developed and successfully implemented without adverse reactions. The patient completed a 2-week course of antibiotic therapy that included linezolid upon finishing the desensitization protocol. Linezolid is useful in treating complicated and uncomplicated skin and soft tissue infections caused by gram-positive bacteria. With precautions, including premedication, a monitored nursing unit, and immediate availability of an emergency anaphylaxis kit, drug desensitization allows patients the ability to safely use medications to which they may have an immediate hypersensitivity reaction. Minimal data exist on linezolid desensitization protocols. Linezolid desensitization can be a viable option in patients requiring antimicrobial therapy for complicated gram-positive skin infections.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-11-01

    Specific binding of /sup 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.

  5. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Milne, J.L.; Coukell, M.B. (York Univ., North York, Ontario (Canada))

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  6. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    Science.gov (United States)

    Liu, F T; Albrandt, K; Robertson, M W

    1988-08-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

  7. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Zhang, Xian; Sun, Mengjiao; Kang, Yue; Xie, Hui; Wang, Xin; Song, Houhui; Li, Xiaoliang; Fang, Weihuan

    2015-11-01

    Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 × 10(9) L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y = -0.6076x + 0.2441, R(2) = 0.9923) with the working range from 2.4 to 23.6 μg/kg, IC50 at 7.6 μg/kg and lower limit of detection at 1.4 μg/kg. The recovery rates in spiked samples were 91.2-110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R(2) = 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA.

  8. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    Science.gov (United States)

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  9. Novel and High Affinity 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues as Atypical Dopamine Transporter Inhibitors

    DEFF Research Database (Denmark)

    Cao, Jianjing; Slack, Rachel D.; Bakare, Oluyomi M.

    2016-01-01

    pharmacological profiles in animal models of cocaine and methamphetamine abuse. Herein, we report a series of modafinil analogues that have an atypical DAT inhibitor profile. We extended SAR by chemically manipulating the oxidation states of the sulfoxide and the amide functional groups, halogenating the phenyl...

  10. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo

    National Research Council Canada - National Science Library

    Yu, Channing; Cantor, Alan B; Yang, Haidi; Browne, Carol; Wells, Richard A; Fujiwara, Yuko; Orkin, Stuart H

    2002-01-01

    .... Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss...

  11. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed......γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...

  12. Eye Movement Desensitization and Reprocessing Integrating with Ego State Therapy for Clients with Trauma Disorders (review)%眼动脱敏再加工与自我状态疗法在创伤障碍性疾病治疗中的整合

    Institute of Scientific and Technical Information of China (English)

    单敬; 田宝; 冯孟哲

    2011-01-01

    Post-traumatic stress disorder (PTSD) has long been clinical practice to use a three-part phased model in the treatment of trauma and the dissociative disorders.The phased treatment is an intrinsic element of the model presented throughout this article, which integrates the eye movement desensitization and reprocessing (EMDR) standard protocol and additional components:ego state therapy, attachment theory, and dissociative disorder treatment interventions.Although the researches on this model are still in its infancy, it is the current standard of care because of its efficacy with traumatized and dissociative-disordered clients.%创伤后应激障碍(PTSD)一直以来临床上采用三阶段疗法诊治创伤、分离性及其他障碍性疾病.三阶段疗法整合了眼动脱敏与再加工(EMDR)诊疗标准以及其他技术方法,包括自我状态治疗等.虽然三阶段疗法的机理研究刚刚起步,但是由于对创伤性、分离性障碍疗效显著,已成为目前的标准治疗方法.

  13. Organogel-assisted topochemical synthesis of multivalent glyco-polymer for high-affinity lectin binding.

    Science.gov (United States)

    Krishnan, Baiju P; Raghu, Sreedevi; Mukherjee, Somnath; Sureshan, Kana M

    2016-12-01

    An organogelator, 2,4-undeca-diynyl-4',6'-O-benzylidene-β-d-galactopyranoside, which aligns its diacetylene upon gelation, has been synthesized. UV irradiation of its gel resulted in topochemical polymerization of the gelator forming polydiacetylene (PDA). We have used this gel-state reaction for the synthesis of surface-immobilized multi-valent glycoclusters, which showed 1000-fold enhanced binding, compared to monomers, with various galactose-binding lectins.

  14. GABAρ1/GABAAα1 receptor chimeras to study receptor desensitization

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Demuro, Angelo; Miledi, Ricardo

    2000-01-01

    γ-Aminobutyrate type C (GABAC) receptors are ligand-gated ion channels that are expressed preponderantly in the vertebrate retina and are characterized, among other things, by a very low rate of desensitization and resistance to the specific GABAA antagonist bicuculline. To examine which structural elements determine the nondesensitizing character of the human homomeric ρ1 receptor, we used a combination of gene chimeras and electrophysiology of receptors expressed in Xenopus oocytes. Two chimeric genes were constructed, made up of portions of the ρ1-subunit and of the α1-subunit of the GABAA receptor. When expressed in Xenopus oocytes, one chimeric gene (ρ1/α1) formed functional homooligomeric receptors that were fully resistant to bicuculline and were blocked by the specific GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and by zinc. Moreover, these chimeric receptors had a fast-desensitizing component, even faster than that of heterooligomeric GABAA receptors, in striking contrast to the almost nil desensitization of wild-type ρ1 (wt ρ1) receptors. To see whether the fast-desensitizing characteristic of the chimera was determined by the amino acids forming the ion channels, we replaced the second transmembrane segment (TM2) of ρ1 by that of the α1-subunit of GABAA. Although the α1-subunit forms fast-desensitizing receptors when coexpressed with other GABAA subunits, the sole transfer of the α1TM2 segment to ρ1 was not sufficient to form desensitizing receptors. All this suggests that the slow-desensitizing trait of ρ1 receptors is determined by a combination of several interacting domains along the molecule. PMID:10725369

  15. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

    Science.gov (United States)

    Ryndak, Michelle B; Wang, Shuishu; Smith, Issar; Rodriguez, G Marcela

    2010-02-01

    Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

  16. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    Science.gov (United States)

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  17. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    Science.gov (United States)

    Himmel, Mirko; Ritter, Anett; Rothemund, Sven; Pauling, Björg V; Rottner, Klemens; Gingras, Alexandre R; Ziegler, Wolfgang H

    2009-05-15

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

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

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte

    2002-01-01

    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......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...... 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...

  19. The occurrence and production of avidin: a new conception of the high-affinity biotin-binding protein.

    Science.gov (United States)

    Elo, H A; Korpela, J

    1984-01-01

    The production of avidin, a high-affinity biotin-binding egg-white protein, is not restricted to the avian, amphibian and reptilian oviducts. In the acute phase of inflammation, avidin is synthesized and secreted by various injured tissues in the domestic fowl, both male and female. Also in other avian species and a lizard, injured tissues produce an avidin-like biotin-binding factor. The non-oviductal production of avidin in domestic fowl has a great variety of inducers, for example acute inflammation caused by mechanical or thermal tissue injury, septic bacterial infection and (toxic) drugs, and even retrovirus-induced cell transformation. In culture, chicken embryo fibroblasts and yolk sac macrophages synthesize and secrete avidin. Besides the albumen, avidin may act as an antibacterial protein also in the tissues.

  20. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    Science.gov (United States)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  1. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  2. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R

    2005-01-01

    acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB...... analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported......Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites...

  3. 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.

  4. Emerging Approaches to Food Desensitization in Children.

    Science.gov (United States)

    Hamad, Ahmad; Burks, Wesley A

    2017-05-01

    The purpose of this review is to highlight the recent advances in food desensitization in children with food allergy. Recent advancements in epicutaneous, sublingual, and oral immunotherapy for food allergy in the future may offer children with food allergy and their families a viable option to reduce risk or severity of anaphylaxis with phase III trials ongoing for two of these treatment modalities. Food allergy prevalence in children is estimated to be up to 8%. These children are at risk of significant allergic reactions and anaphylaxis. Food avoidance and use of antihistamines or epinephrine has been the standard of care for these patients. This approach also has a significant socioeconomic effects on patients and their families. Recent advancements in understanding food allergy have allowed for exploring new methods of treatment. There is an increasing interest in oral immunotherapy, epicutaneous immunotherapy, or sublingual immunotherapy for food allergy. There have been also innovative approaches to immunotherapy by modification of food allergens (to make them less allergenic while maintain their immunogenicity) or adding adjunctive treatments (probiotics, anti-IgE, etc.) to increase efficacy or safety.

  5. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng [Michigan; (HHMI)

    2013-02-12

    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.

  6. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    Science.gov (United States)

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

  7. 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

    Full Text Available BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

  8. 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 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.

  9. 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

    2016-01-01

    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 (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 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. PMID:27754349

  10. 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.

  11. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. (Genentech, Inc., South San Francisco, CA (USA))

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  12. Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

    Science.gov (United States)

    Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

    2012-10-01

    The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

  13. Sertraline and its metabolite desmethylsertraline, but not bupropion or its three major metabolites, have high affinity for P-glycoprotein.

    Science.gov (United States)

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-02-01

    The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood-brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertraline, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic P(i) in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis-Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The V(max)/K(m) values of sertraline (1.6 min(-1) x 10(-3)) and desmethylsertraline (1.4 min(-1) x 10(-3)) were comparable with that of verapamil (1.7 min(-1) x 10(-3)). Bupropion and its three metabolites showed very weak affinity for P-gp, with V(max)/K(m) values lower than 0.01 min(-1) x 10(-3). The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug-drug interactions among antidepressants.

  14. Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

    Directory of Open Access Journals (Sweden)

    Gauri R Nair

    Full Text Available Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3' were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV and avian myeloblastosis virus (AMV and one retrotransposon (Ty3 RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'. In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3' showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

  15. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

    Science.gov (United States)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R; de Lichtenberg, Anne; Nielsen, Birgitte; Frølund, Bente; Brehm, Lotte; Clausen, Rasmus P; Bräuner-Osborne, Hans

    2005-10-01

    Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.

  16. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    Science.gov (United States)

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  17. Desensitization to clopidogrel: a tailor-made protocol.

    Science.gov (United States)

    Barreira, P; Cadinha, S; Malheiro, D; Moreira da Silva, J P

    2014-01-01

    Clopidogrel is an antiplatelet drug widely used for treatment and prevention of a variety of cardiovascular diseases. We report a successful desensitization to clopidogrel in a 70-year-old Caucasian man with delayed hypersensitivity (HS) reaction. He developed lip, hand and foot swelling, erythematous papular non-pruritic lesions and arthralgias 2 weeks after starting treatment with clopidogrel 75 mg/d. A 3-hour desensitization protocol was started, achieving a cumulative dose of 154 mg without any reaction, and a daily dose of 75 mg was recommended. On the 4th day, the patient developed skin lesions similar to the previously described. He was treated with topical steroids and oral antihistamines, and the daily dose of clopidogrel was reduced to 20 mg. A new desensitization protocol was established, with a slow dose increment, according to the patient's response. It was only possible to achieve the dose of 75 mg/d after 2 months. Although well tolerated by most patients, HS reactions with clopidogrel may occur and desensitization is rising as a safe alternative in those patients. In delayed reactions with cutaneous lesions, a slower desensitization protocol may be necessary, as in this case.

  18. Progress in Desensitization of the Highly HLA Sensitized Patient.

    Science.gov (United States)

    Jordan, S C; Choi, J; Kahwaji, J; Vo, A

    2016-04-01

    The presence of HLA antibodies remains a significant and often impenetrable barrier to kidney transplantation, leading to increased morbidity and mortality for patients remaining on long-term dialysis. In recent years, a number of new approaches have been developed to overcome these barriers. Intravenous immunoglobulin (IVIG) remains the lynchpin of HLA desensitization therapy and has been shown in a prospective, randomized, placebo-controlled trial to improve transplantation rates. In addition, IVIG used in low doses with plasma exchange is a reliable protocol for desensitization. Another significant advancement was the addition of rituximab (anti-B-cell therapy) to IVIG and plasma exchange-based desensitization. This approach has significantly improved rates of transplantation and outcomes. There is limited experience with bortezomib (anti-plasma cell therapy) and eculizumab (complement inhibition) for desensitization. However, recent data from a completed trial of eculizumab failed to show a significant benefit for prevention of antibody-mediated rejection compared with standard therapy plus placebo, and bortezomib produced inconsistent results. There is a growing interest in developing new therapeutic agents for desensitization. Newer approaches that address antibody reduction with B-cell depletion are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Observations on the Curative Effect of Acupuncture and Moxibustion plus Systematic Desensitization on Anxiety Neurosis

    Institute of Scientific and Technical Information of China (English)

    LIU Gui-zhen; ZANG Yun-juan; LIU Ai-zhen; WANG Si-you

    2003-01-01

    Purpose To investigate the clinical effect of a combined treatment on anxiety neurosis. Method Eighty cases of anxiety neurosis were treated by acupuncture-moxibustion plus systematic desensitization.The result was compared with simple acupuncture-moxibustion or simple systematic desensitization. Results and conclusion The cure rate was 52.5% in acupuncturemoxibustion plus systematic desensitization, which was significantly different from that in simple acupuncturemoxibustion or in simple systematic desensitization ( P <0. 05).

  20. A Controlled Study to Assess the Clinical Efficacy of Totally Self-Administered Systematic Desensitization

    Science.gov (United States)

    Rosen, Gerald M.; And Others

    1976-01-01

    Highly anxious self-referred snake phobics received either (a) therapist-administered desensitization, (b) self-administered desensitization with weekly therapist phone calls, (c) totally self-administered desensitization, (d) self-administered double-blind placebo control, or (e) no treatment. Pretreatment to posttreatment measures revealed…

  1. Audio-Taped Desensitization for the Modification of Speech and Communication Anxiety.

    Science.gov (United States)

    McManus, Marianne

    The fear of speaking before a group affects a high percentage of American adults. The effectiveness of an audiotape desensitization program was compared to conventional therapist-led group desensitization and no treatment for the modification of speech and communication anxiety behaviors. All desensitization treated subjects demonstrated…

  2. Prolonged Exposure versus Eye Movement Desensitization and Reprocessing (EMDR) for PTSD rape victims.

    Science.gov (United States)

    Rothbaum, Barbara Olasov; Astin, Millie C; Marsteller, Fred

    2005-12-01

    This controlled study evaluated the relative efficacy of Prolonged Exposure (PE) and Eye Movement Desensitization and Reprocessing (EMDR) compared to a no-treatment wait-list control (WAIT) in the treatment of PTSD in adult female rape victims (n = 74). Improvement in PTSD as assessed by blind independent assessors, depression, dissociation, and state anxiety was significantly greater in both the PE and EMDR group than the WAIT group (n = 20 completers per group). PE and EMDR did not differ significantly for change from baseline to either posttreatment or 6-month follow-up measurement for any quantitative scale.

  3. Using operant conditioning and desensitization to facilitate veterinary care with captive reptiles.

    Science.gov (United States)

    Hellmuth, Heidi; Augustine, Lauren; Watkins, Barbara; Hope, Katharine

    2012-09-01

    In addition to being a large component of most zoological collections, reptile species are becoming more popular as family pets. Reptiles have the cognitive ability to be trained to facilitate daily husbandry and veterinary care. Desensitization and operant conditioning can alleviate some of the behavioral and physiological challenges of treating these species. A survey of reptile training programs at zoos in the United States and worldwide reveals that there are many successful training programs to facilitate veterinary care and minimize stress to the animal. Many of the techniques being used to train reptiles in zoological settings are transferable to the exotic pet clinician.

  4. 378 Aspirin Desensitization Treatment: A Case Report

    Science.gov (United States)

    Alvarez, Mirta; Osoria, Lludenich; Ronquillo, Mercedes; Castro, Raúl; Perez, Doguerti; Rodríguez, José; Irarragorri, Catalina

    2012-01-01

    Background Aspirin (ASA) is one of the best known and most widely used drugs in the world. Patients with coronary artery disease require prolonged treatments with this drug, which is denied to those patients with histories of adverse reactions to it. Methods In a rapid desensitization protocol a patient with a history of ASA-induced urticaria-angioedema was treated with escalating doses of aspirin administered orally every 25 minutes. Results The patient completed the desensitization protocol in few hours without complications, and currently is able to take 125 mg of ASA per day without adverse reactions. Conclusions This report describes the first desensitization treatment with aspirin carried out in our hospital. No other case was found in the national bibliography.

  5. Desensitization of delayed-type hypersensitivity in mice: suppressive environment

    Directory of Open Access Journals (Sweden)

    Takashi Katsura

    1993-01-01

    Full Text Available The systemic injection of high doses of antigen into a preimmunized animal results in transient unresponsiveness of cell-mediated immune responses. This phenomenon is known as desensitization. Serum interleukin 2 (IL-2 activity was found transiently in desensitized mice at 3 h after the antigen challenge. These mice could not reveal antigen nonspecific delayed-type hypersensitivity (DTH 1 d after the challenge. Specific suppression of DTH was observed at later stages. Sera from 3 h desensitized mice showed suppressive effects on DTH in preo immunized mice. Administration of recombinant IL-2 into preimmunized mice led to the failure of development of DTH to antigens. These observations suggest that IL-2 plays an important role in the suppressive environment.

  6. Omalizumab facilitates rapid oral desensitization for peanut allergy.

    Science.gov (United States)

    MacGinnitie, Andrew J; Rachid, Rima; Gragg, Hana; Little, Sara V; Lakin, Paul; Cianferoni, Antonella; Heimall, Jennifer; Makhija, Melanie; Robison, Rachel; Chinthrajah, R Sharon; Lee, John; Lebovidge, Jennifer; Dominguez, Tina; Rooney, Courtney; Lewis, Megan Ott; Koss, Jennifer; Burke-Roberts, Elizabeth; Chin, Kimberly; Logvinenko, Tanya; Pongracic, Jacqueline A; Umetsu, Dale T; Spergel, Jonathan; Nadeau, Kari C; Schneider, Lynda C

    2017-03-01

    Peanut oral immunotherapy is a promising approach to peanut allergy, but reactions are frequent, and some patients cannot be desensitized. The anti-IgE medication omalizumab (Xolair; Genentech, South San Francisco, Calif) might allow more rapid peanut updosing and decrease reactions. We sought to evaluate whether omalizumab facilitated rapid peanut desensitization in highly allergic patients. Thirty-seven subjects were randomized to omalizumab (n = 29) or placebo (n = 8). After 12 weeks of treatment, subjects underwent a rapid 1-day desensitization of up to 250 mg of peanut protein, followed by weekly increases up to 2000 mg. Omalizumab was then discontinued, and subjects continued on 2000 mg of peanut protein. Subjects underwent an open challenge to 4000 mg of peanut protein 12 weeks after stopping study drug. If tolerated, subjects continued on 4000 mg of peanut protein daily. The median peanut dose tolerated on the initial desensitization day was 250 mg for omalizumab-treated subjects versus 22.5 mg for placebo-treated subject. Subsequently, 23 (79%) of 29 subjects randomized to omalizumab tolerated 2000 mg of peanut protein 6 weeks after stopping omalizumab versus 1 (12%) of 8 receiving placebo (P desensitized over as little as 8 weeks of peanut oral immunotherapy. In the majority of subjects, this desensitization is sustained after omalizumab is discontinued. Additional studies will help clarify which patients would benefit most from this approach. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  7. Angiotensin II (de)sensitization: Fluid intake studies with implications for cardiovascular control.

    Science.gov (United States)

    Daniels, Derek

    2016-08-01

    Cardiovascular disease is the leading cause of death worldwide and hypertension is the most common risk factor for death. Although many anti-hypertensive pharmacotherapies are approved for use in the United States, rates of hypertension have increased over the past decade. This review article summarizes a presentation given at the 2015 meeting of the Society for the Study of Ingestive Behavior. The presentation described work performed in our laboratory that uses angiotensin II-induced drinking as a model system to study behavioral and cardiovascular effects of the renin-angiotensin system, a key component of blood pressure regulation, and a common target of anti-hypertensives. Angiotensin II (AngII) is a potent dipsogen, but the drinking response shows a rapid desensitization after repeated injections of AngII. This desensitization appears to be dependent upon the timing of the injections, requires activation of the AngII type 1 (AT1) receptor, requires activation of mitogen-activated protein (MAP) kinase family members, and involves the anteroventral third ventricle (AV3V) region as a critical site of action. Moreover, the response does not appear to be the result of a more general suppression of behavior, a sensitized pressor response to AngII, or an aversive state generated by the treatment. More recent studies suggest that the treatment regimen used to produce desensitization in our laboratory also prevents the sensitization that occurs after daily bolus injections of AngII. Our hope is that these findings can be used to support future basic research on the topic that could lead to new developments in treatments for hypertension.

  8. Eye movement desensitization and reprocessing: a conceptual framework.

    Science.gov (United States)

    Menon, Sukanya B; Jayan, C

    2010-07-01

    Eye movement desensitization and reprocessing (EMDR) is a method which was initially used for the treatment of post-traumatic stress disorder. But it is now being used in different therapeutic situations. EMDR is an eight-phase treatment method. History taking, client preparation, assessment, desensitization, installation, body scan, closure and reevaluation of treatment effect are the eight phases of this treatment which are briefly described. A case report is also depicted which indicates the efficacy of EMDR. The areas where EMDR is used and the possible ways through which it is working are also described.

  9. Opioid receptor desensitization: mechanisms and its link to tolerance

    Directory of Open Access Journals (Sweden)

    Stéphane eAllouche

    2014-12-01

    Full Text Available Opioid receptors are part of the class A of G-protein coupled receptors and the target of the opiates, the most powerful analgesic molecules used in clinic. During a protracted use, a tolerance to analgesic effect develops resulting in a reduction of the effectiveness. So understanding mechanisms of tolerance is a great challenge and may help to find new strategies to tackle this side effect. This review will summarize receptor-related mechanisms that could underlie tolerance especially receptor desensitization. We will focus on the latest data obtained on molecular mechanisms involved in opioid receptor desensitization: phosphorylation, receptor uncoupling, internalization and post-endocytic fate of the receptor.

  10. Eye movement desensitization and reprocessing: A conceptual framework

    Directory of Open Access Journals (Sweden)

    Menon Sukanya

    2010-01-01

    Full Text Available Eye movement desensitization and reprocessing (EMDR is a method which was initially used for the treatment of post-traumatic stress disorder. But it is now being used in different therapeutic situations. EMDR is an eight-phase treatment method. History taking, client preparation, assessment, desensitization, installation, body scan, closure and reevaluation of treatment effect are the eight phases of this treatment which are briefly described. A case report is also depicted which indicates the efficacy of EMDR. The areas where EMDR is used and the possible ways through which it is working are also described.

  11. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative......γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...... understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...

  12. Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

    Science.gov (United States)

    Erickson, Jeffrey D

    2017-07-01

    Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K(+) -stimulated MeAIB transport displays an affinity (Km ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca(2+) , and is blocked by inhibition of voltage-gated Ca(2+) channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca(2+) and voltage-gated calcium channels, but is also blocked by the Na(+) channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca(2+) , but on Na(+) ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K(+) -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017

  13. Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Xue-Qiang Zhao; Yong-Guan Zhu; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2007-01-01

    Nitrate is a major nitrogen (N) source for most crops.Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels.Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency.The present study investigated the regulation of the high-affinity nitrate transport system (HATS) by exogenous abscisic acid (ABA) and glutamine (Gin) in wheat (Triticum aestivum L.) roots.Wheat seedlings grown in nutrient solution containing 2 mmollL nitrate as the only nitrogen source for 2 weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h.Treated wheat plants were then divided into two groups.One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L 15 N-labeled nitrate.The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate.Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction.When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced.These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media.Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.

  14. Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

    Science.gov (United States)

    Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

    2017-01-01

    Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and

  15. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  16. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    Science.gov (United States)

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  17. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  18. 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.

  19. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

    Science.gov (United States)

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-01-01

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research. PMID:28266535

  20. 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.

  1. High affinity melatonin receptors in the vertebrate brain: implications for the control of the endogenous oscillatory systems.

    Science.gov (United States)

    Fraschini, F; Stankov, B

    1994-01-01

    Currently, the melatonin receptor is depicted as a membrane-associated protein, linked to a guanine nucleotide-binding protein (G-protein), and thus the melatonin receptor represents a member of a receptor superfamily, acting through G-proteins in the first step of their signal-transduction pathways. Although on a number of occasions specific binding of radioactive melatonin has been demonstrated in a wide variety of tissues and organs, to date, high affinity G-protein-regulated melatonin binding sites, suggestive for a functional melatonin receptor, have been convincingly confirmed in the brain only. There is a significant species variation in the distribution of the melatonin receptor in the vertebrate brain. The limited number of studies prevents any definitive conclusion in terms of phylogeny, though generally speaking, the lower vertebrates' brains tend to express melatonin receptors with wider distribution. Two sites have been consistently found to express high density of melatonin receptors: the pars tuberalis of the adenohypophysis and the hypothalamic suprachiasmatic nuclei (SCN). It must be pointed out, however, that there are some exceptions. Binding in the human pars tuberalis has not been reported, and apparently, the sheep and the mustelids' suprachiasmatic nuclei do not express detectable binding. The function of melatonin in pars tuberalis is unclear, and the control of the synthesis (and release) of paracrine factors that act at site(s) distant from the melatonin target cells, have been suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    Science.gov (United States)

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  3. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes.

    Science.gov (United States)

    Lang, Benjamin; Blot, Nicolas; Bouffartigues, Emeline; Buckle, Malcolm; Geertz, Marcel; Gualerzi, Claudio O; Mavathur, Ramesh; Muskhelishvili, Georgi; Pon, Cynthia L; Rimsky, Sylvie; Stella, Stefano; Babu, M Madan; Travers, Andrew

    2007-01-01

    The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

  4. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    Science.gov (United States)

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  5. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    Science.gov (United States)

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  6. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  7. The high affinity IgE receptor (FcεRI) expression and function in airway smooth muscle.

    Science.gov (United States)

    Redhu, Naresh Singh; Gounni, Abdelilah S

    2013-02-01

    The airway smooth muscle (ASM) is no longer considered as merely a contractile apparatus and passive recipient of growth factors, neurotransmitters and inflammatory mediators signal but a critical player in the perpetuation and modulation of airway inflammation and remodeling. In recent years, a molecular link between ASM and IgE has been established through Fc epsilon receptors (FcεRs) in modulating the phenotype and function of these cells. Particularly, the expression of high affinity IgE receptor (FcεRI) has been noted in primary human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthmatic subjects. The activation of FcεRI on ASM cells suggests a critical yet almost completely ignored network which may modulate ASM cell function in allergic asthma. This review is intended to provide a historical perspective of IgE effects on ASM and highlights the recent updates in the expression and function of FcεRI, and to present future perspectives of activation of this pathway in ASM cells.

  8. Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head*

    Science.gov (United States)

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P.; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E.; Kozyraki, Renata

    2013-01-01

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity. PMID:23592779

  9. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    Science.gov (United States)

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

    2013-06-07

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

  10. The High Affinity IgE Receptor (FcεRI as a Target for Anti-allergic Agents

    Directory of Open Access Journals (Sweden)

    Kyoko Takahashi

    2005-01-01

    Full Text Available Prevention of the effector cell activation via high affinity IgE receptor (FcεRI is thought to be a straightforward strategy for suppressing the allergic reaction. Among the numerous methods to prevent the activation through FcεRI, three versions are described in this article. The first and second ideas involve inhibition of binding between FcεRI and IgE with a soluble form of the FceRI α chain and a humanized antibody directed against the a chain, respectively. Both of these paths involve suppression the histamine release from human peripheral blood basophils in vitro. They also inhibited the allergic reaction in vivo. The soluble α inhibited the anaphylactic reaction in rodents and the Fab fragments of the humanized anti-FcεRI α chain antibody suppressed the dermal response in rhesus monkeys. The third idea involves repression of FcεRI expression by suppressing the transcription of the genes encoding the subunits of FceRI. Although no plausible candidate molecule for actualizing this idea can be identified at present, further analyses of the transcriptional regulatory mechanisms in the human FcεRI α and β chain genes will lead to the discovery of novel targets for developing anti-allergic agents.

  11. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  12. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    Science.gov (United States)

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  13. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

    Science.gov (United States)

    Ogata, Makoto; Chuma, Yasushi; Yasumoto, Yoshinori; Onoda, Takashi; Umemura, Myco; Usui, Taichi; Park, Enoch Y

    2016-01-01

    Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

  14. A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    HE Cheng-yan; ZHAO Lu; LIU Yan-li; XU Li-na; SHANG De-jing; YANG Hong

    2004-01-01

    The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

  15. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  16. 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.

  17. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion (Cornell); (UAB); (Florida)

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  18. Acute and chronic desensitization of penicillin-allergic patients using oral penicillin.

    Science.gov (United States)

    Stark, B J; Earl, H S; Gross, G N; Lumry, W R; Goodman, E L; Sullivan, T J

    1987-03-01

    The efficacy, safety and mechanisms of penicillin desensitization were studied in 24 adults and two children with serious infections that required therapy with a beta-lactam drug. Indications for desensitization included debilitating as well as life-endangering infections. Increasing oral doses of phenoxymethyl penicillin were administered at 15-minute intervals to a cumulative dose of 1.3 million units. Parenteral therapy with the beta-lactam drug of choice was instituted at that point. Immunologic complications of desensitization or therapy, ranging from pruritus to serum sickness, occurred in 12 patients. The appearance of gradually worsening wheezing led to abandonment of the procedure in one subject with cystic fibrosis and severe pulmonary disease. The remaining 25 patients were successfully desensitized and received full-dose parenteral therapy. Chronic desensitization was maintained in seven individuals with twice daily oral penicillins for 3 weeks to more than 2 years. No allergic complications of chronic desensitization or recurrent full-dose parenteral therapy were detected. Skin test reactions to one or all penicillin determinants became negative in 11 of 15 patients retested after acute desensitization. Two desensitized patients became skin test negative, remained skin test negative after cessation of desensitization, and tolerated subsequent beta-lactam therapy without allergic reactions or resensitization. The results of this study provide new evidence that acute and chronic penicillin desensitization is useful and an acceptably safe approach and suggest that antigen-specific mast cell desensitization contributes to the protection against anaphylaxis.

  19. Eye Movement Desensitization and Reprocessing: A Critical Analysis.

    Science.gov (United States)

    Erwin, Terry McVannel

    Since Shapiro's introduction of Eye Movement Desensitization and Reprocessing (EMDR) in 1989, it has been a highly controversial therapeutic technique. Critical reviews of Shapiro's initial study have highlighted many methodological shortcomings in her work. And early empirical research that followed Shapiro's original study has been criticized…

  20. Exposure to Violence and Children's Desensitization Attitudes in Lebanon.

    Science.gov (United States)

    Tarabah, Asma; Badr, Lina Kurdahi; Usta, Jinan; Doyle, John

    2016-11-01

    Children exposed to multiple sources of violence may become desensitized, increasing the possibility of them imitating the aggressive behaviors they watch and considering such behavior as normal. The purpose of this article is to assess the association between exposure to various types of violence (including war) and desensitization in Lebanese children. A cross-sectional design with 207 school-aged children assessed exposure to violence using three surveys: (a) violence in the media (the Media Preference survey), (b) exposure to violence (the KID-SAVE survey), and (c) desensitization attitudes (the Attitude Toward Violence-Child Version). Children were between 8 and 12 years old, 56% were males, and 70%were from middle socioeconomic status (SES) backgrounds. Seventy-six percent of children reported being exposed to violence, with more exposure in males and in the lower SES group. Impact, however, was greater on girls. The predictors of attitude toward violence were "Frequency" of exposure, "Impact" of exposure, and the amount of violence viewed on television. Children are massively exposed to violence in Lebanon resulting in desensitization, which may habituate them to accept violence as normal and put them at risk for imitating violent behaviors. © The Author(s) 2015.

  1. Desensitization to media violence over a short period of time.

    Science.gov (United States)

    Fanti, Kostas A; Vanman, Eric; Henrich, Christopher C; Avraamides, Marios N

    2009-01-01

    This study investigated the desensitization to violence over a short period of time. Participants watched nine violent movie scenes and nine comedy scenes, and reported whether they enjoyed the violent or comedy scenes and whether they felt sympathetic toward the victim of violence. Using latent growth modeling, analyses were carried out to investigate how participants responded to the different scenes across time. The findings of this study suggested that repeated exposure to media violence reduces the psychological impact of media violence in the short term, therefore desensitizing viewers to media violence. As a result, viewers tended to feel less sympathetic toward the victims of violence and actually enjoy more the violence portrayed in the media. Additionally, desensitization to media violence was better represented by a curvilinear pattern, whereas desensitization to comedy scenes was better represented by a linear pattern. Finally, trait aggression was not related to the pattern of change over time, although significant effects were found for initial reports of enjoyment and sympathy.

  2. The Use of Models in a Desensitization Procedure.

    Science.gov (United States)

    Leventhal, Allan M.

    The author describes a counseling technique which: (1) is based upon behavior theory and treats acts and thoughts as operants, and anxiety as a respondent controlled by classical conditioning; (2) entails in vivo desensitization, requiring the construction of a hierarchy of anxiety-producing situations which can be enacted; (3) uses peer- or…

  3. Oral desensitization to milk: how to choose the starting dose!

    Science.gov (United States)

    Mori, Francesca; Pucci, Neri; Rossi, Maria Elisabetta; de Martino, Maurizio; Azzari, Chiara; Novembre, Elio

    2010-01-01

    Mori F, Pucci N, Rossi ME, de Martino M, Azzari C, Novembre E. Oral desensitization to milk: how to choose the starting dose! Pediatr Allergy Immunol 2010: 21: e450–e453. © 2009 John Wiley & Sons A/S A renewed interest in oral desensitization as treatment for food allergy has been observed in the last few years. We studied a novel method based on the end point skin prick test procedure to establish the starting dose for oral desensitization in a group of 30 children higly allergic to milk. The results (in terms of reactions to the first dose administered) were compared with a group of 20 children allergic to milk as well. Such control group started to swallow the same dose of 0.015 mg/ml of milk. None reacted to the first dose when administered according to the end point skin prick test. On the other side, ten out of 20 children (50%) from the control group showed mild allergic reactions to the first dose of milk. In conclusion the end point skin prick test procedure results safe and easy to be performed in each single child in order to find out the starting dose for oral desensitization to milk, also by taking into account the individual variability. PMID:19624618

  4. Emotionally Numb: Desensitization to Community Violence Exposure among Urban Youth

    Science.gov (United States)

    Kennedy, Traci M.; Ceballo, Rosario

    2016-01-01

    Community violence exposure (CVE) is associated with numerous psychosocial outcomes among youth. Although linear, cumulative effects models have typically been used to describe these relations, emerging evidence suggests the presence of curvilinear associations that may represent a pattern of emotional desensitization among youth exposed to…

  5. Agonist mediated fetal muscle-type nicotinic acetylcholine receptor desensitization

    Science.gov (United States)

    The exposure of a developing embryo or fetus to teratogenic alkaloids from plants has the potential to cause developmental defects in livestock due to the inhibition of fetal movement by alkaloids. The mechanism behind the inhibition of fetal movement is the desensitization of fetal muscle-type nico...

  6. Emotional Desensitization to Violence Contributes to Adolescents' Violent Behavior.

    Science.gov (United States)

    Mrug, Sylvie; Madan, Anjana; Windle, Michael

    2016-01-01

    Many adolescents are exposed to violence in their schools, communities and homes. Exposure to violence at high levels or across multiple contexts has been linked with emotional desensitization, indicated by low levels of internalizing symptoms. However, the long-term consequences of such desensitization are unknown. This study examined emotional desensitization to violence, together with externalizing problems, as mediators of the relationship between exposure to violence in pre-adolescence and violent behavior in late adolescence. A community sample of youth (N = 704; 48% female; 76% African American, 22% Caucasian) reported on their exposure to violence in multiple settings at ages 11, 13 and 18. Internalizing and externalizing problems were assessed at ages 11 and 13; violent behavior was measured at age 18. Structural Equation Modeling showed that exposure to high levels of violence at age 11 was associated with lower levels of internalizing problems (quadratic effect) at age 13, as was exposure to violence across multiple contexts (linear effect). In turn, fewer internalizing problems and more externalizing problems at age 13 predicted more violent behavior at age 18. The results suggest that emotional desensitization to violence in early adolescence contributes to serious violence in late adolescence.

  7. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    Science.gov (United States)

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  8. Pharmacokinetics, pharmacodynamics and safety of CEP-26401, a high-affinity histamine-3 receptor antagonist, following single and multiple dosing in healthy subjects.

    Science.gov (United States)

    Spiegelstein, Ofer; Stevens, Jasper; Van Gerven, Joop; Nathan, Pradeep J; Maynard, James P; Mayleben, David W; Hellriegel, Edward; Yang, Ronghua

    2016-10-01

    CEP-26401 is a novel orally active, brain-penetrant, high-affinity histamine H3 receptor (H3R) antagonist, with potential therapeutic utility in cognition enhancement. Two randomized, double-blind, placebo-controlled dose escalation studies with single (0.02 to 5 mg) or multiple administration (0.02 to 0.5 mg once daily) of CEP-26401 were conducted in healthy subjects. Plasma and urine samples were collected to investigate CEP-26401 pharmacokinetics. Pharmacodynamic endpoints included a subset of tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and nocturnal polysomnography. Population pharmacokinetic-pharmacodynamic modeling was conducted on one CANTAB and one polysomnography parameter of interest. CEP-26401 was slowly absorbed (median tmax range 3-6 hours) and the mean terminal elimination half-life ranged from 24-60 hours. Steady-state plasma concentrations were achieved within six days of dosing. CEP-26401 exhibits dose- and time-independent pharmacokinetics, and renal excretion is a major elimination pathway. CEP-26401 had a dose-dependent negative effect on sleep, with some positive effects on certain CANTAB cognitive parameters seen at lower concentrations. The derived three compartment population pharmacokinetic model, with first-order absorption and elimination, accurately described the available pharmacokinetic data. CEP-26401 was generally well tolerated up to 0.5 mg/day with most common treatment related adverse events being headache and insomnia. Further clinical studies are required to establish the potential of low-dose CEP-26401 in cognition enhancement. © The Author(s) 2016.

  9. Acute activation, desensitization and smoldering activation of human acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Barbara G Campling

    Full Text Available The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β22β2 and (α4β22α4. The (α4β22β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β22α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

  10. Taking advantage: high-affinity B cells in the germinal center have lower death rates, but similar rates of division, compared to low-affinity cells.

    Science.gov (United States)

    Anderson, Shannon M; Khalil, Ashraf; Uduman, Mohamed; Hershberg, Uri; Louzoun, Yoram; Haberman, Ann M; Kleinstein, Steven H; Shlomchik, Mark J

    2009-12-01

    B lymphocytes producing high-affinity Abs are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high-affinity B cells are selected during the immune response has never been elucidated. Although it has been shown that high-affinity cells directly outcompete low-affinity cells in the germinal center (GC), whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the GC. By comparing high- and low-affinity B cells for the same Ag, we show here that low-affinity cells have an intrinsically higher death rate than do cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared with lower affinity B cells. Control over survival rather than proliferation of low- and high-affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.

  11. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    in delivering iron to cells during formation of the tubular epithelial cells of the primordial kidney. No cellular receptor for NGAL has been described. We show here that megalin, a member of the low-density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown...

  12. Replacement of the Bryostatin A- and B-Pyran Rings With Phenyl Rings Leads to Loss of High Affinity Binding With PKC.

    Science.gov (United States)

    Petersen, Mark E; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Keck, Gary E

    2016-10-19

    We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring 'binding domain'.

  13. 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 i

  14. 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...

  15. 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 le

  16. 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

    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...

  17. 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...

  18. 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...

  19. Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

    DEFF Research Database (Denmark)

    Cha, Joo Hwan; Zou, Mu-Fa; Adkins, Erika M

    2005-01-01

    Novel fluorescent ligands were synthesized to identify a high-affinity probe that would enable visualization of the dopamine transporter (DAT) in living cells. Fluorescent tags were extended from the N- or 2-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane, using an ethylamino lin...

  20. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

    Science.gov (United States)

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M; Berezhnoy, Alexey

    2015-07-13

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

  1. Perbedaan Penggunaan Bahan Desensitizing dan Tanpa Desensitizing Pasca Bleaching Ekstra-Koronal terhadap Kekerasan Email

    Directory of Open Access Journals (Sweden)

    Wignyo Hadriyanto

    2012-12-01

    Full Text Available Latar Belakang. Pada bleaching ekstrakoronal diketahui terjadi proses demineralisasi sehingga terjadi hiersensitivitas dentin. UltraEZ salah satu bahan desensitizing yang dapat mengurangi hipersensitivitas akibat demineralisasi email pasca bleaching ekstrakoronal terkini. Tujuan penelitian ini bertujuan untuk mengetahui perbedaan kekerasan email pasca pemutihan gigi ekstra-koronal dengan aplikasi bahan desensitizing dan tanpa aplikasi bahan desensitizing. Metode penelitian ini menggunakan 20 gigi premolar permanen pasca pencabutanyang masih utuh dan direndam dalam saliva buatan, kemudian dilakukan pemolesan pada bagian bukal dengan menggunakan pasta profilaksis kemudian gigi dicuci dan dikeringkan. Bahan pemutih Opalescence Xtra Boost diaplikasikan pada semua permukaan bukal gigi premolar kemudian dibagi menjadi 2 kelompok yaitu kelompok I, II, masing-masing kelompok sebanyak 10 gigi. Kelompok I sebagai kelompok control setelah dilakukan pemutihan, tidak dilakukan aplikasi Ultra-EZ, dimasukkan dalam wadah botol dan direndam dalam saliva buatan kemudian disimpan dalam incubator. Mahkota dan akar gigi,kemudian ditanam dalam resin akrilik sesuai kelompok sebelumnya dengan permukaan bukal menghadap ke atas. Semua sampel diuji kekerasannya dengan uji kekerasan Vickers menggunakan beban 100 g selama 15 detik. Permukaan bukal menghadap ke atas, kemudian dijepit dengan alat penjepit pada meja alat Micro Vickers Hardness Tester. Sampel diatur sedemikian rupa sehingga akan terlihat gambar yang dapat diukur panjang diagonalnya langsung dengan micrometer yang ada pada lensa okuler. Nilai kekerasan email dalam Vickers hardness number (VHN juga dapat diperoleh dari table setelah mengetahui rata-rata panjang diagonal, berat badan yang digunakan dan waktu yang digunakan untuk uji kekerasan. Pengujian ini dilakukan pada setiap kelompok. Selanjutnya diuji dengan uji-t. hasil penelitian menunjukkan ada perbedaan yang bermakna antara aplikasi ultraEZ lima menit

  2. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    Science.gov (United States)

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  3. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    Science.gov (United States)

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  4. Gonyautoxin 1/4 aptamers with high-affinity and high-specificity: From efficient selection to aptasensor application.

    Science.gov (United States)

    Gao, Shunxiang; Hu, Bo; Zheng, Xin; Cao, Ying; Liu, Dejing; Sun, Mingjuan; Jiao, Binghua; Wang, Lianghua

    2016-05-15

    Gonyautoxin 1/4 (GTX1/4) are potent marine neurotoxins with significant public health impact. However, the ethical issues and technical defects associated with the currently applied detection methods for paralytic shellfish toxin GTX1/4 are pressing further studies to develop suitable alternatives in a regulatory monitoring system. This work describes the first successful selection, optimization, and characterization of an aptamer that bind with high affinity and specificity to GTX1/4. Compared to the typical MB-SELEX, GO-SELEX, an advanced screening technology, has significant advantages for small molecular aptamer development. Furthermore, we truncated GTX1/4 aptamer and obtained the aptamer core sequence with a higher Kd of 17.7 nM. The aptamer GO18-T-d was then used to construct a label-free and real-time optical BLI aptasensor for the detection of GTX1/4. The aptasensor showed a broad detection range from 0.2 to 200 ng/mL GTX1/4 (linear range from 0.2 to 90 ng/mL), with a low detection limit of 50 pg/mL. Moreover, the aptasensor exhibited a high degree of specificity for GTX1/4 and no cross reactivity to other marine toxins. The aptasensor was then applied to the detection of GTX1/4 in spiked shellfish samples and showed a good reproducibility and stability. We believe that this novel aptasensor offers a promising alternative to traditional analytical methods for the rapid detection of the marine biotoxin GTX1/4. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy.

    Science.gov (United States)

    Kuo, Macus Tien; Fu, Siqing; Savaraj, Niramol; Chen, Helen H W

    2012-09-15

    The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.

  6. Plant High-Affinity Potassium (HKT Transporters Involved in Salinity Tolerance: Structural Insights to Probe Differences in Ion Selectivity

    Directory of Open Access Journals (Sweden)

    Maria Hrmova

    2013-04-01

    Full Text Available High-affinity Potassium Transporters (HKTs belong to an important class of integral membrane proteins (IMPs that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

  7. Plant High-Affinity Potassium (HKT) Transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity.

    Science.gov (United States)

    Waters, Shane; Gilliham, Matthew; Hrmova, Maria

    2013-04-09

    High-affinity Potassium Transporters (HKTs) belong to an important class of integral membrane proteins (IMPs) that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

  8. Effect of repeated nicotine exposure on high-affinity nicotinic acetylcholine receptor density in spontaneously hypertensive rats.

    Science.gov (United States)

    Hohnadel, Elizabeth J; Hernandez, Caterina M; Gearhart, Debra A; Terry, Alvin V

    Spontaneously hypertensive rats (SHRs) are often used as a model of attention deficit hyperactivity disorder (ADHD) and to investigate the effects of hypertension on cognitive function. Further, they appear to have reduced numbers of central nicotinic acetylcholine receptors (nAChRs) and, therefore, may be useful to model certain aspects of Alzheimer's disease (AD) and other forms of dementia given that a decrease in nAChRs is thought to contribute to cognitive decline in these disorders. In the present study, based on reports that chronic nicotine exposure increases nAChRs in several mammalian models, we tested the hypothesis that repeated exposures to a relatively low dose of the alkaloid would ameliorate the receptor deficits in SHR. Thus, young-adult SHRs and age-matched Wistar-Kyoto (WKY) control rats were treated with either saline or nicotine twice a day for 14 days (total daily dose = 0.7 mg/kg nicotine base) and then sacrificed. Quantitative receptor autoradiography with [125I]-IPH, an epibatidine analog, revealed: (1) that high-affinity nAChRs were higher in saline-treated WKY (control) rats compared to saline-treated SHRs in 18 of the 19 brain region measured, although statistically different only in the mediodorsal thalamic nuclei, (2) that nicotine significantly increased nAChR binding in WKY rats in six brain areas including cortical regions and the anterior thalamic nucleus, (3) that there were no cases where nicotine significantly increased nAChR binding in SHRs. These results indicate that subjects deficient in nAChRs may be less sensitive to nAChR upregulation with nicotine than normal subjects and require higher doses or longer periods of exposure.

  9. The effects of desensitizing agents on the hydraulic conductance of human dentin in vitro.

    Science.gov (United States)

    Greenhill, J D; Pashley, D H

    1981-03-01

    The hydrodynamic theory of dentin sensitivity states that a stimulus applied at the orifice of exposed dentinal tubules causes movement of tubular fluid which stimulates nerve receptors. The fluid should obey principles of fluid movement through capillary tubes. Any decrease in the functional radius of the dentinal tubules should greatly reduce the rate of fluid flow, thus reducing dentinal sensitivity. The purpose of this study was to evaluate the ability of agents that have been used previously for clinical dentin desensitization to reduce the rate of fluid flow through dentin in vitro. Dentin discs prepared from extracted human third molars were treated with 50% citric acid to remove debris from tubular orifices. After placing the discs in a split chamber device, the rate at which buffer solution could filter across the dentin under 240 cm of water pressure was measured. The occlusal side of the disc was then treated with an agent thought to desensitize dentin to determine if it reduced fluid flow rate. Discs that had more than a 50% reduction in flow rate were examined by scanning electron microscopy to determine if those agents that decreased fluid flow also partially occluded tubular orifices. This in vitro model provided a useful quantitative method for screening a host of preparations that have been used in the past to decrease dentin sensitivity.

  10. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

  11. BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

    Science.gov (United States)

    Uegaki, Koichi; Kumanogoh, Haruko; Mizui, Toshiyuki; Hirokawa, Takatsugu; Ishikawa, Yasuyuki; Kojima, Masami

    2017-05-12

    Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function

  12. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    Science.gov (United States)

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  13. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    Directory of Open Access Journals (Sweden)

    Kelly N Messingham

    Full Text Available Bullous pemphigoid (BP is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen. Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1 To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2 To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16 with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.

  14. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

    Science.gov (United States)

    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  15. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.

    Science.gov (United States)

    Ieong, Ka-Weng; Pavlov, Michael Y; Kwiatkowski, Marek; Ehrenberg, Måns; Forster, Anthony C

    2014-05-01

    There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

  16. Increasing time interval and decreasing allergen dose interval improves ex vivo desensitization of human blood basophils

    DEFF Research Database (Denmark)

    Witting Christensen, Sara K; Krohn, Inge Kortekaas; Thuraiaiyah, Jani;

    2016-01-01

    BACKGROUND: Desensitization is a method for inducing temporary tolerance to allergen. The mechanism underlying desensitization is yet to be established. METHODS: Basophil granulocytes in whole blood from grass pollen allergic subjects were desensitized ex vivo by sequential addition of increasing...... allergen concentrations. At each step basophil activation (CD193(+) CD63(+) ) was monitored with and without (background activation) allergen challenge at optimal concentration. The sequential desensitization protocol was compared to a single-dose desensitization protocols with threshold and subthreshold...... allergen concentrations. Incubation intervals and allergen concentrations were varied in order to optimise the protocol. RESULTS: Sequential desensitization effectively reduced basophil response. The single-dose subthreshold protocol and single-dose threshold protocols did not reduce basophil activation...

  17. DESENSITIZATION OF ACETYLCHOLINE ON THE INHIBITION EFFECTS OF BLOOD PRESSURE IN ANESTHETIZED CANINE

    Institute of Scientific and Technical Information of China (English)

    陈莉娜; 吕军; 臧伟进; 于晓江; 孙晓东; 高小利

    2004-01-01

    Objective To investigate the desensitization of acetylcholine (Ach) on the inhibition effects of blood pressure (BP) in anesthetized canine and build a model for studying desensitization in vivo. Methods Through changing the intervals (120, 100, 80, 60, 40, 20 seconds) of twice Ach administration (each was 15μg*kg-1,I.v.), the desensitization on the effect of systemic blood pressure of the first Ach injection towards the subsequent Ach administration was observed. Results When Ach administration intervals were 40, 60, 80, 100 seconds, the percentages of desensitization of Ach on systemic blood pressure were significantly increased (P0.05). Conclusion The results indicated that Ach contents in blood might influence the action of next Ach administration. To some extent, the higher the concentration of Ach in blood, the bigger the ratio of desensitization of exogenous Ach is. In addition, this method of twice drug administration could be used as a model of studying desensitization in vivo.

  18. The clinical dilemma of "silent desensitization" in aspirin-exacerbated respiratory disease.

    Science.gov (United States)

    White, Andrew A; Bosso, John V; Stevenson, Donald D

    2013-01-01

    Aspirin desensitization is a treatment option for patients with aspirin-exacerbated respiratory disease (AERD). Some patients with an excellent history of aspirin or nonsteroidal anti-inflammatory drug (NSAID) reactions have negative aspirin challenges/desensitization. This study discusses the clinical entity of silent desensitization in AERD and the dilemma that this presents to the practicing allergist/immunologist. We discuss a series of patients with a strong history of NSAID reactions who initially underwent a negative challenge/silent desensitization. These patients were subsequently proven to have AERD after a second positive aspirin challenge. Silent desensitization is an uncommon but important outcome to recognize in AERD. Clinicians performing aspirin desensitization should understand that this can occur and consider a second confirmatory aspirin challenge in some patients.

  19. Origin, development, and evolution of eye movement desensitization and reprocessing

    Directory of Open Access Journals (Sweden)

    Carolina Marín

    2016-12-01

    Full Text Available Eye movement desensitization and reprocessing (EMDR has led to a great number of studies since its appearance in 1989. The aim of this article is to describe EMDR development and evolution to the present day. With this purpose a search was carried out on MEDLINE and PsycINFO with the entry "eye movement desensitization". After revising the resulting 797 articles, those that because of their relevance explained best the development and evolution of the technique were chosen and shaped into a lifeline graphically representing the history of EMDR. Despite the fact that during the first years the focus of research was on the validation of the technique for post-traumatic disorder (PTSD, it was soon applied to other areas. Only 14% of the articles found account for controlled studies. Up to date, in spite of the effectiveness of EMDR for the treatment of PTSD that has been proven, many different explanatory hypotheses are still up for discussion.

  20. Brentuximab vedotin desensitization in a patient with refractory Hodgkin's lymphoma.

    Science.gov (United States)

    Arora, Anubha; Bhatt, Vijaya Raj; Liewer, Susanne; Armitage, James O; Bociek, R Gregory

    2015-10-01

    Brentuximab vedotin has emerged as a useful treatment option for relapsed or refractory Hodgkin's lymphoma; however, uncommon cases of anaphylactic reactions may require its permanent discontinuation. We report a 29-yr-old woman with refractory Hodgkin's lymphoma, who developed an anaphylactic reaction during the second dose of brentuximab vedotin. A 12-step desensitization protocol was followed; after premedicating with antihistaminic agents, methylprednisolone and montelukast, a total dose of 156 mg of brentuximab vedotin (1.8 mg/kg) was given as three infusions with increasing rate and concentration. Such desensitization protocol can allow safe administration of brentuximab vedotin and may have a broader applicability in managing hypersensitivity reactions with other monoclonal antibodies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Evaluation of ex vivo effectiveness of commercial desensitizing dentifrices

    OpenAIRE

    Mockdeci, Hanny; Polonini, Hudson; Martins, Isadora; Granato, Ana-Paula; Raposo, Nádia; Chaves, Maria das Graças Afonso Miranda

    2017-01-01

    Background Dentin hypersensitivity is a short, severe pain with fast onset. Therapy aims to either prevent or decrease neural transmission or physically occlude the dentinal tubules. This study aimed to evaluate the effectiveness of commercial desensitizing dentifrice by means of an ex vivo method. Material and Methods Samples (n=8 lower human premolars for each group) were randomly allocated into: G1- brushing with Colgate?Sensitive Pro-Relief; G2- brushing with Sensodyne?Rapid Relief; G3- b...

  2. Effect of two desensitizing agents on dentin permeability in vitro

    Science.gov (United States)

    ISHIHATA, Hiroshi; KANEHIRA, Masafumi; FINGER, Werner J.; TAKAHASHI, Hidekazu; TOMITA, Makoto; SASAKI, Keiichi

    2017-01-01

    Abstract Objective The aim of this in vitro study was to investigate the effect of two desensitizing agents and water on hydraulic conductance in human dentin. Material and Methods GLUMA Desensitizer PowerGel (GLU) contains glutaraldehyde (GA) and 2-hydroxyethyl methacrylate (HEMA), and Teethmate Desensitizer (TD) is a powder comprising tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) that is mixed with water. Deionized water was used as a negative control (CTR). Thirty discs with a thickness of 1.2 mm were cut from the coronal dentin of the third molars and cleaned with 0.5 M EDTA (pH 7.4). After being mounted in a split-chamber device, the discs were pressurized with water at 1 kPa and 3 kPa in order to measure flow rates with a highly sensitive micro-flow sensor and to calculate hydraulic conductance as a baseline value (BL). Following the application of GLU, TD, and CTR (n=10), hydraulic conductance was remeasured with intermittent storage in water after 15 min, 1 d, 1 w, and 1 m. Reduction in permeability (PR%) was calculated from hydraulic conductance. Data were statistically analyzed using nonparametric methods (α<0.05). Representative discs were inspected by SEM. Results PR% for GLU and TD were 30-50% 15 min and 1 m after their application. Post hoc tests indicated that PR% of CTR was significantly greater than those of GLU and TD at all time points tested. The PR% of GLU and TD were not significantly different. SEM examinations showed noncollapsed collagen meshes at the tubular entrances after GLU, and crystalline precipitates occluding the tubular orifices after TD, whereas CTR specimens showed typical patterns of etched dentin. Conclusions The present study on hydraulic conductance in dentin discs treated with two chemically different desensitizing agents and water as a control demonstrated that both products may be characterized as effective. PMID:28198974

  3. Surfactants and Desensitizing Wax Substitutes for TNT-Based Systems.

    Science.gov (United States)

    1994-10-01

    36 esters, 25% long chain acids and 10% long chain alcohols, ketones, and hydrocarbons. The resin portion contains terpenes and resinic acids, while...aliphatic and aromatic resins and turpentine for terpene resins (i.e. polymerized B-pinene). The most important commercial products today are the resins...Wax desensitizes by suppressing these processes by: Filling voids that otherwise contain gases Separating individual crystals of explosive Absorbing

  4. Evaluation of Five Different Desensitizers: A Comparative Dentin Permeability and SEM Investigation In Vitro

    OpenAIRE

    Yilmaz, Nasibe Aycan; Ertas, Ertan; Oruco?lu, Hasan

    2017-01-01

    Background/Objective: The purpose of this study was to evaluate the efficacy and durability of five different dentin desensitizers (Gluma Desensitizer Powergel, Bifluorid 12, Gluma Self Etch Bond, D/Sense Crystal, Nupro Sensodyne Prophylaxis Paste with Novamin) on tubule occlusion and dentin permeability reduction in vitro. Method: The quantitative changes in permeability of 100 dentin discs were measured after desensitizer treatments and following post-treatments of 6% citric acid challenge ...

  5. [Acetylsalicylic acid desensitization in the new era of percutaneous coronary intervention].

    Science.gov (United States)

    Fuertes Ferre, Georgina; Ferrer Gracia, Maria Cruz; Calvo Cebollero, Isabel

    2015-09-21

    Dual antiplatelet therapy is essential in patients undergoing percutaneous coronary intervention with stent implantation. Hypersensitivity to acetylsalicylic acid (ASA) limits treatment options. Desensitization to ASA has classically been studied in patients with respiratory tract disease. Over the last years, many protocols have been described about ASA desensitization in patients with ischemic heart disease, including acute coronary syndrome and the need for coronary stent implantation. It is important to know the efficacy and safety of ASA desensitization in these patients.

  6. Evaluation of ex vivo effectiveness of commercial desensitizing dentifrices.

    Science.gov (United States)

    Mockdeci, Hanny; Polonini, Hudson; Martins, Isadora; Granato, Ana-Paula; Raposo, Nádia; Chaves, Maria-das Graças

    2017-04-01

    Dentin hypersensitivity is a short, severe pain with fast onset. Therapy aims to either prevent or decrease neural transmission or physically occlude the dentinal tubules. This study aimed to evaluate the effectiveness of commercial desensitizing dentifrice by means of an ex vivo method. Samples (n=8 lower human premolars for each group) were randomly allocated into: G1- brushing with Colgate®Sensitive Pro-Relief; G2- brushing with Sensodyne®Rapid Relief; G3- brushing with Sensodyne®Repair & Protect; and G4- brushing with Colgate®Maximum Cavity Protection. The test bodies were submitted to simulated toothbrushing and dentifrices were analyzed regarding their hydrodynamic size, polydispersity index (PI) and zeta potential. Specimens were evaluated using: scanning electron microscopy (SEM); spectroscopy energy dispersive X-ray (EDS); and profilometry. A qualitative analysis of the photomicrographs and topographies was performed. The dentifrices showed statistical similar physical and chemical characteristics. They also demonstrated obliteration of dentinal tubules when micrographs were observed. Regarding the chemical elements present in the dentin samples, there was a statistically significant difference between the control and experimental surfaces in the four groups. Joint data analysis shows that the desensitizing dentifrice showed better results with regards to the obliteration of dentinal tubules compared to positive and negative controls. Key words:Dentin hypersensitivity, dentin desensitizing agents, toothpastes.

  7. Evaluation of ex vivo effectiveness of commercial desensitizing dentifrices

    Science.gov (United States)

    Mockdeci, Hanny; Polonini, Hudson; Granato, Ana-Paula; Raposo, Nádia; Chaves, Maria-das Graças

    2017-01-01

    Background Dentin hypersensitivity is a short, severe pain with fast onset. Therapy aims to either prevent or decrease neural transmission or physically occlude the dentinal tubules. This study aimed to evaluate the effectiveness of commercial desensitizing dentifrice by means of an ex vivo method. Material and Methods Samples (n=8 lower human premolars for each group) were randomly allocated into: G1- brushing with Colgate®Sensitive Pro-Relief; G2- brushing with Sensodyne®Rapid Relief; G3- brushing with Sensodyne®Repair & Protect; and G4- brushing with Colgate®Maximum Cavity Protection. The test bodies were submitted to simulated toothbrushing and dentifrices were analyzed regarding their hydrodynamic size, polydispersity index (PI) and zeta potential. Specimens were evaluated using: scanning electron microscopy (SEM); spectroscopy energy dispersive X-ray (EDS); and profilometry. A qualitative analysis of the photomicrographs and topographies was performed. Results The dentifrices showed statistical similar physical and chemical characteristics. They also demonstrated obliteration of dentinal tubules when micrographs were observed. Regarding the chemical elements present in the dentin samples, there was a statistically significant difference between the control and experimental surfaces in the four groups. Conclusions Joint data analysis shows that the desensitizing dentifrice showed better results with regards to the obliteration of dentinal tubules compared to positive and negative controls. Key words:Dentin hypersensitivity, dentin desensitizing agents, toothpastes. PMID:28469813

  8. Antibody desensitization therapy in highly sensitized lung transplant candidates.

    Science.gov (United States)

    Snyder, L D; Gray, A L; Reynolds, J M; Arepally, G M; Bedoya, A; Hartwig, M G; Davis, R D; Lopes, K E; Wegner, W E; Chen, D F; Palmer, S M

    2014-04-01

    As HLAs antibody detection technology has evolved, there is now detailed HLA antibody information available on prospective transplant recipients. Determining single antigen antibody specificity allows for a calculated panel reactive antibodies (cPRA) value, providing an estimate of the effective donor pool. For broadly sensitized lung transplant candidates (cPRA ≥ 80%), our center adopted a pretransplant multi-modal desensitization protocol in an effort to decrease the cPRA and expand the donor pool. This desensitization protocol included plasmapheresis, solumedrol, bortezomib and rituximab given in combination over 19 days followed by intravenous immunoglobulin. Eight of 18 candidates completed therapy with the primary reasons for early discontinuation being transplant (by avoiding unacceptable antigens) or thrombocytopenia. In a mixed-model analysis, there were no significant changes in PRA or cPRA changes over time with the protocol. A sub-analysis of the median fluorescence intensity (MFI) change indicated a small decline that was significant in antibodies with MFI 5000-10,000. Nine of 18 candidates subsequently had a transplant. Posttransplant survival in these nine recipients was comparable to other pretransplant-sensitized recipients who did not receive therapy. In summary, an aggressive multi-modal desensitization protocol does not significantly reduce pretransplant HLA antibodies in a broadly sensitized lung transplant candidate cohort. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

  9. Desensitization with DTP (diphtheria, tetanus and pertussis vaccine

    Directory of Open Access Journals (Sweden)

    Atanasković-Marković Marina

    2003-01-01

    Full Text Available Immunization with DTP vaccine (diphtheria, tetanus and pertussis is a part of the vaccination calendar offered in childhood. Adverse allergic reactions vary from minimal urticarial reactions to life-threatening anaphylaxis. In infancy these reactions usually interrupt the vaccination calendar, but immunization in these children should be done. At the University Children's Hospital of Belgrade, a group of 137 children with suspected allergic anaphylactic reaction to DTP, DT, TT and monopertussis vaccine was studied for the last six years. Skin (prick and intradermal tests were performed with corresponding vaccine. If both tests were negative, the vaccine could be given as a single dose of 0.5 ml. If one of these tests were positive desensitization with vaccine could be done (according to the protocol described by Carey and Meltzer. In one group of 52 children three days before desensitization, premedication with antihistamines, was done, whereas in the other group of 52 children premedication was not done. Two (3.8% children in a group of 52 children with premedication had a minor (local reaction after vaccination and 50 children (96.2% had no reaction after vaccination, whereas no children (0% had systemic reaction after desensitization.

  10. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

    Science.gov (United States)

    Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J; Shanker, Sreenath; Kuscu, Canan; Joshua-Tor, Leemor; Partridge, Janet F

    2009-04-10

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  11. 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

    BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) impacts lipid, bone, and glucose homeostasis. The GIP receptor belongs to G protein-coupled receptor family B1 and signals through GαS. High affinity ligands for in vivo use are needed to elucidate GIP's physiological...... 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......, 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....

  12. Explosion Power and Pressure Desensitization Resisting Property of Emulsion Explosives Sensitized by MgH2

    Science.gov (United States)

    Cheng, Yangfan; Ma, Honghao; Liu, Rong; Shen, Zhaowu

    2014-07-01

    Due to low detonation power and pressure desensitization problems that traditional emulsion explosives encounter in utilization, a hydrogen-based emulsion explosives was devised. This type of emulsion explosives is sensitized by hydrogen-containing material MgH2, and MgH2 plays a double role as a sensitizer and an energetic material in emulsion explosives. Underwater explosion experiments and shock wave desensitization experiments show that an MgH2 emulsion explosives has excellent detonation characteristics and is resistant to pressure desensitization. The pressure desensitization-resistant mechanism of MgH2 emulsion explosives was investigated using scanning electron microscopy.

  13. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  14. Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A.; Lowe, Edward D.; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W.; Cogdell, Richard J.; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  15. Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

    Science.gov (United States)

    Brophy, Megan Brunjes; Nakashige, Toshiki G; Gaillard, Aleth; Nolan, Elizabeth M

    2013-11-27

    Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

  16. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

    Science.gov (United States)

    Burel, Sebastien A; Hart, Christopher E; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-Hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M; Hung, Gene; Dan, Amy; Prakash, T P; Seth, Punit P; Swayze, Eric E; Bennett, C Frank; Crooke, Stanley T; Henry, Scott P

    2016-03-18

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    Science.gov (United States)

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  18. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites.

    Science.gov (United States)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina; Vogensen, Stine B; Lehel, Szabolcs; Thiesen, Louise; Bundgaard, Christoffer; Clausen, Rasmus P; Knudsen, Gitte M; Herth, Matthias M; Wellendorph, Petrine; Frølund, Bente

    2017-01-18

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA.

  19. Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes.

    Science.gov (United States)

    Kumar, Sunil; Xue, Liang; Arya, Dev P

    2011-05-18

    A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT

  20. Cytokine expression before and after aspirin desensitization therapy in aspirin-exacerbated respiratory disease.

    Science.gov (United States)

    Aktas, Ayse; Kurt, Emel; Gulbas, Zafer

    2013-12-01

    Aspirin exacerbated respiratory disease (AERD) is induced by acetylsalicylic acid (ASA) and/or nonsteroidal antiinflammatory drugs (NSAIDs). Effects of desensitization on many mediators have been examined previously, but few studies addressed the influence of desensitization on T lymphocytes and T lymphocyte-derived cytokines. This study was performed to examine peripheral blood lymphocyte (PBL) cytokine expression in aspirin-sensitive patients who have asthma before and after aspirin desensitization. In this study, the release of interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-gamma (IFN-γ) by CD4+ T lymphocytes prior to aspirin desensitization were also measured at intracellular levels, and expression of these cytokines after 1 month aspirin desensitization was evaluated. Twelve patients with AERD were included in the study. Two different control groups were formed, one consisted of 15 healthy people and second 12 aspirin tolerant asthmatic (ATA) patients using aspirin. A blood sample was collected prior to desensitization, and the tests were repeated by taking a second blood sample 1 month after the 4-day desensitization treatment. The proportion of lymphocytes secreting IFN-γ in the study group was 15.61 ± 4.40 % before desensitization and 15.08 ± 5.89 % after desensitization. The rate of IFN-γ secreting CD4+ T lymphocytes was 20.51 ± 4.41 % in the normal control group and 16.07 ± 5.7 % in the ATA group (p = 0.021). The ratio of CD4+ T lymphocyte secreting IFN-γ was reduced in patients with AERD before desensitization compared to normal control group (p = 0.040). The levels of IL-2, IL-4, and the subsets of lymphocyte were not different before and after desensitization compared to control groups.

  1. Desensitization to inhaled aztreonam lysine in an allergic patient with cystic fibrosis using a novel approach.

    Science.gov (United States)

    Guglani, Lokesh; Abdulhamid, Ibrahim; Ditouras, Joanna; Montejo, Jenny

    2012-10-01

    To report the successful desensitization of a highly allergic patient with cystic fibrosis (CF) to inhaled aztreonam lysine using the novel approach of intravenous desensitization followed by full-dose inhaled therapy without any adverse reactions. A 19-year-old woman with CF had persistent Pseudomonas aeruginosa-positive cultures and a history of type I hypersensitivity reactions to multiple medications, including aztreonam and tobramycin (intravenous and inhaled). To start therapy with an inhaled antipseudomonal antibiotic on a chronic basis, she underwent rapid desensitization to intravenous aztreonam followed by initiation of inhaled aztreonam lysine. Following intravenous desensitization with aztreonam, there was no adverse reaction or decline in lung function noted with inhaled aztreonam lysine and the chronic therapy was continued at home, with a modified regimen to maintain desensitization. Aztreonam lysine has been used for treatment of patients with CF with chronic P. aeruginosa colonization. Previous allergic reaction to intravenous aztreonam is considered a contraindication for use of aztreonam lysine. Our patient had a history of hives and facial swelling following administration of intravenous aztreonam (type I hypersensitivity reaction) as well as hypersensitivity to tobramycin. Rapid desensitization can be done for drugs that mediate a type I hypersensitivity reaction, with mast cells and basophils being the cellular targets. There are a few case reports of desensitization to inhaled antibiotics such as tobramycin and colistin, but desensitization to aztreonam lysine has not previously been reported. Desensitization of a patient with CF who is allergic to intravenous aztreonam was successfully accomplished with the novel approach of rapid intravenous desensitization followed by inhaled therapy. As inhaled antibiotics are being increasingly used for patients with CF, this novel strategy can be used for desensitizing allergic patients with CF to

  2. THE RELATION BETWEEN ANTIANAPHYLAXIS AND ANTIBODY BALANCE : II. THE EFFECT OF SPECIFIC DESENSITIZATION UPON RESISTANCE TO INFECTION AND UPON ANTIBODY BALANCE.

    Science.gov (United States)

    Morris, M C

    1936-09-30

    It has been shown that antianaphylaxis is not caused by a partial saturation of cellular or humoral antibodies by the following facts. 1. Guinea pigs passively sensitized with anti-horse or antipneumococcus serum and specifically desensitized do not manifest as great a reactivity upon resensitization with the same antiserum as upon the original sensitization. 2. Guinea pigs passively sensitized with anti-Friedländer Type B serum or antipneumococcus Type II serum and specifically desensitized do not attain the same degree of reactivity as normal animals when passively sensitized with anti-horse serum. 3. Guinea pigs passively sensitized with anti-Friedländer Type B serum and desensitized with the specific carbohydrate remain as resistant to infection with Friedlander's bacillus Type B as undesensitized guinea pigs. Since in this case, at least, it is agreed that type-specific immunity and type-specific hypersensitiveness are due to the same type-specific antibody, a change in anaphylactic response should be accompanied by a change in immune response, provided this change depends on antibody balance. 4. A determination of the antibody content of the serum of sensitized as well as of desensitized guinea pigs by mouse protection tests indicates that a loss of reactivity in desensitized animals cannot be adequately accounted for on the basis of depletion of circulating antibody. These experiments suggest that hypersensitiveness and resistance are different manifestations of the same antigen-antibody reaction while antianaphylaxis is a state of refractoriness which is due neither to excess of circulating antibody nor to antibody depletion, but is the result of secondary changes the true nature of which is still not definitely established.

  3. Desensitizing Addiction : Using Eye Movements to Reduce the Intensity of Substance-Related Mental Imagery and Craving

    NARCIS (Netherlands)

    Littel, M.; van den Hout, M.A.; Engelhard, I.M.

    2016-01-01

    Eye movement desensitization and reprocessing (EMDR) is an effective treatment for posttraumatic stress disorder. During this treatment, patients recall traumatic memories while making horizontal eye movements (EM). Studies have shown that EM not only desensitize negative memories but also positive

  4. Desensitizing Addiction : Using Eye Movements to Reduce the Intensity of Substance-Related Mental Imagery and Craving

    NARCIS (Netherlands)

    Littel, M.; van den Hout, M.A.; Engelhard, I.M.

    2016-01-01

    Eye movement desensitization and reprocessing (EMDR) is an effective treatment for posttraumatic stress disorder. During this treatment, patients recall traumatic memories while making horizontal eye movements (EM). Studies have shown that EM not only desensitize negative memories but also positive

  5. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    LIANG; Tiebing

    2001-01-01

    ., 1996, 255: 373-386.[13]Kim, J. -S., Pabo, C. O., Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants, Proc. Natl. Acad. Sci. USA, 1998, 95: 2812-2817.[14]Wu, H., Yang, W. -P., BarbasIII, C. F., Building zinc fingers by selection: toward a therapeutic application, Proc. Natl. Acad. Sci. USA, 1995, 92: 344-348.[15]Wang, B. S., Pabo, C. O., Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences, Proc. Natl. Acad. Sci. USA, 1999, 96: 9568-9573.[16]Choo, Y., Sánchez-García, I., Klug, A., In vivo repression by a site-specific DNA-binding protein designed against an on-cogenic sequence, Nature, 1994, 372: 642-645.[17]Wolfe, S. A., Greisman, H. A., Ramm, E. I. et al., Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code, J. Mol. Biol., 1999, 285: 1917-1934.[18]Chen, J. -Q., Pongor, S., Simoncsits, A., Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs, Nucleic Acids Research, 1997, 25: 2047-2054.[19]Simoncsits, A., Tj?rnhammar, M. -L., Wang, S. -L. et al., Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites, Nucleic Acids Research, 1999, 27: 3474-3480.[20]Zhou, Y. -H., Busby, S., Ebright, R. H., Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers, Cell, 1993, 73: 375-379.[21]Studier, F. W., Rosenberg, A. H., Dunn, J. J. et al., Use of T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol., 1990, 185: 60-89.[22]Simoncsits, A., Bristulf, J., Tj?rnhammar, M. -L. et al., Deletion mutants of human interleukin 1? with significantly re-duced agonist properties: search for agonist/ antagonist switch in ligands to the interleukin 1

  6. Evaluation of Five Different Desensitizers: A Comparative Dentin Permeability and SEM Investigation In Vitro.

    Science.gov (United States)

    Yilmaz, Nasibe Aycan; Ertas, Ertan; Orucoğlu, Hasan

    2017-01-01

    The purpose of this study was to evaluate the efficacy and durability of five different dentin desensitizers (Gluma Desensitizer Powergel, Bifluorid 12, Gluma Self Etch Bond, D/Sense Crystal, Nupro Sensodyne Prophylaxis Paste with Novamin) on tubule occlusion and dentin permeability reduction in vitro. The quantitative changes in permeability of 100 dentin discs were measured after desensitizer treatments and following post-treatments of 6% citric acid challenge for 1 min or immersion in artificial saliva for 24 hours under hydrostatic pressure generated by a computerised fluid filtration meter. Qualitative SEM analyses were also carried out. Dentin permeability decreased after desensitizer application in all groups. Nevertheless, only the difference between 'Gluma Self Etch Bond' and 'Nupro Sensodyne Prophylaxis Paste with Novamin' groups was significantly different (p0.05). Of all the artificial saliva-subgroups, only the difference between 'D/Sense Crystal' and 'Bifluorid 12' was significantly different (p<0.05). In SEM analysis, morphological changes were detected on the dentin surface and within the tubules following desensitizer treatments and post-treatments. All the desensitizers significantly reduced dentin permeability by changing the morphology of the dentin surface and/or dentinal tubules. Following post-treatments, there was some reduction in the efficacy of the desensitizers which was represented by the reduction in permeability values. SEM analysis revealed some physical changes in the dentin structure which can partly give an explanation to the reduced efficacy of tested desensitizers.

  7. Self-Desensitization and Meditation in the Reduction of Public Speaking Anxiety.

    Science.gov (United States)

    Kirsch, Irving; Henry, David

    1979-01-01

    Speech-anxious students were assigned to self-administered treatment conditions: (1) systematic desensitization, (2) desensitization with meditation replacing progressive relaxation, and (3) meditation only. Treatment manuals included coping-skill instructions. Treatments were equally effective in reducing anxiety and produced a greater reduction…

  8. The Effect of Self and In Vivo Desensitization on Counselor Trainee Anxiety and Performance.

    Science.gov (United States)

    Miller, Thomas V.

    The rationale, planning and implementing of this research is discussed in terms of its three hypotheses: (1) that both self desensitization and in vivo desensitization would result in lower Fear Index and Anxiety Differential scores of counselor trainees just prior to communicating with a role playing client in a counseling room where they are…

  9. Aspirin desensitization in patients undergoing percutaneous coronary intervention: a survey of current practice.

    Science.gov (United States)

    Chapman, Andrew R; Rushworth, Gordon F; Leslie, Stephen J

    2013-01-01

    Aspirin remains the mainstay of anti-platelet therapy in cardiac patients. However, if a patient is allergic to aspirin and dual anti-platelet therapy is indicated - such as with percutaneous coronary intervention (PCI), then there is no clear guidance. One possibility is aspirin desensitization. A variety of protocols exist for the rapid desensitization of patients with aspirin allergy. The aim of this survey was to assess current knowledge and practice regarding aspirin desensitization in the UK. We conducted a UK wide survey of all UK 116 PCI centers and obtained complete responses from 40 (35.4%) centers. Of these, just 7 (17.5%) centers had previously desensitised patients; 29 (87.9%) centers suggested a lack of a local protocol prevented them from desensitizing, with 10 (30.3%) unsure of how to conduct desensitization. Only 5 (12.5%) centers had a local policy for aspirin desensitization although 25 (64.1%) units had a clinical strategy for dealing with aspirin allergy; the majority (72%) giving higher doses of thienopyridine class drugs. In the UK, there appears to be no consistent approach to patients with aspirin allergy. Patients undergoing PCI benefit from dual anti-platelet therapy (including aspirin), and aspirin desensitization in those with known allergy may facilitate this. Sustained effort should be placed on encouraging UK centers to use desensitization as a treatment modality prior to PCI rather than avoiding aspirin altogether.

  10. Rapid desensitization for non-immediate reactions in patients with cystic fibrosis.

    Science.gov (United States)

    Whitaker, P; Shaw, N; Gooi, J; Etherington, C; Conway, S; Peckham, D

    2011-07-01

    Non-immediate hypersensitivity reactions to antibiotics in patients with CF represent a real-life challenge for clinicians. Desensitization is often performed in patients who have exhausted all therapeutic options. Whilst desensitization is an established procedure for immediate reactions we assessed the outcomes and safety of desensitization for non-immediate reactions. We retrospectively reviewed 275 desensitization procedures in 42 patients with a range of non-immediate reactions to six commonly used antibiotics. Desensitization was performed using a 7-step rapid intravenous protocol on a normal medical ward. 250 (91%) of overall desensitization procedures were successful; however, this figure incorporates certain individuals having multiple successful procedures. Individual patient success ranged from 55% with tazocin through to 88% with tobramycin. In the 25 patients who failed desensitization the reactions were mild and the majority occurred within 48 h of starting treatment. Prophylactic anti-histamines and steroids did not reduce the risk of reaction. Whilst the mechanisms remain uncertain we can confirm that rapid desensitization is a safe and effective way of re-introducing an antibiotic to a patient with a non-immediate reaction. Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  11. A practical and successful desensitization protocol for immediate hypersensitivity reactions to iron salts.

    Science.gov (United States)

    Demir, Semra; Olgac, Muge; Unal, Derya; Gelincik, Asli; Colakoglu, Bahauddin; Buyukozturk, Suna

    2014-01-01

    Orally administered iron salts (OAS) are widely used in the management of iron deficiency anemia and hypersensitivity reactions to OAS are not common. If an offending drug is the sole option or is significantly more effective than its alternatives, it can be readministered by desensitization. The oral desensitization protocols for iron published so far concern either desensitization that was completed only over a long period or did not attain the recommended therapeutic dose. We aimed to develop a more effective protocol. We report here on 2 patients who experienced hypersensitivity reactions to OAS. After confirming the diagnosis, both patients were desensitized to oral ferrous (II) glycine sulfate complex according to a 2-day desensitization protocol. A commercial suspension of oral ferrous glycine sulfate, which contains 4 mg of elemental iron in 1 ml, was preferred. We started with a dose as low as 0.1 ml from a 1/100 dilution (0.004 mg elemental iron) of the original suspension and reached the maximum effective dose in 2 days. Both patients were successfully desensitized and they went on to complete the 6-month iron treatment without any adverse effects. Although hypersensitvity reactions to iron are not common, there is no alternative for iron administration. Therefore, desensitization has to be the choice. This easy desensitization protocol seems to be a promising option. © 2014 S. Karger AG, Basel.

  12. Effectiveness of Structured Psychodrama and Systematic Desensitization in Reducing Test Anxiety.

    Science.gov (United States)

    Kipper, David A.; Giladi, Daniel

    1978-01-01

    Students with examination anxiety took part in study of effectiveness of two kinds of treatment, structured psychodrama and systematic desensitization, in reducing test anxiety. Results showed that subjects in both treatment groups significantly reduced test-anxiety scores. Structured psychodrama is as effective as systematic desensitization in…

  13. Violence Exposure in Real-Life, Video Games, Television, Movies, and the Internet: Is There Desensitization?

    Science.gov (United States)

    Funk, Jeanne B.; Baldacci, Heidi Bechtoldt; Pasold; Tracie; Baumgardner, Jennifer

    2004-01-01

    It is believed that repeated exposure to real-life and to entertainment violence may alter cognitive, affective, and behavioral processes, possibly leading to desensitization. The goal of the present study was to determine if there are relationships between real-life and media violence exposure and desensitization as reflected in related…

  14. Prospective iterative trial of proteasome inhibitor-based desensitization.

    Science.gov (United States)

    Woodle, E S; Shields, A R; Ejaz, N S; Sadaka, B; Girnita, A; Walsh, R C; Alloway, R R; Brailey, P; Cardi, M A; Abu Jawdeh, B G; Roy-Chaudhury, P; Govil, A; Mogilishetty, G

    2015-01-01

    A prospective iterative trial of proteasome inhibitor (PI)-based therapy for reducing HLA antibody (Ab) levels was conducted in five phases differing in bortezomib dosing density and plasmapheresis timing. Phases included 1 or 2 bortezomib cycles (1.3 mg/m(2) × 6-8 doses), one rituximab dose and plasmapheresis. HLA Abs were measured by solid phase and flow cytometry (FCM) assays. Immunodominant Ab (iAb) was defined as highest HLA Ab level. Forty-four patients received 52 desensitization courses (7 patients enrolled in multiple phases): Phase 1 (n = 20), Phase 2 (n = 12), Phase 3 (n = 10), Phase 4 (n = 5), Phase 5 (n = 5). iAb reductions were observed in 38 of 44 (86%) patients and persisted up to 10 months. In Phase 1, a 51.5% iAb reduction was observed at 28 days with bortezomib alone. iAb reductions increased with higher bortezomib dosing densities and included class I, II, and public antigens (HLA DRβ3, HLA DRβ4 and HLA DRβ5). FCM median channel shifts decreased in 11/11 (100%) patients by a mean of 103 ± 54 mean channel shifts (log scale). Nineteen out of 44 patients (43.2%) were transplanted with low acute rejection rates (18.8%) and de novo DSA formation (12.5%). In conclusion, PI-based desensitization consistently and durably reduces HLA Ab levels providing an alternative to intravenous immune globulin-based desensitization. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

  15. Desensitized nicotinic receptors that, however, afford cytoprotection in bovine chromaffin cells.

    Science.gov (United States)

    Egea, Javier; Hernández-Guijo, Jesús Miguel; Olivares, Roman; López, Manuela G; García, Antonio G

    2006-01-01

    Neuronal nicotinic receptors for acetylcholine (nAChRs) are among the ionotropic receptors that suffer the most desensitization upon prolonged exposure to their agonists. This is particularly true for the alpha7 subtype of nAChRs, although alpha3beta4 receptors also suffer quick desensitization. This study was planned to test the hypothesis that even after suffering desensitization, a given nAChR might still afford cell protection against a noxious stimulus. Of the many agonists developed for nAChRs, we selected the poorly desensitizing ligand dimethylphenylpiperazinium (DMPP) (Britt and Brenner, 1997) and the highly desensitizing agent epibatidine (EPB) (Marks et al., 1996). We have measured nAChR currents, catecholamine secretory responses, and changes of [Ca2+]c elicited by stimulation of nAChRs with DMPP or EPB. We have also investigated cytoprotection elicited by DMPP and EPB against the cytotoxic effects of veratridine in bovine chromaffin cells.

  16. Sexual violence: psychiatric healing with eye movement reprocessing and desensitization.

    Science.gov (United States)

    Posmontier, Bobbie; Dovydaitis, Tiffany; Lipman, Kenneth

    2010-08-01

    Sexual violence, which affects one in three women worldwide, can result in significant psychiatric morbidity and suicide. Eye movement desensitization and reprocessing (EMDR) offers health care providers the option of a brief psychiatric intervention that can result in psychiatric healing in as few as four sessions. Because health care providers often hear stories of sexual violence from their patients, they are in an ideal position to make recommendations for treatment. The purpose of this article is to introduce health care providers to the technique of EMDR, review safety and appropriateness, and discuss clinical and research implications.

  17. Efficacy, outcomes, and cost-effectiveness of desensitization using IVIG and rituximab.

    Science.gov (United States)

    Vo, Ashley A; Petrozzino, Jeffrey; Yeung, Kai; Sinha, Aditi; Kahwaji, Joseph; Peng, Alice; Villicana, Rafael; Mackowiak, John; Jordan, Stanley C

    2013-03-27

    Transplantation rates are very low for the broadly sensitized patient (panel reactive antibody [PRA]>80%; HS). Here, we examine the efficacy, outcomes, and cost-effectiveness of desensitization using high-dose intravenous immunoglobulin (IVIG) and rituximab to improve transplantation rates in HS patients. From July 2006 to December 2011, 207 HS (56 living donors/151 deceased donors) patients (donor-specific antibody positive, PRA>80%) were desensitized using IVIG and rituximab. After desensitization, responsive patients proceeded to transplantation with an acceptable crossmatch. Cost and outcomes of desensitization were compared with dialysis. Of the 207 treated patients, 146 (71%) were transplanted. At 48 months, patient and graft survival by Kaplan-Meier were 95% and 87.5%, respectively. The total 3-year cost for patients treated in the desensitization arm was $219,914 per patient compared with $238,667 per patient treated in the dialysis arm. Thus, each patient treated with desensitization is estimated to save the U.S. healthcare system $18,753 in 2011 USD. Overall, estimated patient survival at the end of 3 years was 96.6% for patients in the desensitization arm of the model (based on Cedars-Sinai survival rate) compared with 79.0% for an age, end-stage renal disease etiology, and PRA matched group of patients remaining on dialysis during the study period. We conclude that desensitization with IVIG+rituximab is clinically and cost-effective, with both financial savings and an estimated 17.6% greater probability of 3-year survival associated with desensitization versus dialysis alone. However, the benefits of desensitization and transplantation are limited by organ availability and allocation policies.

  18. Safety of aspirin desensitization in patients with reported aspirin allergy and cardiovascular disease.

    Science.gov (United States)

    McMullan, Kathryn L; Wedner, H James

    2013-01-01

    Aspirin (ASA) is the drug of choice in patients with coronary artery disease for primary and secondary prevention. This poses a problem for those patients reporting hypersensitivity to this drug or class of drugs. Desensitization to ASA may be carried out safely and effectively in patients with reported ASA or nonsteroidal anti-inflammatory drug (NSAID) hypersensitivity needing ASA for cardiac indications. Our 7-step protocol is one choice for a rapid desensitization protocol. A retrospective chart review was conducted evaluating ASA desensitization in patients with reported ASA or NSAID hypersensitivity and a cardiac indication for ASA. In 160 evaluations over 15 years, 89 desensitizations were performed in both the inpatient and outpatient setting with only 16 reactions (18%). Eleven of these 16 patients (68.7%) were able to take daily ASA. Twenty-six desensitization procedures were performed with our 7-step rapid desensitization protocol in 10 inpatients and 16 outpatients with 3 reactions (18.75% of reactions). Initial reaction to ASA involving angioedema and reacting to ASA within the past year increased the risk of having a reaction to desensitization. Desensitization may be safely performed in patients with reported ASA or NSAID hypersensitivity and a cardiac indication for ASA. Our 7-step rapid protocol may be used in both the inpatient and outpatient setting to desensitize these patients. Patients who had angioedema with ASA ingestion or a reaction to ASA within the past year are at higher risk for reaction during the desensitization protocol. The authors have no funding, financial relationships, or conflicts of interest to disclose. © 2012 Wiley Periodicals, Inc.

  19. Long-term sinonasal outcomes of aspirin desensitization in aspirin exacerbated respiratory disease.

    Science.gov (United States)

    Cho, Kyu-Sup; Soudry, Ethan; Psaltis, Alkis J; Nadeau, Kari C; McGhee, Sean A; Nayak, Jayakar V; Hwang, Peter H

    2014-10-01

    This study aimed to assess sinonasal outcomes in patients with aspirin exacerbated respiratory disease (AERD) undergoing aspirin desensitization following endoscopic sinus surgery (ESS). Case series with chart review. University hospital. A retrospective review of sinonasal outcomes was conducted for 30 AERD patients undergoing aspirin desensitization and maintenance therapy following ESS. Sinonasal outcomes were prospectively assessed by the Sinonasal Outcomes Test-22 (SNOT-22) and endoscopic polyp grading system. Data were collected preoperatively, 1 and 4 weeks postsurgery (before desensitization), and 1, 6, 12, 18, 24, and 30 months after aspirin desensitization. Twenty-eight of 30 patients (93.3%) successfully completed aspirin desensitization, whereas 2 of 30 (6.7%) were unable to complete desensitization due to respiratory intolerance. Of the 21 patients who successfully completed a minimum of 24 weeks of follow-up, 20 (95.2%) patients demonstrated sustained endoscopic and symptomatic improvement for a median follow-up period of 33 months. After surgical treatment but before desensitization, patients experienced significant reductions in SNOT-22 and polyp grade scores. In the first 6 months after aspirin desensitization, patients experienced further significant reductions in SNOT-22 scores, whereas polyp grade remained stable. The improvements in symptom endoscopic scores were preserved throughout the follow-up period after desensitization. No patients required additional sinus surgery. One patient had to discontinue aspirin therapy due to gastrointestinal side effects. No other adverse reactions to aspirin were noted. Aspirin desensitization following ESS appears to be a well-tolerated and effective adjunctive therapy for long-term control of nasal polyposis in patients with AERD. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

  20. High-affinity binding of southern African HIV type 1 subtype C envelope protein, gp120, to the CCR5 coreceptor.

    Science.gov (United States)

    Fromme, Bernhard J; Coetsee, Marla; Van Der Watt, Pauline; Chan, Mei-Chi; Sperling, Karin M; Katz, Arieh A; Flanagan, Colleen A

    2008-12-01

    HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.

  1. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    Science.gov (United States)

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1996-08-30

    Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

  3. Programmed Death-1 Culls Peripheral Accumulation of High-Affinity Autoreactive CD4 T Cells to Protect against Autoimmunity

    OpenAIRE

    2016-01-01

    Self-reactive CD4 T cells are incompletely deleted during thymic development, and their peripheral seeding highlights the need for additional safeguards to avert autoimmunity. Here, we show an essential role for the coinhibitory molecule programmed death-1 (PD-1) in silencing the activation of high-affinity autoreactive CD4 T cells. Each wave of self-reactive CD4 T cells that escapes thymic deletion autonomously upregulates PD-1 to maintain self-tolerance. By tracking the progeny derived from...

  4. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    Science.gov (United States)

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  5. A new class of fluorescent boronic acids that have extraordinarily high affinities for diols in aqueous solution at physiological pH.

    Science.gov (United States)

    Cheng, Yunfeng; Ni, Nanting; Yang, Wenqian; Wang, Binghe

    2010-12-03

    The boronic acid group is an important recognition moiety for sensor design. Herein, we report a series of isoquinolinylboronic acids that have extraordinarily high affinities for diol-containing compounds at physiological pH. In addition, 5- and 8-isoquinolinylboronic acids also showed fairly high binding affinities towards D-glucose (K(a)=42 and 46 M(-1), respectively). For the first time, weak but encouraging binding of cis-cyclohexanediol was found for these boronic acids. Such binding was coupled with significant fluorescence changes. Furthermore, 4- and 6-isoquinolinylboronic acids also showed the ability to complex methyl α-D-glucopyranose (K(a)=3 and 2 M(-1), respectively).

  6. Immune Desensitization Allows Pediatric Blood Group Incompatible Kidney Transplantation.

    Science.gov (United States)

    Stojanovic, Jelena; Adamusiak, Anna; Kessaris, Nicos; Chandak, Pankaj; Ahmed, Zubir; Sebire, Neil J; Walsh, Grainne; Jones, Helen E; Marks, Stephen D; Mamode, Nizam

    2017-06-01

    Blood group incompatible transplantation (ABOi) in children is rare as pretransplant conditioning remains challenging and concerns persist about the potential increased risk of rejection. We describe the results of 11 ABOi pediatric renal transplant recipients in the 2 largest centers in the United Kingdom, sharing the same tailored desensitization protocol. Patients with pretransplant titers of 1 or more in 8 received rituximab 1 month before transplant; tacrolimus and mycophenolate mofetil were started 1 week before surgery. Antibody removal was performed to reduce titers to 1 or less in 8 on the day of the operation. No routine postoperative antibody removal was performed. Death-censored graft survival at last follow-up was 100% in the ABOi and 98% in 50 compatible pediatric transplants. One patient developed grade 2A rejection successfully treated with antithymocyte globulin. Another patient had a titer rise of 2 dilutions treated with 1 immunoadsorption session. There was no histological evidence of rejection in the other 9 patients. One patient developed cytomegalovirus and BK and 2 others EBV and BK viremia. Tailored desensitization in pediatric blood group incompatible kidney transplantation results in excellent outcomes with graft survival and rejection rates comparable with compatible transplants.

  7. Immunological mechanisms for desensitization and tolerance in food allergy1

    Science.gov (United States)

    Rachid, Rima; Umetsu, Dale T.

    2013-01-01

    Food allergy is a major public health concern in westernized countries, estimated to affect 5% of children and 3-4 % of adults. Allergen specific immunotherapy for food allergy is currently being actively evaluated, but is still experimental. The optimal protocol, in terms of the route of administration of the food, target maintenance dose, duration of maintenance therapy and the optimal patient for these procedures are still being worked out. The mechanisms underlying successful food desensitization are also unclear, in part because there is no standard immunotherapy protocol. The mechanisms involved however, may include mast cell and basophil suppression, development of food-specific IgG4 antibodies, reduction in the food specific IgE/IgG4 ratio, up-regulation and expansion of natural or inducible regulatory T cells, a skewing from a Th2 to a Th1 profile and the development of anergy and/or deletion in antigen specific cells. Additional studies are required to elucidate and understand these mechanisms by which desensitization and tolerance are achieved, and which may reveal valuable biomarkers for evaluating and following food allergic patients on immunotherapy. PMID:22821087

  8. Programmed Death-1 Culls Peripheral Accumulation of High-Affinity Autoreactive CD4 T Cells to Protect against Autoimmunity

    Directory of Open Access Journals (Sweden)

    Tony T. Jiang

    2016-11-01

    Full Text Available Self-reactive CD4 T cells are incompletely deleted during thymic development, and their peripheral seeding highlights the need for additional safeguards to avert autoimmunity. Here, we show an essential role for the coinhibitory molecule programmed death-1 (PD-1 in silencing the activation of high-affinity autoreactive CD4 T cells. Each wave of self-reactive CD4 T cells that escapes thymic deletion autonomously upregulates PD-1 to maintain self-tolerance. By tracking the progeny derived from individual autoreactive CD4 T cell clones, we demonstrate that self-reactive cells with the greatest autoimmune threat and highest self-antigen affinity express the most PD-1. Reciprocally, PD-1 deprivation unleashes high-affinity self-reactive CD4 T cells in target tissues to exacerbate neuronal inflammation and autoimmune diabetes. Reliance on PD-1 to actively maintain self-tolerance may explain why exploiting this pathway by cancerous cells and invasive microbes efficiently subverts protective immunity, and why autoimmune side effects can develop after PD-1-neutralizing checkpoint therapies.

  9. Involvement of nitrogen functional groups in high-affinity copper binding in tomato and wheat root apoplasts: spectroscopic and thermodynamic evidence.

    Science.gov (United States)

    Guigues, Stéphanie; Bravin, Matthieu N; Garnier, Cédric; Masion, Armand; Chevassus-Rosset, Claire; Cazevieille, Patrick; Doelsch, Emmanuel

    2016-03-01

    Carboxylic groups located in plant cell walls (CW) are generally considered to be the main copper binding sites in plant roots, despite the presence of other functional groups. The aim of this study was to investigate sites responsible for copper binding in root apoplasts, i.e. CW and outer surface of the plasma membrane (PM) continuum. Binding sites in root apoplasts were investigated by comparing isolated CW of a monocotyledon (Triticum aestivum L.) and dicotyledon (Solanum lycopersicum L.) crop with their respective whole roots. Copper speciation was examined by X-ray absorption (XAS) and (13)C-nuclear magnetic resonance spectroscopies while the affinity of ligands involved in copper binding was investigated by modeling copper sorption isotherms. Homogeneous speciation and binding of copper was found in wheat and tomato root apoplasts. Only Cu-N and Cu-O bonds were detected in wheat and tomato root apoplasts. Nitrogen/oxygen ligands were identified in slightly higher proportions (40-70%) than single oxygen ligands. Furthermore, low- and high-affinity binding sites contributed in an almost equivalent proportion to copper binding in root apoplasts. The high-affinity N functional groups embedded in root apoplasts participated in copper binding in the same magnitude than the low-affinity carboxylic groups.

  10. 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.

  11. Effect of P Availability on Temporal Dynamics of Carbon Allocation and Glomus intraradices High-Affinity P Transporter Gene Induction in Arbuscular Mycorrhiza

    Science.gov (United States)

    Olsson, Pål Axel; Hansson, Maria C.; Burleigh, Stephen H.

    2006-01-01

    Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment. PMID:16751522

  12. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs......, but not by ¿-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites...

  13. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ......-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [3H...

  14. Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

    Science.gov (United States)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte; Frydenvang, Karla; Dahl, Ivar F; Bräuner-Osborne, Hans; Brehm, Lotte; Frølund, Bente; Clausen, Rasmus P

    2008-12-25

    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 in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

  15. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    Science.gov (United States)

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  16. Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

    Science.gov (United States)

    Polepally, Prabhakar R; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D; Roth, Bryan L; Zjawiony, Jordan K

    2014-10-06

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

  17. Influence of ischemic preconditioning on levels of nerve growth factor, brain-derived neurotrophic factor and their high-affinity receptors in hippocampus following forebrain ischemia.

    Science.gov (United States)

    Lee, Tsong-Hai; Yang, Jen-Tsung; Ko, Yu-Shien; Kato, Hiroyuki; Itoyama, Yasuto; Kogure, Kyuya

    2008-01-02

    Preconditioning of gerbil brain with a sublethal forebrain ischemia is known to protect hippocampal CA1 neurons following a subsequent lethal ischemia (the second ischemia) which usually damages neurons (ischemic tolerance). Present report using a confocal laser scanning microscope demonstrated that the hippocampal cells of sham operation gerbils contained immunofluorescent NGF and BDNF and their high-affinity receptors (TrkA and TrkB). A 2-min ischemia caused little change of these proteins (ANOVA test, PBDNF but not NGF and their high-affinity receptors showed a transient reduction at 4 h (ANOVA test, PBDNF and TrkB decreased transiently from 4 h to 1 day (ANOVA test, PCA3 and dentate gyrus areas, only BDNF decreased significantly at 7 days in the CA3 area without ischemic preconditioning (ANOVA test, PCA3 and dentate gyrus areas with and without ischemic preconditioning. Western blot study showed that in the hippocampal formation with ischemic preconditioning, preconditioning prevented the decline of these protein levels from 1 day to 7 days after the second lethal ischemia (ANOVA test, P>0.05). Results of this study demonstrate that ischemic preconditioning recovers the initial decline in NGF and BDNF and their corresponding receptors in the vulnerable CA1 neurons after the second lethal ischemia, suggesting that growth factors might play a role in the protective mechanism of ischemic preconditioning.

  18. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. (J. Plateaustraat, Gent (Belgium))

    1988-11-01

    Binding studies were performed with two {sup 125}I-labeled Bacillus thuringiensis {delta}-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One {delta}-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 {delta}-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 {delta}-endotoxins active against M. sexta compete for binding of {sup 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.

  19. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    Science.gov (United States)

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

  20. Effectiveness of Eye Movement Desensitization and Reprocessing on Anxiety in Children with Thalassemia in a 12-month follow up

    Directory of Open Access Journals (Sweden)

    A Shahnavazi

    2016-02-01

    Full Text Available Background and  Objective: Thalassemia is the most common genetic diseases in the world and especially in our country. For many reasons, including chronic disease, health care costs, the expected mortality Ray disease states such as anxiety, psychological and social problems in thalassemia. The aim of the present study was to assess the efficacy of Eye Movement Desensitization and Reprocessing (EMDR on anxiety of children with thalassemia in a 12-month follow up..  Methods: It was a clinical trial study, which was performed on 60 patients with Thalassemia in 2013-2014. A total of 60 thalassemic patients based on Beck anxiety inventory, selected using sampling and were randomly divided into experimental and Control groups. in experimental group, eye movement desensitization and reprocessing therapy was perfomed in five sessions and The control group received no intervention. In order to collect information on demographic questionnaire and Beck Anxiety Inventory were used. data were gathered on anxiety symptoms at pretreatment, posttreatment and 12 month follow up and were analyzed using descriptive statistics, repeated measures and chi square. Results: The average age of the participants was 15.22±1.93  years old. This study shows that the mean or average level of anxiety children with thalassemia in the experimental group before and after intervention and in a 12-month follow up group 34.73 ± 5.62, 19.90 ± 2.48 and 19.23 ± 2.48 respectively; repeated measures ANOVA showed significant statistical difference (p.05 Conclusion: This study shows that Method of eye movement desensitization and reprocessing as an alternative method to treat or reduce anxiety, aggressive children with thalassemia .Trained Nurses can use this new method is effective in the treatment of anxiety.

  1. Synaptically released neurotransmitter fails to desensitize postsynaptic GABA(A) receptors in cerebellar cultures.

    Science.gov (United States)

    Mellor, J R; Randall, A D

    2001-05-01

    GABA concentration jump experiments performed on membrane patches predict that postsynaptic GABA(A) receptors will become desensitized following the release of the contents of a single GABA-containing synaptic vesicle. To examine this we used a single synaptic bouton stimulation technique to directly examine whether postsynaptic GABA(A) receptors in cultured cerebellar granule cells exhibit transmitter-induced desensitization. In a large number of recordings, no evidence was found for desensitization of postsynaptic GABA(A) receptors by vesicularly released transmitter. This was the case even when as many as 40 vesicles were released from a single bouton within 1.5 s. In addition, postsynaptic depolarization and application of the benzodiazepine flunitrazepam, manipulations previously shown to enhance desensitization of GABA(A) receptors, failed to unmask transmitter-induced desensitization. In contrast, a single 2- to 3-s application of a high concentration of exogenous GABA was able to depress synaptic responsiveness for up to 70 s. Furthermore, pharmacological depletion of GABA eliminated inhibitory synaptic communication, suggesting that GABA is the transmitter and the desensitization-resistant inhibitory postsynaptic currents are not mediated by a "nondesensitizing" ligand such as beta-alanine. Overall our data indicate that a specific desensitization-resistant population of GABA(A) receptors are present at postsynaptic sites on cultured cerebellar granule cells.

  2. The role of aspirin desensitization in patients with aspirin-exacerbated respiratory disease (AERD).

    Science.gov (United States)

    Spies, Jonas Willian; Valera, Fabiana Cardoso Pereira; Cordeiro, Daniel Loiola; de Mendonça, Taís Nociti; Leite, Marcelo Gonçalves Junqueira; Tamashiro, Edwin; Arruda, Luiza Karla; Anselmo-Lima, Wilma Terezinha

    2016-01-01

    Aspirin-exacerbated respiratory disease (AERD) consists of a classic tetrad: moderate/severe asthma, chronic rhinosinusitis, nasal polyps, and intolerance to aspirin or other nonsteroidal anti-inflammatory drugs. Clinical control with drugs, surgery, and desensitization are treatment options. To evaluate the efficacy and tolerability of aspirin desensitization in patients with AERD. Periodic symptom assessment and endoscopy in patients with AERD undergoing surgery who were desensitized. Seventeen patients were desensitized. Eight patients completed the desensitization and were followed for a minimum of a one-year period (mean 3.1 years). These patients showed improvement in all symptoms. Moreover, surgical reassessment was not indicated in any of these patients and there was a decrease in costs with medication and procedures. Eight patients did not complete desensitization, mainly due to procedure intolerance and uncontrolled asthma, whereas another patient was lost to follow-up. Aspirin desensitization, when tolerated, was effective in patients with AERD and with poor clinical/surgical response. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  3. Rapid drug desensitization for hypersensitivity reactions to chemotherapy and monoclonal antibodies in the 21st century.

    Science.gov (United States)

    Castells Guitart, M C

    2014-01-01

    The frequency of hypersensitivity reactions (HSR) to drugs has risen in the last 10 years owing to increased exposure to better and more allergenic medications including monoclonal antibodies. HSRs prevent patients from using their first-line therapy, leading to decreased quality of life and life expectancy. Although premedication with antihistamines, leukotriene blockers, and corticosteroids can protect against mild-to-moderate HSR, none of these medications has provided protection against anaphylaxis. Rapid drug desensitization is a treatment option for patients with HSR to their first-line medication that protects against anaphylaxis.Although the mechanisms of drug desensitization are not completely understood, in vitro mast cell models of IgE antigen desensitization have led to the design of safe and effective in vivo protocols aimed at protecting highly sensitized patients from hypersensitivity reactions and anaphylaxis. This review provides an insight into the mechanisms of IgE/mast cell desensitization, the principles and practice of drug desensitization, and an overview of the different desensitization protocols and their safety and efficacy profiles. Drug desensitization should only be performed by allergists, trained nurses, and experienced pharmacists, since this high-risk procedure involves reintroducing allergenic medication to highly sensitized patients, with the consequent potential for severe or fatal HSRs.

  4. Influence of an emulsifier on the pressure desensitization of emulsion explosives

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The desensitization degree of emulsion explosives (EE) was calculated with the peak pressure of explosion shock waves tested in water.To an explosive, the less the desensitization degree, the better the compression resistance, so the compression resistance of an explosive can be compared and analyzed quantificationally with the desensitization degree.The influence of an emulsifier on the pressure desensitization of EE was studied, including the content and category of emulsifiers.Three kinds of emulsifiers (Span-80,compound emulsifier, and T-152) were used in the tests.The experimental results show that both the content and category of emulsifiers make a great effect on the pressure desensitization of EE.The desensitization degree of EE reduces with the emulsifier content being increased, but there is an optimal content of an emulsifier for the compression resistance of EE.While the content of Span-80 reaches4wt%, the desensitization degree of EE becomes a minimal value, and augments somewhat if the emulsifier content is increased more.That is to say, the compression resistance of EE becomes the highest while the content of Span-80 is 4wt%, and the compression resistance will decline if the content of Span-80 is increased more.The compression resistance of the explosive emulsified by compound emulsifier is the highest among all the explosives, when the content of the whole components and manufacturing engineering are kept invariable.

  5. A successful desensitization protocol for horse-derived antithymocyte globulin in severe aplastic anemia.

    Science.gov (United States)

    Demir, Esen; Cigerci Günaydın, Nurşen; Karadaş, Nihal; Gülen, Figen; Tanac, Remziye; Yılmaz, Deniz

    2015-03-01

    Horse antithymocyte globulin (h-ATG) (ATGAM(®) ) is the first choice of treatment in very severe patients with aplastic anemia who do not have any HLA matched sibling donor. h-ATG is a heterologous serum that may cause anaphylaxis. Alternative treatment strategies must be planned in case of hypersensitivity. Desensitization must be considered in patients without an alternative treatment of choice. We aimed to present the h-ATG desensitization protocol and consider its effectiveness in patients with aplastic anemia who are hypersensitized with h-ATG and do not have an alternative treatment of choice. Skin prick tests were performed with non-diluted solution in eight very severe patients with aplastic anemia who are followed up in Ege University Children's Hospital. Although skin prick test was found negative in these eight patients, different dilution h-ATG intradermal tests were performed and found positive in all patients. h-ATG desensitization program was started to these hypersensitized patients. Desensitization program was started to six male and two female very severe patients with aplastic anemia whose ages were between seven and 19 yr (median: 12.9 yr). All of the patients completed the desensitization program. While local reaction was seen in two patients, systemic reaction was seen in one patient and late reaction was seen in one patient during and after desensitization program. A successful desensitization program with h-ATG in children with aplastic anemia is presented. Even though there is not an exposure before to such high allergy potential heterologous serum, skin tests should be performed and desensitization must be started to patients who are hypersensitized to h-ATG. As the expected effectiveness of the treatment is so much, the desensitization protocol can be carried out safely and effectively with trained stuff although allergic reactions can be seen. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. 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...... the design and synthesis of the first 125I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites....

  7. Excessive users of violent video games do not show emotional desensitization: an fMRI study.

    Science.gov (United States)

    Szycik, Gregor R; Mohammadi, Bahram; Hake, Maria; Kneer, Jonas; Samii, Amir; Münte, Thomas F; Te Wildt, Bert T

    2016-04-16

    Playing violent video games have been linked to long-term emotional desensitization. We hypothesized that desensitization effects in excessive users of violent video games should lead to decreased brain activations to highly salient emotional pictures in emotional sensitivity brain regions. Twenty-eight male adult subjects showing excessive long-term use of violent video games and age and education matched control participants were examined in two experiments using standardized emotional pictures of positive, negative and neutral valence. No group differences were revealed even at reduced statistical thresholds which speaks against desensitization of emotion sensitive brain regions as a result of excessive use of violent video games.

  8. Desensitization in ABO-Incompatible Kidney Transplantation With Low ABO Iso-Agglutinin Titers.

    Science.gov (United States)

    Gelpi, R; Cid, J; Lozano, M; Revuelta, I; Sanchez-Escuredo, A; Blasco, M; de Souza, E; Esforzado, N; Torregrosa, J V; Cofán, F; Ricart, M J; Campistol, J M; Oppenheimer, F; Diekmann, F

    2015-10-01

    In ABO-incompatible (ABOi) kidney transplantation (KT) with low iso-agglutinin (IG) titers (IGT), standard pre-conditioning treatment might be excessive. To try to answer this question, we evaluated the pre-conditioning requirements of a group of ABOi KT with low ABO IGT in our center. Our main objective was to assess desensitization requirements for ABOi KT with low IGT (desensitization (rituximab and plasma exchange [PE]) requirements for ABOi KT with IGT desensitization protocol in ABOi KT donors with low titers of IG to reduce adverse effects, reduce cost, and simplify pre-transplant logistics. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Successful interferon desensitization in a patient with chronic hepatitis C infection

    Institute of Scientific and Technical Information of China (English)

    Seyed Alireza Taghavi; Ahad Eshraghian

    2009-01-01

    Treatment of hepatitis C, even when absolutely necessary, is almost impossible when interferon cannot be administered for any reason. We report a 65-year-old patient with chronic hepatitis C virus (HCV) infection and fibrosis, who was unable to receive interferon because of systemic hypersensitivity. The patient was desensitized successfully through gradual incremental exposure to interferon, and HCV infection was eradicated after a complete course of treatment,with no further allergic reactions. This case report that describes successful eradication of hepatitis C in a patient with advanced liver disease after desensitization to interferon revealed that desensitization may not necessarily damage the therapeutic efficacy of the drug.

  10. Desensitizing the posterior interosseous nerve alters wrist proprioceptive reflexes.

    Science.gov (United States)

    Hagert, Elisabet; Persson, Jonas K E

    2010-07-01

    The presence of wrist proprioceptive reflexes after stimulation of the dorsal scapholunate interosseous ligament has previously been described. Because this ligament is primarily innervated by the posterior interosseous nerve (PIN) we hypothesized altered ligamento-muscular reflex patterns following desensitization of the PIN. Eight volunteers (3 women, 5 men; mean age, 26 y; range 21-28 y) participated in the study. In the first study on wrist proprioceptive reflexes (study 1), the scapholunate interosseous ligament was stimulated through a fine-wire electrode with 4 1-ms bipolar pulses at 200 Hz, 30 times consecutively, while EMG activity was recorded from the extensor carpi radialis brevis, extensor carpi ulnaris, flexor carpi radialis, and flexor carpi ulnaris, with the wrist in extension, flexion, radial deviation, and ulnar deviation. After completion of study 1, the PIN was anesthetized in the radial aspect of the fourth extensor compartment using 2-mL lidocaine (10 mg/mL) infiltration anesthesia. Ten minutes after desensitization, the experiment was repeated as in study 1. The average EMG results from the 30 consecutive stimulations were rectified and analyzed using Student's t-test. Statistically significant changes in EMG amplitude were plotted along time lines so that the results of study 1 and 2 could be compared. Dramatic alterations in reflex patterns were observed in wrist flexion, radial deviation, and ulnar deviation following desensitization of the PIN, with an average of 72% reduction in excitatory reactions. In ulnar deviation, the inhibitory reactions of the extensor carpi ulnaris were entirely eliminated. In wrist extension, no differences in the reflex patterns were observed. Wrist proprioception through the scapholunate ligament in flexion, radial deviation, and ulnar deviation depends on an intact PIN function. The unchanged reflex patterns in wrist extension suggest an alternate proprioceptive pathway for this position. Routine excision of

  11. 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

  12. 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.).

  13. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    Science.gov (United States)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

  14. Short-term desensitization of G-protein-activated, inwardly rectifying K+ (GIRK) currents in pyramidal neurons of rat neocortex.

    Science.gov (United States)

    Sickmann, Thomas; Alzheimer, Christian

    2003-10-01

    Whole cell recordings from acutely isolated rat neocortical pyramidal cells were performed to study the kinetics and the mechanisms of short-term desensitization of G-protein-activated, inwardly rectifying K+ (GIRK) currents during prolonged application (5 min) of baclofen, adenosine, or serotonin. Most commonly, desensitization of GIRK currents was characterized by a biphasic time course with average time constants for fast and slow desensitization in the range of 8 and 120 s, respectively. The time constants were independent of the agonist used to evoke the current. The biphasic time course was preserved in perforated-patch recordings, indicating that neither component of desensitization is attributable to cell dialysis. Desensitization of GIRK currents displayed a strong heterologous component in that application of a second agonist substantially reduced the responsiveness to a test agonist. Fast desensitization, but not slow desensitization, was lost in cells loaded with GDP, suggesting that the hydrolysis cycle of G proteins might underlie the initial, rapid current decline. Hydrolysis of phosphatidylinositol biphosphate is an unlikely candidate underlying short-term desensitization, because both components of desensitization were preserved in the presence of the phospholipase C inhibitor U73122. We conclude that short-term desensitization does neither result from receptor downregulation nor from altered channel gating but might involve modifications of the G-protein-dependent pathway that serves to translate receptor activation into channel opening.

  15. Hypersensitivity and desensitization to antineoplastic agents: outcomes of 189 procedures with a new short protocol and novel diagnostic tools assessment.

    Science.gov (United States)

    Madrigal-Burgaleta, R; Berges-Gimeno, M P; Angel-Pereira, D; Ferreiro-Monteagudo, R; Guillen-Ponce, C; Pueyo, C; Gomez de Salazar, E; Alvarez-Cuesta, E

    2013-07-01

    Desensitization to antineoplastic agents is becoming a standard of care. Efforts to establish and improve these techniques are being made at many institutions. Our aims are to evaluate a new rapid desensitization protocol designed to be shorter (approximately 4 h) and safer (reducing hazardous drugs exposure risks) and to assess the oxaliplatin-specific immunoglobulin E (IgE) as a novel diagnostic tool. Prospective, observational, longitudinal study with patients who, for a 1-year period, suffered reactions to antineoplastic agents and were referred to the Desensitization Program at Ramon y Cajal University Hospital (RCUH). Patients were included or excluded as desensitization candidates after anamnesis, skin testing, risk assessment, and graded challenge. Specific IgE was determined in oxaliplatin-reactive patients. Candidate patients were desensitized using the new RCUH rapid desensitization protocol. Of 189 intravenous rapid desensitizations, 188 were successfully accomplished in the 23 patients who met inclusion criteria for desensitization (of 58 referred patients). No breakthrough reactions occurred in 94% of desensitizations, and most breakthrough reactions were mild. In 10 oxaliplatin-reactive patients, 38 desensitizations were successfully accomplished. Sensitivity for oxaliplatin-specific IgE was 38% (0.35UI/l cutoff point) and 54% (0.10UI/l cutoff point); specificity was 100% for both cutoff points. In the hands of a Desensitization Program, managed by drug desensitization experts, this new protocol has proven an effective therapeutic tool for hypersensitivity to several antineoplastic agents (oxaliplatin, carboplatin, paclitaxel, docetaxel, cyclophosphamide, and rituximab); moreover, it improves safety handling of hazardous drugs. We report the first large series of oxaliplatin desensitizations. Oxaliplatin-specific IgE determination could be helpful. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    Science.gov (United States)

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

  17. A novel rabbit immunospot array assay on a chip allows for the rapid generation of rabbit monoclonal antibodies with high affinity.

    Directory of Open Access Journals (Sweden)

    Tatsuhiko Ozawa

    Full Text Available Antigen-specific rabbit monoclonal antibodies (RaMoAbs are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10(-12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

  18. Design, Synthesis, Binding and Docking-Based 3D-QSAR Studies of 2-Pyridylbenzimidazoles—A New Family of High Affinity CB1 Cannabinoid Ligands

    Directory of Open Access Journals (Sweden)

    Patricio Iturriaga-Vásquez

    2013-04-01

    Full Text Available A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39 was the most active of the series (KiCB1 = 0.53 nM, while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823.

  19. The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.

    Science.gov (United States)

    Kozyraki, R; Fyfe, J; Kristiansen, M; Gerdes, C; Jacobsen, C; Cui, S; Christensen, E I; Aminoff, M; de la Chapelle, A; Krahe, R; Verroust, P J; Moestrup, S K

    1999-06-01

    Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

  20. Impaired signaling via the high-affinity IgE receptor in Wiskott-Aldrich syndrome protein-deficient mast cells.

    Science.gov (United States)

    Pivniouk, Vadim I; Snapper, Scott B; Kettner, Alexander; Alenius, Harri; Laouini, Dhafer; Falet, Hervé; Hartwig, John; Alt, Frederick W; Geha, Raif S

    2003-12-01

    Wiskott-Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott-Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (FcepsilonRI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include FcepsilonRI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of FcepsilonRIbeta and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase Cgamma and Ca(2+) mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates FcepsilonRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

  1. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  2. A Further Assessment of Attention-Placebo Effects and Demand Characteristics in Studies of Systematic Desensitization

    Science.gov (United States)

    McReynolds, William T.; Tori, Christopher

    1972-01-01

    It was predicted that phobic Ss receiving systematic desensitization would show greater reductions on both fear-related behavior measures and simulated fear measures than Ss receiving relaxation treatment or no treatment at all. The prediction was confirmed. (Author)

  3. Lymphocyte-mediated regulation of platelet activation during desensitization in patients with hymenoptera venom hypersensitivity.

    Science.gov (United States)

    Ledru, E; Pestel, J; Tsicopoulos, A; Joseph, M; Wallaert, B; Tonnel, A B; Capron, A

    1988-01-01

    T cells from peripheral blood of hymenoptera sensitive patients were studied before and after venom desensitization. Before treatment, T cells showed a variable but higher proliferative response to allergen than T cells of treated patients or controls. While before desensitization, T cell products, specifically released after in vitro allergen stimulation, were able to amplify the IgE-dependent platelet activity, we showed that after treatment of the same patients, T cell products strongly reduced platelet activation. Considering the modifications in platelet activation previously observed in patients treated by specific immunotherapy, the present results suggest that, through a modification of T cell reactivity to allergen, T cell functions are modulated by desensitization, and emphasize the involvement of T cell products in the desensitization mechanisms. PMID:3263227

  4. Anaphylactic reaction to polyethylene-glycol conjugated-asparaginase: premedication and desensitization may not be sufficient.

    Science.gov (United States)

    Sahiner, Umit M; Yavuz, S Tolga; Gökce, Muge; Buyuktiryaki, Betul; Altan, Ilhan; Aytac, Selin; Tuncer, Murat; Tuncer, Ayfer; Sackesen, Cansin

    2013-08-01

    In hypersensitive reactions to native L-asparaginase, either premedication and desensitization or substitution with polyethylene glycol conjugated asparaginase (PEG-ASP) is preferred. Anaphylaxis with PEG-ASP is rare. An 8-year-old girl and a 2.5-year-old boy, both diagnosed as having acute lymphoblastic leukemia, presented with native L-asparaginase hypersensitivity and substitution with PEG-ASP was preferred. They received a premedication (methylprednisolone, hydroxyzine and ranitidine) followed by desensitization with PEG-ASP infusion. Both patients developed anaphylaxis with peg-asparaginase. These are the first reported cases of anaphylactic reaction to PEG-ASP, despite the application of both premedication and desensitization. Anaphylaxis with PEG-ASP is very rare and premedication and desensitization protocols may not prevent these hypersensitive reactions. © 2013 The Authors. Pediatrics International © 2013 Japan Pediatric Society.

  5. Effects of applying glutaraldehyde-containing desensitizer formulations on reducing dentin permeability

    Directory of Open Access Journals (Sweden)

    Hiroshi Ishihata

    2012-06-01

    Conclusions: The liquid and the gel desensitizing agents both significantly reduced dentin permeability. The obvious advantage of a gel formulation is the controlled application, limited to the hypersensitive tooth area, thus avoiding inadvertent contact with adjacent gingival tissues.

  6. Efficacy of Eye Movements Desensitization and Reprocessing on the Quality of Life of the Patients with Myocardial Infarction.

    Science.gov (United States)

    Salehian, Tahmineh; Saeedinejad, Saidzaker; Behnammoghadam, Mohammad; Shafiee, Mohsen; Mohammadhossini, Sima; Behnammoghadam, Zargham; Behnammoghadam, Aziz; Ebrahimpour, Soheil; Paymard, Akvan

    2016-10-01

    Myocardial infarction causes limitations in the physical activity and perturbation of quality of life.The aim of this study was to evaluate the effect of eye movements desensitization and reprocessing (EMDR) on the quality of life of these patients. This study was conducted in two groups as the before and after while the effect of eye movements desensitization and reprocessing on the quality of life of the patients with Myocardial infarction. Sampling was done based on the purposive sampling. Patients were randomly divided into two experimental and control groups (30 patients in each group). Samples were assigned through randomized allocation. In the experimental group, the EMDR method was carried out on the patients in five 90-minute sessions over a two week period. In the control group no intervention was received. Data of Quality of life, pre-treatment, post-treatment were analyzed using SPSS. The results showed that the quality of life increase in all its dimensions of the experimental group, after performing the EMDR therapy significantly (P=0.001). Treatment what has already been stated, was effective on the quality of life in patients. Treatment team members can use this method as an effective intervention in order to improve the quality of life of their patients.

  7. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    Science.gov (United States)

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

  8. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    Energy Technology Data Exchange (ETDEWEB)

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  9. Crystallization and preliminary X-ray structural studies of a high-affinity CD8αα co-receptor to pMHC

    Energy Technology Data Exchange (ETDEWEB)

    Cole, David K. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom); Rizkallah, Pierre J., E-mail: p.j.rizkallah@dl.ac.uk [CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD (United Kingdom); Sami, Malkit; Lissin, Nikolai M.; Gao, Feng [Avidex Ltd, 57c Milton Park, Abingdon, Oxon OX14 4RX (United Kingdom); Bell, John I. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom); Boulter, Jonathan M. [Medical Biochemistry and Immunology, Henry Wellcome Building, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN,Wales (United Kingdom); Glick, Meir [Novartis Pharmaceuticals, One Health Plaza, East Hanover, NJ 07936 (United States); Vuidepot, Anne-Lise; Jakobsen, Bent K., E-mail: p.j.rizkallah@dl.ac.uk [Avidex Ltd, 57c Milton Park, Abingdon, Oxon OX14 4RX (United Kingdom); Gao, George F. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom)

    2005-03-01

    A high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR–pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6{sub 4}22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 Å. V{sub M} calculations indicated one molecule per asymmetric unit. A 2 Å data set was collected and the structure is currently being determined using molecular replacement.

  10. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    Science.gov (United States)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  11. High affinity binding of /sup 125/I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

    Energy Technology Data Exchange (ETDEWEB)

    Aguet, M.; Blanchard, B.

    1981-12-01

    Direct ligand-binding studies with highly purified /sup 125/I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of /sup 125/I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using /sup 125/I- and /sup 127/I-labeled interferon provided strong evidence that the fraction of /sup 125/I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of /sup 125/I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37/sup o/ bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of /sup 125/I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated, separated, and identified as mouse ..cap alpha.. and ..beta.. interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material thus providing evidence for a common receptor site for mouse interferon.

  12. Pitocin and autism: An analysis of oxytocin receptor desensitization in the fetus.

    Science.gov (United States)

    Gottlieb, Mark M

    2016-02-01

    The risk of Pitocin as a cause of autism attributable to oxytocin receptor desensitization in the brain of the fetus is evaluated in terms of a mathematical model. A composite unit, D, for oxytocin receptor desensitization levels is established with the form ((IU-h)/ml)E-3, where IU is the international unit for oxytocin. The desensitization values for oxytocin receptor desensitization at a concentration of 10 nmol of oxytocin per liter for 3, 4.2 and 6h corresponding to 0%, 50% and 100% desensitization are calculated to be 15 D, 21 D, and 30 D, respectively. The permeability of the blood-brain barrier in the fetus to oxytocin is discussed, and the upper limit of the concentration of Pitocin in the placenta, and its possible diffusion into the blood and brain of the fetus, is calculated for a routine dose of 6 milli U per minute of Pitocin over a 12h labor. This dose of Pitocin is shown to result in a desensitization value in units of D that is more than a factor of 10 below the 0% desensitization value of 15 D. This indicates that routine doses of Pitocin are not a significant cause of autism attributable to oxytocin receptor desensitization. This is consistent with the findings of a major epidemiological study of the association of Pitocin with autism in Denmark entitled, "Oxytocin-augmented labor and risk for males", Behavioral Brain Research, May 1, 2015; 284:207-212, which found no association between the use of Pitocin during labor and the incidence of autism for females, and a modest association for males.

  13. Effect of desensitizer application on shear bond strength of composite resin to bleached enamel

    Directory of Open Access Journals (Sweden)

    Maryam Khoroushi

    2013-01-01

    Full Text Available Objective: Tooth sensitivity is common after vital tooth bleaching. The aim of this study is to evaluate the effect of a desensitizing agent on shear bond strength of composite resin to bleached enamel; and determine whether a delay of one or two weeks in bonding procedure is sufficient subsequent to bleaching/desensitizer regimen. Materials and Methods: Buccal enamel surfaces of ninety-six human sound molars were prepared and divided into eight groups. The surfaces of specimens in Group 1 as negative control group were bonded by composite resin using the single bond adhesive. Specimens in Groups 2-4 were bleached with an at-home bleaching agent (Daywhite ACP. Relief ACP desensitizing gel alone was applied in Group 5. In Groups 6-8, specimens were bleached same as in Group 2 and relief ACP desensitizing gel was applied same as inGroup 5 subsequent to each bleaching session. Composite cylinders were bonded after 24 h, 7 days and 14 days in Groups 2-4, respectively, and also in Groups 6-8, respectively. The shear bond strengths of the cylinders were tested and data was analyzed using two-way ANOVA and Tukey test (α = 0.05. Results: The results showed that bleaching and bleaching/desensitizer regimens significantly reduced the bond strength of composite resin to enamel. However, desensitizer alone did not reduce bond strength. No statistically significant differences were found between bleaching and bleaching/desensitizer regarding bond strength. Conclusion: Bleaching or bleaching/desensitizer treatment significantly decreases bond strength of composite resin to enamel. In both regimens, adhesive bonding is recommended after two weeks.

  14. Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors.

    Science.gov (United States)

    Madadi, Nikhil Reddy; Penthala, Narsimha Reddy; Brents, Lisa K; Ford, Benjamin M; Prather, Paul L; Crooks, Peter A

    2013-04-01

    A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R=R(2)=H, R(1)=F) and 13 (R=COOCH3, R(1)=R(2)=H) exhibited high affinity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.

  15. Effect of desensitizing treatments on bond strength of resin composites to dentin - an in vitro study

    Directory of Open Access Journals (Sweden)

    Sameer Makkar

    2014-01-01

    Full Text Available Objectives: Hypersensitivity is a common clinical multietiological problem. Many desensitizing treatments are there to overcome hypersensitivity. The aim of this study was to evaluate the effect of different dentin-desensitizing treatments on the tensile bond strength of composite restoration. Materials and Methods: Twenty-four sound human molars were used. Enamel was wet abraded to expose flat dentin surfaces, polished with sandpaper. The specimens were then divided into three groups (n = 8 based on the type of dentin-desensitizing treatment given. The first group: G1 was the control group where no desensitizing agent was used. The second group: G2 was treated with desensitizing dentifrice containing a combination of potassium nitrate, triclosan, and sodium monoflorophosphate. The third group: G3 was treated with Er:YAG laser. Afterwards, the desensitized specimens were treated with one step self-etch adhesive according to manufacturer′s instructions and composite microcylinders were packed. The specimens were then examined for tensile bond strength using universal tensile machine (KMI TM . Results: Statistical analysis of the data obtained revealed the mean values for the tensile bond strengths were 10.2613 MPa, 5.9400 MPa and 6.3575 MPa for groups 1, 2 and 3, respectively. These values were statistically significantly different between groups pretreated with laser or dentifrice as compared to control group. Conclusions: Dentifrice and Laser pre-treated dentin has lower tensile bond strength with resin composites as compared to dentin that is untreated.

  16. A SAFE PROTOCOL FOR RAPID DESENSITIZATION IN PATIENTS WITH CYSTIC FIBROSIS AND ANTIBIOTIC HYPERSENSITIVITY

    Science.gov (United States)

    Legere, Henry J.; Palis, Ross I.; Bouza, Tito Rodriguez; Uluer, Ahmet Z.; Castells, Mariana C.

    2009-01-01

    Background CF patients often demonstrate hypersensitivity to one or multiple antibiotics due to frequent and repeated exposures. Attempts at antibiotic desensitization in this population are historically complicated by higher reaction rates, failure to complete the procedure and consequent withholding of first-line therapy. This study evaluates the outcomes of a rapid desensitization protocol developed at our institution. Methods We retrospectively reviewed the medical records of 15 patients undergoing 52 rapid antibiotic desensitizations at Brigham and Women’s Hospital and Children’s Hospital Boston utilizing our protocol. Results Mean FEV1 % predicted was 44.1 (SD 16.5), with two patients at desensitized during bilateral lung transplantation. Adverse reactions during desensitization occurred in 13.4%, and most were mild. 100% of patients completed the protocol and ultimately tolerated subsequent full-strength antibiotic courses. Conclusions CF patients with antibiotic hypersensitivity can safely receive first-line antibiotics via our rapid desensitization protocol, including those with severe obstructive lung disease. PMID:19740711

  17. An Hourly Dose-Escalation Desensitization Protocol for Aspirin-Exacerbated Respiratory Disease.

    Science.gov (United States)

    Chen, Justin R; Buchmiller, Brett L; Khan, David A

    2015-01-01

    Aspirin desensitization followed by maintenance therapy effectively improves symptom control in patients with aspirin exacerbated respiratory disease (AERD). The majority of current desensitization protocols use 3-hour dosing intervals and often require 2 to 3 days to complete. We evaluated hourly dose escalations in a subset of patients with chronic rhinosinusitis, nasal polyps, and asthma who historically reacted to aspirin within 1 hour or were avoiding aspirin with the goal of developing a safe and efficient desensitization protocol. Fifty-seven aspirin desensitizations were performed under the hourly protocol. All patients had refractory nasal polyposis as an indication for aspirin desensitization. The clinical characteristics of each subject were analyzed in relation to aspects of his or her reactions during the procedure. Ninety-eight percent of study patients were successfully treated under the hourly protocol, including those with a history of severe reactions and intubation. None required further medication than is available in an outpatient allergy clinic. A total of 96% of reactors recorded a bronchial or naso-ocular reaction within 1 hour of the preceding dose. Of the total patients on this protocol, 40% were able to complete the procedure in a single day, and 60% within 2 days. Patients with AERD who have a history of symptoms less than 1 hour after aspirin exposure can be safely desensitized with a 1-hour dose-escalation protocol that can often be completed in a single day. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  18. Perioperative Desensitization Improves Outcomes Among Crossmatch Positive Recipients of Deceased Donor Renal Transplants.

    Science.gov (United States)

    Sharma, Amit; King, Anne; Kumar, Dhiren; Behnke, Martha; McDougan, Felecia; Kimball, Pamela M

    2016-06-01

    Graft failure due to chronic rejection is greater among renal transplant patients with donor-specific antibody (DSA) than among DSA-free patients. For patients dependent on deceased donor transplantation, preoperative desensitization to eliminate DSAs may be impractical. We speculated that perioperative desensitization might eliminate preexisting DSAs and prevent de novo DSAs and improve graft outcomes. We report that brief perioperative desensitization using either intravenous immunoglobulin (IVIG) or plasmapheresis/IVIG (PP/IVIG) treatment improves clinical outcomes among patients with positive crossmatches. Immediately following deceased donor transplantation, 235 renal recipients were assigned points for PRA and flow crossmatches (FCXM): delayed graft function (DGF) ≤ 1 point received standard therapy; 2 points received high-dose IVIG; and ≥3 points received PP/IVIG. The DSAs were serially monitored by single antigen bead luminex for 1 year. Five-year clinical outcomes were determined from the chart review. All desensitized patients had preoperatively positive FCXM with DSA. Rejection was more common (P desensitized than nonsensitized groups. However, overall graft survivals were similar between the groups (P = not significant) and superior to historic untreated patients (P 90% in all desensitizated patients with DSA elimination as well as PP/IVIG patients with residual DSA. In contrast, IVIG patients with persistent DSA had poorer graft survival (45%, P desensitization improved overall graft survival of sensitized patients compared to historic untreated patients. Plasmapheresis/IVIG had greater impact on DSA eradication and graft survival than IVIG alone. © 2016, NATCO.

  19. The effect of improvisation-assisted desensitization, and music-assisted progressive muscle relaxation and imagery on reducing pianists' music performance anxiety.

    Science.gov (United States)

    Kim, Youngshin

    2008-01-01

    The purpose of this study was to investigate the effects of two music therapy approaches, improvisation-assisted desensitization, and music-assisted progressive muscle relaxation and imagery on ameliorating the symptoms of music performance anxiety (MPA) among student pianists. Thirty female college pianists (N = 30) were randomly assigned to one of two conditions: (a) improvised music-assisted desensitization group (n = 15), or (b) music-assisted progressive muscle relaxation (PMR) and imagery group (n = 15). All participants received 6 weekly music therapy sessions according to their assigned group. Two lab performances were provided; one before and one after the 6 music therapy sessions, as the performance stimuli for MPA. All participants completed pretest and posttest measures that included four types of visual analogue scales (MPA, stress, tension, and comfort), the state portion of Spielberger's State-Trait Anxiety Inventory (STAI), and the Music Performance Anxiety Questionnaire (MPAQ) developed by Lehrer, Goldman, and Strommen (1990). Participants' finger temperatures were also measured. When results of the music-assisted PMR and imagery condition were compared from pretest to posttest, statistically significant differences occurred in 6 out of the 7 measures-MPA, tension, comfort, STAI, MPAQ, and finger temperature, indicating that the music-assisted PMR and imagery treatment was very successful in reducing MPA. For the improvisation-assisted desensitization condition, the statistically significant decreases in tension and STAI, with increases in finger temperature indicated that this approach was effective in managing MPA to some extent. When the difference scores for the two approaches were compared, there was no statistically significant difference between the two approaches for any of the seven measures. Therefore, no one treatment condition appeared more effective than the other. Although statistically significant differences were not found between

  20. Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

    Science.gov (United States)

    Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

    2013-06-01

    Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  1. Renal transplantation across the donor-specific antibody barrier: Graft outcome and cancer risk after desensitization therapy

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    Ching-Yao Yang

    2016-06-01

    Conclusion: When compared to renal transplantation without DSA, desensitization therapy for DSA resulted in equivalent renal transplant outcome but potentially increased risk of urothelial carcinoma after transplantation.

  2. Design and Investigation of a [(18)F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging.

    Science.gov (United States)

    Yang, Dongzhi; Comeau, Anthony; Bowen, Wayne D; Mach, Robert H; Ross, Brian D; Hong, Hao; Van Dort, Marcian E

    2017-03-06

    High overexpression of sigma (σ) receptors (σ1 and σ2 subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ2 receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor subtype affinity (σ1, 6.3 nM; σ2, 10.2 nM) was radiolabeled with fluorine-18 ((18)F) to give [(18)F]3a and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [(18)F]3a was localized to cell surface σ receptors, while ∼10% of [(18)F]3a was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [(18)F]3a was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (n = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [(18)F]3a was σ receptor-mediated. Histology analysis confirmed similar expression of σ1 and σ2 in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [(18)F]3a. Prominent σ receptor-specific uptake of [(18)F]3a in

  3. An HIV-1 encoded peptide mimics the DNA binding loop of NF-{kappa}B and binds thioredoxin with high affinity

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    Su Guoping [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: gsu@u.washington.edu; Wang Min [Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023 (United States)]. E-mail: wang.min@yale.edu; Taylor, Ethan Will [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: wtaylor@rx.uga.edu

    2005-11-11

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-{kappa}B), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-{kappa}B via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-{kappa}B/Rel family of transcription factors by the putative HIV-1 pro-fs protein.

  4. High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen.

    Science.gov (United States)

    Rutgers, A; Meyers, K E; Canziani, G; Kalluri, R; Lin, J; Madaio, M P

    2000-07-01

    Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation

  5. REM Desensitization as a New Therapeutic Method for Post- Traumatic Stress Disorder: A Randomized Controlled Trial

    Directory of Open Access Journals (Sweden)

    Khodabakhsh Ahmadi

    2016-05-01

    Full Text Available Aim: to evaluate potential efficacy of a new therapeutic approach in posttraumatic stress disorder in comparison with eye movement desensitization and reprocessing (EMDR, a standard treatment approach and controls. Methods: the study was designed using a randomized controlled trial methodology. Participants were recruited from military servicemen aged between 25 to 50 years who were admitting hospitals of Bushehr, Iran, with the final diagnosis of PTSD. Finally 33 male patients were devided into three subgroups: G1: EMDR; G2: REM Desensitization; and group 3: controls who received no therapy. Mississippi Scale for Posttraumatic Stress Disorder, Pittsburgh Sleep Quality Index (PSQI and a 37 item death anxiety questionnaire were used for measures. Results: multiple comparisons showed that intrusive thoughts were significantly more likely to improve with REM Desensitization versus EMDR (P=0.03, while depression was more responsive to EMDR (p=0.03. Among the Pittsburgh scale for the quality of sleep items, sleep quality (p=0.02, sleep duration (p=0.001, and total sleep quality score (p=0.002 were significantly more likely to improve in the REM Desensitization group. Change in the absolute death anxiety scores was not different between subgroups excepting EMDR versus control group (p=0.05. Conclusion: REM, desensitization, the new therapeutic approach to PTSD is a highly effective strategy, even more than EMDR, the standard treatment, in most of the evaluated subjects, with special emphasis on sleep symptoms, and also in the management of intrusive thoughts. Depression is the only factor in which, REM Desensitization was significantly less likely to represent a superior therapeutic effect than EMDR. Key words: post traumatic stress disorder (PTSD, eye movement desensitization and reprocessing, new treatment.

  6. Infectious complications in living-donor kidney transplant recipients undergoing multi-modal desensitization.

    Science.gov (United States)

    Turza, Kristin C; Shafique, Michael; Lobo, Peter I; Sawyer, Robert G; Keith, Douglas S; Brayman, Kenneth L; Agarwal, Avinash

    2014-06-01

    Pre-existing humoral barriers challenge the transplantation of living donor kidneys (LDK) into highly sensitized ABO- and human leukocyte antigen (HLA)-incompatible recipients. Conditioning these LDK recipients' immune systems is required before they undergo transplantation. We hypothesized that medical desensitization would yield higher post-transplantation rates of infection. We conducted a study in which matched controls consisting of non-desensitized (NDS) LDK recipients were compared with desensitized (DS) receipients. Pre-transplantation desensitization included treatment with rituximab and mycophenolate mofetil followed by intravenous immunoglobulin (IVIg) and plasmapheresis. All participants in the study underwent induction therapy and maintenance immunosuppression. Primary outcomes included infection (opportunistic, local, systemic) within 12 mo after transplantation. Twenty-five patients underwent desensitization and LDK transplantation. Graft survival in the DS and NDS groups of patients was 96% and 98%, respectively. The mean 3- and 12-mo serum creatinine concentrations in the DS and NDS groups were 1.1±0.2 mg/dL and 1.2±0.3 mg/dL and 0.95±0.4 mg/dL and 0.73±0.8 mg/dL (p=0.3 and p=0.01), respectively. Thirty-six percent of the patients in the DS group had one or more infections, vs. 28% of those in the NDS group (p=0.1). No difference was observed in the frequency of opportunistic or systemic infections in the two groups. Local infections were statistically significantly more frequent in the DS group (60% vs. 30%, respectively; p=0.02). Pre-operative desensitization in highly sensitized LDK recipients is followed by a similar incidence of opportunistic and systemic infections as in NDS patients. Local infections were significantly more frequent in the DS than in the NDS patients in the study. With careful monitoring of infectious complications, pre-transplant desensitization permits LDK transplantation into highly sensitized patients.

  7. Does PKC activation increase the homologous desensitization of μ opioid receptors?

    Science.gov (United States)

    Arttamangkul, Seksiri; Birdsong, William; Williams, John T

    2015-01-01

    This study examined the role of agents known to activate PKC on morphine-induced desensitization of μ-opioid receptors (MOP receptors) in brain slices containing locus coeruleus neurons. Intracellular recordings were obtained from rat locus coeruleus neurons. Two measurements were used to characterize desensitization, the decline in hyperpolarization induced by application of a saturating concentration of agonist (acute desensitization) and the decrease in hyperpolarization induced by a subsaturating concentration of [Met](5) enkephalin (ME) following washout of the saturating concentration (sustained desensitization). Internalization of MOP receptors was studied in brain slices prepared from transgenic mice expressing Flag-MOP receptors. The subcellular distribution of activated PKC was examined using a novel fluorescent sensor of PKC in HEK293 cells. The phorbol esters (PMA and PDBu) and muscarine increased acute desensitization induced by a saturating concentration of morphine and ME. These effects were not sensitive to staurosporine. Staurosporine did not block the decline in hyperpolarization induced by muscarine. PDBu and muscarine did not affect sustained desensitization induced by ME nor did phorbol esters or muscarine change the trafficking of MOP receptors induced by morphine or ME. The distribution of activated PKC measured in HEK293 cells differed depending on which phorbol ester was applied. This study demonstrates a distinct difference in two measurements that are often used to evaluate desensitization. The measure of decline correlated well with the reduction in peak amplitudes caused by PKC activators implicating the modification of other factors rather than MOP receptors. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  8. Trigeminal sensitization and desensitization in the nasal cavity: a study of cross interactions.

    Science.gov (United States)

    Jacquot, Laurence; Monnin, Julie; Lucarz, Annie; Brand, Gérard

    2005-06-01

    Chemical irritation in the human nasal cavity is poorly documented. In this field, an important issue concerns the differential responses produced by successive stimulation. Repeated identical chemical irritant stimuli can produce increases or decreases in responses (two phenomena known as self-sensitization or self-desensitization). In the same way, different molecules can interact and produce cross-sensitization or cross-desensitization. The aim of this study was to contribute to this question using two specific molecules, acetic acid (AA) and allyl isothiocyanate (AIC). As the self-sensitization and -desensitization for AIC is known, a first experiment in the present work investigated the response, acute effects and time course of sensitization or desensitization to acetic acid. A second experiment tested the responses of acetic acid after a previous stimulation with allyl isothiocyanate (mustard oil) and inversely with a short inter-stimulus interval (ISI of 45 s). A third experiment similar to the second used a long inter-stimulus interval (ISI of 3 min 30). Twelve healthy subjects participated in the study using psychophysical (intensity ratings) and psychophysiological (skin conductance response) measurements. Firstly, the results showed that repeated nasal stimulation with acetic acid produced a self-desensitization whatever the ISI. Secondly, the results showed a cross-desensitization of allyl isothiocyanate by previous acetic acid stimulation. In contrast, previous stimulation with allyl isothiocyanate had no effect on the following acetic acid response. These findings confirm that trigeminal sensitization and desensitization in the nasal cavity do not follow the same processes in relation to molecules used.

  9. Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization.

    Directory of Open Access Journals (Sweden)

    Nicole K McKinnon

    Full Text Available 5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4 and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-A(n = 2-7. Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC(50's were at most 5-fold greater than wild-type (WT. The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A(2, 5-HT3A-A(4, 5-HT3A-A(6, and 5-HT3A-A(7 were similar to WT. In contrast, 5-HT3A-A(3 and 5-HT3A-A(5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A(1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.

  10. Hypersensitivity to antineoplastic agents: mechanisms and treatment with rapid desensitization.

    Science.gov (United States)

    Castells, Mariana; Sancho-Serra, Maria del Carmen; Simarro, Maria

    2012-09-01

    Hypersensitivity reactions (HSRs) to chemotherapy drugs, such as taxanes and platins, and to monoclonal antibodies limit their therapeutic use due to the severity of some reactions and the fear of inducing a potentially lethal reaction in highly sensitized patients. Patients who experience hypersensitivity reactions face the prospect of abandoning first-line treatment and switching to a second-line, less effective therapy. Some of these reactions are mast cell-mediated hypersensitivity reactions, a subset of which occur through an immunoglobulin (IgE)-dependent mechanism, and are thus true allergies. Others involve mast cells without a demonstrable IgE mechanism. Whether basophils can participate in these reactions has not been demonstrated. Rapid drug desensitization (RDD) is a procedure that induces temporary tolerance to a drug, allowing a medication allergic patient to receive the optimal agent for his or her disease. Through RDD, patients with IgE and non-IgE HSRs can safely be administered important medications while minimizing or completely inhibiting adverse reactions. Due to the clinical expansion and success of RDD, the molecular mechanisms inducing the temporary tolerization have been investigated and are partially understood, allowing for safer and more effective protocols. This article reviews the current literature on molecular mechanisms of RDD with an emphasis in our recent contributions to this field as well as the indications, methods and outcomes of RDD for taxanes, platins, and monoclonal antibodies.

  11. Physiological correlates of eye movement desensitization and reprocessing.

    Science.gov (United States)

    Elofsson, Ulf O E; von Schèele, Bo; Theorell, Töres; Söndergaard, Hans Peter

    2008-05-01

    Eye movement desensitization and reprocessing (EMDR) is an established treatment for post-traumatic stress disorder (PTSD). However, its working mechanism remains unclear. This study explored physiological correlates of eye movements during EMDR in relation to current hypotheses; distraction, conditioning, orienting response activation, and REM-like mechanisms. During EMDR therapy, fingertip temperature, heart rate, skin conductance, expiratory carbon dioxide level, and blood pulse oximeter oxygen saturation, were measured in male subjects with PTSD. The ratio between the low and high frequency components of the heart rate power spectrum (LF/HF) were computed as measures of autonomic balance. Respiratory rate was calculated from the carbon dioxide trace. Stimulation shifted the autonomic balance as indicated by decreases in heart rate, skin conductance and LF/HF-ratio, and an increased finger temperature. The breathing frequency and end-tidal carbon dioxide increased; oxygen saturation decreased during eye movements. In conclusion, eye movements during EMDR activate cholinergic and inhibit sympathetic systems. The reactivity has similarities with the pattern during REM-sleep.

  12. Eye movement desensitization and reprocessing (EMDR): a meta-analysis.

    Science.gov (United States)

    Davidson, P R; Parker, K C

    2001-04-01

    Eye movement desensitization and reprocessing (EMDR), a controversial treatment suggested for posttraumatic stress disorder (PTSD) and other conditions, was evaluated in a meta-analysis of 34 studies that examined EMDR with a variety of populations and measures. Process and outcome measures were examined separately. and EMDR showed an effect on both when compared with no treatment and with therapies not using exposure to anxiety-provoking stimuli and in pre post EMDR comparisons. However, no significant effect was found when EMDR was compared with other exposure techniques. No incremental effect of eye movements was noted when EMDR was compared with the same procedure without them. R. J. DeRubeis and P. Crits-Christoph (1998) noted that EMDR is a potentially effective treatment for noncombat PTSD. but studies that examined such patient groups did not give clear support to this. In sum, EMDR appears to be no more effective than other exposure techniques, and evidence suggests that the eye movements integral to the treatment, and to its name, are unnecessary.

  13. Eye movement desensitization and reprocessing for adolescent depression.

    Science.gov (United States)

    Bae, Hwallip; Kim, Daeho; Park, Yong Chon

    2008-03-01

    While cognitive behavior therapy is considered to be the first-line therapy for adolescent depression, there are limited data on whether other psychotherapeutic techniques are also effective in treating adolescents with depression. This report suggests the potential application of eye movement desensitization and reprocessing (EMDR) for treatment of depressive disorder related, not to trauma, but to stressful life events. At present, EMDR has only been empirically validated for only trauma-related disorders such as posttraumatic stress disorder. Two teenagers with major depressive disorder (MDD) underwent three and seven sessions of EMDR aimed at memories of stressful life events. After treatment, their depressive symptoms decreased to the level of full remission, and the therapeutic gains were maintained after two and three months of follow up. The effectiveness of EMDR for depression is explained by the model of adaptive information processing. Given the powerful effects observed within a brief period of time, the authors suggest that further investigation of EMDR for depressive disorders is warranted.

  14. Clinical evaluation of desensitizing treatments for cervical dentin hypersensitivity

    Directory of Open Access Journals (Sweden)

    Ana Cecilia Corrêa Aranha

    2009-09-01

    Full Text Available The aim of this study was to compare different treatments for dentin hypersensitivity in a 6-month follow-up. One hundred and one teeth exhibiting non carious cervical lesions were selected. The assessment method used to quantify sensitivity was the cold air syringe, recorded by the visual analogue scale (VAS, prior to treatment (baseline, immediately after topical treatment, after 1 week, 1, 3 and 6 months. Teeth were randomly assigned to five groups (n = 20: G1: Gluma Desensitizer (GD; G2: Seal&Protect (SP; G3: Oxa-gel (OG; G4: Fluoride (F; G5: Low intensity laser-LILT (660 nm/3.8 J/cm²/15 mW. Analysis was based on the non-parametric Kruskal-Wallis test that demonstrated statistical differences immediately after the treatment (p = 0.0165. To observe the individual effects of each treatment, data was submitted to Friedman test. It was observed that GD and SP showed immediate effect after application. Reduction in the pain level throughout the six-month follow-up was also observed. In contrast, LILT presented a gradual reduction of hypersensitivity. OG and F showed effects as of the first and third month respectively. It can be concluded that, after the 6-month clinical evaluation, all therapies showed lower VAS sensitivity values compared with baseline, independently of their different modes of action.

  15. Eye Movement Desensitization and Reprocessing (EMDR as a Neurorehabilitation Method

    Directory of Open Access Journals (Sweden)

    Afsaneh Zarghi

    2013-01-01

    Full Text Available   A variety of nervous system components such as medulla, pons, midbrain, cerebellum, basal ganglia, parietal, frontal and occipital lobes have role in Eye Movement Desensitization and Reprocessing (EMDR processes. The eye movement is done simultaneously for attracting client's attention to an external stimulus while concentrating on a certain internal subject. Eye movement guided by therapist is the most common attention stimulus. The role of eye movement has been documented previously in relation with cognitive processing mechanisms. A series of systemic experiments have shown that the eyes’ spontaneous movement is associated with emotional and cognitive changes and results in decreased excitement, flexibility in attention, memory processing, and enhanced semantic recalling. Eye movement also decreases the memory's image clarity and the accompanying excitement. By using EMDR, we can reach some parts of memory which were inaccessible before and also emotionally intolerable. Various researches emphasize on the effectiveness of EMDR in treating and curing phobias, pains, and dependent personality disorders. Consequently, due to the involvement of multiple neural system components, this palliative method of treatment can also help to rehabilitate the neuro-cognitive system.

  16. Eye movement desensitization in fibromyalgia: a pilot study.

    Science.gov (United States)

    Friedberg, Fred

    2004-11-01

    The purpose of this study was to investigate the effectiveness of eye movement desensitization (EMD) for the relief of pain, fatigue and anxiety and depression in fibromyalgia patients. Six Caucasian female patients (mean age=43.2 yr) participated in two treatment sessions. Outcome assessments included the Fibromyalgia Impact Questionnaire, Fatigue Scale, Beck Anxiety Inventory, and Beck Depression Inventory. In-session process measures included thermal biofeedback monitoring and subjective units of discomfort ratings of pain, stress, and fatigue. Four out of six subjects were considered treatment responders. Thermal biofeedback monitoring revealed an average increase in hand temperature of 5.4 degrees indicating a relaxation effect. At treatment termination, average scores decreased on the measures of anxiety (28.6%), depression (29.9%), fibromyalgia impact (12.6%), and fatigue (11.5%). At the 3-month follow-up assessment, total reductions in average scores from pre-treatment baseline reflected further improvements on measures of anxiety (45.8%), depression (31.6%), fibromyalgia impact (19.2%), and fatigue (26.7%). Because EMD produced a somewhat automatic relaxation response with minimal patient participation, it may be especially useful when standard relaxation techniques fail.

  17. Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

    Science.gov (United States)

    Liot, Quentin; Constant, Philippe

    2016-02-01

    The Streptomyces avermitilis genome encodes a putative high-affinity [NiFe]-hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase-saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP-binding cassette-transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival-mixotrophic energy mode and discuss important ecological implications of this finding.

  18. Inhibition of human high-affinity copper importer Ctr1 orthologous in the nervous system of Drosophila ameliorates Aβ42-induced Alzheimer's disease-like symptoms.

    Science.gov (United States)

    Lang, Minglin; Fan, Qiangwang; Wang, Lei; Zheng, Yajun; Xiao, Guiran; Wang, Xiaoxi; Wang, Wei; Zhong, Yi; Zhou, Bing

    2013-11-01

    Disruption of copper homeostasis has been implicated in Alzheimer's disease (AD) during the last 2 decades; however, whether copper is a friend or a foe is controversial. Within a genetically tractable Drosophila AD model, we manipulated the expression of human high-affinity copper importer orthologous in Drosophila to explore the in vivo roles of copper ions in the development of AD. We found that inhibition of Ctr1C expression by RNAi in Aβ-expressing flies significantly reduced copper accumulation in the brains of the flies as well as ameliorating neurodegeneration, enhancing climbing ability, and prolonging lifespan. Interestingly, Ctr1C inhibition led to a significant increase in higher-molecular-weight Aβ42 forms in brain lysates, whereas it was accompanied by a trend of decreased expression of amyloid-β degradation proteases (including NEP1-3 and IDE) with age and reduced Cu-Aβ interaction-induced oxidative stress in Ctr1C RNAi flies. Similar results were obtained from inhibiting another copper importer Ctr1B and overexpressing a copper exporter DmATP7 in the nervous system of AD flies. These results imply that copper may play a causative role in developing AD, as either Aβ oligomers or aggregates were less toxic in a reduced copper environment or one with less copper binding. Early manipulation of brain copper uptake can have a great effect on Aβ pathology.

  19. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

    Science.gov (United States)

    Xu, Qiang; Wang, Yanshu; Dabdoub, Alain; Smallwood, Philip M; Williams, John; Woods, Chad; Kelley, Matthew W; Jiang, Li; Tasman, William; Zhang, Kang; Nathans, Jeremy

    2004-03-19

    Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

  20. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

    Energy Technology Data Exchange (ETDEWEB)

    Klein, M.; Canoll, P.D.; Musacchio, J.M. (New York Univ. Medical Center, New York, NY (USA))

    1991-01-01

    The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.

  1. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

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    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  2. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

    Science.gov (United States)

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-04-23

    AMT1-3 encodes the high affinity NH₄⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  3. H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups.

    Science.gov (United States)

    Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G; Constant, Philippe

    2016-01-01

    Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.

  4. Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria

    Science.gov (United States)

    Porcherie, Adeline; Mathieu, Cedric; Peronet, Roger; Schneider, Elke; Claver, Julien; Commere, Pierre-Henri; Kiefer-Biasizzo, Hélène; Karasuyama, Hajime; Milon, Geneviève; Dy, Michel; Kinet, Jean-Pierre; Louis, Jacques; Blank, Ulrich

    2011-01-01

    The role of the IgE–FcεRI complex in malaria severity in Plasmodium falciparum–hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell–deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI+ neutrophil population, which is not observed in mice hosting a non–ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI+ neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI+ neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity. PMID:21967768

  5. Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG.

    Science.gov (United States)

    Minamihata, Kosuke; Goto, Masahiro; Kamiya, Noriho

    2015-01-01

    Cross-linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody-binding protein possessing high affinity to IgG by conjugating a number of antibody-binding proteins using the horseradish peroxidase (HRP)-mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y-tag) to the C-terminus of the model protein, a chimera protein of protein G and protein A (pG2 pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2 pA conjugate, composed of mainly two to three units of pG2 pA. The cross-linking occurred site specifically at the Tyr residue in the Y-tag and introduction of the Y-tag showed no effect on the function of pG2 pA. The affinity of the Y-tagged pG2 pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross-linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.

  6. High-affinity Near-infrared Fluorescent Small-molecule Contrast Agents for In Vivo Imaging of Prostate-specific Membrane Antigen

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    Valerie Humblet

    2005-10-01

    Full Text Available Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM, single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

  7. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

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    Cheryl M Vahling-Armstrong

    Full Text Available The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

  8. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice.

    Science.gov (United States)

    Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia; Banala, Ashwini K; Burzynski, Caitlin; Pommier, Elie; Keck, Thomas M; Donthamsetti, Prashant; Javitch, Jonathan A; Rais, Rana; Slusher, Barbara S; Xi, Zheng-Xiong; Newman, Amy Hauck

    2015-08-13

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice.

  9. The P2’ residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity

    Energy Technology Data Exchange (ETDEWEB)

    Salameh, M.A.; Soares, A.; Hockla, A.; Radisky, D. C.; Radisky, E. S.

    2011-11-15

    PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumor progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P'{sub 2} position. We find that bulky and charged residues strongly disfavor binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P{sub 1} and P'{sub 2} residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K{sub i} of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

  10. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Directory of Open Access Journals (Sweden)

    Aili Bao

    2015-04-01

    Full Text Available AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  11. New insights in the structure and biology of the high affinity receptor for IgE (Fc epsilon RI) on human epidermal Langerhans cells.

    Science.gov (United States)

    Bieber, T; Kraft, S; Jürgens, M; Strobel, I; Haberstok, J; Tomov, H; Regele, D; de la Salle, H; Wollenberg, A; Hanau, D

    1996-10-01

    The recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.

  12. Tityus gamma toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane.

    Science.gov (United States)

    Barhanin, J; Ildefonse, M; Rougier, O; Sampaio, S V; Giglio, J R; Lazdunski, M

    1984-01-01

    Toxin gamma from the venom of Tityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3 X 10(-10) M. Hyperpolarization to potentials more negative than the resting potential (E = -90 mV) reduces or abolishes the effect of the toxin. Radioiodinated toxin gamma binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1 X 10(-11) M. Data obtained with both rabbit and frog muscle indicate that toxin gamma is specific for Na+ channels in surface membranes. Toxin gamma does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is one Tityus gamma toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed that Tityus gamma toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II from Centruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel than Tityus gamma toxin.

  13. Tityus serrulatus venom contains two classes of toxins. Tityus gamma toxin is a new tool with a very high affinity for studying the Na+ channel.

    Science.gov (United States)

    Barhanin, J; Giglio, J R; Léopold, P; Schmid, A; Sampaio, S V; Lazdunski, M

    1982-11-10

    The interaction of TiTx gamma, the major toxin in the venom of the scorpion Tityus serrulatus, with its receptor in excitable membranes was studied with the use of 125I-TiTx gamma. This derivative retains biological activity, and its specific binding to both brain synaptosomes and electroplaque membranes from Electrophorus electricus is characterized by a dissociation constant equal to that of the native toxin-receptor complex, about 2 to 5 pM. This very high affinity results mainly from a very slow rate of dissociation, equivalent to a half-life longer than 10 h at 4 degrees C. There is a 1:1 stoichiometry between TiTx gamma binding and tetrodotoxin binding to the membranes, but neither tetrodotoxin nor any of 7 other neurotoxins that are representative of 4 different classes of effectors of the Na+ channel interfere with TiTx gamma binding. Similarly, local anesthetics and other molecules that affect other types of ionic channels or neurotransmitter receptors have no effect on TiTx gamma binding. However, toxin II from Centruroides suffusus suffusus does compete with TiTx gamma, though its affinity for the receptor is much lower. Since the Centruroides toxin II is known to affect Na+ channel function, these two scorpion toxins must be put into a fifth class of Na+ channel effectors.

  14. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    Science.gov (United States)

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β.

  15. Time-resolved investigation of molecular components involved in the induction of NO3- high affinity transport system in maize roots

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    Youry Pii

    2016-11-01

    Full Text Available The induction, i.e. the rapid increase of nitrate (NO3- uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3- treatment were characterized in terms of changes in NO3- uptake rate and plasma membrane (PM H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3 and PM H+-ATPase gene families. The behaviour of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼ 150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3- treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A nondenaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  16. Isolation of a high affinity neutralizing monoclonal antibody against 2009 pandemic H1N1 virus that binds at the 'Sa' antigenic site.

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    Nachiket Shembekar

    Full Text Available Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K(D = 2.1±0.4 pM murine MAb, MA2077 that binds specifically to the hemagglutinin (HA surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC(50 = 0.08 µg/ml. MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein 'Sa' and 'Sb' sites were independently mutated, localized the binding site of MA2077 within the 'Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

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

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    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.

  18. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    Science.gov (United States)

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stre