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Sample records for soluble ligand receptor

  1. Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

    Science.gov (United States)

    Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

    2008-01-01

    The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

  2. Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein

    DEFF Research Database (Denmark)

    Sennels, H P; Jacobsen, Søren; Jensen, T

    2007-01-01

    Objective. Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high-sensitivity C-reactive protein (hsCRP) has recently attracted increased interest. The purpose...

  3. Computer program for Scatchard analysis of protein: Ligand interaction - use for determination of soluble and nuclear steroid receptor concentrations

    International Nuclear Information System (INIS)

    Leake, R.; Cowan, S.; Eason, R.

    1998-01-01

    Steroid receptor concentration may be determined routinely in biopsy samples of breast and endometrial cancer by the competition method. This method yields data for both the soluble and nuclear fractions of the tissue. The data are usually subject to Scatchard analysis. This Appendix describes a computer program written initially for a PDP-11. It has been modified for use with IBM, Apple Macintosh and BBC microcomputers. The nature of the correction for competition is described and examples of the printout are given. The program is flexible and its use for different receptors is explained. The program can be readily adapted to other assays in which Scatchard analysis is appropriate

  4. AMPA receptor ligands

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Mellor, Ian

    2004-01-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player in the f......Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player...... in the formation of memory. Hence, ligands affecting AMPARs are highly important for the study of the structure and function of this receptor, and in this regard polyamine-based ligands, particularly polyamine toxins, are unique as they selectively block Ca2+ -permeable AMPARs. Indeed, endogenous intracellular...

  5. Soluble ligands for the NKG2D receptor are released during endometriosis and correlate with disease severity.

    Directory of Open Access Journals (Sweden)

    Iñaki González-Foruria

    Full Text Available Endometriosis is a benign gynaecological disease. Abundant bulk of evidence suggests that patients with endometriosis have an immunity dysfunction that enables ectopic endometrial cells to implant and proliferate. Previous studies show that natural killer cells have a pivotal role in the immune control of endometriosis.This is a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n= 202 during surgery for benign gynaecological conditions. After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free controls women were enrolled. Patients with endometriosis were classified according to a surgical classification in three different types of endometriosis: superficial peritoneal endometriosis (SUP, ovarian endometrioma (OMA and deep infiltrating endometriosis (DIE. Peritoneal fluid samples were obtained from all study participants during the surgery in order to detect soluble NKG2D ligands (MICA, MICB and ULBP-2. When samples with undetectable peritoneal fluid levels of MICA, MICB and ULBP-2 were excluded, MICA ratio levels were significantly higher in endometriosis patients than in controls (median, 1.1 pg/mg; range, 0.1-143.5 versus median, 0.6 pg/mg; range, 0.1-3.5; p=0.003. In a similar manner peritoneal fluid MICB levels were also increased in endometriosis-affected patients compared with disease-free women (median, 4.6 pg/mg; range, 1.2-4702 versus median, 3.4 pg/mg; range, 0.7-20.1; p=0.001. According to the surgical classification, peritoneal fluid soluble MICA, MICB and ULBP-2 ratio levels were significantly increased in DIE as compared to controls (p=0.015, p=0.003 and p=0.045 respectively. MICA ratio levels also correlated with dysmenorrhea (r=0.232; p=0.029, total rAFS score (r=0.221; p=0.031 and adhesions rAFS score (r=0.221; p=0

  6. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA...

  7. A compartment model of VEGF distribution in humans in the presence of soluble VEGF receptor-1 acting as a ligand trap.

    Directory of Open Access Journals (Sweden)

    Florence T H Wu

    Full Text Available Vascular endothelial growth factor (VEGF, through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis--new capillary growth from existing microvasculature--at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1--a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains--has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis-dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage; and molecularly-detailed binding interactions between the ligand isoforms VEGF(121 and VEGF(165, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1, as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 - acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization

  8. Synthesis, characterization and biodistribution of neutral and lipid-soluble 99mTc-bisaminoethanethiol spiperone derivatives: Possible ligands for receptor imaging with SPECT

    International Nuclear Information System (INIS)

    Samnick, Samuel; Brandau, Wolfgang; Sciuk, Joachim; Steinstrasser, Axel; Schober, Otmar

    1995-01-01

    Using parts of the molecular structure of spiperone, two new ligand systems for complexation with [ 99m Tc]technetium were prepared in order to develop potential receptor imaging agents for single photon emission computer tomography (SPECT). The bis-aminoethanethiols (BAT): 1-benzyl-4-(2-mercapto-2-methyl-4-aza-pentyl)-4-(2-mercapto-2-methyl- propylamino)-piperidine (benzylpiperidyl-BAT, BP-BAT) and 1-[3-(4-fluorobenzoyl)-propyl]-4-(2-mercapto-2-methyl-4-aza-pentyl)-4-(2- mercapto-2-methyl-propylamino)-piperidine (butyrophenoylpiperidyl-BAT, BUP-BAT) form stable, neutral and lipid soluble complexes with [ 99m Tc]technetium at pH ≥ 11 using SnCl 2 as reducing agent in nearly quantitative radiochemical yields. Biodistribution of 99m Tc-BP-BAT and 99m Tc-BUP-BAT in rats showed a moderate clearance from blood and low uptake and retention in the liver, whereas brain uptake was moderate, however with prolonged brain retention. On the other hand, significant accumulations and retentions were observed in heart, kidney and lung with increasing oxygen/blood ratios up to 24 h. Within 24 h p.i. 22 and 29% of the injected dose (i.d.) of 99m Tc-BP-BAT and 99m Tc-BUP-BAT were eliminated by hepatobiliary excretion whereas 22% i.d. of both 99m Tc-BAT complexes were excreted into the urine. Although first biodistribution studies of 99m Tc-BP-BAT and 99m Tc-BUP-BAT in rats showed relatively low brain uptake, the high uptake in peripheral, receptor rich organs indicates that compounds of this type may be used as a basis for further structural modification to develop agents with optimal properties for cerebral or peripheral receptor imaging with SPECT

  9. Circulating osteoprotegerin and soluble receptor activator of nuclear factor κB ligand in polycystic ovary syndrome: relationships to insulin resistance and endothelial dysfunction.

    Science.gov (United States)

    Pepene, Carmen Emanuela; Ilie, Ioana Rada; Marian, Ioan; Duncea, Ileana

    2011-01-01

    There is plenty of evidence that osteoprotegerin (OPG) is linked to subclinical vascular damage and predicts cardiovascular disease in high-risk populations. Our aim is to investigate the relationships of OPG/free soluble receptor activator of nuclear factor κB ligand (sRANKL) to insulin resistance, brachial artery flow-mediated vasodilation (FMD), and the carotid artery intima-media thickness (CIMT) in polycystic ovary syndrome (PCOS), a disorder characterized by hyperandrogenism, impaired glucose control, and endothelial injury. A cross-sectional, observational study. Hormonal and metabolic profiles, FMD, CIMT, serum OPG, and ampli-sRANKL were assessed in 64 young PCOS patients and 20 controls of similar age. Body composition was measured by dual energy X-ray absorptiometry. OPG was significantly lower in PCOS and related negatively to free testosterone and positively to estradiol (E(2)) levels. In multivariate analysis, OPG but not ampli-sRANKL correlated positively to fasting insulin, insulin sensitivity indices, and FMD. Neither OPG nor ampli-sRANKL was associated with CIMT. Significantly lower adjusted FMD values were demonstrated in women in the upper OPG quartile group (>2.65 pmol/l) compared with all other quartile groups together (P=0.012). In PCOS, multiple regression analysis retained E(2)/sex hormone-binding globulin ratio, fat mass, and homeostasis model assessment of insulin resistance as independent predictors of OPG. In PCOS, circulating OPG is related to both endothelial dysfunction and insulin resistance, independent of obesity and androgen excess, suggesting OPG as a useful biomarker of these effects. Further studies are needed to evaluate OPG in relation to cardiovascular events and cardiovascular mortality in PCOS.

  10. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Science.gov (United States)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  11. Radioiodinated ligands for dopamine receptors

    International Nuclear Information System (INIS)

    Kung, H.F.

    1994-01-01

    The dopamine receptor system is important for normal brain function; it is also the apparent action site for various neuroleptic drugs for the treatment of schizophrenia and other metal disorders. In the past few years radioiodinated ligands for single photon emission tomography (SPECT) have been successfully developed and tested in humans: [ 123 I]TISCH for D1 dopamine receptors; [ 123 I]IBZM, epidepride, IBF and FIDA2, four iodobenzamide derivatives, for D2/D3 dopamine receptors. In addition, [ 123 I]β-CIT (RTI-55) and IPT, cocaine derivatives, for the dopamine reuptake site are potentially useful for diagnosis of loss of dopamine neurons. The first iodinated ligand, (R)trans-7-OH-PIPAT, for D3 dopamine receptors, was synthesized and characterized with cloned cell lines (Spodoptera frugiperda, Sf9) expressing the D2 and D3 dopamine receptors and with rat basal forebrain membrane preparations. Most of the known iodobenzamides displayed similar potency in binding to both D2 and D3 dopamine receptors expressed in the cell lines. Initial studies appear to suggest that by fine tuning the structures it may be possible to develop agents specific for D2 and D3 dopamine receptors. It is important to investigate D2/D3 selectivity for this series of potent ligands

  12. Soluble NKG2D ligands: prevalence, release, and functional impact.

    Science.gov (United States)

    Salih, Helmut Rainer; Holdenrieder, Stefan; Steinle, Alexander

    2008-05-01

    Natural Killer (NK) cells are capable to recognize and eliminate malignant cells. Anti-tumor responses of NK cells are promoted by the tumor-associated expression of cell stress-inducible ligands of the activating NK receptor NKG2D. Current evidence suggests that established tumors subvert NKG2D-mediated tumor immunosurveillance by releasing NKG2D ligands (NKG2DL). Release of NKG2DL has been observed in a broad variety of human tumor entities and is thought to interfere with NKG2D-mediated tumor immunity in several ways. Further, levels of soluble NKG2DL (sNKG2DL) were also found to be elevated under various non-malignant conditions, although the functional implications remain largely unclear. Here we review and discuss the available data on the prevalence, release, functional impact, and potential clinical value of sNKG2DL.

  13. Senescent cells re-engineered to express soluble programmed death receptor-1 for inhibiting programmed death receptor-1/programmed death ligand-1 as a vaccination approach against breast cancer.

    Science.gov (United States)

    Chen, Zehong; Hu, Kang; Feng, Lieting; Su, Ruxiong; Lai, Nan; Yang, Zike; Kang, Shijun

    2018-06-01

    Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent-cell-based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple-negative breast cancer. However, the programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor-1 (sPD1)-expressing senescent cells to overcome PD-L1/PD-1-mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T-cell activation. In the present study, sPD1-expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1-expressing senescent tumor cell vaccine (STCV/sPD-1) treatment attracted more mature DC and fewer exhausted-PD1 + T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD-1 than for control treatments. STCV/sPD-1 pre-injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD-1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD-1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  14. Labeled receptor ligands for spect

    International Nuclear Information System (INIS)

    Kung, H.F.

    1989-01-01

    Receptor specific imaging agents for single photon emission computed tomography (SPECT) can potentially be useful in the understanding of basic biochemistry and pharmacology of receptors. SPECT images may also provide tools for evaluation of density and binding kinetics of a specific receptor, information important for diagnosis and patient management. Basic requirements for receptor imaging agents are: (a) they are labeled with short-lived isotopes, (b) they show high selectivity and specific uptake, (c) they exhibit high target/background ratio, and (d) they can be modeled to obtain quantitative information. Several good examples of CNS receptor specific ligands labeled with I-123 have been developed, including iodoQNB, iodoestrogen iodobenzadiazepine, iodobenazepine, iodobenzamides for muscarinic, estrogen benzadiazepine, D-1 and D-2 dopamine receptors. With the advent of newer and faster SPECT imaging devices, it may be feasible to quantitate the receptor density by in vivo imaging techniques. These new brain imaging agents can provide unique diagnostic information, which may not be available through other imaging modalities, such as CT and MRI

  15. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    surface-linked ligands to diffuse freely in two dimensions. Ligands can become reorganized beneath cells, by reaction-diffusion processes, and may also adopt spatial configurations reflecting those of their cognate receptors on the cell surface (Figure 1B). This provides a significant benefit over conventional cell signaling and culturing systems that present inflexible distributions of signaling molecules. In this study, we observe marked differences in the response of cells to membrane surface displayed soluble ligands as a function of membrane fluidity. Tethering of soluble signaling molecules to fluid supported membranes opens up opportunities to use already developed membrane fabrication technologies to present soluble components within a surface array format.

  16. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    International Nuclear Information System (INIS)

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2007-01-01

    A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

  17. Characteristic molecular vibrations of adenosine receptor ligands.

    Science.gov (United States)

    Chee, Hyun Keun; Yang, Jin-San; Joung, Je-Gun; Zhang, Byoung-Tak; Oh, S June

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Water-soluble diphosphadiazacyclooctanes as ligands for aqueous organometallic catalysis

    KAUST Repository

    Boulanger, Jérôme

    2012-12-01

    Two new water-soluble diphosphacyclooctanes been synthesized and characterized by NMR and surface tension measurements. Both phosphanes proved to coordinate rhodium in a very selective way as well-defined bidentates were obtained. When used in Rh-catalyzed hydroformylation of terminal alkenes, both ligands positively impacted the reaction chemoselectivity. © 2012 Elsevier B.V.

  19. Water-soluble diphosphadiazacyclooctanes as ligands for aqueous organometallic catalysis

    KAUST Repository

    Boulanger, Jé rô me; Bricout, Hervé ; Tilloy, Sé bastien; Fihri, Aziz; Len, Christophe; Hapiot, Fré dé ric; Monflier, É ric

    2012-01-01

    Two new water-soluble diphosphacyclooctanes been synthesized and characterized by NMR and surface tension measurements. Both phosphanes proved to coordinate rhodium in a very selective way as well-defined bidentates were obtained. When used in Rh-catalyzed hydroformylation of terminal alkenes, both ligands positively impacted the reaction chemoselectivity. © 2012 Elsevier B.V.

  20. Ligand-receptor Interactions by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Novak. P.

    2008-04-01

    Full Text Available Today NMR spectroscopy is a method of choice for elucidation of interactions between biomolecules and the potential ligands. Knowledge on these interactions is an essential prerequisite for the rational drug design. The most important contribution of NMR to drug design a few years ago was the 3D structure determination of proteins. Besides delivering the 3D structures of the free proteins as a raw material for the modeling studies on ligand binding, NMR can directly yield valuable experimental data on the biologically important protein-ligand complexes. In addition to X-ray diffraction, NMR spectroscopy can provide information on the internal protein dynamics ordynamics of intermolecular interactions. Changes in NMR parameters allow us to detect ("SAR by NMR" and quantitatively determine binding affinities (titration, diffusion NMR experiments, etc. of potential ligands. Also, it is possible to determine the binding site and conformations of ligands, receptors and receptor-ligand complexes with the help of NMR methods such as tr-NOESY. Epitopes or functional groups responsible for binding of ligands to the receptor can be identified by employing STD or WaterLOGSY experiments. In this review are described some of the most frequent NMR methods for the characterization of the interactions between biomolecules and ligands, together with their advantages and disadvantages.

  1. Sigma-2 receptor ligands QSAR model dataset

    Directory of Open Access Journals (Sweden)

    Antonio Rescifina

    2017-08-01

    Full Text Available The data have been obtained from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB and refined according to the QSAR requirements. These data provide information about a set of 548 Sigma-2 (σ2 receptor ligands selective over Sigma-1 (σ1 receptor. The development of the QSAR model has been undertaken with the use of CORAL software using SMILES, molecular graphs and hybrid descriptors (SMILES and graph together. Data here reported include the regression for σ2 receptor pKi QSAR models. The QSAR model was also employed to predict the σ2 receptor pKi values of the FDA approved drugs that are herewith included.

  2. Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy

    NARCIS (Netherlands)

    Ochodnicky, Peter; Lattenist, Lionel; Ahdi, Mohamed; Kers, Jesper; Uil, Melissa; Claessen, Nike; Leemans, Jaklien C.; Florquin, Sandrine; Meijers, Joost C. M.; Gerdes, Victor E. A.; Roelofs, Joris J. T. H.

    2017-01-01

    TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3,

  3. Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity.

    Directory of Open Access Journals (Sweden)

    Sophie Daburon

    Full Text Available Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.

  4. Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jørgensen, M U; Emr, S D; Winther, Jakob R.

    1999-01-01

    Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand bind...

  5. Novel Somatostatin Receptor Ligands Therapies for Acromegaly

    Directory of Open Access Journals (Sweden)

    Rosa Maria Paragliola

    2018-03-01

    Full Text Available Surgery is considered the treatment of choice in acromegaly, but patients with persistent disease after surgery or in whom surgery cannot be considered require medical therapy. Somatostatin receptor ligands (SRLs octreotide (OCT, lanreotide, and the more recently approved pasireotide, characterized by a broader receptor ligand binding profile, are considered the mainstay in the medical management of acromegaly. However, in the attempt to offer a more efficacious and better tolerated medical approach, recent research has been aimed to override some limitations related to the use of currently approved drugs and novel SRLs therapies, with potential attractive features, have been proposed. These include both new formulation of older molecules and new molecules. Novel OCT formulations are aimed in particular to improve patients’ compliance and to reduce injection discomfort. They include an investigational ready-to-use subcutaneous depot OCT formulation (CAM2029, delivered via prefilled syringes and oral OCT that uses a “transient permeability enhancer” technology, which allows for OCT oral absorption. Another new delivery system is a long-lasting OCT implant (VP-003, which provide stable doses of OCT throughout a period of several months. Finally, a new SRL DG3173 (somatoprim seems to be more selective for GH secretion, suggesting possible advantages in the presence of hyperglycemia or diabetes. How much these innovations will actually be beneficial to acromegaly patients in real clinical practice remains to be seen.

  6. Substrate coated with receptor and labelled ligand for assays

    International Nuclear Information System (INIS)

    1980-01-01

    Improvements in the procedures for assaying ligands are described. The assay consists of a polystyrene tube on which receptors are present for both the ligand to be assayed and a radioactively labelled form of the ligand. The receptors on the bottom portion of the tube are also coated with labelled ligands, thus eliminating the necessity for separate addition of the labelled ligand and sample during an assay. Examples of ligands to which this method is applicable include polypeptides, nucleotides, nucleosides and proteins. Specific examples are given in which the ligand to be assayed is digoxin, the labelled form of the ligand is 3-0-succinyl digoxyigenin tyrosine ( 125 I) and the receptor is digoxin antibody. (U.K.)

  7. Novel photoaffinity ligands for the GA-receptor

    International Nuclear Information System (INIS)

    Suttle, J.C.; Hultstrand, J.F.; Tanaka, F.S.

    1990-01-01

    Previous studies from this laboratory have shown that certain N-substituted phthalimides (NSPs) exhibit GA-like activity in a range of specific bioassays and that bioactive NSPs compete with [ 3 H]-GA 4 for soluble binding sites in cucumber homogenates. As such, these compounds may prove useful in the purification and characterization of GA receptor proteins. To this end, five azido-NSPs have been synthesized and are currently being screened for biological activity and photochemical stability. Three azido-NSPs elicit α-amylase production in barley half-seeds and stimulate tissue elongation in d 5 maize, lettuce, sunflower, and soybean. Further evaluations are in progress and these data as well as the utility of these compounds as photo-affinity ligands will be discussed

  8. Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

    Directory of Open Access Journals (Sweden)

    Tamiru N Alkie

    Full Text Available Natural or synthetic Toll-like receptor (TLR ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide polymer nanoparticles (PLGA NPs was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4 induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN-γ and IFN-β until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1β until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

  9. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  10. Biological significance of soluble IL-2 receptor

    Directory of Open Access Journals (Sweden)

    Calogero Caruso

    1993-01-01

    Full Text Available A NUMBER of receptors for growth factors and differentiation antigens have been found to be secreted or released by cells. Following mononuclear cell (MNC activation and interleukin-2 receptor (IL-2R expression, a soluble form of the Alpha;-chain of IL-2R (sIL-2R is released. The sIL-2R has been shown to be present in the culture supernatants of activated MNCs as well as in normal sera and, in higher amounts, in sera from subjects affected by several diseases including neoplastic, infectious and autoimmune ones, and in sera from transplanted patients suffering allograft rejection. The blood sIL-2R levels depend on the number of producing cells and the number of molecules per cell, so that sIL-2R blood values may represent an index of the number and the functional state of producing cells, both normal and neoplastic. Thus, monitoring of the immune system, mostly T-cells and haematological malignancies might be targets for the measurement of sIL-2R. Since many conditions may influence sIL-2R production, little diagnostic use may result from these measurements. However, since blood sIL-2R levels may correlate with disease progression and/or response to therapy, their measurement may be a useful index of activity and extent of disease. The precise biological role of the soluble form of the IL-2R is still a matter of debate. However, we know that increased sIL-2R levels may be observed in association with several immunological abnormalities and that sIL-2R is able to bind IL-2. It is conceivable then that in these conditions the excess sIL-2R released in vivo by activated lymphoid cells or by neoplastic cells may somehow regulate IL-2-dependent processes. On the other hand, it cannot exclude that sIL-2R is a by-product without biological significance. Finally, it is puzzling that in many conditions in which an increase of blood sIL-2R values has been observed, MNCs display a decreased in vitro capacity to produce sIL-2R. These seemingly contrasting

  11. Ligands specify estrogen receptor alpha nuclear localization and degradation

    Directory of Open Access Journals (Sweden)

    Caze-Subra Stéphanie

    2010-12-01

    Full Text Available Abstract Background The estrogen receptor alpha (ERα is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate. Results A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα. Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2, and pure antagonists (selective estrogen regulator disruptor; SERD, ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM, 4-hydroxytamoxifen (OHT or RU39,411, diffuse nuclear staining persisted. Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Conclusions Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on

  12. Ligand recognition by RAR and RXR receptors: binding and selectivity.

    Science.gov (United States)

    Sussman, Fredy; de Lera, Angel R

    2005-10-06

    Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

  13. Epibatidine-derivatives: ligands for the neuronal nicotinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Westera, G.; Patt, J.T.; Jankowski, K.; Bertrand, D.; Spang, J.; Schubiger, P.A.

    1997-01-01

    Epibatidine, isolated from the Ecuadorian frog Epipedobates tricolar, has been synthesized. 11 C-N-methyl derivate is investigated as useful nicotinergic receptor ligand by electrophysiological methods and in vivo mice experiments. (author) 2 figs., 7 refs

  14. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  15. Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

    Directory of Open Access Journals (Sweden)

    Xavier Charest-Morin

    Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

  16. Ligand Receptor-Mediated Regulation of Growth in Plants.

    Science.gov (United States)

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  17. Tissue distribution of the death ligand TRAIL and its receptors

    NARCIS (Netherlands)

    Spierings, DC; de Vries, EG; Vellenga, E; van den Heuvel, FA; Koornstra, JJ; Wesseling, J; Hollema, H; de Jong, S

    Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor

  18. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation

    NARCIS (Netherlands)

    Liu, Fahui; Teodorowicz, Gosia; Wichers, Harry J.; Boekel, van Tiny; Hettinga, Kasper A.

    2016-01-01

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE

  19. Dehydroepiandrosterone: an ancestral ligand of neurotrophin receptors.

    Science.gov (United States)

    Pediaditakis, Iosif; Iliopoulos, Ioannis; Theologidis, Ioannis; Delivanoglou, Nickoleta; Margioris, Andrew N; Charalampopoulos, Ioannis; Gravanis, Achille

    2015-01-01

    Dehydroepiandosterone (DHEA), the most abundant steroid in humans, affects multiple cellular functions of the endocrine, immune, and nervous systems. However, up to quite recently, no receptor has been described specifically for it, whereas most of its physiological actions have been attributed to its conversion to either androgens or estrogens. DHEA interacts and modulate a variety of membrane and intracellular neurotransmitter and steroid receptors. We have recently reported that DHEA protects neuronal cells against apoptosis, interacting with TrkA, the high-affinity prosurvival receptor of the neurotrophin, nerve growth factor. Intrigued by its pleiotropic effects in the nervous system of a variety of species, we have investigated the ability of DHEA to interact with the other two mammalian neurotrophin receptors, ie, the TrkB and TrkC, as well as their invertebrate counterparts (orthologs) in mollusks Lymnaea and Aplysia and in cephalochordate fish Amphioxus. Amazingly, DHEA binds to all Trk receptors, although with lower affinity by 2 orders of magnitude compared with that of the polypeptidic neurotrophins. DHEA effectively induced the first step of the TrkA and TrkC receptors activation (phosphorylation at tyrosine residues), including the vertebrate neurotrophin nonresponding invertebrate Lymnaea and Aplysia receptors. Based on our data, we hypothesize that early in evolution, DHEA may have acted as a nonspecific neurotrophic factor promoting neuronal survival. The interaction of DHEA with all types of neurotrophin receptors offers new insights into the largely unidentified mechanisms of its actions on multiple tissues and organs known to express neurotrophin receptors.

  20. Monocyte and plasma expression of TAM ligand and receptor in renal failure: Links to unregulated immunity and chronic inflammation.

    Science.gov (United States)

    Lee, Iris J; Hilliard, Brendan A; Ulas, Mehriban; Yu, Daohai; Vangala, Chandan; Rao, Swati; Lee, Jean; Gadegbeku, Crystal A; Cohen, Philip L

    2015-06-01

    Chronic inflammation is increased in patients with chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality. Specific immune mechanisms and pathways that drive and maintain chronic inflammation in CKD are not well described. The TAM ligands (Gas6 and protein S) and receptors (Axl and Mer) have been recently recognized as playing a prominent role in immune regulation. The receptors exist in both soluble and cell-bound forms; the soluble receptors (sAxl and sMer) are believed to compete with the bound receptors and thus inhibit their function. In this study, we determined the expression of cell-bound and soluble TAM proteins in patients with CKD. CKD patients had significantly lower expression of Mer in monocytes, yet increased expression of soluble TAM receptors sAxl and sMer in plasma compared to controls. The metalloproteinase ADAM 17, responsible for cleavage of Mer to its soluble form, was increased in patient monocytes. Elevated levels of soluble TAM receptors were more evident in patients with progressive renal failure. These observations suggest that functional deficiency of TAM receptor-mediated regulation of inflammation may contribute to chronic inflammation in patients with CKD. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    A. Beaudet

    1998-11-01

    Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

  2. Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

    DEFF Research Database (Denmark)

    Madsen, Mette; Møller, Holger J; Nielsen, Marianne Jensby

    2004-01-01

    CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163...... truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies...... to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant...

  3. Evolution of ligand specificity in vertebrate corticosteroid receptors

    Directory of Open Access Journals (Sweden)

    Deitcher David L

    2011-01-01

    Full Text Available Abstract Background Corticosteroid receptors include mineralocorticoid (MR and glucocorticoid (GR receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear. Results We used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC] to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (Mus musculus and the midshipman fish (Porichthys notatus, a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (Neolamprologus pulcher, another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences. Conclusion The mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.

  4. Receptor-ligand binding sites and virtual screening.

    Science.gov (United States)

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  5. DMPD: Endogenous ligands of Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15178705 Endogenous ligands of Toll-like receptors. Tsan MF, Gao B. J Leukoc Biol. ...2004 Sep;76(3):514-9. Epub 2004 Jun 3. (.png) (.svg) (.html) (.csml) Show Endogenous ligands of Toll-like re...ceptors. PubmedID 15178705 Title Endogenous ligands of Toll-like receptors. Authors Tsan MF, Gao B. Publicat

  6. Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

    Science.gov (United States)

    Eble, Johannes A

    2018-02-15

    The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

  7. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    Science.gov (United States)

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  8. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1

    NARCIS (Netherlands)

    Zwijsen, R.M.L.; Buckle, R.S.; Hijmans, E.M.; Loomans, C.J.M.; Bernards, R.A.

    1998-01-01

    The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast

  9. Non-conventional Frizzled ligands and Wnt receptors.

    Science.gov (United States)

    Hendrickx, Marijke; Leyns, Luc

    2008-05-01

    The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

  10. Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

    Directory of Open Access Journals (Sweden)

    Giulia Falivelli

    Full Text Available The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As or a transmembrane segment (ephrin-Bs, which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

  11. Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor.

    Science.gov (United States)

    Singer, M S; Shepherd, G M

    1994-06-02

    Olfactory receptors belong to the superfamily of seven transmembrane domain, G protein-coupled receptors. In order to begin analysis of mechanisms of receptor activation, a computer model of the OR5 olfactory receptor has been constructed and compared with other members of this superfamily. We have tested docking of the odor molecule lyral, which is known to activate the OR5 receptor. The results point to specific ligand-binding residues on helices III through VII that form a binding pocket in the receptor. Some of these residues occupy sequence positions identical to ligand-binding residues conserved among other superfamily members. The results provide new insights into possible molecular mechanisms of odor recognition and suggest hypotheses to guide future experimental studies using site-directed mutagenesis.

  12. 5D-QSAR for spirocyclic sigma1 receptor ligands by Quasar receptor surface modeling.

    Science.gov (United States)

    Oberdorf, Christoph; Schmidt, Thomas J; Wünsch, Bernhard

    2010-07-01

    Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptor surface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptor surface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  13. Steroid receptors and their ligands: Effects on male gamete functions

    International Nuclear Information System (INIS)

    Aquila, Saveria; De Amicis, Francesca

    2014-01-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  14. Steroid receptors and their ligands: Effects on male gamete functions

    Energy Technology Data Exchange (ETDEWEB)

    Aquila, Saveria; De Amicis, Francesca, E-mail: francesca.deamicis@unical.it

    2014-11-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  15. Levels of soluble delta-like ligand 1 in the serum and cerebrospinal fluid of tuberculous meningitis patients

    Institute of Scientific and Technical Information of China (English)

    Jinghong Li; Jinyi Li; Yanjie Jia

    2012-01-01

    In this study, the levels of soluble delta-like ligand 1 in cerebrospinal fluid and serum of 50 patients with tuberculous meningitis, 30 patients with viral meningitis, 20 patients with purulent meningitis and 40 subjects without central nervous system disease were determined using an enzyme-linked immunosorbent assay. The mean levels of soluble delta-like ligand 1 in both cerebrospinal fluid and serum from patients with tuberculous meningitis were significantly higher compared with those from patients with viral meningitis or purulent meningitis or from subjects without central nervous system disease. Meanwhile, the level of soluble delta-like ligand 1 gradually decreased as tuberculous meningitis patients recovered. If patients deteriorated after treatment, the level of soluble delta-like ligand 1 in cerebrospinal fluid gradually increased. There was no correlation between the level of soluble delta-like ligand 1 and the protein level/cell number in cerebrospinal fluid. Our findings in-dicate that the levels of soluble delta-like ligand 1 in cerebrospinal fluid and serum are reliable markers for the diagnosis of tuberculous meningitis and for monitoring treatment progress. At the same time, this index is not influenced by protein levels or cell numbers in cerebrospinal fluid.

  16. Soluble Receptor for Advanced Glycation End Product: A Biomarker for Acute Coronary Syndrome

    Directory of Open Access Journals (Sweden)

    Louise J. N. Jensen

    2015-01-01

    Full Text Available The receptor of advanced glycation end products (RAGE and its ligands are linked to the pathogenesis of coronary artery disease (CAD, and circulating soluble receptor of advanced glycation end products (sRAGE, reflecting the RAGE activity, is suggested as a potential biomarker. Elevated sRAGE levels are reported in relation to acute ischemia and this review focuses on the role of sRAGE as a biomarker for the acute coronary syndrome (ACS. The current studies demonstrated that sRAGE levels are elevated in relation to ACS, however during a very narrow time period, indicating that the time of sampling needs attention. Interestingly, activation of RAGE may influence the pathogenesis and reflection in sRAGE levels in acute and stable CAD differently.

  17. Transition Metal Complexes Coordinated by Water Soluble Phosphane Ligands: How Cyclodextrins Can Alter the Coordination Sphere?

    Directory of Open Access Journals (Sweden)

    Michel Ferreira

    2017-01-01

    Full Text Available The behaviour of platinum(II and palladium(0 complexes coordinated by various hydrosoluble monodentate phosphane ligands has been investigated by 31P{1H} NMR spectroscopy in the presence of randomly methylated β-cyclodextrin (RAME-β-CD. This molecular receptor can have no impact on the organometallic complexes, induce the formation of phosphane low-coordinated complexes or form coordination second sphere species. These three behaviours are under thermodynamic control and are governed not only by the affinity of RAME-β-CD for the phosphane but also by the phosphane stereoelectronic properties. When observed, the low-coordinated complexes may be formed either via a preliminary decoordination of the phosphane followed by a complexation of the free ligand by the CD or via the generation of organometallic species complexed by CD which then lead to expulsion of ligands to decrease their internal steric hindrance.

  18. Differential expression of VEGF ligands and receptors in prostate cancer.

    Science.gov (United States)

    Woollard, David J; Opeskin, Kenneth; Coso, Sanja; Wu, Di; Baldwin, Megan E; Williams, Elizabeth D

    2013-05-01

    Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer. Copyright © 2012 Wiley Periodicals, Inc.

  19. Efficient Route to Highly Water-Soluble Aromatic Cyclic Hydroxamic Acid Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Seitz, Michael; Raymond, Kenneth N.

    2008-02-06

    2-Hydroxyisoquinolin-1-one (1,2-HOIQO) is a new member of the important class of aromatic cyclic hydroxamic acid ligands which are widely used in metal sequestering applications and metal chelating therapy. The first general approach for the introduction of substituents at the aromatic ring of the chelating moiety is presented. As a useful derivative, the highly water-soluble sulfonic acid has been synthesized by an efficient route that allows general access to 1,2-HOQIO 3-carboxlic acid amides, which are the most relevant for applications.

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

    Science.gov (United States)

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

    2010-12-01

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

  1. Levels of the soluble LDL receptor-relative LR11 decrease in overweight individuals with type 2 diabetes upon diet-induced weight loss

    NARCIS (Netherlands)

    K.A.C. Berk (Kirsten); R. Vongpromek (Ranitha); M. Jiang (Meizi); W.J. Schneider (Wolfgang); R. Timman (Reinier); A.J.M. Verhoeven; H. Bujo (Hideaki); E.J.G. Sijbrands (Eric); M.T. Mulder (Monique)

    2016-01-01

    markdownabstract__Background and aims__ Cardiovascular disease (CVD) is a major complication in patients with type 2 diabetes (T2D), especially in those with obesity. Plasma soluble low density lipoprotein receptor-relative with 11 ligand-binding repeats (sLR11) plays a role in the development of

  2. Progress on the application of ligand receptor binding assays in radiopharmaceuticals

    International Nuclear Information System (INIS)

    Zhou Xue; Qian Jinping; Kong Aiying; Zhu Lin

    2010-01-01

    Receptor binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide the information of the relative binding affinity of ligand-receptor. The imaging of many radiopharmaceuticals is based on highly selective radioligand-receptor binding. The technique plays an important role in the design and screening of receptor-targeting radiopharmaceuticals. (authors)

  3. MIPs are ancestral ligands for the sex peptide receptor.

    Science.gov (United States)

    Kim, Young-Joon; Bartalska, Katarina; Audsley, Neil; Yamanaka, Naoki; Yapici, Nilay; Lee, Ju-Youn; Kim, Yong-Chul; Markovic, Milica; Isaac, Elwyn; Tanaka, Yoshiaki; Dickson, Barry J

    2010-04-06

    Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, postmating behaviors are triggered by sex peptide (SP), which is produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors via sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the CNS. Although required for postmating responses only in these female sensory neurons, SPR is expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. These observations suggest that SPR may have additional ligands and functions. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes, and Aplysia SPRs in vitro, yet are unable to trigger postmating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for postmating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors.

  4. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    Science.gov (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  5. Killer Cell Immunoglobulin-like Receptors and their Ligands

    Directory of Open Access Journals (Sweden)

    Tajik N.

    2010-09-01

    Full Text Available The Natural killer (NK cells are a subset of lymphocytes comprising around 10% of total lymphocytes in peripheral blood. Due to their role in the innate response, NK cells provide a ‘first line of defense’ against infectious agents and cancer and are also thought to play a role in autoimmunity. The killer cell immunoglobulin-like receptors (KIR are regulatory surface molecules, found on NK cells and on a subset of T lymphocytes. The genes for KIR are present on chromosome 19 in the leukocyte receptor complex and show a major difference for both the type and number of KIR genes present among different ethnic groups. They have been divided into two groups of 2D or 3D, depending on the number of external immunoglobulin domains. The presence of a long cytoplasmic tail with two immune tyrosine-based inhibitory motifs (ITIM allows the transduction of inhibitory signals and characterizes the inhibitory KIRs (2DL and 3DL, whereas the presence of short cytoplasmic tails corresponds to the activating KIR receptors (2DS and 3DS.These polymorphic receptors interact with specific motifs on human leukocyte antigen (HLA class I molecules, modulate NK cytolytic activity. Some KIRs are known to interact with HLA-C molecules of target cells, HLA-Bw4 molecules and HLA-A3/11. For some KIRs the corresponding ligands are still unknown.

  6. Involvement of direct inhibition of NMDA receptors in the effects of sigma-receptor ligands on glutamate neurotoxicity in vitro.

    Science.gov (United States)

    Nishikawa, H; Hashino, A; Kume, T; Katsuki, H; Kaneko, S; Akaike, A

    2000-09-15

    This study was performed to examine the roles of the N-methyl-D-aspartate (NMDA) receptor/phencyclidine (PCP) channel complex in the protective effects of sigma-receptor ligands against glutamate neurotoxicity in cultured cortical neurons derived from fetal rats. A 1-h exposure of cultures to glutamate caused a marked loss of viability, as determined by Trypan blue exclusion. This acute neurotoxicity of glutamate was prevented by NMDA receptor antagonists. Expression of sigma(1) receptor mRNA in cortical cultures was confirmed by reverse transcription polymerase chain reaction (RT-PCR). sigma Receptor ligands with affinity for NMDA receptor channels including the PCP site, such as (+)-N-allylnormetazocine ((+)-SKF10,047), haloperidol, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane ((-)-PPAP), prevented glutamate neurotoxicity in a concentration-dependent manner. In contrast, other sigma-receptor ligands without affinity for NMDA receptors, such as carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), did not show neuroprotective effects. Putative endogenous sigma receptor ligands such as pregnenolone, progesterone, and dehydroepiandrosterone did not affect glutamate neurotoxicity. The protective effects of (+)-SKF10,047, haloperidol, and (-)-PPAP were not affected by the sigma(1) receptor antagonist rimcazole. These results suggested that a direct interaction with NMDA receptors but not with sigma receptors plays a crucial role in the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.

  7. Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor

    International Nuclear Information System (INIS)

    Imai, Y.; Singer, M.S.; Fennie, C.; Lasky, L.A.; Rosen, S.D.

    1991-01-01

    Lymphocyte attachment to high endothelial venules within lymph nodes is mediated by the peripheral lymph node homing receptor (pnHR), originally defined on mouse lymphocytes by the MEL-14 mAb. The pnHR is a calcium-dependent lectin-like receptor, a member of the LEC-CAM family of adhesion proteins. Here, using a soluble recombinant form of the homing receptor, we have identified an endothelial ligand for the pnHR as an ∼ 50-kD sulfated, fucosylated, and sialylated glycoprotein, which we designate Sgp50 (sulfated glycoprotein of 50 kD). Recombinant receptor binding to this lymph node-specific glycoprotein requires calcium and is inhibitable by specific carbohydrates and by MEL-14 mAb. Sialylation of the component is required for binding. Additionally, the glycoprotein is precipitated by MECA-79, an adhesion-blocking mAb reactive with lymph node HEV. A related glycoprotein of ∼ 90 kD (designated as Sgp90) is also identified

  8. Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat

    NARCIS (Netherlands)

    Gispen, W.H.; Adan, R.A.H.; Szklarczyk, A.W.; Oosterom, J.; Brakkee, J.H.; Nijenhuis, W.A.; Schaaper, W.M.; Meloen, R.H.

    1999-01-01

    Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined

  9. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    NARCIS (Netherlands)

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  10. The future of type 1 cannabinoid receptor allosteric ligands.

    Science.gov (United States)

    Alaverdashvili, Mariam; Laprairie, Robert B

    2018-02-01

    Allosteric modulation of the type 1 cannabinoid receptor (CB1R) holds great therapeutic potential. This is because allosteric modulators do not possess intrinsic efficacy, but instead augment (positive allosteric modulation) or diminish (negative allosteric modulation) the receptor's response to endogenous ligand. Consequently, CB1R allosteric modulators have an effect ceiling which allows for the tempering of CB1R signaling without the desensitization, tolerance, dependence, and psychoactivity associated with orthosteric compounds. Pain, movement disorders, epilepsy, obesity are all potential therapeutic targets for CB1R allosteric modulation. Several challenges exist for the development of CB1R allosteric modulators, such as receptor subtype specificity, translation to in vivo systems, and mixed allosteric/agonist/inverse agonist activity. Despite these challenges, elucidation of crystal structures of CB1R and compound design based on structure-activity relationships will advance the field. In this review, we will cover recent progress for CB1R allosteric modulators and discuss the future promise of this research.

  11. Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

    Energy Technology Data Exchange (ETDEWEB)

    Li, E L.F.; Perdue, J F [Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada

    1980-10-01

    A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously /sup 125/I- and /sup 32/P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific (/sup 125/I)insulin and (/sup 125/I)thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 ..mu..g of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating /sup 125/I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

  12. Tetrasubstituted phenanthrolines as highly potent, water-soluble, and selective g-quadruplex ligands

    DEFF Research Database (Denmark)

    Larsen, Anders Foller; Nielsen, Mads Corvinius; Ulven, Trond

    2012-01-01

    Small molecules capable of stabilizing the G-quadruplex (G4) structure are of interest for the development of improved anticancer drugs. Novel 4,7-diamino-substituted 1,10-phenanthroline-2,9-dicarboxamides that represent hybrid structures of known phenanthroline-based ligands have been designed....... An efficient synthetic route to the compounds has been developed and their interactions with various G4 sequences have been evaluated by Förster resonance energy transfer (FRET) melting assays, fluorescent intercalator displacement (FID), electrospray ionization mass spectrometry (ESI-MS), and circular...... dichroism (CD) spectroscopy. The preferred compounds have high aqueous solubility and are strong and potent G4 binders with a high selectivity over duplex DNA; thus, they represent a significant improvement over the lead compounds. Two of the compounds are inhibitors of HeLa and HT1080 cell proliferation....

  13. Identification of VDR Antagonists among Nuclear Receptor Ligands Using Virtual Screening

    Directory of Open Access Journals (Sweden)

    Kelly Teske

    2014-04-01

    Full Text Available Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR antagonists among nuclear receptor (NR ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available “Binding Database.” Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2D3 and 25(OH2D3. The first virtual screen identified 32 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 μM. The second screen identified 162 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ERα/β ligands (26%, TRα/β ligands (7%, and LxRα/β ligands (7%. The binding between VDR and ERα ligand H6036 as well as TRα/β ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.

  14. Imaging of a glioma using peripheral benzodiazepine receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Starosta-Rubinstein, S.; Ciliax, B.J.; Penney, J.B.; McKeever, P.; Young, A.B.

    1987-02-01

    Two types of benzodiazepine receptors have been demonstrated in mammalian tissues, one which is localized on neuronal elements in brain and the other, on glial cells and in peripheral tissues such as kidney. In vivo administration of /sup 3/H-labeled PK 11195 (1-(2-chlorophenyl-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide) or (/sup 3/H)flunitrazepam with 5 mg of clonazepam per kg to rats with intracranial C6 gliomas resulted in high levels of tritiated-drug binding to the tumor as shown by quantitative autoradiography. Pharmacological studies indicated that the bound drugs labeled the peripheral benzodiazepine binding site. Binding to the peripheral benzodiazepine site was confirmed primarily to malignant cells with little binding to adjacent normal brain tissue or to necrotic tissue. Tumor cell binding was completely inhibited by preadministration of the peripheral benzodiazepine blocking agent PK 11195 at 5 mg/kg. The centrally selective benzodiazepine ligand clonazepam had no effect on PK 11195 binding to the tumor cells. When binding to other tumor cell lines grown in nude mice and nude athymic rats was evaluated, little or no peripheral benzodiazepine binding was detected on human pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high densities of peripheral benzodiazepine binding sites were observed on tumors derived from a human glioma cell line (ATCC HTB 14, U-87 MG). The presence of high concentrations of specific peripheral benzodiazepine receptors on glial tumors suggests that human primary central nervous system tumors could be imaged and diagnosed using peripheral benzodiazepine ligands labeled with positron- or gamma-emitting isotopes.

  15. Development of radioiodinated receptor ligands for cerebral single photon emission tomography

    International Nuclear Information System (INIS)

    Knapp, F.F. Jr.; McPherson, D.W.

    1992-01-01

    In the last decade the use of radiolabeled ligands for the imaging of cerebral receptors by emission computed tomography (ECT) has seen rapid growth. The opportunity to routinely perform cerebral single photon emission tomography (SPET) with iodine-123-labeled ligands depends on the availability of receptor ligands into which iodine can be introduced without decreasing the required high target receptor specificity. The use of iodine-123-labeled receptor-specific ligands also depends on the availability of high purity iodine-123 at reasonable costs and the necessary imaging instrumentation. In this paper, the development and current stage of evaluation of various iodine-123-labeled ligands for SPET imaging of dopaminergic, serotonergic and muscarinic acetylcholinergic receptor classes are discussed

  16. RAGE receptor and its soluble isoforms in diabetes mellitus complications

    Directory of Open Access Journals (Sweden)

    Mauren Isfer Anghebem Oliveira

    2013-04-01

    Full Text Available Chronic hyperglycemia, which is present in all types of diabetes, increases the formation of advanced glycation end-products (AGEs. The interaction of AGEs with receptor of advanced glycation end-products (RAGE initiates a cascade of pro-inflammatory and pro-coagulant processes that result in oxidative stress, stimulating the formation and accumulation of more AGE molecules. This cyclic process, denominated metabolic memory, may explain the persistency of diabetic vascular complications in patients with satisfactory glycemic control. The RAGE found in several cell membranes is also present in soluble isoforms (esRAGE and cRAGE, which are generated by alternative deoxyribonucleic acid splicing or by proteolytic cleavage. This review focuses on new research into these mediators as potential biomarkers for vascular complications in diabetes.

  17. Muscarinic acetylcholine receptors: location of the ligand binding site

    International Nuclear Information System (INIS)

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-01-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

  18. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    International Nuclear Information System (INIS)

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced 155 Eu: 3+ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor

  19. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    DEFF Research Database (Denmark)

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  20. Engineering of Olfactory Receptor OlfCc1 for Directed Ligand Sensitivity

    OpenAIRE

    Berke, Allison Paige

    2013-01-01

    Abstract Engineering of Olfactory Receptor OlfCc1 for Directed Ligand Sensitivityby Allison Paige Berke Joint Doctor of Philosophywith the University of California San FranciscoUniversity of California, Berkeley Professor Song Li, ChairDue to structural similarity, OlfCc1and its mammalian analogue V2R2 are hypothesized to respond to amino acid ligands in a calcium-mediated fashion. By analyzing receptor structure and making targeted mutations, the specificity and sensitivity of the receptor s...

  1. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

    Science.gov (United States)

    Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

    2016-01-01

    ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

  2. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    Science.gov (United States)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  3. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  4. Divergent Ah Receptor Ligand Selectivity during Hominin Evolution.

    Science.gov (United States)

    Hubbard, Troy D; Murray, Iain A; Bisson, William H; Sullivan, Alexis P; Sebastian, Aswathy; Perry, George H; Jablonski, Nina G; Perdew, Gary H

    2016-10-01

    We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150-1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  6. Supercritical Carbon Dioxide-Soluble Ligands for Extracting Actinide Metal Ions from Porous Solids

    International Nuclear Information System (INIS)

    Dietz, Mark L.

    2001-01-01

    Numerous types of actinide-bearing waste materials are found throughout the DOE complex. Most of these wastes consist of large volumes of non-hazardous materials contaminated with relatively small quantities of actinide elements. Separation of these wastes into their inert and radioactive components would dramatically reduce the costs of stabilization and disposal. For example, the DOE is responsible for decontaminating concrete within 7000 surplus contaminated buildings. The best technology now available for removing surface contamination from concrete involves removing the surface layer by grit blasting, which produces a large volume of blasting residue containing a small amount of radioactive material. Disposal of this residue is expensive because of its large volume and fine particulate nature. Considerable cost savings would result from separation of the radioactive constituents and stabilization of the concrete dust. Similarly, gas diffusion plants for uranium enrichment contain valuable high-purity nickel in the form of diffusion barriers. Decontamination is complicated by the extremely fine pores in these barriers, which are not readily accessible by most cleaning techniques. A cost-effective method for the removal of radioactive contaminants would release this valuable material for salvage. The objective of this project is to develop novel, substituted diphosphonic acid ligands that can be used for supercritical carbon dioxide extraction of actinide ions from solid wastes. Specifically, selected diphosphonic acids, which are known to form extremely stable complexes with actinides in aqueous and organic solution, are to be rendered carbon dioxide-soluble by the introduction of appropriate alkyl- or silicon-containing substituents. The metal complexation chemistry of these new ligands in SC-CO2 will then be investigated and techniques for their use in actinide extraction from porous solids developed

  7. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

    Andersen, P S; Menné, C; Mariuzza, R A

    2001-01-01

    determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated...

  8. Total soluble and endogenous secretory receptor for advanced glycation endproducts (RAGE) in IBD.

    Science.gov (United States)

    Meijer, Berrie; Hoskin, Teagan; Ashcroft, Anna; Burgess, Laura; Keenan, Jacqueline I; Falvey, James; Gearry, Richard B; Day, Andrew S

    2014-06-01

    Recruitment and activation of neutrophils, with release of specific proteins such as S100 proteins, is a feature of inflammatory bowel disease (IBD). Soluble forms of the receptor for advanced glycation endproducts (sRAGE), and variants such as endogenous secretory (esRAGE), can act as decoy receptors by binding ligands, including S100A12. The aims of this study were to determine total sRAGE and esRAGE concentrations in patients with IBD and correlate these with C-reactive protein (CRP), endoscopic scores and clinical disease activity scores. EDTA-plasma was collected from patients undergoing colonoscopy including those with Crohn's disease (CD: n=125), ulcerative colitis (UC: n=79) and control patients without endoscopic signs of inflammation (non-IBD: n=156). Concentrations of sRAGE and esRAGE were determined by enzyme-linked immunosorbent assay and plasma CRP concentrations measured. Standard clinical disease activity and endoscopic severity scores were defined for all subjects. Plasma sRAGE concentrations were lower in UC (but not CD) than non-IBD subjects (pdefine the significance of sRAGE and esRAGE in IBD. Copyright © 2013 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

  9. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  10. Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB.

    Science.gov (United States)

    Lättig, Jens; Oksche, Alexander; Beyermann, Michael; Rosenthal, Walter; Krause, Gerd

    2009-07-01

    The molecular basis for recognition of peptide ligands endothelin-1, -2 and -3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET(A) or ET(B) is not clearly resolved. We derived sequence-structure-function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10-fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET(A) is restrictive for a selected group of peptide ligands' N-termini, whereas a broad funnel-shaped entrance in ET(B) accepts a variety of different shapes and properties of ligands.

  11. Monitoring ligand-receptor interactions by photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jeney, Sylvia [M E Mueller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056 (Switzerland); Mor, Flavio; Forro, Laszlo [Laboratory of Complex Matter Physics (LPMC), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland); Koszali, Roland [Institute for Information and Communication Technologies (IICT), University of Applied Sciences of Western Switzerland (HEIG-VD), Rue Galilee 15, CH 1401 Yverdon-les-bains (Switzerland); Moy, Vincent T, E-mail: sylvia.jeney@unibas.ch, E-mail: vmoy@miami.edu [Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136 (United States)

    2010-06-25

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  12. Mutual inactivation of Notch receptors and ligands facilitates developmental patterning.

    Directory of Open Access Journals (Sweden)

    David Sprinzak

    2011-06-01

    Full Text Available Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

  13. Monitoring ligand-receptor interactions by photonic force microscopy

    International Nuclear Information System (INIS)

    Jeney, Sylvia; Mor, Flavio; Forro, Laszlo; Koszali, Roland; Moy, Vincent T

    2010-01-01

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  14. Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

    OpenAIRE

    Levoye, Angélique; Dam, Julie; Ayoub, Mohammed A; Guillaume, Jean-Luc; Jockers, Ralf

    2006-01-01

    G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observ...

  15. Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

    Science.gov (United States)

    Do, Bich Hang; Nguyen, Minh Tan; Song, Jung-A; Park, Sangsu; Yoo, Jiwon; Jang, Jaepyeong; Lee, Sunju; So, Seoungjun; Yoon, Yejin; Kim, Inki; Lee, Kyungjin; Jang, Yeon Jin; Choe, Han

    2017-12-28

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli . In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

  16. Reduction reactions of water soluble cyano-cobalt(III)-porphyrins: Metal versus ligand centered processes

    International Nuclear Information System (INIS)

    Mosseri, S.; Neta, P.; Harriman, A.; Hambright, P.

    1990-01-01

    Reduction reactions of dicyano-cobalt(III)-porphyrins [potential in vivo cyanide scavenger drugs] were studied by radiolytic and electrochemical methods using the water soluble tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP). For [(CN)2CoIIITPPS]-, reduction occurs stepwise to the CoII, CoI, and finally to the phlorin anion. This behavior is similar to that of the cobalt porphyrins in the absence of cyanide, except that the cyanide ligand shifts the reduction potentials to much more negative values. On the other hand, under radiolytic conditions, [(CN)2CoIIITMPyP]- is reduced on the porphyrin macrocycle by one electron to give the CoIII pi-radical anion, which disproportionates into the initial complex and the two-electron ring reduced CoIII phlorin. The radical anion is also formed by intramolecular electron transfer subsequent to the reaction of CoIITMPyP and cyanide. The results are compared with the chemistry of Vitamin B-12

  17. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    Science.gov (United States)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  18. Involvement of the Soluble Urokinase Receptor in Chondrosarcoma Cell Mobilization

    Directory of Open Access Journals (Sweden)

    Katia Bifulco

    2011-01-01

    Full Text Available High levels of urokinase receptor (uPAR in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR88-92 sequence, since the DII88-183 or DIIDIIR88-284 uPAR domains retain motogen effect whereas DI1-87 or DIII184-284 domains, both lacking the uPAR88-92 sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR88-92 signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.

  19. Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

    Science.gov (United States)

    Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

    2001-08-03

    Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

  20. Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

    Science.gov (United States)

    Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points.

  1. Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2009-12-01

    Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

  2. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    LENUS (Irish Health Repository)

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  3. Is the isolated ligand binding domain a good model of the domain in the native receptor?

    Science.gov (United States)

    Deming, Dustin; Cheng, Qing; Jayaraman, Vasanthi

    2003-05-16

    Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.

  4. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    Science.gov (United States)

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  5. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  6. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  7. Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Efange, S M.N.; Khare, A B; Langason, R B

    1995-05-01

    Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-indene-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4'- piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)]-3,4-dihydrospiro[naphthalene- 1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)-5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex.

  8. Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

    International Nuclear Information System (INIS)

    Efange, S.M.N.; Khare, A.B.; Langason, R.B.

    1995-01-01

    Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-indene-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4'- piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4-dihydrospiro[naphthalene- 1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)-5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex

  9. A macrocyclic ligand as receptor and Zn(II)-complex receptor for anions in water: binding properties and crystal structures.

    Science.gov (United States)

    Ambrosi, Gianluca; Formica, Mauro; Fusi, Vieri; Giorgi, Luca; Macedi, Eleonora; Micheloni, Mauro; Paoli, Paola; Pontellini, Roberto; Rossi, Patrizia

    2011-02-01

    Binding properties of 24,29-dimethyl-6,7,15,16-tetraoxotetracyclo[19.5.5.0(5,8).0(14,17)]-1,4,9,13,18,21,24,29-octaazaenatriaconta-Δ(5,8),Δ(14,17)-diene ligand L towards Zn(II) and anions, such as the halide series and inorganic oxoanions (phosphate (Pi), sulfate, pyrophosphate (PPi), and others), were investigated in aqueous solution; in addition, the Zn(II)/L system was tested as a metal-ion-based receptor for the halide series. Ligand L is a cryptand receptor incorporating two squaramide functions in an over-structured chain that connects two opposite nitrogen atoms of the Me(2)[12]aneN(4) polyaza macrocyclic base. It binds Zn(II) to form mononuclear species in which the metal ion, coordinated by the Me(2)[12]aneN(4) moiety, lodges inside the three-dimensional cavity. Zn(II)-containing species are able to bind chloride and fluoride at the physiologically important pH value of 7.4; the anion is coordinated to the metal center but the squaramide units play the key role in stabilizing the anion through a hydrogen-bonding network; two crystal structures reported here clearly show this aspect. Free L is able to bind fluoride, chloride, bromide, sulfate, Pi, and PPi in aqueous solution. The halides are bound at acidic pH, whereas the oxoanions are bound in a wide range of pH values ranging from acidic to basic. The cryptand cavity, abundant in hydrogen-bonding sites at all pH values, allows excellent selectivity towards Pi to be achieved mainly at physiological pH 7.4. By joining amine and squaramide moieties and using this preorganized topology, it was possible, with preservation of the solubility of the receptor, to achieve a very wide pH range in which oxoanions can be bound. The good selectivity towards Pi allows its discrimination in a manner not easily obtainable with nonmetallic systems in aqueous environment. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  11. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    Science.gov (United States)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  12. Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy.

    Science.gov (United States)

    Ochodnicky, Peter; Lattenist, Lionel; Ahdi, Mohamed; Kers, Jesper; Uil, Melissa; Claessen, Nike; Leemans, Jaklien C; Florquin, Sandrine; Meijers, Joost C M; Gerdes, Victor E A; Roelofs, Joris J T H

    2017-09-01

    TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion 300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n = 9) and controls (n = 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. Ligand Activation of TAM Family Receptors-Implications for Tumor Biology and Therapeutic Response.

    Science.gov (United States)

    Davra, Viralkumar; Kimani, Stanley G; Calianese, David; Birge, Raymond B

    2016-11-29

    The TAM family of receptors (i.e., Tyro3, Axl, and Mertk), and their ligands Growth arrest specific factor 6 (Gas6) and Protein S (Pros1) contribute to several oncogenic processes, such as cell survival, invasion, migration, chemo-resistance, and metastasis, whereby expression often correlates with poor clinical outcomes. In recent years, there has been great interest in the study of TAM receptors in cancer, stemming both from their roles as oncogenic signaling receptors, as well as their roles in tumor immunology. As a result, several classes of TAM inhibitors that include small molecule tyrosine kinase inhibitors, monoclonal antibodies, decoy receptors, as well as novel strategies to target TAM ligands are being developed. This paper will review the biology of TAM receptors and their ligands with a focus on cancer, as well as evidence-based data for the continued pursuit of TAM/Gas6 inhibitors in clinical practice.

  14. [Serum leptin levels and soluble leptin receptors in female patients with anorexia nervosa].

    Science.gov (United States)

    Jiskra, J; Haluzík, M; Svobodová, J; Haluzíková, D; Nedvídková, J; Parízková, J; Kotrlíková, E

    2000-10-25

    Leptin action in peripheral tissues is enabled by an interaction with specific transmembrane receptors. Several of leptin receptor isoforms were identified, including soluble leptin receptor isoform structurally identical to extracellular domain of the the long leptin receptor isoform. The soluble receptor isoform is released to the circulation and acts probably as leptin-binding factor. The aim of our study was to measure serum concentrations of the soluble leptin receptor in patients with anorexia nervosa and in the control group of healthy women. Relationships of soluble leptin receptor levels to body mass index (BMI), body fat content, serum leptin, TNF-alpha and insulin levels were also studied. 16 patients with anorexia nervosa and 16 age-matched lean healthy women were included into the study. All of the subjects were measured and weighed, the body fat content was estimated from the skinfold thickness measurement. The blood for the determination of leptin, soluble leptin receptor and other hormonal parameters was obtained from all subjects after the overnight fasting. BMI, body fat content, serum leptin and insulin levels in patients with anorexia nervosa were significantly lower than in the control group (BMI: 14.98 +/- 2.32 vs. 22.21 +/- 2.48, p anorexia nervosa were significantly higher compared the to control group (24.67 +/- 8.3 U.ml-1 vs. 15.71 +/- 2.79 U.ml-1, p anorexia nervosa were significantly higher in comparison with the healthy subjects. Except of the negative correlation between serum soluble leptin receptor levels and BMI no statistically significant relationships between serum soluble leptin receptor and the rest of parameters studied were found.

  15. Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis.

    Science.gov (United States)

    García-Vallejo, Juan J; van Kooyk, Yvette

    2009-07-01

    C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.

  16. Differential effects of EGFR ligands on endocytic sorting of the receptor

    DEFF Research Database (Denmark)

    Roepstorff, Kirstine; Grandal, Michael Vibo; Henriksen, Lasse

    2009-01-01

    signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic...... trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor...

  17. A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

    Science.gov (United States)

    Lenkei, Z; Beaudet, A; Chartrel, N; De Mota, N; Irinopoulou, T; Braun, B; Vaudry, H; Llorens-Cortes, C

    2000-11-01

    Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

  18. Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

    International Nuclear Information System (INIS)

    Dietz, Marina S; Haße, Daniel; Ferraris, Davide M; Göhler, Antonia; Niemann, Hartmut H; Heilemann, Mike

    2013-01-01

    The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

  19. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  20. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    DEFF Research Database (Denmark)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  1. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Shengchen; Han, Ying; Shi, Yuzhe; Rong, Hui; Zheng, Songyang; Jin, Shikan; Lin, Shu-Yong; Lin, Sheng-Cai; Li, Yong (Pitt); (Xiamen)

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

  2. Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

    Science.gov (United States)

    Dolenc, Jožica; Riniker, Sereina; Gaspari, Roberto; Daura, Xavier; van Gunsteren, Wilfred F

    2011-08-01

    Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

  3. Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands

    NARCIS (Netherlands)

    Weterings, Josephus Johannes

    2008-01-01

    Toll-like receptors (TLRs) are receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. TLRs can be found at cells that play a role in the immune system. Binding of the TLR with its corresponding ligand

  4. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

  5. PET and Hormone Receptor Ligands in Breast Cancer

    National Research Council Canada - National Science Library

    Gemignani, Mary

    2006-01-01

    .... To investigate this further, this project's objectives are: To evaluate the use of estrogen-like ligands labeled with positron emitters in preoperatively determining the ER status of breast cancer using PET...

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

    Science.gov (United States)

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

    2017-11-22

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

  7. Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

    Science.gov (United States)

    Vandenbulcke, F; Nouel, D; Vincent, J P; Mazella, J; Beaudet, A

    2000-09-01

    The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

  8. Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

    Science.gov (United States)

    Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

    2009-01-01

    Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

  9. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    OpenAIRE

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its na...

  10. NNAlign: a platform to construct and evaluate artificial neural network models of receptor-ligand interactions

    DEFF Research Database (Denmark)

    Nielsen, Morten; Andreatta, Massimo

    2017-01-01

    Peptides are extensively used to characterize functional or (linear) structural aspects of receptor-ligand interactions in biological systems, e.g. SH2, SH3, PDZ peptide-recognition domains, the MHC membrane receptors and enzymes such as kinases and phosphatases. NNAlign is a method for the ident...... with insertions and deletions, encoding of receptor pseudo-sequences, and custom alphabets for the training sequences. The server is available at http://www.cbs.dtu.dk/services/NNAlign-2.0....

  11. Toxicity of copper to the marine amphipod Allorchestes compressa in the presence of water- and lipid-soluble ligands

    Energy Technology Data Exchange (ETDEWEB)

    Ahsanullah, M; Florence, T M

    1984-12-01

    Acute toxicity tests were carried out on the marine amphipod Allorchestes compressa Dana using copper sulfate and some organocopper complexes. For copper sulfate, 96 h LC50 values of 0.11 and 0.5 mg Cu/liter were determined for the juveniles and the adults, respectively. Juveniles were about 4.5 times more sensitive to copper than adults Organocopper complexes were tested on adults only. The three water soluble ligands nitrotriacetic acid, 8-hydroxyquinoline-5-sulfoic acid and tannic acid ameliorated copper toxicity by decreasing the concentration of free ionic copper while lipid-soluble ligands such as oxine and potassium ethylxanthogenate increased copper toxicity, presumably as a result of the complexes diffusing through the cell membrane and participating in injurious reaction. The copper complex with 2,9-dimethyl-1,10-phenanthroline was the most toxic complex tested. It is suggested that the presence of these ligands in the receiving water should be taken into consideration when establishing water quality criteria. 19 references, 2 figures, 3 tables.

  12. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Science.gov (United States)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  13. ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers

    International Nuclear Information System (INIS)

    Wellstein, Anton

    2012-01-01

    The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

  14. Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

    Science.gov (United States)

    Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

    1998-04-10

    The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

  15. Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

    Science.gov (United States)

    Khashan, Raed S

    2015-01-01

    As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

  16. Mix-and-match: ligand-receptor pairs in stomatal development and beyond.

    Science.gov (United States)

    Torii, Keiko U

    2012-12-01

    Stomata are small valves on the plant epidermis balancing gas exchange and water loss. Stomata are formed according to positional cues. In Arabidopsis, two EPIDERMAL PATTERNING FACTOR (EPF) peptides, EPF1 and EPF2, are secreted from stomatal precursors enforcing proper stomatal patterning. Here, I review recent studies revealing the ligand-receptor pairs and revising the previously predicted relations between receptors specifying stomatal patterning: ERECTA-family and TOO MANY MOUTHS (TMM). Furthermore, EPF-LIKE9 (EPFL9/Stomagen) promotes stomatal differentiation from internal tissues. Two EPFL peptides specify inflorescence architecture, a process beyond stomatal development, as ligands for ERECTA. Thus, broadly expressed receptor kinases may regulate multiple developmental processes through perceiving different peptide ligands, each with a specialized expression pattern. TMM in the epidermis may fine-tune multiple EPF/EPFL signals to prevent signal interference. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Importance of the pharmacological profile of the bound ligand in enrichment on nuclear receptors: toward the use of experimentally validated decoy ligands.

    Science.gov (United States)

    Lagarde, Nathalie; Zagury, Jean-François; Montes, Matthieu

    2014-10-27

    The evaluation of virtual ligand screening methods is of major importance to ensure their reliability. Taking into account the agonist/antagonist pharmacological profile should improve the quality of the benchmarking data sets since ligand binding can induce conformational changes in the nuclear receptor structure and such changes may vary according to the agonist/antagonist ligand profile. We indeed found that splitting the agonist and antagonist ligands into two separate data sets for a given nuclear receptor target significantly enhances the quality of the evaluation. The pharmacological profile of the ligand bound in the binding site of the target structure was also found to be an additional critical parameter. We also illustrate that active compound data sets for a given pharmacological activity can be used as a set of experimentally validated decoy ligands for another pharmacological activity to ensure a reliable and challenging evaluation of virtual screening methods.

  18. Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

    Science.gov (United States)

    Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J

    2017-08-15

    Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

  19. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    DEFF Research Database (Denmark)

    Nguyen, E.D.; Meiler, J.; Norn, C.

    2013-01-01

    screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone...... extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves...

  20. Photoaffinity ligand for dopamine D2 receptors: azidoclebopride

    International Nuclear Information System (INIS)

    Niznik, H.B.; Guan, J.H.; Neumeyer, J.L.; Seeman, P.

    1985-01-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [ 3 H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol

  1. Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Szymańska, Ewa; Chałupnik, Paulina; Johansen, Tommy Nørskov

    2017-01-01

    in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of 4.6 µM for native AMPA receptors and almost 5-fold lower affinity...

  2. Genomic organization, annotation, and ligand-receptor inferences of chicken chemokines and chemokine receptor genes based on comparative genomics

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2005-03-01

    Full Text Available Abstract Background Chemokines and their receptors play important roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this report, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to systematically identify chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Results Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. All of the chicken chemokines contained a conserved CC, CXC, CX3C, or XC motif, whereas all the chemokine receptors had seven conserved transmembrane helices, four extracellular domains with a conserved cysteine, and a conserved DRYLAIV sequence in the second intracellular domain. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. Conclusion The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. All identified chicken chemokines and their cognate receptors were identified in the chicken genome except CCR9, whose ligand was not identified in this study. The organization of these genes suggests that there were a substantial number of these genes present before divergence between aves and mammals and more gene duplications of CC, CXC, CCR, and CXCR subfamilies in mammals than in aves after the divergence.

  3. Characterization of the structure of the erythropoietin receptor by ligand blotting

    International Nuclear Information System (INIS)

    Atkins, H.L.; Broudy, V.C.; Papayannopoulou, T.

    1991-01-01

    Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd ± 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding

  4. Breast and other cancer dormancy as a therapeutic endpoint: speculative recombinant T cell receptor ligand (RTL) adjuvant therapy worth considering?

    International Nuclear Information System (INIS)

    Bakács, Tibor; Mehrishi, Jitendra N

    2010-01-01

    Most individuals who died of trauma were found to harbour microscopic primary cancers at autopsies. Surgical excision of the primary tumour, unfortunately, seems to disturb tumour dormancy in over half of all metastatic relapses. A recently developed immune model suggested that the evolutionary pressure driving the creation of a T cell receptor repertoire was primarily the homeostatic surveillance of the genome. The model is based on the homeostatic role of T cells, suggesting that molecular complementarity between the positively selected T cell receptors and the self peptide-presenting major histocompatibility complex molecules establishes and regulates homeostasis, strictly limiting variations of its components. The repertoire is maintained by continuous peripheral stimulation via soluble forms of self-peptide-presenting major histocompatibility complex molecules governed by the law of mass action. The model states that foreign peptides inhibit the complementary interactions between the major histocompatibility complexes and T cell receptors. Since the vast majority of clinically detected cancers present self-peptides the model assumes that tumour cells are, paradoxically, under homeostatic T cell control. The novelty of our hypothesis therefore is that resection of the primary tumour mass is perceived as loss of 'normal' tissue cells. Consequently, T cells striving to reconstitute homeostasis stimulate rather than inhibit the growth of dormant tumour cells and avascular micrometastases. Here we suggest that such kick-start growths could be prevented by a recombinant T cell receptor ligand therapy that modifies T cell behaviour through a partial activation mechanism. The homeostatic T cell regulation of tumours can be tested in a tri-transgenic mice model engineered to express potent oncogenes in a doxycycline-dependent manner. We suggest seeding dissociated, untransformed mammary cells from doxycycline naïve mice into the lungs of two mice groups: one

  5. Nanoparticle-based receptors mimic protein-ligand recognition

    OpenAIRE

    Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; Biasi, Federico De; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

    2017-01-01

    Summary The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the...

  6. Heart Failure Therapeutics on the Basis of a Biased Ligand of the Angiotensin-2 Type 1 Receptor Rationale and Design of the BLAST-AHF Study (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure)

    NARCIS (Netherlands)

    Felker, G. Michael; Butler, Javed; Collins, Sean P.; Cotter, Gad; Davison, Beth A.; Ezekowitz, Justin A.; Filippatos, Gerasimos; Levy, Phillip D.; Metra, Marco; Ponikowski, Piotr; Soergel, David G.; Teerlink, John R.; Violin, Jonathan D.; Voors, Adriaan A.; Pang, Peter S.

    The BLAST-AHF (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure) study is designed to test the efficacy and safety of TRV027, a novel biased ligand of the angiotensin-2 type 1 receptor, in patients with acute heart failure (AHF). AHF remains a major public health problem, and

  7. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  8. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  9. GluVII:06--a highly conserved and selective anchor point for non-peptide ligands in chemokine receptors

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Schwartz, Thue W

    2006-01-01

    ligands and that the two peripheral chemical moieties of the ligands from this central point in the receptor structure explore each of the two halves of the main ligand binding pocket. It is envisioned that knowledge of this binding mode can be exploited in structure-based discovery and design of novel...

  10. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    DEFF Research Database (Denmark)

    Madsen, U; Sløk, F A; Stensbøl, T B

    2000-01-01

    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...

  11. Soluble tumor necrosis factor receptor-1 in preterm infants with chronic lung disease.

    Science.gov (United States)

    Sato, Miho; Mori, Masaaki; Nishimaki, Shigeru; An, Hiromi; Naruto, Takuya; Sugai, Toshiyuki; Shima, Yoshio; Seki, Kazuo; Yokota, Shumpei

    2010-04-01

    It is clear that inflammation plays an important role in developing chronic lung disease in preterm infants. The purpose of the present study is to investigate changes of serum soluble tumor necrosis factor receptor-1 levels over time in infants with chronic lung disease. The serum levels of soluble tumor necrosis factor receptor-1 were measured after delivery, and at 7, 14, 21 and 28 days of age in 10 infants with chronic lung disease and in 18 infants without chronic lung disease. The serum level of soluble tumor necrosis factor receptor-1 was significantly higher in infants with chronic lung disease than in infants without chronic lung disease after delivery. The differences between these two groups remained up to 28 days of age. Prenatal inflammation with persistence into postnatal inflammation may be involved in the onset of chronic lung disease.

  12. Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

    Science.gov (United States)

    Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

    2017-03-15

    Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Adrian E. Roitberg

    2008-06-01

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

  14. Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

    Science.gov (United States)

    Lew, Erin D; Oh, Jennifer; Burrola, Patrick G; Lax, Irit; Zagórska, Anna; Través, Paqui G; Schlessinger, Joseph; Lemke, Greg

    2014-09-29

    The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

  15. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Nahoum, Virginie; Lipski, Alexandra; Quillard, Fabien; Guichou, Jean-François [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France); Boublik, Yvan [CNRS, UMR5237, Centre de Recherche de Biochimie Macromoléculaire (CRBM), 34293 Montpellier (France); Pérez, Efrèn [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Germain, Pierre [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), BP 10142, 67404 Illkirch CEDEX (France); Lera, Angel R. de [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Bourguet, William, E-mail: bourguet@cbs.cnrs.fr [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France)

    2008-07-01

    Attempts have been made to crystallize the ligand-binding domain of the human retinoid X receptor in complex with a variety of newly synthesized ligands. An inverse correlation was observed between the ‘crystallizability’ and the structural dynamics of the various receptor–ligand complexes. Crystallization trials of the human retinoid X receptor α ligand-binding domain (RXRα LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRα LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRα LBD–ligand–CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

  16. LiCABEDS II. Modeling of ligand selectivity for G-protein-coupled cannabinoid receptors.

    Science.gov (United States)

    Ma, Chao; Wang, Lirong; Yang, Peng; Myint, Kyaw Z; Xie, Xiang-Qun

    2013-01-28

    The cannabinoid receptor subtype 2 (CB2) is a promising therapeutic target for blood cancer, pain relief, osteoporosis, and immune system disease. The recent withdrawal of Rimonabant, which targets another closely related cannabinoid receptor (CB1), accentuates the importance of selectivity for the development of CB2 ligands in order to minimize their effects on the CB1 receptor. In our previous study, LiCABEDS (Ligand Classifier of Adaptively Boosting Ensemble Decision Stumps) was reported as a generic ligand classification algorithm for the prediction of categorical molecular properties. Here, we report extension of the application of LiCABEDS to the modeling of cannabinoid ligand selectivity with molecular fingerprints as descriptors. The performance of LiCABEDS was systematically compared with another popular classification algorithm, support vector machine (SVM), according to prediction precision and recall rate. In addition, the examination of LiCABEDS models revealed the difference in structure diversity of CB1 and CB2 selective ligands. The structure determination from data mining could be useful for the design of novel cannabinoid lead compounds. More importantly, the potential of LiCABEDS was demonstrated through successful identification of newly synthesized CB2 selective compounds.

  17. Interaction of cadmium with atrial natriuretic factor receptors: Ligand binding and cellular processing

    International Nuclear Information System (INIS)

    Giridhar, J.; Rathinavelu, A.; Isom, G.E.

    1990-01-01

    ANF is a peptide hormone secreted by the heart and produces potent diuresis and vascular smooth muscle relaxation. It is well known that Cd produces cardiovascular toxicity and is implicated in the pathogenesis of hypertension. Hence the effects of Cd on ANF receptor dynamics and ligand binding were studied in PC12 cells. Receptor internalization using 125 I-ANF as the ligand at 37 degree C displayed a decrease in endocytic rate constants (ERC) when either preincubated with Cd (500 μM for 30 min, ERC = 0.183/min) or coincubated with Cd (500 μM, ERC = 0.196) when compared to control value (ERC = 0.259/min). Ligand binding ( 125 I-ANF) was changed by Cd as reflected by a decrease in the number of binding sites/cell in both Cd preincubated (Kd = 3.81 x 10 -10 M, B max = 1 x 10 -10 M, binding sites/cell = 9333) and coincubated cells (Kd = 1.76 x 10 -10 M, B max = 3.92 x 10 -11 M, binding sites/cell = 5960) from control (Kd = 3.87 x 10 -10 M, B max = 9.58 x 10 -11 M, binding sites/cell = 12141). Photoaffinity labelling with 125 I-ANF as the ligand was used to measure receptor subtype binding. Coincubation of cells with Cd (500 μM) and ligand decreased both high and low mol. wt. receptor binding, whereas preincubation with Cd (500μM) for 60 min produced a slight decrease in binding of both receptor subtypes. These results indicate that the cardiovascular toxicity of Cd may be partially mediated by altered ANF receptor function

  18. Histamine H3 receptor ligands in the group of (homo)piperazine derivatives.

    Science.gov (United States)

    Szczepanska, Katarzyna; Kuder, Kamil; Kiec-Kononowicz, Katarzyna

    2017-11-23

    Since its' discovery in 1983, followed by gene cloning in 1999, the histamine H3 receptor served as an outstanding target for drug discovery. The wide spectrum of possible therapeutic implications make H3R's one of the most researched areas in the vast GPCR ligands field - started from imidazole containing ligands, through various successful imidazole replacements, with recent introduction of Wakix® to pharmaceutical market. One of such replacements is piperazine moiety, a significant versatile scaffold in rational drug design for most of the GPCR ligands. Therefore, herein we review ligands built on piperazine, as well as its seven membered analogue azepine, that target H3R's and their potential therapeutical applications, in order to elucidate the current state of the art in this vast field. Due to a high level of structural divergence among compounds described herein, we decided to divide them into groups, where the key division element was the position of nitrogen basicity decreasing moieties in (homo)piperazine ring. Paying attention to a number of published structures and their overall high biological activity, one can realize that the (homo)piperazine scaffold bids a versatile template also for histamine H3 receptor ligands. With two possible substitution sites and therefore a number of possible structural combinations, piperazine derivatives stand as one of the largest group of high importance among H3R ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R

    2002-01-01

    Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology...... with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent...

  20. Imaging GABAc Receptors with Ligand-Conjugated Quantum Dots

    Directory of Open Access Journals (Sweden)

    Ian D. Tomlinson

    2007-01-01

    Full Text Available We report a methodology for labeling the GABAc receptor on the surface membrane of intact cells. This work builds upon our earlier work with serotonin-conjugated quantum dots and our studies with PEGylated quantum dots to reduce nonspecific binding. In the current approach, a PEGylated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide. These conjugates were used to image GABAC receptors heterologously expressed in Xenopus laevis oocytes.

  1. Major advances in the development of histamine H4 receptor ligands.

    Science.gov (United States)

    Smits, Rogier A; Leurs, Rob; de Esch, Iwan J P

    2009-08-01

    The search for new and potent histamine H4 receptor ligands is leading to a steadily increasing number of scientific publications and patent applications. Several interesting and structurally diverse compounds have been found, but fierce IP competition for a preferred 2-aminopyrimidine scaffold is becoming apparent. Recent investigations into the role of the histamine H(4)R in (patho)physiology and the use of H4R ligands in in vivo disease models reveal enormous potential in the field of inflammation and allergy, among others. The development of ligands that display activity at two or more histamine receptor (HR) subtypes is another clinical opportunity that is currently being explored. Taken together, the histamine H4R field is gearing up for clinical studies and has the potential to deliver another generation of blockbuster drugs.

  2. Exogenous or endogenous Toll-like receptor ligands: which is the MVP in tumorigenesis?

    Science.gov (United States)

    Yu, Li; Wang, Liantang; Chen, Shangwu

    2012-03-01

    Toll-like receptors (TLRs) are a class of pattern recognition receptors sensing microbial components and triggering an immune response against pathogens. In addition to their role in anti-infection immunity, increasing evidence indicates that engagement of TLRs can promote cancer cell survival and proliferation, induce tumor immune evasion, and enhance tumor metastasis and chemoresistance. Recent studies have demonstrated that endogenous molecules or damage-associated molecular patterns released from damaged/necrotic tissues are capable of activating TLRs and that the endogenous ligands-mediated TLR signaling is implicated in the tumor development and affects the therapeutic efficacy of tumors. Since both exogenous and endogenous TLR ligands can initiate TLR signaling, which is the most valuable player in tumor development becomes an interesting question. Here, we summarize the effect of TLR signaling on the development and progression of tumors, and discuss the role of exogenous and endogenous TLR ligands in the tumorigenesis.

  3. A multistep continuous-flow system for rapid on-demand synthesis of receptor ligands

    DEFF Research Database (Denmark)

    Petersen, Trine P; Ritzén, Andreas; Ulven, Trond

    2009-01-01

    A multistep continuous-flow system for synthesis of receptor ligands by assembly of three variable building blocks in a single unbroken flow is described. The sequence consists of three reactions and two scavenger steps, where a Cbz-protected diamine is reacted with an isocyanate, deprotected, an......, and reacted further with an alkylating agent....

  4. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    DEFF Research Database (Denmark)

    Hudson, Brian D; Shimpukade, Bharat; Milligan, Graeme

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands ...

  5. Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation

    NARCIS (Netherlands)

    Mangoni, R; Roccatano, D; Di Nola, A

    1999-01-01

    In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al, (Di Nola et al,, Proteins 1994;19:174-182), The

  6. Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Ahern, Christopher A

    2013-01-01

    -edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences...

  7. Aryl hydrocarbon receptor ligand effects in RBL2H3 cells

    DEFF Research Database (Denmark)

    Maaetoft-Udsen, Kristina; Shimoda, Lori M. N.; Frøkiær, Hanne

    2012-01-01

    The aryl hydrocarbon receptor (AHR) mediates toxic effects of dioxin and xenobiotic metabolism. AHR has an emerging role in the immune system, but its physiological ligands and functional role in immunocytes remain poorly understood. Mast cells are immunocytes that are central to inflammatory...

  8. Peroxisome Proliferator-Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies.

    Science.gov (United States)

    Yousefi, Bahman; Samadi, Nasser; Baradaran, Behzad; Shafiei-Irannejad, Vahid; Zarghami, Nosratollah

    2016-07-01

    Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail. © 2016 John Wiley & Sons A/S.

  9. Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition.

    Science.gov (United States)

    Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; De Biasi, Federico; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

    2017-07-13

    The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the monolayer itself and with the solvent. We further describe how the nature and plasticity of these interactions modulate nanoparticle-based chemosensing. Importantly, we found that self-organization of coating thiols can induce the formation of binding pockets in AuNPs. These transient cavities can accommodate small molecules, mimicking protein-ligand recognition, which could explain the selectivity and sensitivity observed for different organic analytes in NMR chemosensing experiments. Thus, our findings advocate for the rational design of tailored coating groups to form specific recognition binding sites on monolayer-protected AuNPs.

  10. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-ht2 receptors

    DEFF Research Database (Denmark)

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine...... about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure...... but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure...

  11. Elimination of a ligand gating site generates a supersensitive olfactory receptor.

    Science.gov (United States)

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

    2016-06-21

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

  12. Kinetic modeling of receptor-ligand binding applied to positron emission tomographic studies with neuroleptic tracers

    Energy Technology Data Exchange (ETDEWEB)

    Logan, J; Wolf, A P; Shiue, C Y; Fowler, J S

    1987-01-01

    Positron emission tomography (PET) with labeled neuroleptics has made possible the study of neurotransmitter-receptor systems in vivo. In this study we investigate the kinetics of the 3,4-dihydroxyphenylethylamine (dopamine) receptor-ligand binding using PET data from a series of experiments in the baboon with the /sup 18/F-labeled drugs spiperone, haloperidol, and benperidol. Models used to describe these systems are based on first-order kinetics which applies at high specific activity (low receptor occupancy). The parameters governing the uptake and loss of drug from the brain were found by fitting PET data from regions with little or no receptor concentration (cerebellum) and from experiments in which specific binding was blocked by pretreatment with the drug (+)-butaclamol. Receptor constants were determined by fitting data from receptor-containing structures. Correcting the arterial plasma activities (the model driving function) for the presence of drug metabolites was found to be important in the modeling of these systems.

  13. Structural basis of ligand recognition in 5-HT(3) receptors

    NARCIS (Netherlands)

    Kesters, D.; Thompson, A.J.; Brams, M.; van Elk, R.; Spurny, R.; Geitmann, M.; Villalgordo, J.M.; Guskov, A.; Danielson, U.H.; Lummis, S.C.R.; Smit, A.B.; Ulens, C.

    2013-01-01

    The 5-HT 3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist

  14. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    Science.gov (United States)

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  15. Synthesis of water-soluble mono- and ditopic imidazoliums for carbene ligands

    Energy Technology Data Exchange (ETDEWEB)

    Anstey, Mitchell [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division; Murtagh, Dustin [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division; Cordaro, Joseph Gabriel [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division; Stavila, Vitalie [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division; Feng, Patrick L. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division; Mengesha, Wondwosen [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Chemistry Division

    2015-09-01

    Synthesis of ditopic imidazoliums was achieved using a modular step-wise procedure. The procedure itself is amenable to a wide array of functional groups that can be incorporated into the imidazolium architecture. The resulting compounds range from ditopic zwitterions to highly-soluble dicationic aromatics

  16. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    , and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E....... At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes...... in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded...

  17. Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

    Science.gov (United States)

    Huang, Xiao X; McCaughan, Geoffrey W; Shackel, Nicholas A; Gorrell, Mark D

    2007-09-01

    Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

  18. Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells.

    Science.gov (United States)

    Kishimoto, Seishi; Muramatsu, Mayumi; Gokoh, Maiko; Oka, Saori; Waku, Keizo; Sugiura, Takayuki

    2005-02-01

    2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

  19. Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

    Science.gov (United States)

    Marchetti, Laura; Luin, Stefano; Bonsignore, Fulvio; de Nadai, Teresa; Beltram, Fabio; Cattaneo, Antonino

    2015-01-01

    Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells. PMID:25603178

  20. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Science.gov (United States)

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  1. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Directory of Open Access Journals (Sweden)

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  2. Soluble receptors for tumor necrosis factor as markers of disease activity in visceral leishmaniasis

    NARCIS (Netherlands)

    Zijlstra, E. E.; van der Poll, T.; Mevissen, M.

    1995-01-01

    Serum concentrations of soluble receptors for tumor necrosis factor (sTNFRs) were measured before and after antimony therapy in 25 Sudanese patients with active visceral leishmaniasis (VL). Both sTNFR types I and II were significantly elevated in patients with VL compared with healthy controls from

  3. Total soluble and endogenous secretory receptor for advanced glycation endproducts (RAGE) in IBD

    NARCIS (Netherlands)

    Meijer, Berrie; Hoskin, Teagan; Ashcroft, Anna; Burgess, Laura; Keenan, Jacqueline I.; Falvey, James; Gearry, Richard B.; Day, Andrew S.

    2014-01-01

    Recruitment and activation of neutrophils, with release of specific proteins such as S100 proteins, is a feature of inflammatory bowel disease (IBD). Soluble forms of the receptor for advanced glycation endproducts (sRAGE), and variants such as endogenous secretory (esRAGE), can act as decoy

  4. A soluble form of the transcobalamin receptor CD320 can be detected in human serum

    DEFF Research Database (Denmark)

    Arendt, Johan Frederik Berg; Quadros, Edward V.; Christensen, Anna Lisa

    2010-01-01

    Background: Recently, the cell-surface receptor involved in the internalisation of the cobalamin(vitamin B12, Cbl) transporting protein, transcobalamin(TC), was described, and was found to be CD320(1). So far, it remains unsolved whether CD320 is present in a soluble form (sCD320) in serum. Our aim...

  5. The soluble urokinase plasminogen activator receptor and its fragments in venous ulcers

    DEFF Research Database (Denmark)

    Ahmad, Anwar; Saha, Prakash; Evans, Colin

    2015-01-01

    OBJECTIVE: Activation of proteolytic mechanisms at the cell surface through the activity of urokinase-type plasminogen activator (uPA) bound to its receptor, uPAR, is an important process in wound healing. The soluble forms of uPAR (suPAR and its fragments I, II, and III) have nonproteolytic func...

  6. Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening

    DEFF Research Database (Denmark)

    Kjellman, A; Akre, O; Gustafsson, O

    2011-01-01

    BACKGROUND: The urokinase plasminogen activator (uPA) system is involved in tissue remodelling processes and is up-regulated in many types of malignancies. We investigated whether serum levels of different forms of soluble uPA receptor (suPAR) are associated with survival and in particular with p...

  7. Soluble urokinase-type plasminogen activator receptor forms in plasma as markers of atherosclerotic plaque vulnerability

    DEFF Research Database (Denmark)

    Olson, Fredrik J; Thurison, Tine; Ryndel, Mikael

    2009-01-01

    OBJECTIVES:: To test if circulating forms of the soluble urokinase-type plasminogen activator receptor (suPAR) are potential biomarkers of plaque vulnerability. DESIGN AND METHODS:: Plasma concentrations of suPAR(I-III), suPAR(II-III) and uPAR(I) were measured by time-resolved fluorescence immuno...

  8. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    Determann, Rogier M.; Weisfelt, Martijn; de Gans, Jan; van der Ende, Arie; Schultz, Marcus J.; van de Beek, Diederik

    2006-01-01

    OBJECTIVE: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. DESIGN: Retrospective study of diagnostic accuracy. SETTING AND PATIENTS: CSF was

  9. Synthesis of freestanding water-soluble indium oxide nanocrystals capped by alanine nitric acid via ligand exchange for thin film transistors and effects of ligands on the electrical properties

    International Nuclear Information System (INIS)

    Choi, Jin-Kyu; Koh, Ye-Seul; Jeong, Hyun-Dam

    2015-01-01

    We demonstrate synthesis of freestanding water-soluble indium oxide nanocrystals (In 2 O 3 NCs) by ligand exchange to β-alanine nitric acid (Ala·HNO 3 ) and its application for active channel layer in thin film transistors (TFTs), with investigation of the effect of curing temperatures on the TFT properties in terms of thermal behaviour of the ligand molecules at 150, 300, and 350 °C. After ligand exchange from long alkyl ligand (myristic acid, MA) to short Ala·HNO 3 , the mobility of NC TFTs cured at 150 °C increased by over 1 order of magnitude, from 1.3 × 10 −4 cm 2 V -1 s −1 to 4.5 × 10 −3 cm 2 V -1 s −1 , due to enhanced tunnelling rate (Γ) between adjective NCs. Higher curing temperatures such as 300 and 350 °C, inducing thermal decomposition of the organic ligands, led to further enhancement of the mobility, particularly up to 2.2 cm 2 V -1 s −1 for the In 2 O 3 NC-Ala·HNO 3 TFT cured at 350 °C. It is also found that the ligand exchange of In 2 O 3 NC in acidic condition (e.g. HNO 3 ) would be simple and effective to reduce the surface defects by surface etching, which may lead to better device performances. - Graphical abstract: Display Omitted - Highlights: • Freestanding water-soluble In 2 O 3 nanocrystals (NCs) were synthesized by ligand exchange. • Thin film transistors (TFTs) of colloidal NCs were fabricated by spin-coating method. • Water-soluble In 2 O 3 NC TFTs showed higher mobilities due to shorter ligand length. • Surface defects of NCs were notably reduced by surface etching during ligand exchange

  10. Application of 3D-QSAR in the rational design of receptor ligands and enzyme inhibitors.

    Science.gov (United States)

    Mor, Marco; Rivara, Silvia; Lodola, Alessio; Lorenzi, Simone; Bordi, Fabrizio; Plazzi, Pier Vincenzo; Spadoni, Gilberto; Bedini, Annalida; Duranti, Andrea; Tontini, Andrea; Tarzia, Giorgio

    2005-11-01

    Quantitative structure-activity relationships (QSARs) are frequently employed in medicinal chemistry projects, both to rationalize structure-activity relationships (SAR) for known series of compounds and to help in the design of innovative structures endowed with desired pharmacological actions. As a difference from the so-called structure-based drug design tools, they do not require the knowledge of the biological target structure, but are based on the comparison of drug structural features, thus being defined ligand-based drug design tools. In the 3D-QSAR approach, structural descriptors are calculated from molecular models of the ligands, as interaction fields within a three-dimensional (3D) lattice of points surrounding the ligand structure. These descriptors are collected in a large X matrix, which is submitted to multivariate analysis to look for correlations with biological activity. Like for other QSARs, the reliability and usefulness of the correlation models depends on the validity of the assumptions and on the quality of the data. A careful selection of compounds and pharmacological data can improve the application of 3D-QSAR analysis in drug design. Some examples of the application of CoMFA and CoMSIA approaches to the SAR study and design of receptor or enzyme ligands is described, pointing the attention to the fields of melatonin receptor ligands and FAAH inhibitors.

  11. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    Science.gov (United States)

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  12. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    International Nuclear Information System (INIS)

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-01-01

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  13. A multi-protein receptor-ligand complex underlies combinatorial dendrite guidance choices in C. elegans

    Science.gov (United States)

    Zou, Wei; Shen, Ao; Dong, Xintong; Tugizova, Madina; Xiang, Yang K; Shen, Kang

    2016-01-01

    Ligand receptor interactions instruct axon guidance during development. How dendrites are guided to specific targets is less understood. The C. elegans PVD sensory neuron innervates muscle-skin interface with its elaborate dendritic branches. Here, we found that LECT-2, the ortholog of leukocyte cell-derived chemotaxin-2 (LECT2), is secreted from the muscles and required for muscle innervation by PVD. Mosaic analyses showed that LECT-2 acted locally to guide the growth of terminal branches. Ectopic expression of LECT-2 from seam cells is sufficient to redirect the PVD dendrites onto seam cells. LECT-2 functions in a multi-protein receptor-ligand complex that also contains two transmembrane ligands on the skin, SAX-7/L1CAM and MNR-1, and the neuronal transmembrane receptor DMA-1. LECT-2 greatly enhances the binding between SAX-7, MNR-1 and DMA-1. The activation of DMA-1 strictly requires all three ligands, which establishes a combinatorial code to precisely target and pattern dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.18345.001 PMID:27705746

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  15. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  16. [3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    International Nuclear Information System (INIS)

    Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

    1999-01-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  17. Multiplex bead-based immunoassay for the free soluble forms of the HLA-G receptors, ILT2 and ILT4

    DEFF Research Database (Denmark)

    Wu, Ching-Lien; Svendsen, Signe Goul; Riviere, Adrien

    2016-01-01

    in the plasma of healthy controls, but that elevated levels of plasmatic sILT2 were present in non-muscle-infiltrating bladder cancer patients. This demonstrated that the titration test is indeed working, and that soluble ILT2 molecules do exist in pathological contexts, which relevance may now be sought......Human leukocyte antigen (HLA)-G is an immune-inhibitory molecule that exerts its function via interaction with two main inhibitory receptors: ILT2 and ILT4. This interaction is considered to be an immune checkpoint. HLA-G can be found as a soluble molecule, but it is not known if its receptors can...... reveals that it specifically detects the free soluble forms of sILT2 and sILT4, and not those complexed to HLA Class I molecules such as their ligand of highest affinity HLA-G. A study on two small cohorts of cancer patients demonstrated that soluble ILT2 and ILT4 molecules were of low abundance...

  18. Metabotropic glutamatergic receptors and their ligands in drug addiction.

    Science.gov (United States)

    Pomierny-Chamioło, Lucyna; Rup, Kinga; Pomierny, Bartosz; Niedzielska, Ewa; Kalivas, Peter W; Filip, Małgorzata

    2014-06-01

    Glutamatergic excitatory transmission is implicated in physiological and pathological conditions like learning, memory, neuronal plasticity and emotions, while glutamatergic abnormalities are reported in numerous neurological and psychiatric disorders, including neurodegenerative diseases, epilepsy, stroke, traumatic brain injury, depression, anxiety, schizophrenia and pain. Also, several lines of evidence have accumulated indicating a pivotal role for glutamatergic neurotransmission in mediating addictive behaviors. Among the proteins regulating glutamatergic transmission, the metabotropic glutamate receptors (mGluR) are being developed as pharmacological targets for treating many neuropsychiatric disorders, including drug addiction. In this review we describe the molecular structure of mGluRs and their distribution, physiology and pharmacology in the central nervous system, as well as their use as targets in preclinical studies of drug addiction. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  20. Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats.

    Science.gov (United States)

    Miszkiel, Joanna; Przegaliński, Edmund

    2013-01-01

    Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.

  1. Preparation and evaluation of an astatine-211-labeled sigma receptor ligand for alpha radionuclide therapy

    International Nuclear Information System (INIS)

    Ogawa, Kazuma; Mizuno, Yoshiaki; Washiyama, Kohshin; Shiba, Kazuhiro; Takahashi, Naruto; Kozaka, Takashi; Watanabe, Shigeki; Shinohara, Atsushi; Odani, Akira

    2015-01-01

    Introduction: Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated 131 I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[ 131 I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[ 131 I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[ 211 At]pAtV, an 211 At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. Methods: The radiolabeled sigma receptor ligand (+)-[ 211 At]pAtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[ 211 At]pAtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[ 211 At]pAtV and (+)-[ 125 I]pIV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. Results: The lipophilicity of (+)-[ 211 At]pAtV was similar to that of (+)-[ 125 I]pIV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1 h post-injection were also similar between (+)-[ 211 At]pAtV and (+)-[ 125 I]pIV. Namely, (+)-[ 211 At]pAtV demonstrated high uptake and retention in tumor via binding to sigma receptors. Conclusion: These results indicate that (+)-[ 211 At]pAtV could function as an new agent for alpha-radionuclide receptor therapy.

  2. Preparation and evaluation of an astatine-211-labeled sigma receptor ligand for alpha radionuclide therapy.

    Science.gov (United States)

    Ogawa, Kazuma; Mizuno, Yoshiaki; Washiyama, Kohshin; Shiba, Kazuhiro; Takahashi, Naruto; Kozaka, Takashi; Watanabe, Shigeki; Shinohara, Atsushi; Odani, Akira

    2015-11-01

    Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated (131)I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[(131)I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[(131)I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[(211)At]pAtV, an (211)At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. The radiolabeled sigma receptor ligand (+)-[(211)At]pAtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[(211)At]pAtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[(211)At]pAtV and (+)-[(125)I]pIV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. The lipophilicity of (+)-[(211)At]pAtV was similar to that of (+)-[(125)I]pIV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1h post-injection were also similar between (+)-[(211)At]pAtV and (+)-[(125)I]pIV. Namely, (+)-[(211)At]pAtV demonstrated high uptake and retention in tumor via binding to sigma receptors. These results indicate that (+)-[(211)At]pAtV could function as an new agent for alpha-radionuclide receptor therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Novel chalcone-based fluorescent human histamine H3 receptor ligands as pharmacological tools

    Directory of Open Access Journals (Sweden)

    Holger eStark

    2012-03-01

    Full Text Available Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R have been designed, synthesized and characterized. Compounds described are non-imidazole analogues of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like that of the reference antagonist ciproxifan (hH3R pKi value of 7.2. Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be taken to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues.

  4. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Directory of Open Access Journals (Sweden)

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  5. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...... about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic...

  6. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    Science.gov (United States)

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Antidiabetic actions of a phosphatidylcholine ligand for nuclear receptor LRH-1

    Science.gov (United States)

    Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L.; Busby, Scott A.; Griffin, Patrick R.; Pathak, Manish C.; Ortlund, Eric A.; Moore, David D.

    2011-01-01

    Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis. PMID:21614002

  8. submitter Emerging importance of chemokine receptor CXCR3 and its ligands in cardiovascular diseases

    CERN Document Server

    Altara, R; Brandao, R D; Zeidan, A; Booz, G W; Zouein, F A

    2016-01-01

    The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the deve...

  9. Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

    Science.gov (United States)

    Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

    2004-01-01

    In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

  10. Labeling of receptor ligands with bromine radionuclides. Progress report, March 1, 1981-February 28, 1982

    International Nuclear Information System (INIS)

    Welch, M.J.

    1981-10-01

    In recent years there has been an interest in the use of various radioisotopes of bromine as labels for radiopharmaceuticals. Although radioisotopes of iodine have been used extensively as radiopharmaceutical labels, there are several advantages associated with the use of radiobromine as a label, due primarily to increased stability of bonds to the radiohalide and smaller steric perturbation resulting from substitution of the radiohalide. Methods of attaching radiobromine to receptor ligands with the potential of mapping estrogen receptors in mammary tumors and uteri were studied. Two ligands were studied extensively in vitro and in animal models; preliminary studies were also carried out in humans. To date, the only radioisotope of bromine used was bromine-77. In addition, a series of model compounds were labeled with bromine-77 using a recently described method for rapid bromination; the scope and limitations of this new rapid radiobromination technique were evaluated

  11. Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

    1989-09-01

    Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

  12. Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

    Science.gov (United States)

    Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

    2013-03-05

    AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    International Nuclear Information System (INIS)

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  14. Synthesis and radiofluorination of putative NMDA receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, U

    2011-01-15

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[{sub 18}F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent

  15. Synthesis and radiofluorination of putative NMDA receptor ligands

    International Nuclear Information System (INIS)

    Kronenberg, U.

    2011-01-01

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[ 18 F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent, but no

  16. Rapid, portable detection of endocrine disrupting chemicals through ligand-nuclear hormone receptor interactions.

    Science.gov (United States)

    Hunt, J Porter; Schinn, Song-Min; Jones, Matthew D; Bundy, Bradley C

    2017-12-04

    Endocrine disrupting chemicals (EDC) are structurally diverse compounds that can interact with nuclear hormone receptors, posing significant risk to human and ecological health. Unfortunately, many conventional biosensors have been too structure-specific, labor-intensive or laboratory-oriented to detect broad ranges of EDC effectively. Recently, several technological advances are providing more rapid, portable, and affordable detection of endocrine-disrupting activity through ligand-nuclear hormone receptor interactions. Here, we overview these recent advances applied to EDC biosensors - including cell lyophilization, cell immobilization, cell-free systems, smartphone-based signal detection, and improved competitive binding assays.

  17. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter Johannes

    2011-01-01

    The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we...... with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases...

  18. Quantitative autoradiography of ligands for dopamine receptors and transporters in brain of Göttingen minipig

    DEFF Research Database (Denmark)

    Minuzzi, Luciano; Olsen, Aage Kristian; Bender, Dirk

    2006-01-01

    The pig has been used as animal model for positron emission tomography (PET) studies of dopamine (DA) receptors and pharmacological perturbations of DA neurotransmission. However, the binding properties of DA receptors and transporters in pig brain have not been characterized in vitro. Therefore...... in young and old pigs, and were close to those reported for rat and human brain. Furthermore, gradients in the concentrations of D1 and D2/3 sites in striatum measured in vitro agreed with earlier findings in PET studies. However, the dopamine transporter (DAT) ligand [3H]GBR12935 did not bind in pig brain...

  19. Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

    International Nuclear Information System (INIS)

    Takahashi, Miki; Kanayama, Tomohiko; Yashiro, Takuya; Kondo, Hidehiko; Murase, Takatoshi; Hase, Tadashi; Tokimitsu, Ichiro; Nishikawa, Jun-ichi; Sato, Ryuichiro

    2008-01-01

    In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism

  20. Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

    Science.gov (United States)

    Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

    2014-01-01

    The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. © 2014 Elsevier B.V. All rights reserved.

  1. Microchemical synthesis of the serotonin receptor ligand, 125I-LSD

    International Nuclear Information System (INIS)

    Hartig, P.R.; Krohn, A.M.; Hirschman, S.A.

    1985-01-01

    The synthesis and properties of 2-[ 125 I]-lysergic acid diethylamide, the first 125 I-labeled serotonin receptor ligand, are described. A novel microsynthesis apparatus was developed for this synthesis. The apparatus employs a micromanipulator and glass micro tools to handle microliter to nanoliter volumes on a microscope stage. This apparatus should be generally useful for the synthesis of radioligands and other compounds when limited amounts of material must be handled in small volumes

  2. Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

    Science.gov (United States)

    Cotnoir-White, David; El Ezzy, Mohamed; Boulay, Pierre-Luc; Rozendaal, Marieke; Bouvier, Michel; Gagnon, Etienne; Mader, Sylvie

    2018-03-13

    There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

  3. The Prelude on Novel Receptor and Ligand Targets Involved in the Treatment of Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Venu Gopal Jonnalagadda

    2014-05-01

    Full Text Available Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  4. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

    Directory of Open Access Journals (Sweden)

    John G Yamauchi

    2011-01-01

    Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

  5. General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

    Science.gov (United States)

    Fei, Xiang; Zavorka, Megan E; Malik, Guillaume; Connelly, Christopher M; MacDonald, Richard G; Berkowitz, David B

    2017-08-18

    A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

  6. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    International Nuclear Information System (INIS)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-01-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for [ 3 H]diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with [ 3 H]flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines

  7. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    Science.gov (United States)

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  8. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    Science.gov (United States)

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Masayuki Yarimizu

    2015-01-01

    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  10. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  11. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  12. Measurement of biologically active interleukin-1 by a soluble receptor binding assay

    International Nuclear Information System (INIS)

    Riske, F.; Chizzonite, R.; Nunes, P.; Stern, A.S.

    1990-01-01

    A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants

  13. Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

    Directory of Open Access Journals (Sweden)

    Marcia eHiriart

    2014-06-01

    Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

  14. Effects of cypermethrin on the ligand-independent interaction between androgen receptor and steroid receptor coactivator-1

    International Nuclear Information System (INIS)

    Pan, Chen; Liu, Ya-Peng; Li, Yan-Fang; Hu, Jin-Xia; Zhang, Jin-Peng; Wang, Hong-Mei; Li, Jing; Xu, Li-Chun

    2012-01-01

    The pyrethroid insecticide, cypermethrin has been considered as an environmental anti-androgen by interfering with the androgen receptor (AR) transactivation. In order to clarify the effects of cypermethrin on the ligand-independent interaction between the AR and SRC-1, the mammalian two-hybrid assay has been developed in the study. The AR N-terminal domain 1–660 amino acid residues were subcloned into the plasmid pVP16 to construct the vector pVP16-ARNTD. The SRC-1 C-terminal domain 989–1240 amino acid residues were subcloned into the plasmid pM to construct the vector pM-SRC-1. The fusion vectors pVP16-ARNTD, pM-SRC-1 and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The AR AF1 interacted with SRC-1 in the absence of exogenous ligand 5α-dihydrotestosterone (DHT). Furthermore, DHT did not enhance the interaction between AR AF-1 and SRC-1 at the concentrations from 10 −10 M to 10 −8 M. Cypermethrin inhibited the interaction between the AR AF1 and SRC-1, and the significant reduction was detected at the concentration of 10 −5 M. It is suggested that the interaction between the AR AF1 and SRC-1 is ligand-independent. Cypermethrin inhibits AR activity by disrupting the ligand-independent AR–SRC-1 interaction.

  15. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Christiansen, Elisabeth; Tikhonova, Irina G

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL...... on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering...

  16. Synthesis of the possible receptor Ligand [125I]-spiperone for D2-dopamine receptor and in-vivo biodistribution

    International Nuclear Information System (INIS)

    Amin, A.M.; Shoukry, M.; Abd EL-Bary, A.

    2009-01-01

    The spiperone is a selective D2-dopamine receptor antagonist radioiodination of spiperone is of interest for dopamine (DA) receptor studies both in vivo and in vitro. The labeling of spiperone with iodine-125 was extremely done in a neutral ph 7, using chloramine-T as oxidizing agent via heating the reaction mixture at 70 C (degree) for 10 - 15 minutes producing radiochemical yield of 97 %. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compound. The brain uptake of 125 I-Spiperone was high and equal to 3.5, 3.25,2.75 and 1.7 % per gram tissue at 5, 30, 60 and 120 minutes post injection, respectively. 125 I-Spiperone binds with high affinity to dopamine receptors in vivo. Specific binding is about 65% of the total binding as is displaced stereo-specifically by clozapine. 125 I-spiperone may prove to be a useful ligand in studies examining D2-dopamine receptors. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123 I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.

  17. Novel aza-analogous ergoline derived scaffolds as potent serotonin 5-HT6 and dopamine D2 receptor ligands

    DEFF Research Database (Denmark)

    Krogsgaard-Larsen, Niels; Jensen, Anders A.; Schrøder, T.J.

    2014-01-01

    By introducing distal substituents on a tetracyclic scaffold resembling the ergoline structure, two series of analogues were achieved exhibiting subnanomolar receptor binding affinities for the dopamine D2 and serotonin 5-HT6 receptor subtype, respectively. While the 5-HT6 ligands were antagonists......, the D2 ligands displayed intrinsic activities ranging from full agonism to partial agonism with low intrinsic activity. These structures could potentially be interesting for treatment of neurological diseases such as schizophrenia, Parkinson’s disease, and cognitive deficits....

  18. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    OpenAIRE

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leuko...

  19. Evaluation of 3-Ethyl-3-(phenylpiperazinylbutyl)oxindoles as PET Ligands for the Serotonin 5-HT7 Receptor

    DEFF Research Database (Denmark)

    Herth, Matthias M; Andersen, Valdemar L; Hansen, Hanne D

    2015-01-01

    We have investigated several oxindole derivatives in the pursuit of a 5-HT7 receptor PET ligand. Herein the synthesis, chiral separation, and pharmacological profiling of two possible PET candidates toward a wide selection of CNS-targets are detailed. Subsequent (11)C-labeling and in vivo evaluat...... evaluation in Danish landrace pigs showed that both ligands displayed high brain uptake. However, neither of the radioligands could be displaced by the 5-HT7 receptor selective inverse agonist SB-269970....

  20. Soluble transferrin receptor: a differentiating marker between iron deficiency anaemia and anaemia of chronic disorders

    International Nuclear Information System (INIS)

    Saboor, M.; Moinuddin, A.; Naureen, A.

    2012-01-01

    Background: Iron deficiency anaemia and anaemia of chronic disorders are the two major causes of microcytic and hypochromic anaemia. Many times the diagnosis of these conditions becomes difficult through conventional laboratory tests. Determination of soluble transferrin receptors is a helpful laboratory test for the differential diagnosis of these conditions. The study was conducted to evaluate the role of soluble transferrin receptors in the differential diagnosis between iron deficiency anaemia and anaemia of chronic disorders. Methods: A total of 80 blood samples were evaluated, i.e., 20 samples from normal adult male, 20 samples from normal adult female, 20 samples from iron deficiency anaemia group and 20 samples from patients with anaemia of chronic disorders. Soluble transferrin receptors were determined by ELISA technique using Quantikine IVD kit (R and D Systems). Results: There was significant difference in the levels of sTfR in iron deficiency anaemia and anaemia of chronic disorders. Statistically non-significant difference was observed between the levels of sTfR in patients with anaemia of chronic disorders as compared to normal control group. Conclusion: The sTfR determination can be used as a reliable differentiating marker in the diagnosis of iron deficiency anaemia and anaemia of chronic disorders. (author)

  1. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Energy Technology Data Exchange (ETDEWEB)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Rodrigues, Michele A. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Department of General Pathology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Gomes, Dawidson A., E-mail: dawidson@ufmg.br [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil)

    2016-09-09

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  2. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    International Nuclear Information System (INIS)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.; Rodrigues, Michele A.; Gomes, Dawidson A.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  3. Increased concentrations of soluble CD40 ligand platelet in patients with primary antiphospholipidic syndrome.

    Science.gov (United States)

    Galicia López, Aida; Olguín Ortega, Lourdes; Saavedra, Miguel A; Méndez Cruz, René; Jimenez Flores, Rafael; García de la Peña, Maximiliano

    2013-01-01

    To determine the concentrations of sCD40L in patients with PAPS, and establish its association with the number of thrombosis. We included patients with PAPS and healthy controls of the same age and sex. For analysis, patients with PAPS were divided into 2 groups: 1) patients with 1 thrombosis, and 2) patients with >1 thrombosis. Soluble CD40L concentrations were determined by ELISA method. sCD40L concentrations were significantly higher in patients with PAPS compared with the controls (9.72 ng ± 11.23 ng/ml vs. 4.69 ± 4.04 ng/ml) (P=.04) There was no association between serum levels of sCD40L and the number of thrombosis (1 thrombosis: 9.81 ± 9.87 ng/ml vs 9.63 ± 12.75 ng/ml in ≥ 1thrombosis (P=.13). In women with pregnancy and abortions, (13 patients) concentrations of sCD40L were higher than in those patients without a history of abortion (26 patients) but without statically significant difference (12.11 ± 16.46 ng/ml vs. 8.80 ± 8.61 ng/ml) (P=.33). There was no correlation between levels of sCD40L and the total number of thrombosis. Patients with PAPS have higher concentrations of sCD40L compared with healthy subjects, although this is not associated with a greater number of thrombosis. Among patients with PAPS, there is a tendency to higher concentrations of sCD40L in women with pregnancy and history of abortion. Since the platelet is the main cellular source of sCD40L, is possible that this pathway plays a pathogenic role in patients with PAPS. Copyright © 2012 Elsevier España, S.L. All rights reserved.

  4. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    Science.gov (United States)

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

    Science.gov (United States)

    Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

    2008-04-25

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

  6. Serum soluble Fas ligand (sFasL) in patients with primary squamous cell carcinoma of the esophagus

    International Nuclear Information System (INIS)

    Kozlowski, M.; Sulewska, A.; Kowalczuk, O.

    2007-01-01

    Esophageal carcinomas have been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. Circulating soluble FasL (sFasL) has been suggested to provide protection from Fas-mediated apoptosis. The aim of this study was to assess serum sFasL levels in esophageal cancer. The pretreatment levels of sFasL in the serum of 100 patients with esophageal squamous cell cancer and 41 healthy volunteers were determined by ELISA. Probability of survival was calculated according to the method of Kaplan-Meier. The prognostic influence of high and low level of sFasL was analyzed with the log-rank test. The mean serum level of sFasL in patients with esophageal cancer was significantly higher than that in healthy donors (1.567±1.786 vs 0.261±0.435, p<0.0001). The levels of serum sFasL were significantly higher in advanced stages (II vs IV p<0.034; III vs IV p<0.041; except II vs III p=0.281), patients with lymph node (N0 vs N1 p<0.0389) or distant (M0 vs. M1 p<0.0388) metastases and significantly lower in patients with well differentiated tumors (G1 vs G2 p<0.0272). The serum levels of soluble FasL were not related to gender, age, tumor size, Tstage, tobacco smoking and history of chronic alcohol intake. The survival difference between pretreatment high and low level of sFasL in surgery and chemo- and/or radiotherapy group was not statistically significant (p=0.525; p=0.840). Our results indicate that elevated serum sFasL levels might be associated with a disease progression in patients with esophageal squamous cell carcinoma. (authors)

  7. Serum soluble Fas ligand (sFasL in patients with primary squamous cell carcinoma of the esophagus.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2007-10-01

    Full Text Available Esophageal carcinomas have been shown to express Fas ligand (FasL and down-regulate Fas to escape from host immune surveillance. Circulating soluble FasL (sFasL has been suggested to provide protection from Fas-mediated apoptosis. The aim of this study was to assess serum sFasL levels in esophageal cancer. The pretreatment levels of sFasL in the serum of 100 patients with esophageal squamous cell cancer and 41 healthy volunteers were determined by ELISA. Probability of survival was calculated according to the method of Kaplan-Meier. The prognostic influence of high and low level of sFasL was analyzed with the log-rank test. The mean serum level of sFasL in patients with esophageal cancer was significantly higher than that in healthy donors (1.567+/-1.786 vs 0.261+/-0.435, p<0.0001. The levels of serum sFasL were significantly higher in advanced stages (II vs IV p<0.034; III vs IV p<0.041; except II vs III p=0.281, patients with lymph node (N0 vs N1 p<0.0389 or distant (M0 vs. M1 p<0.0388 metastases and significantly lower in patients with well differentiated tumors (G1 vs G2 p<0.0272. The serum levels of soluble FasL were not related to gender, age, tumor size, T-stage, tobacco smoking and history of chronic alcohol intake. The survival difference between pretreatment high and low level of sFasL in surgery and chemio- and/or radiotherapy group was not statistically significant (p=0.525; p=0.840. Our results indicate that elevated serum sFasL levels might be associated with a disease progression in patients with esophageal squamous cell carcinoma.

  8. The role of TAM family receptors and ligands in the nervous system: From development to pathobiology.

    Science.gov (United States)

    Shafit-Zagardo, Bridget; Gruber, Ross C; DuBois, Juwen C

    2018-03-04

    Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K-dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K-dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6 -/- and TAM mutant mice models. Copyright © 2018. Published by Elsevier Inc.

  9. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

    Directory of Open Access Journals (Sweden)

    Akiyoshi Takahashi

    2016-06-01

    Full Text Available This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016 [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

  10. Endogenous hallucinogens as ligands of the trace amine receptors: a possible role in sensory perception.

    Science.gov (United States)

    Wallach, J V

    2009-01-01

    While the endogenous hallucinogens, N,N-dimethyltryptamine, 5-hydroxy-N,N-dimethyl-tryptamine and 5-methoxy-N,N-dimethyltryptamine, have been acknowledged as naturally occurring components of the mammalian body for decades, their biological function remains as elusive now as it was at the time of their discovery. The recent discovery of the trace amine associated receptors and the activity of DMT and other hallucinogenic compounds at these receptor sites leads to the hypothesis that the endogenous hallucinogens act as neurotransmitters of a subclass of these trace amine receptors. Additionally, while activity at the serotonin 5-HT2A receptor has been proposed as being responsible for the hallucinogenic affects of administered hallucinogens, in their natural setting the 5-HT2A receptor may not interact with the endogenous hallucinogens at all. Additionally 5-HT2A agonist activity is unable to account for the visual altering effects of many of the administered hallucinogens; these effects may be mediated by one of the endogenous hallucinogen trace amine receptors rather than the serotonin 5-HT2A receptor. Therefore, activity at the trace amine receptors, in addition to serotonin receptors, may play a large role in the sensory altering effects of administered hallucinogens and the trace amine receptors along with their endogenous hallucinogen ligands may serve an endogenous role in mediating sensory perception in the mammalian central nervous system. Thus the theory proposed states that these compounds act as true endogenous hallucinogenic transmitters acting in regions of the central nervous system involved in sensory perception.

  11. Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

    Science.gov (United States)

    Cote, Christopher; Welkos, Susan; Manchester, Marianne; Young, John A. T.

    2012-01-01

    Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. PMID:22511955

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  13. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

  14. Circulating Ghrelin, Leptin, and Soluble Leptin Receptor Concentrations and Cardiometabolic Risk Factors in a Community-Based Sample

    OpenAIRE

    Ingelsson, Erik; Larson, Martin G.; Yin, Xiaoyan; Wang, Thomas J.; Meigs, James B.; Lipinska, Izabella; Benjamin, Emelia J.; Keaney, John F.; Vasan, Ramachandran S.

    2008-01-01

    Context: The conjoint effects and relative importance of ghrelin, leptin, and soluble leptin receptor (sOB-R), adipokines involved in appetite control and energy expenditure in mediating cardiometabolic risk, is unknown.

  15. Peroxisome proliferator-activated receptor α ligands and modulators from dietary compounds: Types, screening methods and functions.

    Science.gov (United States)

    Yang, Haixia; Xiao, Lei; Wang, Nanping

    2017-04-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a key role in lipid metabolism and glucose homeostasis and a crucial role in the prevention and treatment of metabolic diseases. Natural dietary compounds, including nutrients and phytochemicals, are PPARα ligands or modulators. High-throughput screening assays have been developed to screen for PPARα ligands and modulators in our diet. In the present review, we discuss recent advances in our knowledge of PPARα, including its structure, function, and ligand and modulator screening assays, and summarize the different types of dietary PPARα ligands and modulators. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

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

    Science.gov (United States)

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

    2003-01-01

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

  17. Predictive value of elevated soluble CD40 ligand in patients undergoing primary angioplasty for ST-segment elevation myocardial infarction.

    Science.gov (United States)

    Pusuroglu, Hamdi; Akgul, Ozgur; Erturk, Mehmet; Uyarel, Huseyin; Bulut, Umit; Akkaya, Emre; Buturak, Ali; Surgit, Ozgur; Fuat, Ali; Cetin, Mustafa; Yldrm, Aydn

    2014-11-01

    The aim of this study was to evaluate the prognostic value of soluble CD40 ligand (sCD40L) in patients with ST-segment elevation myocardial infarction (STEMI) undergoing a primary percutaneous coronary intervention (PCI). The prognostic value of sCD40L has been documented in patients with acute coronary syndrome; however, its value in acute STEMI remains unclear. We prospectively enrolled 499 consecutive STEMI patients (397 men, 102 women) undergoing primary PCI. The study population was divided into tertiles on the basis of admission sCD40L values. The high sCD40L group (n=168) included patients with a value in the third tertile (≥0.947 mg/l) and the low sCD40L group (n=331) included patients with a value in the lower two tertiles (0.947 mg/l) is a powerful independent predictor of 1-year all-cause mortality (odds ratio: 3.68; 95% confidence interval: 1.54-8.77; P=0.003). The results of this study suggest that a high sCD40L level at admission is associated with increased in-hospital and 1-year all-cause mortality rates in patients with STEMI undergoing primary PCI.

  18. Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

    Directory of Open Access Journals (Sweden)

    Maria J. Abrey Recalde

    2017-10-01

    Full Text Available Shiga toxin (Stx, produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS, which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L, which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

  19. Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

    Science.gov (United States)

    Abrey Recalde, Maria J.; Alvarez, Romina S.; Alberto, Fabiana; Mejias, Maria P.; Ramos, Maria V.; Fernandez Brando, Romina J.; Bruballa, Andrea C.; Exeni, Ramon A.; Alconcher, Laura; Ibarra, Cristina A.; Amaral, María M.; Palermo, Marina S.

    2017-01-01

    Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions. PMID:29068360

  20. Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

    Science.gov (United States)

    Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

    2016-05-09

    Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

  1. Simple Ligand-Receptor Interaction Descriptor (SILIRID) for alignment-free binding site comparison.

    Science.gov (United States)

    Chupakhin, Vladimir; Marcou, Gilles; Gaspar, Helena; Varnek, Alexandre

    2014-06-01

    We describe SILIRID (Simple Ligand-Receptor Interaction Descriptor), a novel fixed size descriptor characterizing protein-ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs) by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor) and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion). Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein-ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91). SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM): sc-PDB SILIRID data form clusters corresponding to different protein types.

  2. α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

    Science.gov (United States)

    Sánchez-Soto, Marta; Casadó-Anguera, Verònica; Yano, Hideaki; Bender, Brian Joseph; Cai, Ning-Sheng; Moreno, Estefanía; Canela, Enric I; Cortés, Antoni; Meiler, Jens; Casadó, Vicent; Ferré, Sergi

    2018-03-18

    The poor norepinephrine innervation and high density of Gi/o-coupled α 2A - and α 2C -adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D 2 -like receptor ligands, such as the D 3 receptor agonist 7-OH-PIPAT and the D 4 receptor agonist RO-105824, to α 2 -adrenoceptors in cortical and striatal tissue, which express α 2A -adrenoceptors and both α 2A - and α 2C -adrenoceptors, respectively. The affinity of dopamine for α 2 -adrenoceptors was found to be similar to that for D 1 -like and D 2 -like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α 2A - and α 2C -adrenoceptors. Their ability to activate Gi/o proteins through α 2A - and α 2C -adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α 2 -adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α 2A - and α 2C -adrenoceptors was nearly identical to its binding to the crystallized D 3 receptor. Therefore, we provide conclusive evidence that α 2A - and α 2C -adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D 2 -like receptor ligands, which calls for revisiting previous studies with those ligands.

  3. Collagen Type I as a Ligand for Receptor-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Iris Boraschi-Diaz

    2017-05-01

    Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  4. New pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors.

    Science.gov (United States)

    Greig, Nigel H; Reale, Marcella; Tata, Ada M

    2013-08-01

    The cholinergic system is expressed in neuronal and in non-neuronal tissues. Acetylcholine (ACh), synthesized in and out of the nervous system can locally contribute to modulation of various cell functions (e.g. survival, proliferation). Considering that the cholinergic system and its functions are impaired in a number of disorders, the identification of new pharmacological approaches to regulate cholinergic system components appears of great relevance. The present review focuses on recent pharmacological drugs able to modulate the activity of cholinergic receptors and thereby, cholinergic function, with an emphasis on the muscarinic receptor subtype, and additionally covers the cholinesterases, the main enzymes involved in ACh hydrolysis. The presence and function of muscarinic receptor subtypes both in neuronal and non-neuronal cells has been demonstrated using extensive pharmacological data emerging from studies on transgenic mice. The possible involvement of ACh in different pathologies has been proposed in recent years and is becoming an important area of study. Although the lack of selective muscarinic receptor ligands has for a long time limited the definition of therapeutic treatment based on muscarinic receptors as targets, some muscarinic ligands such as cevimeline (patents US4855290; US5571918) or xanomeline (patent, US5980933) have been developed and used in pre-clinical or in clinical studies for the treatment of nervous system diseases (Alzheimer' and Sjogren's diseases). The present review focuses on the potential implications of muscarinic receptors in different pathologies, including tumors. Moreover, the future use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that some muscarinic antagonists currently used in the treatment of genitourinary disease (e.g. darifenacin, patent, US5096890; US6106864) have also been demonstrated to arrest tumor progression in nude mice. The involvement of muscarinic

  5. MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

    Science.gov (United States)

    Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

    2012-01-17

    The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor

  6. Synthesis of freestanding water-soluble indium oxide nanocrystals capped by alanine nitric acid via ligand exchange for thin film transistors and effects of ligands on the electrical properties

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jin-Kyu; Koh, Ye-Seul; Jeong, Hyun-Dam, E-mail: hdjeong@chonnam.ac.kr

    2015-07-15

    We demonstrate synthesis of freestanding water-soluble indium oxide nanocrystals (In{sub 2}O{sub 3} NCs) by ligand exchange to β-alanine nitric acid (Ala·HNO{sub 3}) and its application for active channel layer in thin film transistors (TFTs), with investigation of the effect of curing temperatures on the TFT properties in terms of thermal behaviour of the ligand molecules at 150, 300, and 350 °C. After ligand exchange from long alkyl ligand (myristic acid, MA) to short Ala·HNO{sub 3}, the mobility of NC TFTs cured at 150 °C increased by over 1 order of magnitude, from 1.3 × 10{sup −4} cm{sup 2}V{sup -1}s{sup −1} to 4.5 × 10{sup −3} cm{sup 2}V{sup -1}s{sup −1}, due to enhanced tunnelling rate (Γ) between adjective NCs. Higher curing temperatures such as 300 and 350 °C, inducing thermal decomposition of the organic ligands, led to further enhancement of the mobility, particularly up to 2.2 cm{sup 2}V{sup -1}s{sup −1} for the In{sub 2}O{sub 3} NC-Ala·HNO{sub 3} TFT cured at 350 °C. It is also found that the ligand exchange of In{sub 2}O{sub 3} NC in acidic condition (e.g. HNO{sub 3}) would be simple and effective to reduce the surface defects by surface etching, which may lead to better device performances. - Graphical abstract: Display Omitted - Highlights: • Freestanding water-soluble In{sub 2}O{sub 3} nanocrystals (NCs) were synthesized by ligand exchange. • Thin film transistors (TFTs) of colloidal NCs were fabricated by spin-coating method. • Water-soluble In{sub 2}O{sub 3} NC TFTs showed higher mobilities due to shorter ligand length. • Surface defects of NCs were notably reduced by surface etching during ligand exchange.

  7. Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

    Science.gov (United States)

    Basilico, Cristina; Modica, Chiara; Maione, Federica; Vigna, Elisa; Comoglio, Paolo M

    2018-04-25

    MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMET K842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMET K842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience." © 2018 UICC.

  8. Emerging Roles for CSF-1 Receptor and its Ligands in the Nervous System

    Science.gov (United States)

    Chitu, Violeta; Gokhan, Solen; Nandi, Sayan; Mehler, Mark F.; Stanley, E. Richard

    2016-01-01

    The colony stimulating factor-1 receptor (CSF-1R) kinase regulates tissue macrophage homeostasis, osteoclastogenesis, and Paneth cell development. However, recent studies in mice have revealed that CSF-1R signaling directly controls the development and maintenance of microglia, and cell autonomously regulates neuronal differentiation and survival. While the CSF-1R-cognate ligands, CSF-1 and interleukin-34 (IL-34), compete for binding to the CSF-1R, they are expressed in a largely non-overlapping manner by mature neurons. The recent identification of a dominantly inherited, adult-onset, progressive dementia associated with inactivating mutations in the CSF-1R highlights the importance of CSF-1R signaling in the brain. We review the roles of the CSF-1R and its ligands in microglial and neural development and function, and their relevance to our understanding of neurodegenerative disease. PMID:27083478

  9. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Directory of Open Access Journals (Sweden)

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

  10. Regulation of Epithelial Morphogenesis by the G-Protein Coupled Receptor Mist and its Ligand Fog*

    Science.gov (United States)

    Manning, Alyssa J.; Peters, Kimberly A.; Peifer, Mark; Rogers, Stephen L.

    2014-01-01

    Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells, and used RNAi-based screening to identify Mist, a Drosophila G protein-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes. PMID:24222713

  11. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

    Science.gov (United States)

    Wang, Kai; Li, Yan; Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S; Song, Jia-Sheng; Zheng, Jing

    2013-10-28

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. The relaxin family peptide receptors and their ligands : new developments and paradigms in the evolution from jawless fish to mammals

    NARCIS (Netherlands)

    Yegorov, Sergey; Bogerd, Jan; Good, Sara V

    2014-01-01

    Relaxin family peptide receptors (Rxfps) and their ligands, relaxin (Rln) and insulin-like (Insl) peptides, are broadly implicated in the regulation of reproductive and neuroendocrine processes in mammals. Most placental mammals harbour genes for four receptors, namely rxfp1, rxfp2, rxfp3 and rxfp4.

  13. Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3))

    NARCIS (Netherlands)

    Jongsma, M.; van Unen, J.; van Loenen, P. B.; Michel, M. C.; Peters, S. L. M.; Alewijnse, A. E.

    2009-01-01

    Recently, some ligands targeting the sphingosine-1-phosphate receptor subtype 3 (S1P(3)) have become available. The characterization of these compounds was mainly based on one functional read-out system, although S1P(3) receptors are known to activate different signal transduction pathways.

  14. Estrogen and progesterone receptor assay using I-125 estradiol and H-3 promegestone as ligands: Results in female mammary carcinoma

    International Nuclear Information System (INIS)

    Glaubitt, D.; Hienz, H.A.; Bettges, G.; Carmanns, B.; Lichtenberg, T.; Akademisches Lehrkrankenhaus, Krefeld

    1984-01-01

    The determination of estrogen and progesterone receptors in the cytosol of carcinoma of the female breast has predictive value as to the success treatment of the patient. An improved estrogen and progesterone receptor assay using 1-125 labelled estradiol and a H-3 tagged synthetic gestagen (H-3 promegestone) as ligands proved to be highly praticable, especially time-saving. (orig.)

  15. Characterization of chicken thrombocyte responses to Toll-like receptor ligands.

    Directory of Open Access Journals (Sweden)

    Michael St Paul

    Full Text Available Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR, to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS, which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL-1β, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

  16. Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors.

    Science.gov (United States)

    Chen, Min; Georgeson, Keith E; Harmon, Carroll M; Haskell-Luevano, Carrie; Yang, Yingkui

    2006-11-01

    The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.

  17. Generation of signaling specificity in Arabidopsis by spatially restricted buffering of ligand-receptor interactions.

    Science.gov (United States)

    Abrash, Emily B; Davies, Kelli A; Bergmann, Dominique C

    2011-08-01

    Core signaling pathways function in multiple programs during multicellular development. The mechanisms that compartmentalize pathway function or confer process specificity, however, remain largely unknown. In Arabidopsis thaliana, ERECTA (ER) family receptors have major roles in many growth and cell fate decisions. The ER family acts with receptor TOO MANY MOUTHS (TMM) and several ligands of the EPIDERMAL PATTERNING FACTOR LIKE (EPFL) family, which play distinct yet overlapping roles in patterning of epidermal stomata. Here, our examination of EPFL genes EPFL6/CHALLAH (CHAL), EPFL5/CHALLAH-LIKE1, and EPFL4/CHALLAH-LIKE2 (CLL2) reveals that this family may mediate additional ER-dependent processes. chal cll2 mutants display growth phenotypes characteristic of er mutants, and genetic interactions are consistent with CHAL family molecules acting as ER family ligands. We propose that different classes of EPFL genes regulate different aspects of ER family function and introduce a TMM-based discriminatory mechanism that permits simultaneous, yet compartmentalized and distinct, function of the ER family receptors in growth and epidermal patterning.

  18. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    Science.gov (United States)

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

  19. Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

    International Nuclear Information System (INIS)

    Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

    1989-01-01

    The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

  20. Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Pedro Reis Rodrigues

    2016-06-01

    Full Text Available Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-β ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans.

  1. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  2. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  3. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  4. Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

    DEFF Research Database (Denmark)

    Li, Hongbing; Panina, Svetlana; Kaur, Amandeep

    2012-01-01

    Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins....... In the present study the role of Ca2+/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca2+ chelator inhibited...

  5. Synthesis of 123I-labelled analogues of imidazobenzodiazepine receptor ligands

    International Nuclear Information System (INIS)

    Katsifis, A.G.; Mattner, F.; McPhee, M.E.; Ridley, D.D.

    1999-01-01

    Reaction of bromo- or iodo-substituted isatoic anhydrides with N-methylglycine, L-proline or D-proline afforded bromo- or iodo-substituted 1,4-benzodiazepinediones which on condensation with ethyl or t-butyl isocyanoacetates gave ethyl or t-butyl bromo- or iodo-imidazobenzodiazepine carboxylates. These aryl halides were converted into the corresponding tributylstannanes with bis(tributyltin) in the presence of (triphenylphosphine)palladium(0), and the stannanes were treated with sodium ( 123 I)iodide in the presence of chloramine-T to give the required 123 I-labelled analogues of the imidazobenzodiazepine receptor ligands flumazenil and bretazenil. Copyright (1999) CSIRO Australia

  6. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    Science.gov (United States)

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPLW515L, MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but was not phosphorylated. We generated disulfide-bonded MPLW515L homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPLW515L with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPLW515L/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling. PMID:19261614

  7. Leptin, soluble leptin receptor, and free leptin index in patients with metabolic syndrome

    Directory of Open Access Journals (Sweden)

    Elena N. Smirnova

    2017-06-01

    Full Text Available Aim. To assess the levels of leptin, its soluble receptor, and index of the formation of free leptin in metabolic syndrome (MS. Materials and methods. The study included 110 individuals with obesity and overweight. The group 1 consisted of 70 patients with MS (IDF, 2005, the average body mass index (BMI 38.4 ± 4.4 kg/m2, aged 48.2 ± 2.4 years, with arterial hypertension (AH 1–2 degree, without regular antihypertensive therapy. Group 2 – "healthy" obesity accounted for 40 patients aged 38.4 ± 6.2 years, BMI 36.0 ± 5.5 kg/m2 without hypertension and metabolic disorders. Group 3 consisted of 30 healthy persons, BMI 27.1 ± 1.3 kg/m2. All patients were evaluated for insulin, HOMA index, leptin, leptin receptor, leptin free index (calculated as the ratio of leptin (ng/ml to the leptin receptor (ng/ml, multiplied by 100. Results: In patients with MS as compared to other two groups there were higher levels of HOMA IR index, leptin and free leptin index. Values of leptin receptor in groups 1 and 2 did not differ significantly and were lower than in healthy persons. The free leptin index was significantly higher in MS group relative to the group 2 and 15 times higher than in the healthy individuals. Free leptin index correlated with values of BMI (R = 0.32; p = 0.02, blood pressure (R = 0.3; p = 0.04, uric acid (R = 0.27; p = 0.04, triglycerides (R = 0.42; p = 0.02, index HOMA-IR (R = 0.45; p = 0.02. Conclusions: Reduction of soluble leptin receptor, depending on the degree of abdominal obesity, may cause progression of leptin resistance in patients with MS. The levels of leptin and soluble leptin receptor appears to have dramatical gender differences. Calculation of free leptin index should be used for the objective evaluation of leptin resistance, regardless of gender, degree of obesity, and other metabolic parameters.

  8.  Dehydroepiandrosterone sulfate, osteoprotegerin and its soluble ligand sRANKL and bone metabolism in girls with anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Zofia Ostrowska

    2012-09-01

    Full Text Available  Background:Only scarce data exist concerning the relationship between dehydroepiandrosterone (DHEA and/or its sulfate form DHEAS and bone status in adolescents with anorexia nervosa (AN.Aim:We investigated whether a relationship existed between DHEAS and bone metabolism (as assessed based on serum osteocalcin [OC], and collagen type I cross-linked carboxy-terminal telopeptide [CTx]. We also aimed to establish whether the above mentioned relationship might be affected by osteoprotegerin (OPG and its soluble ligand sRANKL.Material/Methods:Fifty-six female patients with AN and 21 healthy female subjects aged 13 to 16 years participated in the study. Serum DHEAS, OC, CTx, OPG and sRANKL were measured by ELISA.Results:Our female patients with AN demonstrated significant suppression of DHEAS and bone markers, an increase in OPG and sRANKL levels, and a reduction of the OPG/sRANKL ratio. DHEAS, CTx and the OPG/sRANKL ratio correlated positively with BMI. A significant positive correlation was also observed between DHEAS and the OPG/sRANKL ratio, OC and the OPG/sRANKL ratio, and CTx and sRANKL. The correlation was negative in the case of DHEAS and CTx, DHEAS and sRANKL, CTx and the OPG/sRANKL ratio, and sRANKL and the OPG/sRANKL ratio.Discussion/DHEAS suppression in girls with anorexia nervosa was associated with a decrease in the levels of bone markers, an increase in OPG and sRANKL concentrations and a significant decrease in the OPG/sRANKL ratio. DHEAS suppression in girls with anorexia nervosa might have a harmful effect on their bone tissue, probably via a shift in the OPG/RANKL ratio toward a functional excess of sRANKL.

  9. Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

    Science.gov (United States)

    Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni; Lupia, Enrico

    2017-01-01

    CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

  10. Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

    Directory of Open Access Journals (Sweden)

    Sophie Doublier

    Full Text Available CD40/CD40 ligand (CD40L dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs. We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS, and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS, and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

  11. Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

    Science.gov (United States)

    Cardoso, João C R; Félix, Rute C; Trindade, Marlene; Power, Deborah M

    2014-12-01

    The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyes and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was

  12. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    Science.gov (United States)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  13. Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

    DEFF Research Database (Denmark)

    Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

    2002-01-01

    enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the s...... a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia....

  14. Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320)

    DEFF Research Database (Denmark)

    Hoffmann-Lücke, Elke; Arendt, Johan F B; Nissen, Peter H

    2013-01-01

    deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-transcobalamin antibodies, we precipitated both...... transcobalamin and part of sCD320. CONCLUSIONS: The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation....

  15. The soluble mannose receptor is released from the liver in cirrhotic patients, but is not associated with bacterial translocation

    DEFF Research Database (Denmark)

    Laursen, Tea L; Rødgaard-Hansen, Sidsel; Møller, Holger J

    2017-01-01

    BACKGROUND & AIMS: Intestinal bacterial translocation is involved in activation of liver macrophages in cirrhotic patients. Macrophages play a key role in liver inflammation and are involved in the pathogenesis of cirrhosis and complications. Bacterial translocation may be determined by presence...... receptor level was elevated in the hepatic vein compared with the portal vein (0.57(interquartile range 0.31) vs 0.55(0.40) mg/L, P=.005). The soluble mannose receptor levels were similar in bacterial DNA-positive and -negative patients. The soluble mannose receptor level in the portal and hepatic veins...

  16. In vivo evaluation of [11C]SA4503 as a PET ligand for mapping CNS sigma1 receptors

    International Nuclear Information System (INIS)

    Kawamura, Kazunori; Ishiwata, Kiichi; Tajima, Hisashi; Ishii, Shin-Ichi; Matsuno, Kiyoshi; Homma, Yoshio; Senda, Michio

    2000-01-01

    The potential of the 11 C-labeled selective sigma 1 receptor ligand 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine ([ 11 C]SA4503) was evaluated in vivo as a positron emission tomography (PET) ligand for mapping sigma 1 receptors in rats. SA4503 is known to have a high affinity (IC 50 17.4 nM) and a higher selectivity (sigma 1 /sigma 2 =103) for the sigma 1 receptor. A high and increasing brain uptake of [ 11 C]SA4503 was found. Pre-, co- and postinjection of cold SA4503 significantly decreased uptake of [ 11 C]SA4503 in the brain, spleen, heart, lung, and kidney in which sigma receptors are present as well as in the skeletal muscle. In the blocking study with one of four sigma receptor ligands including haloperidol, (+)-pentazocine, SA4503, and (-)-pentazocine (in the order of their affinity for sigma 1 receptor subtype), SA4503 and haloperidol significantly reduced the brain uptake of [ 11 C]SA4503 to approximately 30% of the control, but the other two benzomorphans did not. A high specific uptake of [ 11 C]SA4503 by the brain was also confirmed by ex vivo autoradiography (ARG) and PET. Ex vivo ARG showed a higher uptake in the vestibular nucleus, temporal cortex, cingulate cortex, inferior colliculus, thalamus, and frontal cortex, and a moderate uptake in the parietal cortex and caudate putamen. Peripherally, the blocking effects of the four ligands depended on their affinity for sigma 1 receptors. No 11 C-labeled metabolite was detected in the brain 30 min postinjection, whereas approximately 20% of the radioactivity was found as 11 C-labeled metabolites in plasma. These results have demonstrated that the 11 C-labeled sigma 1 receptor ligand [ 11 C]SA4503 has a potential for mapping sigma 1 receptors in the central nervous system and peripheral organs

  17. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

    Directory of Open Access Journals (Sweden)

    Fernanda A H Batista

    Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

  18. Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

    Directory of Open Access Journals (Sweden)

    Jimenez-Perez Miriam I

    2012-02-01

    Full Text Available Abstract Background Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. Results We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT. Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. Conclusions Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

  19. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co...

  20. Stereochemistry of charged nitrogen-aromatic interactions and its involvement in ligand-receptor binding

    Science.gov (United States)

    Verdonk, Marcel L.; Boks, Gertjan J.; Kooijman, Huub; Kanters, Jan A.; Kroon, Jan

    1993-04-01

    Recently, new evidence was found for the involvement of charged nitrogen-aromatic interactions in ligand-receptor binding. In this study we report two favourable orientations of a phenyl ring with respect to a R-N+(CH3)3 group, based on crystal structure statistics from the Cambridge Structural Database. In the first orientation, the phenyl ring is situated in between the substituents at about 4.5 Å from the nitrogen atom, and the ring is approximately oriented on the sphere around the nitrogen atom. In the second orientation, the phenyl ring is situated in the same direction as one of the N-C bonds at about 6.0 Å from the nitrogen atom, and the ring is tilted with respect to the sphere around the nitrogen atom. The same two orientations were also found in the crystal structures of three ligand-receptor complexes, which implies that these orientations probably play a major role in molecular recognition mechanisms.

  1. The GABAA receptor complex in hepatic encephalopathy. Autoradiographic evidence for the presence of elevated levels of a benzodiazepine receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Basile, A.S.; Ostrowski, N.L.; Gammal, S.H.; Jones, E.A.; Skolnick, P. (National Institutes of Health, Bethesda, MD (USA))

    1990-02-01

    Autoradiographic analysis was used to examine radioligand binding to benzodiazepine (BZ) and GABAA receptors in the brains of rabbits with hepatic encephalopathy (HE). Thin sections of whole brain from normal rabbits and rabbits with HE were mounted on slides and subdivided into two groups. One group was washed before incubation with radioligand, while the second group was not prewashed. (3H)Flunitrazepam binding to BZ receptors was decreased by 22% to 42% (p less than 0.05) in the cerebral cortex, superior and inferior colliculi, and cerebellum of unwashed sections from rabbits with HE compared to all other groups. The binding of (3H)Ro 15-1788 to unwashed sections from rabbits with HE was reduced by a similar degree (18% to 37%, p less than 0.05) in the cerebral cortex, hippocampus, superior colliculus, and cerebellar cortex. Incubation of sections with the GABA-mimetic muscimol and NaCl produced an additional decrease in (3H)flunitrazepam binding to the cortex and hippocampus (25% to 31%, p less than 0.05) in unwashed HE rabbit brain, but increased radioligand binding (27% to 71%, p less than 0.05) to several regions in control rabbits. No changes in radioligand binding to either GABAA or peripheral benzodiazepine receptors was observed between HE and control rabbit sections. These findings are consistent with previous electrophysiologic and neurochemical observations indicating no significant changes in either the function or density of GABAA or BZ receptors in this model of HE. Further, they indicate that a reversible BZ receptor ligand with agonist properties is present in the brain in HE. This substance may contribute to the enhancement of GABAergic tone observed in this syndrome.

  2. Soluble intercellular adhesion molecule-1 (sICAM-1) and soluble interleukin-2 receptors (sIL-2R) in scleroderma skin

    DEFF Research Database (Denmark)

    Søndergaard, Klaus; Deleuran, Mette; Heickendorff, Lene

    1998-01-01

    In order to investigate whether soluble intercellular adhesion molecule-1 (sICAM-1) and soluble interleukin-2 receptors (sIL-2R) were present in scleroderma skin, and to compare their levels to concentrations measured in plasma and clinical parameters, we examined suction blister fluid and plasma...... from 13 patients with systemic sclerosis and 11 healthy volunteers. Suction blisters and biopsies were from the transition zone between normal skin and scleroderma, and uninvolved abdominal skin. The levels of sICAM-1 and sIL-2R were significantly increased in both plasma and suction blister fluid from...

  3. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor

    International Nuclear Information System (INIS)

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao; Han, Xiang Hua; Lee, Dong-Ho; Lee, Hak-Ju; Hwang, Bang Yeon; Lee, Sung-Joon

    2012-01-01

    Highlights: ► Catalposide is a novel ligand for PPARα. ► Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid β-oxidation and synthesis. ► Catalposdie reduces hepatic triacylglycerides. ► Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPARα agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPARα agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPARα. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  4. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Han, Xiang Hua [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Dong-Ho [Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Lee, Hak-Ju [Division of Green Business Management, Department of Forest Resources Utilization, Korean Forest Research Institute, Seoul 130-712 (Korea, Republic of); Hwang, Bang Yeon, E-mail: byhwang@chungbuk.ac.kr [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Sung-Joon, E-mail: junelee@korea.ac.kr [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  5. Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

    Science.gov (United States)

    Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

    2012-04-05

    The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    Directory of Open Access Journals (Sweden)

    Tang Baozhen

    2012-10-01

    Full Text Available Abstract Background The diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM. In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists to their targets (ecdysone receptors leads to an adaptive response (resistance, is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

  7. Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

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    Chen Zhang

    2016-09-01

    Full Text Available Ca2+-sensing receptors (CaSRs play a central role in regulating extracellular calcium concentration ([Ca2+]o homeostasis and many (pathophysiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

  8. Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

    International Nuclear Information System (INIS)

    Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E.

    1991-01-01

    The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation

  9. Analysis of TNF-related apoptosis-inducing ligand and receptors and implications in thymus biology and myasthenia gravis.

    Science.gov (United States)

    Kanatli, Irem; Akkaya, Bahar; Uysal, Hilmi; Kahraman, Sevim; Sanlioglu, Ahter Dilsad

    2017-02-01

    Myasthenia Gravis is an autoantibody-mediated, neuromuscular junction disease, and is usually associated with thymic abnormalities presented as thymic tumors (~10%) or hyperplastic thymus (~65%). The exact role of thymus in Myasthenia Gravis development is not clear, yet many patients benefit from thymectomy. The apoptotic ligand TNF-Related Apoptosis-Inducing Ligand is thought to be involved in the regulation of thymocyte counts, although conflicting results are reported. We investigated differential expression profiles of TNF-Related Apoptosis-Inducing Ligand and its transmembrane receptors, Nuclear Factor-kB activation status, and apoptotic cell counts in healthy thymic tissue and pathological thymus from Myasthenia Gravis patients. All tissues expressed TNF-Related Apoptosis-Inducing Ligand and its receptors, with hyperplastic tissue having the highest expression levels of death receptors DR4 and DR5. No detectable Nuclear Factor-kB activation, at least via the canonical Protein Kinase A-mediated p65 Ser276 phosphorylation, was evident in any of the tissues studied. Apoptotic cell counts were higher in MG-associated tissue compared to the normal thymus. Possible use of the TNF-Related Apoptosis-Inducing Ligand within the concept of an apoptotic ligand-mediated medical thymectomy in thymoma- or thymic hyperplasia-associated Myasthenia Gravis is also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Synthesis and Evaluation of Orexin-1 Receptor Antagonists with Improved Solubility and CNS Permeability.

    Science.gov (United States)

    Perrey, David A; Decker, Ann M; Zhang, Yanan

    2018-03-21

    Orexins are hypothalamic neuropeptides playing important roles in many functions including the motivation of addictive behaviors. Blockade of the orexin-1 receptor has been suggested as a potential strategy for the treatment of drug addiction. We have previously reported OX 1 receptor antagonists based on the tetrahydroisoquinoline scaffold with excellent OX 1 potency and selectivity; however, these compounds had high lipophilicity (clogP > 5) and low to moderate solubility. In an effort to improve their properties, we have designed and synthesized a series of analogues where the 7-position substituents known to favor OX 1 potency and selectivity were retained, and groups of different nature were introduced at the 1-position where substitution was generally tolerated as demonstrated in previous studies. Compound 44 with lower lipophilicity (clogP = 3.07) displayed excellent OX 1 potency ( K e = 5.7 nM) and selectivity (>1,760-fold over OX 2 ) in calcium mobilization assays. In preliminary ADME studies, 44 showed excellent kinetic solubility (>200 μM), good CNS permeability ( P app = 14.7 × 10 -6 cm/sec in MDCK assay), and low drug efflux (efflux ratio = 3.3).

  11. Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ activation function 1 (AF1 domain.

    Directory of Open Access Journals (Sweden)

    Rolf Diezko

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1 domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

  12. Comparative metabolomics reveals endogenous ligands of DAF-12, a nuclear hormone receptor regulating C. elegans development and lifespan

    Science.gov (United States)

    Mahanti, Parag; Bose, Neelanjan; Bethke, Axel; Judkins, Joshua C.; Wollam, Joshua; Dumas, Kathleen J.; Zimmerman, Anna M.; Campbell, Sydney L.; Hu, Patrick J.; Antebi, Adam; Schroeder, Frank C.

    2014-01-01

    SUMMARY Small-molecule ligands of nuclear hormone receptors (NHRs) govern the transcriptional regulation of metazoan development, cell differentiation, and metabolism. However, the physiological ligands of many NHRs remain poorly characterized primarily due to lack of robust analytical techniques. Using comparative metabolomics, we identified endogenous steroids that act as ligands of the C. elegans NHR, DAF-12, a vitamin-D and liver-X receptor homolog regulating larval development, fat metabolism, and lifespan. The identified molecules feature unexpected chemical modifications and include only one of two DAF-12 ligands reported earlier, necessitating a revision of previously proposed ligand biosynthetic pathways. We further show that ligand profiles are regulated by a complex enzymatic network including the Rieske oxygenase DAF-36, the short-chain dehydrogenase DHS-16, and the hydroxysteroid dehydrogenase, HSD-1. Our results demonstrate the advantages of comparative metabolomics over traditional candidate-based approaches and provide a blueprint for the identification of ligands for other C. elegans and mammalian NHRs. PMID:24411940

  13. Treatment with soluble activin type IIB-receptor improves bone mass and strength in a mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Puolakkainen, Tero; Ma, Hongqian; Kainulainen, Heikki; Pasternack, Arja; Rantalainen, Timo; Ritvos, Olli; Heikinheimo, Kristiina; Hulmi, Juha J; Kiviranta, Riku

    2017-01-19

    Inhibition of activin/myostatin pathway has emerged as a novel approach to increase muscle mass and bone strength. Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to progressive muscle degeneration and also high incidence of fractures. The aim of our study was to test whether inhibition of activin receptor IIB ligands with or without exercise could improve bone strength in the mdx mouse model for DMD. Thirty-two mdx mice were divided to running and non-running groups and to receive either PBS control or soluble activin type IIB-receptor (ActRIIB-Fc) once weekly for 7 weeks. Treatment of mdx mice with ActRIIB-Fc resulted in significantly increased body and muscle weights in both sedentary and exercising mice. Femoral μCT analysis showed increased bone volume and trabecular number (BV/TV +80%, Tb.N +70%, P treatment of mdx mice with the soluble ActRIIB-Fc results in a robust increase in bone mass, without any additive effect by voluntary running. Thus ActRIIB-Fc could be an attractive option in the treatment of musculoskeletal disorders.

  14. Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

    Science.gov (United States)

    Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

    2015-12-01

    We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Lipid domain formation and ligand-receptor distribution in lipid bilayer membranes investigated by atomic force microscopy

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, O.G.; Jørgensen, K.

    2002-01-01

    A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid-supported l......A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid...

  16. sigma receptor ligands attenuate N-methyl-D-aspartate cytotoxicity in dopaminergic neurons of mesencephalic slice cultures.

    Science.gov (United States)

    Shimazu, S; Katsuki, H; Takenaka, C; Tomita, M; Kume, T; Kaneko, S; Akaike, A

    2000-01-28

    We investigated the potential neuroprotective effects of several sigma receptor ligands in organotypic midbrain slice cultures as an excitotoxicity model system. When challenged with 100-microM N-methyl-D-aspartate (NMDA) for 24 h, dopaminergic neurons in midbrain slice cultures degenerated, and this was prevented by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5, 10-imine (MK-801; 1-10 microM). Concomitant application of ifenprodil (1-10 microM) or haloperidol (1-10 microM), both of which are high-affinity sigma receptor ligands, significantly attenuated the neurotoxicity of 100 microM NMDA. The sigma(1) receptor-selective ligand (+)-N-allylnormetazocine ((+)-SKF 10047; 1-10 microM) was also effective in attenuating the toxicity of NMDA. The effect of R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ((-)-PPAP), a sigma receptor ligand with negligible affinity for the phencyclidine site of NMDA receptors, was also examined. (-)-PPAP (3-100 microM) caused a concentration-dependent reduction of NMDA cytotoxicity, with significant protection at concentrations of 30 and 100 microM. In contrast, (+)-SKF 10047 (10 microM) and (-)-PPAP (100 microM) showed no protective effects against cell death induced by the Ca(2+) ionophore ionomycin (1-3 microM). These results indicate that sigma receptor ligands attenuate the cytotoxic effects of NMDA on midbrain dopaminergic neurons, possibly via inhibition of NMDA receptor functions.

  17. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  18. Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

    Science.gov (United States)

    Watson, Brittany M; Oliveria, John Paul; Nusca, Graeme M; Smith, Steven G; Beaudin, Sue; Dua, Benny; Watson, Rick M; Assayag, Evelynne Israël; Cormier, Yvon F; Sehmi, Roma; Gauvreau, Gail M

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses. © 2015 S. Karger AG, Basel.

  19. Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation

    Directory of Open Access Journals (Sweden)

    Byron Carpenter

    2017-12-01

    Full Text Available Adenosine receptors (ARs comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs. ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR, making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.

  20. Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during Plasmodium falciparum invasion of erythrocytes.

    Directory of Open Access Journals (Sweden)

    Greta E Weiss

    2015-02-01

    Full Text Available During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1 an early heparin-blockable interaction which weakly deforms the erythrocyte, 2 EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3 a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4 an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.

  1. Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells.

    LENUS (Irish Health Repository)

    O'Callaghan, G

    2012-02-03

    Fas ligand (FasL\\/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).

  2. SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands

    Science.gov (United States)

    Huber, Sylwia; Casagrande, Fabio; Hug, Melanie N.; Wang, Lisha; Heine, Philipp; Kummer, Lutz; Plückthun, Andreas; Hennig, Michael

    2017-01-01

    The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments. PMID:28510609

  3. A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

    Science.gov (United States)

    Tiefenbach, Jens; Moll, Pamela R; Nelson, Meryl R; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M

    2010-03-22

    Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

  4. A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

    Directory of Open Access Journals (Sweden)

    Jens Tiefenbach

    2010-03-01

    Full Text Available Nuclear receptors (NRs belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio. The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1 respond as expected to endogenous zebrafish hormones and cofactors, 2 facilitate efficient receptor and cofactor purification, 3 respond robustly to NR hormones and drugs and 4 yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

  5. 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.

  6. Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor.

    Science.gov (United States)

    Delanoue, Renald; Meschi, Eleonora; Agrawal, Neha; Mauri, Alessandra; Tsatskis, Yonit; McNeill, Helen; Léopold, Pierre

    2016-09-30

    Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. Here, we identify Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. We further show, using genetic and ex vivo organ culture experiments, that the Mth ligand Stunted (Sun) is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients. Copyright © 2016, American Association for the Advancement of Science.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  8. Synthesis of the sup 11 C-labelled. beta. -adrenergic receptor ligands atenolol, metoprolol and propanolol

    Energy Technology Data Exchange (ETDEWEB)

    Antoni, G.; Ulin, J.; Laangstroem, B. (Uppsala Univ. (Sweden). Dept. of Organic Chemistry)

    1989-01-01

    The {sup 11}C-labelled {beta}-adrenergic receptor ligands atenolol 1, metoprolol 2 and propranolol 3 have been synthesized by an N-alkylation reaction using (2-{sup 11}C)isopropyl iodide. The labelled isopropyl iodide was prepared in a one-pot reactor system from ({sup 11}C)carbon dioxide and obtained in 40% radiochemical yield within 14 min reaction time. The total reaction times for compounds 1-3, counted from the start of the isopropyl iodide synthesis and including purification were 45-55 min. The products were obtained in 5-15% radiochemical yields and with radiochemical purities higher than 98%. The specific activity ranged from 0.4 to 4 GBq/{mu}mol. In a typical experiment starting with 4 GBq around 75 MBq of product was obtained. (author).

  9. Development of Fluorinated Non-Peptidic Ghrelin Receptor Ligands for Potential Use in Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Rareş-Petru Moldovan

    2017-04-01

    Full Text Available The ghrelin receptor (GhrR is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S-6-(4-bromo-2-fluorophenoxy-3-((1-isopropylpiperidin-3-ylmethyl-2-methylpyrido[3,2-d]pyrimidin-4(3H-one ((S-9 has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S-9, (R-9, and (S-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET radiotracers to enable future investigation of GhrR in the brain.

  10. Modulation of seizure activity in mice by metabotropic glutamate receptor ligands

    DEFF Research Database (Denmark)

    Dalby, Nils Ole; Thomsen, C

    1996-01-01

    The anticonvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in different seizure models by use of intracerebroventricular infusion. The mGluR1a antagonist/mGluR2 agonist, (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] dose-dependently antagonized...... pentylenetetrazol- and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-2-carboxylate (DMCM)-induced clonic convulsions in mice with ED50 values of 400 and 180 nmol/mice, respectively. A modest increase in electrical seizure threshold was observed in mice injected with (S)-4C3HPG. No effect on seizures induced...... by systemic administration of N-methyl-D-aspartate was observed by prior intracerebroventricular infusion of (S)-4C3HPG. The more selective (but less potent) mGluR1a antagonist, (S)-4-carboxyphenylglycine, was a weak anticonvulsant in similar seizure models with the exception of convulsions induced...

  11. AMP and adenosine are both ligands for adenosine 2B receptor signaling.

    Science.gov (United States)

    Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

    2018-01-15

    Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Binding-Site Compatible Fragment Growing Applied to the Design of β2-Adrenergic Receptor Ligands.

    Science.gov (United States)

    Chevillard, Florent; Rimmer, Helena; Betti, Cecilia; Pardon, Els; Ballet, Steven; van Hilten, Niek; Steyaert, Jan; Diederich, Wibke E; Kolb, Peter

    2018-02-08

    Fragment-based drug discovery is intimately linked to fragment extension approaches that can be accelerated using software for de novo design. Although computers allow for the facile generation of millions of suggestions, synthetic feasibility is however often neglected. In this study we computationally extended, chemically synthesized, and experimentally assayed new ligands for the β 2 -adrenergic receptor (β 2 AR) by growing fragment-sized ligands. In order to address the synthetic tractability issue, our in silico workflow aims at derivatized products based on robust organic reactions. The study started from the predicted binding modes of five fragments. We suggested a total of eight diverse extensions that were easily synthesized, and further assays showed that four products had an improved affinity (up to 40-fold) compared to their respective initial fragment. The described workflow, which we call "growing via merging" and for which the key tools are available online, can improve early fragment-based drug discovery projects, making it a useful creative tool for medicinal chemists during structure-activity relationship (SAR) studies.

  13. Role of Killer Immunoglobulin-Like Receptor and Ligand Matching in Donor Selection

    Directory of Open Access Journals (Sweden)

    Meral Beksaç

    2012-01-01

    Full Text Available Despite all efforts to improve HLA typing and immunosuppression, it is still impossible to prevent severe graft versus host disease (GVHD which can be fatal. GVHD is not always associated with graft versus malignancy and can prevent stem cell transplantation from reaching its goals. Overall T-cell alloreactivity is not the sole mechanism modulating the immune defense. Innate immune system has its own antigens, ligands, and mediators. The bridge between HLA and natural killer (NK cell-mediated reactions is becoming better understood in the context of stem cell transplantation. Killer immunoglobulin-like receptors (KIRs constitute a wide range of alleles/antigens segregated independently from the HLA alleles and classified into two major haplotypes which imprints the person's ability to suppress or to amplify T-cell alloreactivity. This paper will summarize the impact of both activating and inhibitory KIRs and their ligands on stem cell transplantation outcome. The ultimate goal is to develop algorithms based on KIR profiles to select donors with maximum antileukemic and minimum antihost effects.

  14. CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands.

    Science.gov (United States)

    Lamas, Bruno; Richard, Mathias L; Leducq, Valentin; Pham, Hang-Phuong; Michel, Marie-Laure; Da Costa, Gregory; Bridonneau, Chantal; Jegou, Sarah; Hoffmann, Thomas W; Natividad, Jane M; Brot, Loic; Taleb, Soraya; Couturier-Maillard, Aurélie; Nion-Larmurier, Isabelle; Merabtene, Fatiha; Seksik, Philippe; Bourrier, Anne; Cosnes, Jacques; Ryffel, Bernhard; Beaugerie, Laurent; Launay, Jean-Marie; Langella, Philippe; Xavier, Ramnik J; Sokol, Harry

    2016-06-01

    Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9(-/-) mice are more susceptible to colitis. The microbiota is altered in Card9(-/-) mice, and transfer of the microbiota from Card9(-/-) to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9(-/-) mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

  15. Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Babich, J.W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States); Graham, W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States); Fischman, A.J. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States)

    1997-05-01

    The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [{sup 125}I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were with varying concentrations of {alpha}-agonist (clonidine, methoxamine, and xylazine), {alpha}-antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), {beta}-antagonist (propranolol, atenolol), {beta}-agonist (isoprenaline and salbutamol), mixed {alpha}/{beta} antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [{sup 125}I]MIBG (0.1 {mu}M). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [{sup 125}I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [{sup 125}I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. In conclusion, the results of this study establish that selected adrenergic ligands can significantly influence the pattern of uptake and storage of MIBG in cultured neuroblastoma cells, most likely through inhibition of uptake or through noncompetitive inhibition. The potential inplications of these findings justify further study. (orig./VHE). With 4 figs., 1 tab.

  16. Conformational study on cyclic melanocortin ligands and new insight into their binding mode at the MC4 receptor.

    Science.gov (United States)

    Grieco, Paolo; Brancaccio, Diego; Novellino, Ettore; Hruby, Victor J; Carotenuto, Alfonso

    2011-09-01

    The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. Understanding the basis of the ligand-receptor interactions is crucial for the design of potent and selective ligands for these receptors. The conformational preferences of the cyclic melanocortin ligands MTII (Ac-Nle(4)-c[Asp(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and SHU9119 (Ac-Nle(4)-c[Asp(5)-His(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), which show agonist and antagonist activity at the h-MC4R, respectively, were comprehensively investigated by solution NMR spectroscopy in different environments. In particular, water and water/DMSO (8:2) solutions were used as isotropic solutions and an aqueous solution of DPC (dodecylphosphocholine) micelles was used as a membrane mimetic environment. NMR-derived conformations of these two ligands were docked within h-MC4R models. NMR and docking studies revealed intriguing differences which can help explain the different activities of these two ligands. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Convergent [18]F-labeling and evaluation of N-benzyl-phenethylamines as 5-HT2A receptor PET ligands

    DEFF Research Database (Denmark)

    Petersen, Ida Nymann; Villadsen, Jonas; Hansen, Hanne Demant

    2016-01-01

    Positron emission tomography (PET) investigations of the 5-HT2A receptor (5-HT2AR) system can be used as a research tool in diseases such as depression, Alzheimer's disease and schizophrenia. We have previously developed a (11)C-labeled agonist PET ligand ([(11)C]Cimbi-36), and the aim of this st......Positron emission tomography (PET) investigations of the 5-HT2A receptor (5-HT2AR) system can be used as a research tool in diseases such as depression, Alzheimer's disease and schizophrenia. We have previously developed a (11)C-labeled agonist PET ligand ([(11)C]Cimbi-36), and the aim...... of this study was to identify a (18)F-labeled analogue of this PET-ligand. Thus, we developed a convergent radiochemical approach giving easy access to 5 different (18)F-labeled ligands structurally related to Cimbi-36 from a common (18)F-labeled intermediate. After intravenous injection, all ligands entered...... the pig brain. However, since within-scan intervention with ketanserin, a known orthosteric 5-HT2A receptor antagonist, did not result in significant blocking, the radioligands seem unsuitable for neuroimaging of the 5-HT2AR in vivo....

  18. Quantitative ligand and receptor binding studies reveal the mechanism of interleukin-36 (IL-36) pathway activation.

    Science.gov (United States)

    Zhou, Li; Todorovic, Viktor; Kakavas, Steve; Sielaff, Bernhard; Medina, Limary; Wang, Leyu; Sadhukhan, Ramkrishna; Stockmann, Henning; Richardson, Paul L; DiGiammarino, Enrico; Sun, Chaohong; Scott, Victoria

    2018-01-12

    IL-36 cytokines signal through the IL-36 receptor (IL-36R) and a shared subunit, IL-1RAcP (IL-1 receptor accessory protein). The activation mechanism for the IL-36 pathway is proposed to be similar to that of IL-1 in that an IL-36R agonist (IL-36α, IL-36β, or IL-36γ) forms a binary complex with IL-36R, which then recruits IL-1RAcP. Recent studies have shown that IL-36R interacts with IL-1RAcP even in the absence of an agonist. To elucidate the IL-36 activation mechanism, we considered all possible binding events for IL-36 ligands/receptors and examined these events in direct binding assays. Our results indicated that the agonists bind the IL-36R extracellular domain with micromolar affinity but do not detectably bind IL-1RAcP. Using surface plasmon resonance (SPR), we found that IL-1RAcP also does not bind IL-36R when no agonist is present. In the presence of IL-36α, however, IL-1RAcP bound IL-36R strongly. These results suggested that the main pathway to the IL-36R·IL-36α·IL-1RAcP ternary complex is through the IL-36R·IL-36α binary complex, which recruits IL-1RAcP. We could not measure the binding affinity of IL-36R to IL-1RAcP directly, so we engineered a fragment crystallizable-linked construct to induce IL-36R·IL-1RAcP heterodimerization and predicted the binding affinity during a complete thermodynamic cycle to be 74 μm The SPR analysis also indicated that the IL-36R antagonist IL-36Ra binds IL-36R with higher affinity and a much slower off rate than the IL-36R agonists, shedding light on IL-36 pathway inhibition. Our results reveal the landscape of IL-36 ligand and receptor interactions, improving our understanding of IL-36 pathway activation and inhibition. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ.

    Directory of Open Access Journals (Sweden)

    Yassmine Chebaro

    Full Text Available Retinoic acid (RA plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs, which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD, and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E, which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340 are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

  20. Ligand receptor dynamics at streptavidin-coated particle surfaces: A flow cytometric and spectrofluorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Buranda, T. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Univ. of New Mexico, Albuquerque, NM (United States); Jones, G.M. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States); Nolan, J.P.; Keij, J. [Los Alamos National Labs., NM (United States); Lopez, G.P. [Univ. of New Mexico, Albuquerque, NM (United States); Sklar, L.A. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Los Alamos National Lab., NM (United States)

    1999-04-29

    The authors have studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 {micro}m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. They have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. The authors estimate the site number to be {approx}1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is {approx}(1.3 {+-} 0.3) {times} 10{sup 7} 1{sup {minus}1} S{sup {minus}1}. The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s{sup {minus}1} shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

  1. Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

    International Nuclear Information System (INIS)

    Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

    1986-01-01

    Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

  2. Complete relaxation and conformational exchange matrix (CORCEMA) analysis of intermolecular saturation transfer effects in reversibly forming ligand-receptor complexes.

    Science.gov (United States)

    Jayalakshmi, V; Krishna, N Rama

    2002-03-01

    A couple of recent applications of intermolecular NOE (INOE) experiments as applied to biomolecular systems involve the (i) saturation transfer difference NMR (STD-NMR) method and (ii) the intermolecular cross-saturation NMR (ICS-NMR) experiment. STD-NMR is a promising tool for rapid screening of a large library of compounds to identify bioactive ligands binding to a target protein. Additionally, it is also useful in mapping the binding epitopes presented by a bioactive ligand to its target protein. In this latter application, the STD-NMR technique is essentially similar to the ICS-NMR experiment, which is used to map protein-protein or protein-nucleic acid contact surfaces in complexes. In this work, we present a complete relaxation and conformational exchange matrix (CORCEMA) theory (H. N. B. Moseley et al., J. Magn. Reson. B 108, 243-261 (1995)) applicable for these two closely related experiments. As in our previous work, we show that when exchange is fast on the relaxation rate scale, a simplified CORCEMA theory can be formulated using a generalized average relaxation rate matrix. Its range of validity is established by comparing its predictions with those of the exact CORCEMA theory which is valid for all exchange rates. Using some ideal model systems we have analyzed the factors that influence the ligand proton intensity changes when the resonances from some protons on the receptor protein are saturated. The results show that the intensity changes in the ligand signals in an intermolecular NOE experiment are very much dependent upon: (1) the saturation time, (2) the location of the saturated receptor protons with respect to the ligand protons, (3) the conformation of the ligand-receptor interface, (4) the rotational correlation times for the molecular species, (5) the kinetics of the reversibly forming complex, and (6) the ligand/receptor ratio. As an example of a typical application of the STD-NMR experiment we have also simulated the STD effects for a

  3. Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jacob Lauwring, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Schrøder, Tenna Juul; Christensen, Søren [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Strandbygård, Dorthe [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Pallesen, Lone Tjener [Aarhus University, Ole Worms Allé 3, 8000 Aarhus C (Denmark); García-Alai, Maria Marta [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Lindberg, Samsa; Langgård, Morten; Eskildsen, Jørgen Calí; David, Laurent; Tagmose, Lena; Simonsen, Klaus Baek; Maltas, Philip James; Rønn, Lars Christian Biilmann; Jong, Inge E. M. de; Malik, Ibrahim John; Egebjerg, Jan; Karlsson, Jens-Jacob [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Uppalanchi, Srinivas; Sakumudi, Durga Rao; Eradi, Pradheep [GVK BioScience, Plot No. 28 A, IDA Nacharam, Hyderabad 500 076 (India); Watson, Steven P., E-mail: jla@mb.au.dk [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Thirup, Søren, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark)

    2014-02-01

    The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.

  4. Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

    International Nuclear Information System (INIS)

    Andersen, Jacob Lauwring; Schrøder, Tenna Juul; Christensen, Søren; Strandbygård, Dorthe; Pallesen, Lone Tjener; García-Alai, Maria Marta; Lindberg, Samsa; Langgård, Morten; Eskildsen, Jørgen Calí; David, Laurent; Tagmose, Lena; Simonsen, Klaus Baek; Maltas, Philip James; Rønn, Lars Christian Biilmann; Jong, Inge E. M. de; Malik, Ibrahim John; Egebjerg, Jan; Karlsson, Jens-Jacob; Uppalanchi, Srinivas; Sakumudi, Durga Rao; Eradi, Pradheep; Watson, Steven P.; Thirup, Søren

    2014-01-01

    The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine

  5. Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

    Science.gov (United States)

    Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

    2017-01-01

    Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

  6. The soluble transcobalamin receptor (sCD320) in relation to Alzheimer's disease and cognitive scores

    DEFF Research Database (Denmark)

    Abuyaman, Omar; Combrinck, Marc; Smith, A David

    2017-01-01

    The soluble transcobalamin receptor (sCD320) is present in cerebrospinal fluid and correlates with the dementia-related biomarkers phospho-tau and total-tau. Here we present data on the relation of sCD320 to Alzheimer's disease and scores of cognitive tests. Lumbar cerebrospinal fluid samples from...... 42 pathologically-confirmed cases of Alzheimer's disease and 25 non-demented controls were analyzed for sCD320 employing an in-house ELISA. The participants' cognitive functions were tested using the Cambridge Cognition Examination (CAMCOG) and the Mini-Mental State Examination (MMSE...... be employed as a biomarker for differentiating Alzheimer dementia patients from controls. Further studies are warranted to explore the non-linear correlations between sCD320 and scores of cognitive function....

  7. Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Patients With Schizophrenia

    DEFF Research Database (Denmark)

    Nielsen, Jimmi; Røge, Rasmus; Pristed, Sofie Gry

    2015-01-01

    PAR) is a protein that can be measured in blood samples and reflects the levels of inflammatory activity. It has been associated with mortality and the development of type 2 diabetes and cardiovascular disease. METHODS: suPAR levels in patients with schizophrenia were compared to healthy controls from the Danish......BACKGROUND: The etiology of schizophrenia remains largely unknown but alterations in the immune system may be involved. In addition to the psychiatric symptoms, schizophrenia is also associated with up to 20 years reduction in life span. Soluble urokinase-type plasminogen activator receptor (su...... Blood Donor Study. SuPAR levels were dichotomized at >4.0 ng/ml, which is considered the threshold for low grade inflammation. A multiple logistic regression model was used and adjusted for age, sex, and current smoking. RESULTS: In total we included 1009 subjects, 105 cases with schizophrenia (10...

  8. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    International Nuclear Information System (INIS)

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni; Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin; Jiang, Tao

    2006-01-01

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents

  9. Fluorescent-labeled ligands for the benzodiazepine receptor - Part 1 : Synthesis and characterization of fluorescent-labeled benzodiazepines

    NARCIS (Netherlands)

    Janssen, M.J; Hulst, A.J R L; Kellogg, R.M; Hendriks, M.M W B; Ensing, K; de Zeeuw, R.A

    Because radioactive labeled ligands in receptor assays have several disadvantages, we synthesized a number of fluorescent-labeled benzodiazepines. Several fluorophores were attached at different positions of 1,4-benzodiazepine molecules in order to assess the impact of the fluorophores and their

  10. Combining Stochastic Deformation/Relaxation and Intermolecular Contacts Analysis for Extracting Pharmacophores from Ligand-Receptor Complexes.

    Science.gov (United States)

    Hatmal, Ma'mon M; Taha, Mutasem O

    2018-04-23

    We previously combined molecular dynamics (classical or simulated annealing) with ligand-receptor contacts analysis as a means to extract valid pharmacophore model(s) from single ligand-receptor complexes. However, molecular dynamics methods are computationally expensive and time-consuming. Here we describe a novel method for extracting valid pharmacophore model(s) from a single crystallographic structure within a reasonable time scale. The new method is based on ligand-receptor contacts analysis following energy relaxation of a predetermined set of randomly deformed complexes generated from the targeted crystallographic structure. Ligand-receptor contacts maintained across many deformed/relaxed structures are assumed to be critical and used to guide pharmacophore development. This methodology was implemented to develop valid pharmacophore models for PI3K-γ, RENIN, and JAK1. The resulting pharmacophore models were validated by receiver operating characteristic (ROC) analysis against inhibitors extracted from the CHEMBL database. Additionally, we implemented pharmacophores extracted from PI3K-γ to search for new inhibitors from the National Cancer Institute list of compounds. The process culminated in new PI3K-γ/mTOR inhibitory leads of low micromolar IC 50 s.

  11. Novel mixed ligand technetium complexes as 5-HT1A receptor imaging agents

    International Nuclear Information System (INIS)

    Leon, A.; Rey, A.; Mallo, L.; Pirmettis, I.; Papadopoulos, M.; Leon, E.; Pagano, M.; Manta, E.; Incerti, M.; Raptopoulou, C.; Terzis, A.; Chiotellis, E.

    2002-01-01

    The synthesis, characterization and biological evaluation of two novel 3 + 1 mixed ligand 99m Tc-complexes, bearing the 1-(2-methoxyphenylpiperazine) moiety, a fragment of the true 5-HT 1A antagonist WAY 100635, is reported. Complexes at tracer level 99m TcO[(CH 3 CH 2 ) 2 NCH 2 CH 2 N(CH 2 CH 2 S) 2 ][o-CH 3 OC 6 H 4 N(CH 2 CH 2 ) 2 NCH 2 CH 2 S], 99m Tc-1, and 99m TcO[((CH 3 ) 2 CH) 2 NCH 2 CH 2 N(CH 2 CH 2 S) 2 ][o-CH 3 OC 6 H 4 N (CH 2 CH 2 ) 2 NCH 2 CH 2 S], 99m Tc-2, were prepared using 99m Tc-glucoheptonate as precursor. For structural characterization, the analogous oxorhenium complexes, Re-1 and Re-2, were prepared by ligand exchange reaction using ReOCl 3 (PPh 3 ) 2 as precursor, and characterized by elemental analysis and spectroscopic methods. Complex Re-1 was further characterized by crystallographic analysis. Labeling was performed with high yield (>85%) and radiochemical purity (>90%) using very low ligand concentration. The structure of 99m Tc complexes was established by comparative HPLC using the well-characterized oxorhenium analogues as references. In vitro binding assays demonstrated the affinity of these complexes for 5-HT 1A receptors (IC 50 : 67 and 45 nM for Re-1 and Re-2 respectively). Biological studies in mice showed the ability of 99m Tc-1 and 99m Tc-2 complexes to cross the intact blood-brain barrier (1.4 and 0.9% dose/g, respectively at 1 min post-inj.). The distribution of these complexes in various regions in rat brain is inhomogeneous. The highest ratio between areas reach and poor in 5-HT 1A receptors was calculated for complex Tc-1 at 60 min p.i. (hippocampus/cerebellum = 1.7).

  12. Novel mixed ligand technetium complexes as 5-HT1A receptor imaging agents.

    Science.gov (United States)

    León, A; Rey, A; Mallo, L; Pirmettis, I; Papadopoulos, M; León, E; Pagano, M; Manta, E; Incerti, M; Raptopoulou, C; Terzis, A; Chiotellis, E

    2002-02-01

    The synthesis, characterization and biological evaluation of two novel 3 + 1 mixed ligand 99mTc-complexes, bearing the 1-(2-methoxyphenylpiperazine) moiety, a fragment of the true 5-HT1A antagonist WAY 100635, is reported. Complexes at tracer level 99mTcO[(CH3CH2)2NCH2CH2N(CH2CH2S)2][o-CH3OC6H4N(CH2CH2)2NCH2CH2S], 99mTc-1, and 99mTcO[((CH3)2CH)2NCH2CH2N(CH2CH2S)2][o-CH3OC6H4N (CH2CH2)2NCH2CH2S], 99mTc-2, were prepared using 99mTc-glucoheptonate as precursor. For structural characterization, the analogous oxorhenium complexes, Re-1 and Re-2, were prepared by ligand exchange reaction using ReOCl3(PPh3)2 as precursor, and characterized by elemental analysis and spectroscopic methods. Complex Re-1 was further characterized by crystallographic analysis. Labeling was performed with high yield (>85%) and radiochemical purity (>90%) using very low ligand concentration. The structure of 99mTc complexes was established by comparative HPLC using the well-characterized oxorhenium analogues as references. In vitro binding assays demonstrated the affinity of these complexes for 5-HT1A receptors (IC50 : 67 and 45 nM for Re-1 and Re-2 respectively). Biological studies in mice showed the ability of 99mTc-1 and 99mTc-2 complexes to cross the intact blood-brain barrier (1.4 and 0.9% dose/g, respectively at 1 min post-inj.). The distribution of these complexes in various regions in rat brain is inhomogeneous. The highest ratio between areas reach and poor in 5-HT1A receptors was calculated for complex Tc-1 at 60 min p.i. (hippocampus/cerebellum = 1.7).

  13. Novel mixed ligand technetium complexes as 5-HT{sub 1A} receptor imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Leon, A.; Rey, A. E-mail: arey@bilbo.edu.uy; Mallo, L.; Pirmettis, I.; Papadopoulos, M.; Leon, E.; Pagano, M.; Manta, E.; Incerti, M.; Raptopoulou, C.; Terzis, A.; Chiotellis, E

    2002-02-01

    The synthesis, characterization and biological evaluation of two novel 3 + 1 mixed ligand {sup 99m}Tc-complexes, bearing the 1-(2-methoxyphenylpiperazine) moiety, a fragment of the true 5-HT{sub 1A} antagonist WAY 100635, is reported. Complexes at tracer level {sup 99m}TcO[(CH{sub 3}CH{sub 2}){sub 2}NCH{sub 2}CH{sub 2}N(CH{sub 2}CH{sub 2}S){sub 2}][o-CH{sub 3}OC{sub 6}H{sub 4}N(CH{sub 2}CH{sub 2}){sub 2}NCH{sub 2}= CH{sub 2}S], {sup 99m}Tc-1, and {sup 99m}TcO[((CH{sub 3}){sub 2}CH){sub 2}NCH{sub 2}CH{sub 2}N(CH{sub 2}CH{sub 2}S){sub 2}][o-CH{sub 3}OC{sub 6}H{sub 4}N (CH{sub 2}CH{sub 2}){sub 2}NCH{sub 2}CH{sub 2}S], {sup 99m}Tc-2, were prepared using {sup 99m}Tc-glucoheptonate as precursor. For structural characterization, the analogous oxorhenium complexes, Re-1 and Re-2, were prepared by ligand exchange reaction using ReOCl{sub 3}(PPh{sub 3}){sub 2} as precursor, and characterized by elemental analysis and spectroscopic methods. Complex Re-1 was further characterized by crystallographic analysis. Labeling was performed with high yield (>85%) and radiochemical purity (>90%) using very low ligand concentration. The structure of {sup 99m}Tc complexes was established by comparative HPLC using the well-characterized oxorhenium analogues as references. In vitro binding assays demonstrated the affinity of these complexes for 5-HT{sub 1A} receptors (IC{sub 50} : 67 and 45 nM for Re-1 and Re-2 respectively). Biological studies in mice showed the ability of {sup 99m}Tc-1 and {sup 99m}Tc-2 complexes to cross the intact blood-brain barrier (1.4 and 0.9% dose/g, respectively at 1 min post-inj.). The distribution of these complexes in various regions in rat brain is inhomogeneous. The highest ratio between areas reach and poor in 5-HT{sub 1A} receptors was calculated for complex Tc-1 at 60 min p.i. (hippocampus/cerebellum = 1.7)

  14. Decreased levels of soluble Toll-like Receptor 2 in patients with asthma

    Directory of Open Access Journals (Sweden)

    Mohsen Tehrani

    2012-10-01

    Full Text Available Background: Recently, reports have indicated a role for the membrane form of Toll-like Receptor 2 (TLR2 in asthma pathogenesis. In this study we examined soluble TLR2 levels in serum and sputum of asthmatic and healthy subjects. Methods: Serum and sputum samples were obtained from 33 asthmatic and 19 healthy subjects. The asthmatics were classified into four groups according to the Global Initiative for Asthma. A sandwich ELISA was developed to measure soluble TLR2 (sTLR2 in serum and sputum. TLR2 mRNA expression was determined by semi-quantitative RT-PCR of all sputum samples. Results: The mean sTLR2 levels from serum and sputum of asthmatics were significantly lower than those from healthy subjects. Moreover, sTLR2 concentration decreased concomitantly with asthma severity. The differences observed, however, were not statistically significant. TLR2/GAPDH mRNA of sputum leukocytes was also significantly lower in asthmatics than in healthy subjects. Conclusion: This study demonstrated for the first time thatsTLR2 levels are lower in serum and sputum samples from asthmatic than from healthy subjects, and this could be an indicator of TLR2 expression. We also found that sTLR2 concentration in serum decreased concomitantly with an increase of asthma severity clinical score.

  15. Proinflammatory Soluble Interleukin-15 Receptor Alpha Is Increased in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Ana Cecilia Machado Diaz

    2012-01-01

    Full Text Available Rheumatoid arthritis (RA is an autoimmune and inflammatory disease in which many cytokines have been implicated. In particular, IL-15 is a cytokine involved in the inflammatory processes and bone loss. The aim of this study was to investigate the existence in synovial fluid of soluble IL-15Rα, a private receptor subunit for IL-15 which may act as an enhancer of IL-15-induced proinflammatory cytokines. Soluble IL-15Rα was quantified by a newly developed enzyme-linked immunosorbent assay (ELISA in samples of synovial fluid from patients with RA and osteoarthritis (OA. The levels of IL-15Rα were significantly increased in RA patients compared to OA patients. Also, we studied the presence of membrane-bound IL-15 in cells from synovial fluids, another element necessary to induce pro-inflammatory cytokines through reverse signaling. Interestingly, we found high levels of IL-6 related to high levels of IL-15Rα in RA but not in OA. Thus, our results evidenced presence of IL-15Rα in synovial fluids and suggested that its pro-inflammatory effect could be related to induction of IL-6.

  16. Changes in plasma cytokines and their soluble receptors in complex regional pain syndrome.

    Science.gov (United States)

    Alexander, Guillermo M; Peterlin, B Lee; Perreault, Marielle J; Grothusen, John R; Schwartzman, Robert J

    2012-01-01

    Complex Regional Pain Syndrome (CRPS) is a chronic and often disabling pain disorder. There is evidence demonstrating that neurogenic inflammation and activation of the immune system play a significant role in the pathophysiology of CRPS. This study evaluated the plasma levels of cytokines, chemokines, and their soluble receptors in 148 subjects afflicted with CRPS and in 60 gender- and age-matched healthy controls. Significant changes in plasma cytokines, chemokines, and their soluble receptors were found in subjects with CRPS as compared with healthy controls. For most analytes, these changes resulted from a distinct subset of the CRPS subjects. When the plasma data from the CRPS subjects was subjected to cluster analysis, it revealed 2 clusters within the CRPS population. The category identified as most important for cluster separation by the clustering algorithm was TNFα. Cluster 1 consisted of 64% of CRPS subjects and demonstrated analyte values similar to the healthy control individuals. Cluster 2 consisted of 36% of the CRPS subjects and demonstrated significantly elevated levels of most analytes and in addition, it showed that the increased plasma analyte levels in this cluster were correlated with disease duration and severity. The identification of biomarkers that define disease subgroups can be of great value in the design of specific therapies and of great benefit to the design of clinical trials. It may also aid in advancing our understanding of the mechanisms involved in the pathophysiology of CRPS, which may lead to novel treatments for this very severe condition. Copyright © 2012 American Pain Society. Published by Elsevier Inc. All rights reserved.

  17. Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages

    International Nuclear Information System (INIS)

    Schlepper-Schaefer, J.; Huelsmann, D.; Djovkar, A.; Meyer, H.E.; Herbertz, L.; Kolb, H.; Kolb-Bachofen, V.

    1986-01-01

    The intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues is rat liver was followed. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au 5 ), 17 nm (Au 17 ), 50 nm (Au 50 ) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au 5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc

  18. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    Science.gov (United States)

    Sarlati, Fatemeh; Sattari, Mandana; Razzaghi, Shilan; Nasiri, Malihe

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods: In this analytical study, GCF was obtained from healthy (n = 10), mild chronic periodontitis (n = 18), moderate chronic periodontitis (n = 18), severe chronic periodontitis (n = 20), and generalized aggressive periodontitis (n = 20) subjects. RANKL and OPG concentrations were measured by enzyme-linked immunosorbent assay. Statistical tests used were Kruskal–Wallis test, Mann–Whitney U rank sum test, and Spearman's rank correlation analysis. The level of statistical significance was set at P chronic periodontitis (mild, moderate, severe), and aggressive periodontitis (P = 0.41). There was statistically significant correlation between the concentration of sRANKL and Clinical Attachment Level (CAL) in moderate chronic periodontitis patients (R = 0.48, P = 0.04). There was also negative correlation between OPG concentration and CAL in moderate chronic periodontitis patients, although not significant (R = −0.13). Conclusion: RANKL was prominent in periodontitis sites, especially in moderate periodontitis patients, whereas OPG was not detectable in some diseased sites with bleeding on probing, supporting the role of these two molecules in the bone loss developed in this disease. PMID:23559954

  19. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    Science.gov (United States)

    Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-08-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

  20. Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between endodermis and phloem.

    Science.gov (United States)

    Uchida, Naoyuki; Lee, Jin Suk; Horst, Robin J; Lai, Hung-Hsueh; Kajita, Ryoko; Kakimoto, Tatsuo; Tasaka, Masao; Torii, Keiko U

    2012-04-17

    Multicellular organisms achieve final body shape and size by coordinating cell proliferation, expansion, and differentiation. Loss of function in the Arabidopsis ERECTA (ER) receptor-kinase gene confers characteristic compact inflorescence architecture, but its underlying signaling pathways remain unknown. Here we report that the expression of ER in the phloem is sufficient to rescue compact er inflorescences. We further identified two Epidermal Patterning Factor-like (EPFL) secreted peptide genes, EPFL4 and EPFL6/CHALLAH (CHAL), as redundant, upstream components of ER-mediated inflorescence growth. The expression of EPFL4 or EPFL6 in the endodermis, a layer adjacent to phloem, is sufficient to rescue the er-like inflorescence of epfl4 epfl6 plants. EPFL4 and EPFL6 physically associate with ER in planta. Finally, transcriptome analysis of er and epfl4 epfl6 revealed a potential downstream component as well as a role for plant hormones in EPFL4/6- and ER-mediated inflorescence growth. Our results suggest that intercell layer communication between the endodermis and phloem mediated by peptide ligands and a receptor kinase coordinates proper inflorescence architecture in Arabidopsis.

  1. Water-soluble tetrapodal N, O ligands incorporating soft N-heterocycles for the selective complexation of Am(III) over Ln(III)

    Energy Technology Data Exchange (ETDEWEB)

    Heitzmann, M.; Gateau, Ch.; Delangle, P. [CEA Grenoble, Inac, Service de Chimie Inorganique et Biologique, UMR E 3 CEA UJF, FRE CNRS 3200, F-38054 Grenoble (France); Chareyre, L.; Miguirditchian, M.; Charbonnel, M.Ch. [CEA Marcoule, DEN, DRCP, SCPS, F-30207, Bagnols-sur-Ceze (France)

    2010-07-01

    A series of four water-soluble N, O-tetrapodal ligands derived from ethylenediamine, bearing hard acetate groups and soft N-heterocycles, either pyridine or pyrazine, was developed to study the impact of the softness of N-donors on the complexation properties with trivalent f ions. Two novel ligands of enhanced soft character, bearing three pyridines (L{sup 3py}) or three pyrazines (L{sup 3pz}), were synthesized and the related lanthanide complexes were studied in solution. The ligand containing three pyridyl-methyl moieties L{sup 3py} gives complexes with a coordination similar to EDTA, i.e. a hexa-dentate coordination mode as indicated by NMR and luminescence decays (q = 3) and stability constants in the range log {beta}{sub 110} = 6.99-9.3 (La-Lu). On the other hand, the softest molecule L{sup 3pz} forms much less stable complexes with log {beta}{sub 110} = 4.0-4.4 (La-Eu). The selective back-extraction of Am(III) from organic solutions containing 4f and 5f elements was tested with the four water-soluble complexing agents. The ligand L{sup 3pz} demonstrates poor stripping ability and selectivity. In contrast, the three ligands L{sup py}, L{sup pz} and L{sup 3py} give interesting back-extraction results with Eu/Am separation factors ranging from 36 to 46, which are significantly higher than with HEDTA. This exemplifies the role of the N-hetero-cycle softness in enhancing the separation between Am(III) and Eu(III). Interestingly, the pyrazine-based ligand, L{sup pz}, demonstrates the best stripping properties, with a distribution factor that approaches that of HEDTA in the same conditions (D{sub Am{approx}}0.3). This molecule is a good compromise between softness and hardness and forms complexes still stable at pH 3 due to its low basicity. (authors)

  2. Selective modulation of Wnt ligands and their receptors in adipose tissue by chronic hyperadiponectinemia.

    Directory of Open Access Journals (Sweden)

    Nobuhiko Wada

    Full Text Available BACKGROUND: Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue. MATERIALS AND METHODS: We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells. RESULTS: The Wnt5b, Wnt6, Frizzled 6 (Fzd6, and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6 were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII and phosphorylated Jun N-terminal kinase (p-JNK were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin. CONCLUSION: Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors

  3. Synthetic water soluble di-/tritopic molecular receptors exhibiting Ca2+/Mg2+ exchange.

    Science.gov (United States)

    Lavie-Cambot, Aurélie; Tron, Arnaud; Ducrot, Aurélien; Castet, Frédéric; Kauffmann, Brice; Beauté, Louis; Allouchi, Hassan; Pozzo, Jean-Luc; Bonnet, Célia S; McClenaghan, Nathan D

    2017-05-23

    Structural integration of two synthetic water soluble receptors for Ca 2+ and Mg 2+ , namely 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and o-aminophenol-N,N,O-triacetic acid (APTRA), respectively, gave novel di- and tritopic ionophores (1 and 2). As Mg 2+ and Ca 2+ cannot be simultaneously complexed by the receptors, allosteric control of complexation results. Potentiometric measurements established stepwise protonation constants and showed high affinity for Ca 2+ (log K = 6.08 and 8.70 for 1 and 2, respectively) and an excellent selectivity over Mg 2+ (log K = 3.70 and 5.60 for 1 and 2, respectively), which is compatible with magnesium-calcium ion exchange. While ion-exchange of a single Mg 2+ for a single Ca 2+ is possible in both 1 and 2, the simultaneous binding of two Mg 2+ by 2 appears prohibitive for replacement of these two ions by a single Ca 2+ . Ion-binding and exchange was further rationalized by DFT calculations.

  4. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

    Science.gov (United States)

    Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

    2013-01-01

    Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo. PMID:24089666

  5. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Jiexia Wen

    2013-01-01

    Full Text Available Canine parvovirus (CPV disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

  6. Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro

    International Nuclear Information System (INIS)

    Leysen, J.E.

    1981-01-01

    In vitro binding studies to serotoninergic receptors were performed using 3 H-LSD, 3 H-5-HT and 3 H-spiperone. An overwiew is given on findings using these three ligands with respect to the following: localization of specific binding sites, in various animal species, the regional distribution in the brain and periphery, the subcellular and cellular distribution. Properties of the binding sites, influence of the composition of the assay medium, binding kinetic properties, receptor regulation in vivo. Identity of the binding sites, differences between site for various 3 H-ligands, pharmacological specificity of the membranous binding sites, chemical composition of the macromolecular complex constituting the binding site. Function of the receptor. Binding affinities of 44 compounds were measured in binding assays using 3 H-spiperone and 3 H-LSD with rat frontal cortex membrane preparations and using 3 H-5-HT and 3 H-LSD with rat hippocampal membrane preparations

  7. Controlled-Deactivation CB1 Receptor Ligands as a Novel Strategy to Lower Intraocular Pressure

    Directory of Open Access Journals (Sweden)

    Sally Miller

    2018-05-01

    Full Text Available Nearly half a century has passed since the demonstration that cannabis and its chief psychoactive component Δ9-THC lowers intraocular pressure (IOP. Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a condition that places millions at risk of blindness. It is likely that Δ9-THC exerts much of its IOP-lowering effects via the activation of CB1 cannabinoid receptors. However, the initial promise of CB1 as a target for treating glaucoma has not thus far translated into a credible therapeutic strategy. We have recently shown that blocking monoacylglycerol lipase (MAGL, an enzyme that breaks the endocannabinoid 2-arachidonoyl glycerol (2-AG, substantially lowers IOP. Another strategy is to develop cannabinoid CB1 receptor agonists that are optimized for topical application to the eye. Recently we have reported on a controlled-deactivation approach where the “soft” drug concept of enzymatic deactivation was combined with a “depot effect” that is commonly observed with Δ9-THC and other lipophilic cannabinoids. This approach allowed us to develop novel cannabinoids with a predictable duration of action and is particularly attractive for the design of CB1 activators for ophthalmic use with limited or no psychoactive effects. We have tested a novel class of compounds using a combination of electrophysiology in autaptic hippocampal neurons, a well-characterized model of endogenous cannabinoid signaling, and measurements of IOP in a mouse model. We now report that AM7410 is a reasonably potent and efficacious agonist at CB1 in neurons and that it substantially (30% lowers IOP for as long as 5 h after a single topical treatment. This effect is absent in CB1 knockout mice. Our results indicate that the direct targeting of CB1 receptors with controlled-deactivation ligands is a viable approach to lower IOP in a murine model and merits further study in other model systems.

  8. Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase.

    Science.gov (United States)

    Wu, Gang; Liu, Wen; Berka, Vladimir; Tsai, Ah-Lim

    2017-09-01

    To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O 2 , including K D , k on , and k off , of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The K D (NO) and K D (CO) of each bacterial H-NOX or sGC follow the "sliding scale rule"; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  9. Soluble TNF-Alpha-Receptors I Are Prognostic Markers in TIPS-Treated Patients with Cirrhosis and Portal Hypertension

    DEFF Research Database (Denmark)

    Trebicka, Jonel; Krag, Aleksander; Gansweid, Stefan

    2013-01-01

    TNFα levels are increased in liver cirrhosis even in the absence of infection, most likely owing to a continuous endotoxin influx into the portal blood. Soluble TNFα receptors (sTNFR type I and II) reflect release of the short-lived TNFα, because they are cleaved from the cells after binding...

  10. NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1

    Czech Academy of Sciences Publication Activity Database

    Lyukmanova, E. N.; Shenkarev, Z. O.; Shulepko, M. A.; Mineev, K. S.; D´Hoedt, D.; Kasheverov, I. E.; Filkin, S. Yu.; Krivolapova, A. P.; Janíčková, Helena; Doležal, Vladimír; Dolgikh, D. A.; Arseniev, A. S.; Bertrand, D.; Tsetlin, V.I.; Kirpichnikov, M. P.

    2011-01-01

    Roč. 286, č. 12 (2011), s. 10618-10627 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : NMR structure * nicotinic acetylcholine receptor * water-soluble domain Subject RIV: FH - Neurology Impact factor: 4.773, year: 2011

  11. Serum levels of soluble urokinase plasminogen activator receptor is associated with parasitemia in children with acute Plasmodium falciparum malaria infection

    DEFF Research Database (Denmark)

    Perch, M; Kofoed, P; Fischer, TK

    2004-01-01

    Serum levels of soluble urokinase plasminogen activator receptor (suPAR) are significantly elevated and of prognostic value in patients suffering from serious infectious diseases such as HIV and tuberculosis. Our objective was to investigate suPAR levels during symptomatic malaria infection and 7...

  12. Soluble Receptor for Advanced Glycation End Product Ameliorates Chronic Intermittent Hypoxia Induced Renal Injury, Inflammation, and Apoptosis via P38/JNK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Xu Wu

    2016-01-01

    Full Text Available Obstructive sleep apnea (OSA associated chronic kidney disease is mainly caused by chronic intermittent hypoxia (CIH triggered tissue damage. Receptor for advanced glycation end product (RAGE and its ligand high mobility group box 1 (HMGB1 are expressed on renal cells and mediate inflammatory responses in OSA-related diseases. To determine their roles in CIH-induced renal injury, soluble RAGE (sRAGE, the RAGE neutralizing antibody, was intravenously administered in a CIH model. We also evaluated the effect of sRAGE on inflammation and apoptosis. Rats were divided into four groups: (1 normal air (NA, (2 CIH, (3 CIH+sRAGE, and (4 NA+sRAGE. Our results showed that CIH accelerated renal histological injury and upregulated RAGE-HMGB1 levels involving inflammatory (NF-κB, TNF-α, and IL-6, apoptotic (Bcl-2/Bax, and mitogen-activated protein kinases (phosphorylation of P38, ERK, and JNK signal transduction pathways, which were abolished by sRAGE but p-ERK. Furthermore, sRAGE ameliorated renal dysfunction by attenuating tubular endothelial apoptosis determined by immunofluorescence staining of CD31 and TUNEL. These findings suggested that RAGE-HMGB1 activated chronic inflammatory transduction cascades that contributed to the pathogenesis of the CIH-induced renal injury. Inhibition of RAGE ligand interaction by sRAGE provided a therapeutic potential for CIH-induced renal injury, inflammation, and apoptosis through P38 and JNK pathways.

  13. Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-alpha antibody

    NARCIS (Netherlands)

    Jansen, J.; van der Poll, T.; Levi, M. [=Marcel M.; ten Cate, H.; Gallati, H.; ten Cate, J. W.; van Deventer, S. J.

    1995-01-01

    The role of tumor necrosis factor-alpha in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor-alpha

  14. Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction.

    Science.gov (United States)

    Xu, L; Hu, F; Zhu, H; Liu, X; Shi, L; Li, Y; Zhong, H; Su, Y

    2018-04-01

    The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme-linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In-vitro osteoclast differentiation assay showed further that tartrate-resistant acid phosphatase (TRAP) + osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis. © 2017 British Society for Immunology.

  15. Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds.

    Directory of Open Access Journals (Sweden)

    Michael Behr

    Full Text Available Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK. This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

  16. Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

    Science.gov (United States)

    Behr, Michael; Kaufmann, Johanna K.; Ketzer, Patrick; Engelhardt, Sarah; Mück-Häusl, Martin; Okun, Pamela M.; Petersen, Gabriele; Neipel, Frank; Hassel, Jessica C.; Ehrhardt, Anja; Enk, Alexander H.; Nettelbeck, Dirk M.

    2014-01-01

    Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis. PMID:24760010

  17. Structure-Guided Screening for Functionally Selective D2 Dopamine Receptor Ligands from a Virtual Chemical Library.

    Science.gov (United States)

    Männel, Barbara; Jaiteh, Mariama; Zeifman, Alexey; Randakova, Alena; Möller, Dorothee; Hübner, Harald; Gmeiner, Peter; Carlsson, Jens

    2017-10-20

    Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D 2 dopamine receptor (D 2 R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D 2 R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D 2 R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13 000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D 2 R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and β-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated β-arrestin recruitment (EC 50 = 320 nM, E max = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.

  18. Drug of the year: programmed death-1 receptor/programmed death-1 ligand-1 receptor monoclonal antibodies.

    Science.gov (United States)

    Robert, Caroline; Soria, Jean-Charles; Eggermont, Alexander M M

    2013-09-01

    Programmed death-1 receptor (PD-1)/its ligand (PD-L1) antibodies have changed the landscape in oncology in 2013. The most mature results have been obtained in advanced melanoma patients. They indicate important response rates and high quality responses or prolonged duration. Also in renal cancer and in lung cancer remarkable activity has been demonstrated. Thus it is clear that these antibodies have a very broad potential and trials in many tumour types are being initiated. Breaking tolerance at the tumour site is a potent phenomenon and the potential for synergy with other checkpoint inhibitors such as ipilimumab have also been demonstrated in 2013. Long term tumour control now seems achievable and thus the concept of a clinical cure is emerging by modulation of the immune system. These antibodies bring immunotherapy to the forefront and indicate that immune-modulation will be a key component of therapeutic strategies from now on. Because of all these reasons PD-1/PD-L1 antibodies are considered 'drug of the year'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB₂R selective benzimidazoles reveal unexpected intrinsic properties.

    Science.gov (United States)

    Nimczick, Martin; Pemp, Daniela; Darras, Fouad H; Chen, Xinyu; Heilmann, Jörg; Decker, Michael

    2014-08-01

    The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB2R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Clofibric acid, a peroxisome proliferator-activated receptor alpha ligand, inhibits growth of human ovarian cancer.

    Science.gov (United States)

    Yokoyama, Yoshihito; Xin, Bing; Shigeto, Tatsuhiko; Umemoto, Mika; Kasai-Sakamoto, Akiko; Futagami, Masayuki; Tsuchida, Shigeki; Al-Mulla, Fahd; Mizunuma, Hideki

    2007-04-01

    Recent reports have shown that peroxisome proliferator-activated receptor (PPAR)alpha ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPARalpha on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E(2) (PGE(2)) to PGF(2alpha), in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE(2) level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3-tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE(2) level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs.

  1. Nicotine facilitates nicotinic acetylcholine receptor targeting to mitochondria but makes them less susceptible to selective ligands.

    Science.gov (United States)

    Uspenska, Kateryna; Lykhmus, Olena; Gergalova, Galyna; Chernyshov, Volodymyr; Arias, Hugo R; Komisarenko, Sergiy; Skok, Maryna

    2017-08-24

    Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200μL/L) for 7days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca 2+ or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1nM), dihydro-β-erythroidine (DhβE, 1μM), PNU-120596 (0.3, 3, or 10μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Safety, tolerability, and biomarkers of the treatment of mice with aerosolized toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Victoria eAlfaro

    2014-02-01

    Full Text Available We have previously discovered a synergistically therapeutic combination of two Toll-like receptor (TLR ligands, an oligodeoxynucleotide (ODN and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 hours after treatment, reached a peak at 48 hours, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines IL-6, TNF and CXCL2 rose several hundred-fold in lung lavage fluid 4 hours after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose five-fold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose-response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to 8-fold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage

  3. Towards a Novel Class of Multitarget-Directed Ligands: Dual P2X7–NMDA Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Olga Karoutzou

    2018-01-01

    Full Text Available Multi-target-directed ligands (MTDLs offer new hope for the treatment of multifactorial complex diseases such as Alzheimer’s Disease (AD. Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein—which is open to further optimization—represents the first member of a new class of MTDLs.

  4. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  5. Cannabinoid Type 1 Receptor (CB1) Ligands with Therapeutic Potential for Withdrawal Syndrome in Chemical Dependents of Cannabis sativa.

    Science.gov (United States)

    Ferreira, Jaderson V; Chaves, Gisele A; Marino, Bianca L B; Sousa, Kessia P A; Souza, Lucilene R; Brito, Maiara F B; Teixeira, Hueldem R C; da Silva, Carlos H T P; Santos, Cleydson B R; Hage-Melim, Lorane I S

    2017-08-22

    Cannabis sativa withdrawal syndrome is characterized mainly by psychological symptoms. By using computational tools, the aim of this study was to propose drug candidates for treating withdrawal syndrome based on the natural ligands of the cannabinoid type 1 receptor (CB1). One compound in particular, 2-n-butyl-5-n-pentylbenzene-1,3-diol (ZINC1730183, also known as stemphol), showed positive predictions as a human CB1 ligand and for facile synthetic accessibility. Therefore, ZINC1730183 is a favorable candidate scaffold for further research into pharmacotherapeutic alternatives to treat C. sativa withdrawal syndrome. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Lysosomal membrane permeabilization is an early event in Sigma-2 receptor ligand mediated cell death in pancreatic cancer.

    Science.gov (United States)

    Hornick, John R; Vangveravong, Suwanna; Spitzer, Dirk; Abate, Carmen; Berardi, Francesco; Goedegebuure, Peter; Mach, Robert H; Hawkins, William G

    2012-05-02

    Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD

  7. Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Hornick John R

    2012-05-01

    Full Text Available Abstract Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282 localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco. Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a

  8. NKG2D and its ligands in cancer.

    Science.gov (United States)

    Dhar, Payal; Wu, Jennifer D

    2018-04-01

    NKG2D is an activating immune receptor expressed by NK and effector T cells. Induced expression of NKG2D ligand on tumor cell surface during oncogenic insults renders cancer cells susceptible to immune destruction. In advanced human cancers, tumor cells shed NKG2D ligand to produce an immune soluble form as a means of immune evasion. Soluble NKG2D ligands have been associated with poor clinical prognosis in cancer patients. Harnessing NKG2D pathway is considered a viable avenue in cancer immunotherapy over recent years. In this review, we will discuss the progress and perspectives. Copyright © 2018. Published by Elsevier Ltd.

  9. Novel Chemical Strategies for Labeling Small Molecule Ligands for Androgen, Progestin, and Peroxisome Proliferator-Activated Receptors for Imaging Prostate and Breast Cancer and the Heart

    International Nuclear Information System (INIS)

    Katzenellenbogen, John A.

    2007-01-01

    Summary of Progress The specific aims of this project can be summarized as follows: Aim 1: Prepare and evaluate radiolabeled ligands for the peroxisome proliferator-activated receptor γ (PPARγ), a new nuclear hormone receptor target for tumor imaging and hormone therapy. Aim 2: Prepare steroids labeled with a cyclopentadienyl tricarbonyl technetium or rhenium unit. Aim 3: Prepare and evaluate other organometallic systems of novel design as ligand mimics and halogenated ligands for nuclear hormone receptor-based tumor imaging. As is described in detail in the report, we made excellent progress on all three of these aims; the highlights of our progress are the following: (1) we have prepared the first fluorine-18 labeled analogs of ligands for the PPARγ receptor and used these in tissue distribution studies in rats; (2) we have developed three new methods for the synthesis of cyclopentadienyltricarbonyl rhenium and technetium (CpRe(CO)3 and CpTc(CO)3) systems and we have adapted these to the synthesis of steroids labeled with these metals, as well as ligands for other receptor systems; (3) we have prepared a number of fluorine-18 labeled steroidal and non-steroidal androgens and measured their tissue distribution in rats; (4) we have prepared iodine and bromine-labeled progestins with high progesterone receptor binding affinity; and (5) we have prepared inorganic metal tricarbonyl complexes and steroid receptor ligands in which the metal tricarbonyl unit is an integral part off the ligand core

  10. Synthesis and in vivo evaluation of [11C]SA6298 as a PET sigma1 receptor ligand

    International Nuclear Information System (INIS)

    Kawamura, Kazunori; Ishiwata, Kiichi; Tajima, Hisashi; Ishii, Shin-Ichi; Shimada, Yuhei; Matsuno, Kiyoshi; Homma, Yoshio; Senda, Michio

    1999-01-01

    The potential of a 11 C-labeled selective sigma 1 receptor ligand, 1-(3,4-dimethoxyphenethyl)-4-[3-(3,4-dichlorophenyl)propyl]piperazine ([ 11 C]SA6298), was evaluated as a positron emission tomography (PET) ligand for mapping sigma 1 receptors in the central nervous system and peripheral organs. [ 11 C]SA6298 was synthesized by methylation of the desmethyl SA6298 with [ 11 C]CH 3 I, with the decay-corrected radiochemical yield of 39±5% based on [ 11 C]CH 3 I and with the specific activity of 53±17 TBq/mmol within 20 min from end of bombardment (EOB). In mice, the uptake of [ 11 C]SA6298 was significantly decreased by carrier loading in the brain, liver, spleen, heart, lung, small intestine, and kidney in which sigma receptors are present as well as in the skeletal muscle. Pretreatment with SA6298 also blocked the uptake of [ 11 C]SA6298 by these organs except for the small intestine, but significant displacement of [ 11 C]SA6298 by posttreatment with SA6298 was observed only in the heart, lung, and muscle. In the blocking study with one of the eight sigma receptor ligands, including haloperidol, SA6298, NE-100, (+)-pentazocine, SA4503, (-)-pentazocine, (+)-3-PPP, and (+)-SKF 10,047 (in the order of the affinity for sigma 1 receptor subtype), only SA6298 and an analog SA4503 significantly reduced the brain uptake of [ 11 C]SA6298 to approximately 80% of the control, but the other six ligands did not. Peripherally, the uptake of [ 11 C]SA6298 by the organs described above was decreased predominantly by SA6298 or SA4503, but the blocking effects of the other five ligands except for NE-100 depended on their affinity for sigma 1 receptors. The saturable brain uptake of [ 11 C]SA6298, approximately 20%, was also observed by tissue dissection method in rats and by PET in a cat. Ex vivo autoradiography of the rat brain showed a high uptake in the cortex and thalamus. In the cat brain a relatively high uptake was found in the cortex, thalamus, striatum, and cerebellum

  11. Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Kubas, Holger; Schaefer, Martin; Bauder-Wuest, Ulrike; Eder, Matthias; Oltmanns, Doerte [Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg (Germany); Eisenhut, Michael, E-mail: m.eisenhut@dkfz.d [Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

    2010-11-15

    Peptides involving the RGD motive (arginine-glycine-aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for {alpha}{sub v{beta}3} integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG{sub n}) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG{sub n} linkers were investigated on their binding properties to cultured {alpha}{sub v{beta}3} integrin-expressing U87MG cells. Methods: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with {sup 125}I-labeled echistatin. Results: While c(RGDyK) function is a relative weak competitor against [{sup 125}I]echistatin (K{sub i}, 329{+-}18 nM) for {alpha}{sub v{beta}3} integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K{sub i}, 64{+-}23 nM). This effect was even more pronounced with the RGD trimers (K{sub i}, 40{+-}7 nM) and tetramers (K{sub i}, 26{+-}9 nM). The introduction of EG{sub n} spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [{sup 125}I]echistatin. The EG{sub 6} group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). Conclusion: The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [{sup 125}I]echistatin with the introduction of EG{sub n} spacers. This effect

  12. Binding Mode Prediction of 5-Hydroxytryptamine 2C Receptor Ligands by Homology Modeling and Molecular Docking Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Asif; Nagarajan, Shanthi; Doddareddy, Munikumar Reddy; Cho, Yong Seo; Pae, Ae Nim [Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    2011-06-15

    Serotonin or 5-hydroxytryptamine subtype 2C (5-HT{sub 2C}) receptor belongs to class A amine subfamily of Gprotein- coupled receptor (GPCR) super family and its ligands has therapeutic promise as anti-depressant and -obesity agents. So far, bovine rhodopsin from class A opsin subfamily was the mostly used X-ray crystal template to model this receptor. Here, we explained homology model using beta 2 adrenergic receptor (β2AR), the model was energetically minimized and validated by flexible ligand docking with known agonists and antagonists. In the active site Asp134, Ser138 of transmembrane 3 (TM3), Arg195 of extracellular loop 2 (ECL2) and Tyr358 of TM7 were found as important residues to interact with agonists. In addition to these, V208 of ECL2 and N351 of TM7 was found to interact with antagonists. Several conserved residues including Trp324, Phe327 and Phe328 were also found to contribute hydrophobic interaction. The predicted ligand binding mode is in good agreement with published mutagenesis and homology model data. This new template derived homology model can be useful for further virtual screening based lead identification.

  13. Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods

    International Nuclear Information System (INIS)

    Nonaka, Yosuke; Hiramoto, Takeshi; Fujita, Norihisa

    2005-01-01

    Endogenous ligands acting on a human P2Y 12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y 12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca 2+ assay using the CHO cells stably expressing P2Y 12 -G 16 α fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y 12 receptor as agonists with the EC 50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y 12 , P2Y 13 , P2Y 14 , GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions

  14. [{sup 3}H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    Energy Technology Data Exchange (ETDEWEB)

    Benke, D.; Honer, M.; Mohler, H. [Institute of Pharmacology, ETH and University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland); Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C. [NS Research, Novartis Pharma AG, CH-4002 Basle (Switzerland)

    1999-02-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [{sup 3}H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [{sup 3}H]CGP 61594 labeled with high affinity (K{sub D}=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [{sup 3}H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [{sup 3}H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [{sup 3}H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  15. Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor.

    Science.gov (United States)

    Malo, Marcus; Persson, Ronnie; Svensson, Peder; Luthman, Kristina; Brive, Lars

    2013-03-01

    Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β1 adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D2 receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D2 receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.

  16. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    International Nuclear Information System (INIS)

    Cui Jianzhou; Shen Xueyan; Yan Zuowei; Zhao Haobin; Nagahama, Yoshitaka

    2009-01-01

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  17. Preassembly and ligand-induced restructuring of the chains of the IFN-gamma receptor complex: the roles of Jak kinases, Stat1 and the receptor chains.

    Science.gov (United States)

    Krause, Christopher D; Lavnikova, Natasha; Xie, Junxia; Mei, Erwen; Mirochnitchenko, Olga V; Jia, Yiwei; Hochstrasser, Robin M; Pestka, Sidney

    2006-01-01

    We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-gamma) receptor complex is preassembled (1). In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-gammaR1 results in a conformational change localized to IFN-gammaR1. Jak1 but not Jak2 is required for the two chains of the IFN-gamma receptor complex (IFN-gammaR1 and IFN-gammaR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-gammaR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-gammaR2 with IFN-gammaR1. These results agree with a detailed model of the IFN-gamma receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

  18. Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand-mediated internalization and down-regulation

    DEFF Research Database (Denmark)

    Allevato, G; Billestrup, N; Goujon, L

    1995-01-01

    The functional significance of growth hormone (GH) receptor (GHR) internalization is unknown; therefore, we have analyzed domains and individual amino acids in the cytoplasmic region of the rat GHR required for ligand-mediated receptor internalization, receptor down-regulation, and transcriptiona...

  19. Serum level of soluble receptor for advanced glycation end products in asthmatic children and its correlation to severity and pulmonary functions.

    Science.gov (United States)

    El-Seify, Magda Y Hussein; Fouda, Eman Mahmoud; Nabih, Enas Samir

    2014-01-01

    Soluble receptor for advanced glycation end products (sRAGE) acts as a decoy receptor for RAGE which has several distinct pro-inflammatory ligands in the extracellular compartment, and is believed to afford protection against inflammation and cell injury. This study was conducted to measure serum sRAGE in asthmatic children and to assess its correlation with clinical and functional severity and to asthma phenotype according to sputum cytology. The study was conducted on 60 asthmatic children from the Pediatric Chest Clinic, Children's Hospital, Ain Shams University. The patients were divided according to asthma control and severity. sRAGE showed statistically significant lower levels in asthmatic patients (899.1 +/- 399.8 pg/mL) compared to the control group (1406.7 +/- 474.3 pg/mL, p = 0.000), with a cut off value of asthma diagnosis of 1080.4 pg/mL with a sensitivity and specificity of 77% and 75%, respectively. Uncontrolled and severe asthmatic subgroups showed lower levels of sRAGE, cut off value of sRAGE for the severity of asthma was 829 pg/mL with 89% sensitivity and 81% specificity. Asthmatic patients stratified according to sputum cytology revealed that those with > 2% eosinophils and > or = 40% neutrophils showed lower levels of sRAGE (710 +/- 258 pg/mL) compared to those with > 2% eosinophils and functionally. It may be a target of future therapeutic interventions.

  20. Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

    NARCIS (Netherlands)

    Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2010-01-01

    Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

  1. Macrophage-related serum biomarkers soluble CD163 (sCD163) and soluble mannose receptor (sMR) to differentiate mild liver fibrosis from cirrhosis in patients with chronic hepatitis C

    DEFF Research Database (Denmark)

    Andersen, E S; Rødgaard-Hansen, S; Moessner, B

    2014-01-01

    Macrophages regulate the fibrotic process in chronic liver disease. The aim of the present pilot study was to evaluate two new macrophage-specific serum biomarkers [soluble CD163 (sCD163) and soluble mannose receptor (sMR, sCD206)] as potential fibrosis markers in patients chronically infected wi...

  2. One-pot synthesis of water soluble iron nanoparticles using rationally-designed peptides and ligand release.

    Science.gov (United States)

    Papst, Stefanie; Cheong, Soshan; Banholzer, Moritz J; Brimble, Margaret A; Williams, David E; Tilley, Richard D

    2013-05-18

    Herein we report the rational design of new phosphopeptides for control of nucleation, growth and aggregation of water-soluble, superparamagnetic iron-iron oxide core-shell nanoparticles. The use of the designed peptides enables a one-pot synthesis that avoids utilizing unstable or toxic iron precursors, organic solvents, and the need for exchange of capping agent after synthesis of the NPs.

  3. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    Science.gov (United States)

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. Impact of genetic variations in C-C chemokine receptors and ligands on infectious diseases.

    Science.gov (United States)

    Qidwai, Tabish; Khan, M Y

    2016-10-01

    Chemokine receptors and ligands are crucial for extensive immune response against infectious diseases such as malaria, leishmaniasis, HIV and tuberculosis and a wide variety of other diseases. Role of chemokines are evidenced in the activation and regulation of immune cell migration which is important for immune response against diseases. Outcome of disease is determined by complex interaction among pathogen, host genetic variability and surrounding milieu. Variation in expression or function of chemokines caused by genetic polymorphisms could be associated with attenuated immune responses. Exploration of chemokine genetic polymorphisms in therapeutic response, gene regulation and disease outcome is important. Infectious agents in human host alter the expression of chemokines via epigenetic alterations and thus contribute to disease pathogenesis. Although some fragmentary data are available on chemokine genetic variations and their contribution in diseases, no unequivocal conclusion has been arrived as yet. We therefore, aim to investigate the association of CCR5-CCL5 and CCR2-CCL2 genetic polymorphisms with different infectious diseases, transcriptional regulation of gene, disease severity and response to therapy. Furthermore, the role of epigenetics in genes related to chemokines and infectious disease are also discussed. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  5. Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

    Science.gov (United States)

    Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

    1998-08-01

    In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

  6. The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

    Science.gov (United States)

    Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

  7. The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

    Science.gov (United States)

    Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-11-13

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

  8. Forced unbinding of GPR17 ligands from wild type and R255I mutant receptor models through a computational approach

    Directory of Open Access Journals (Sweden)

    Fantucci Piercarlo

    2010-03-01

    Full Text Available Abstract Background GPR17 is a hybrid G-protein-coupled receptor (GPCR activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs, and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I to Ile. Results MD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast. Conclusions These data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.

  9. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  10. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  11. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    by chemical cross-linking, but quantitative binding/competition studies showed that the apparent ligand affinity was 100- to 1000-fold lower than that of the intact suPAR. This loss of affinity was comparable with the loss found after cleavage between the first domain (D1) and D(2 + 3), using chymotrypsin...

  12. Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

    Science.gov (United States)

    Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

    2015-01-01

    Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

  13. Exploring G Protein-Coupled Receptors (GPCRs) Ligand Space via Cheminformatics Approaches: Impact on Rational Drug Design

    Science.gov (United States)

    Basith, Shaherin; Cui, Minghua; Macalino, Stephani J. Y.; Park, Jongmi; Clavio, Nina A. B.; Kang, Soosung; Choi, Sun

    2018-01-01

    The primary goal of rational drug discovery is the identification of selective ligands which act on single or multiple drug targets to achieve the desired clinical outcome through the exploration of total chemical space. To identify such desired compounds, computational approaches are necessary in predicting their drug-like properties. G Protein-Coupled Receptors (GPCRs) represent one of the largest and most important integral membrane protein families. These receptors serve as increasingly attractive drug targets due to their relevance in the treatment of various diseases, such as inflammatory disorders, metabolic imbalances, cardiac disorders, cancer, monogenic disorders, etc. In the last decade, multitudes of three-dimensional (3D) structures were solved for diverse GPCRs, thus referring to this period as the “golden age for GPCR structural biology.” Moreover, accumulation of data about the chemical properties of GPCR ligands has garnered much interest toward the exploration of GPCR chemical space. Due to the steady increase in the structural, ligand, and functional data of GPCRs, several cheminformatics approaches have been implemented in its drug discovery pipeline. In this review, we mainly focus on the cheminformatics-based paradigms in GPCR drug discovery. We provide a comprehensive view on the ligand– and structure-based cheminformatics approaches which are best illustrated via GPCR case studies. Furthermore, an appropriate combination of ligand-based knowledge with structure-based ones, i.e., integrated approach, which is emerging as a promising strategy for cheminformatics-based GPCR drug design is also discussed. PMID:29593527

  14. Elevated soluble urokinase plasminogen activator receptor (suPAR) predicts mortality in Staphylococcus aureus bacteremia

    DEFF Research Database (Denmark)

    Mölkänen, T; Ruotsalainen, E; Thorball, C W

    2011-01-01

    The soluble form of urokinase-type plasminogen activator receptor (suPAR) is a new inflammatory marker. High suPAR levels have been shown to associate with mortality in cancer and in chronic infections like HIV and tuberculosis, but reports on the role of suPAR in acute bacteremic infections...... are scarce. To elucidate the role of suPAR in a common bacteremic infection, the serum suPAR levels in 59 patients with Staphylococcus aureus bacteremia (SAB) were measured using the suPARnostic ELISA assay and associations to 1-month mortality and with deep infection focus were analyzed. On day three, after...... the first positive blood culture for S. aureus, suPAR levels were higher in 19 fatalities (median 12.3; range 5.7-64.6 ng/mL) than in 40 survivors (median 8.4; range 3.7-17.6 ng/mL, p = 0.002). This difference persisted for 10 days. The presence of deep infection focus was not associated with elevated su...

  15. Soluble Urokinase Plasminogen Activator Receptor Levels in Tuberculosis Patients at High Risk for Multidrug Resistance

    Directory of Open Access Journals (Sweden)

    Tri Yudani Mardining Raras

    2012-01-01

    Full Text Available The soluble urokinase plasminogen activator receptor (suPAR has been shown to be a strong prognostic biomarker for tuberculosis (TB. In the present study, the profiles of plasma suPAR levels in pulmonary TB patients at high risk for multidrug resistance were analyzed and compared with those in multidrug resistant (MDR-TB patients. Forty patients were prospectively included, consisting of 10 MDR-TB patients and 30 TB patients at high risk for MDR, underwent clinical assesment. Plasma suPAR levels were measured using ELISA (SUPARnostic, Denmark and bacterial cultures were performed in addition to drug susceptibility tests. All patients of suspected MDR-TB group demonstrated significantly higher suPAR levels compared with the healthy TB-negative group (1.79 ng/mL. Among the three groups at high risk for MDR-TB, only the relapse group (7.87 ng/mL demonstrated suPAR levels comparable with those of MDR-TB patients (7.67 ng/mL. suPAR levels in the two-month negative acid-fast bacilli conversion group (9.29 ng/mL were higher than positive control, whereas levels in the group consisting of therapy failure patients (5.32 ng/mL were lower. Our results strongly suggest that suPAR levels enable rapid screening of suspected MDR-TB patients, but cannot differentiate between groups.

  16. Soluble (Prorenin Receptor and Obstructive Sleep Apnea Syndrome: Oxidative Stress in Brain?

    Directory of Open Access Journals (Sweden)

    Kazuhiro Takahashi

    2017-06-01

    Full Text Available (Prorenin receptor ((PRR is a multi-functional molecule that is related to both the renin-angiotensin system (RAS and vacuolar H+-ATPase (v-ATPase, an ATP-dependent multi-subunit proton pump. Soluble (PRR (s(PRR, which consists of the extracellular domain of (PRR, is present in blood and urine. Elevated plasma s(PRR concentrations are reported in patients with chronic kidney disease and pregnant women with hypertension or diabetes mellitus. In addition, we have shown that plasma s(PRR concentrations are elevated in patients with obstructive sleep apnea syndrome (OSAS. Interestingly, the levels are elevated in parallel with the severity of OSAS, but are not related to the presence of hypertension or the status of the circulating RAS in OSAS. It is known that v-ATPase activity protects cells from endogenous oxidative stress, and loss of v-ATPase activity results in chronic oxidative stress. We hypothesize that hypoxia and subsequent oxidative stress, perhaps in the brain, may be one of the factors that elevate plasma s(PRR levels in OSAS.

  17. Soluble Triggering Receptor Expressed on Myeloid Cells-1 as a Novel Marker for Abdominal Sepsis.

    Science.gov (United States)

    Song, Xiaofei; Song, Yucheng; Zhang, Xuedong; Xue, Huanzhou

    2017-07-01

    The aim of the study was to investigate the concentration and diagnostic significance of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in acute abdominal conditions. Plasma specimens were obtained from 68 patients with abdominal sepsis, 60 patients with systemic inflammatory response syndrome (SIRS), and 60 healthy individuals. The sepsis group was divided into the survival and death groups according to the 28-d outcome. Plasma sTREM-1, procalcitonin (PCT), C-reactive protein (CRP), and white blood cell (WBC) count were measured. A receiver operating characteristic curve (ROC) was used to compare the diagnostic values of sTREM-1, PCT, CRP, and WBC count. In addition, the correlation between plasma sTREM-1 and the Acute Physiology and Chronic Health Evaluation (APACHE) II score in the sepsis group was assessed by Spearman correlation analysis. The plasma concentration of sTREM-1 in the sepsis group was significantly higher than that in the SIRS and healthy groups (both p sepsis vs. SIRS showed that the area under the curve of sTREM-1 (0.82) was greater than that of PCT (0.77), CRP (0.72), and WBC count (0.70). Additionally, in the sepsis group, the plasma sTREM-1 concentration correlated positively with the APACHE II score (r = 0.41; p sepsis.

  18. [Soluble interleukin 2 receptor as activity parameter in serum of systemic and discoid lupus erythematosus].

    Science.gov (United States)

    Blum, C; Zillikens, D; Tony, H P; Hartmann, A A; Burg, G

    1993-05-01

    The evaluation of disease activity in systemic lupus erythematosus (SLE) is important for selection of the appropriate therapeutic regimen. In addition to the clinical picture, various laboratory parameters are taken into account. However, no validated criteria for the evaluation of the disease activity in SLE have yet been established. Recently, serum levels of soluble interleukin-2 receptor (sIL-2R) have been proposed as a potential parameter for disease activity in SLE. However, the studies reported on this subject so far have focused mainly on certain subsets of the disease, and the evaluation of the disease activity was based on a very limited number of parameters. In the present study, we determined serum levels of sIL-2R in 23 patients with SLE and 30 patients with discoid LE (DLE). Evaluation of disease activity in SLE was based on a comprehensive scale which considered numerous clinical signs and laboratory parameters. In SLE, serum levels of sIL-2R showed a better correlation with disease activity than all the other parameters investigated, including proteinuria, erythrocyte sedimentation rate, serum globulin concentration, titre of antibodies against double-stranded DNA, serum albumin concentration, serum complement levels and white blood cell count. For the first time, we report on elevated serum levels of sIL-2R in DLE, which also correlated with disease activity.

  19. Effects of Polyethylene Glycol Spacer Length and Ligand Density on Folate Receptor Targeting of Liposomal Doxorubicin In Vitro

    Directory of Open Access Journals (Sweden)

    Kumi Kawano

    2011-01-01

    Full Text Available The folate receptor is an attractive target for selective tumor delivery of liposomal doxorubicin (DXR because it is abundantly expressed in a large percentage of tumors. This study examined the effect of polyethylene glycol (PEG spacer length and folate ligand density on the targeting ability of folate-modified liposomes. Liposomes were modified with folate-derivatized PEG-distearoylphosphatidylethanolamine with PEG molecular weights of 2000, 3400, or 5000. The association of DXR-loaded liposomes with KB cells, which overexpress the folate receptor, was evaluated by flow cytometry at various ratios of folate modification. A low ratio of folate modification with a sufficiently long PEG chain showed the highest folate receptor-mediated association with the cells, but did not show the highest in vitro cytotoxicity. DXR release from folate-modified liposomes in endosomes might be different. These findings will be useful for designing folate receptor-targeting carriers.

  20. Chemogenomic analysis of G-protein coupled receptors and their ligands deciphers locks and keys governing diverse aspects of signalling.

    Directory of Open Access Journals (Sweden)

    Jörg D Wichard

    Full Text Available Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic or inhibitory (antagonistic ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences.

  1. Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

    Science.gov (United States)

    Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J; van der Mei, Henny C

    2015-09-29

    Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the

  2. Synthesis and evaluation of 17 alpha-(carboranylalkyl)estradiols as ligands for estrogen receptors alpha and beta

    Czech Academy of Sciences Publication Activity Database

    Sedlák, David; Eignerová, Barbara; Dračínský, Martin; Janoušek, Zbyněk; Bartůněk, Petr; Kotora, Martin

    2013-01-01

    Roč. 747, 1.12.2013 (2013), s. 178-183 ISSN 0022-328X R&D Projects: GA MŠk(CZ) LC06070; GA MŠk(CZ) LC06077; GA MŠk LM2011022; GA ČR GA204/09/1905 Institutional support: RVO:68378050 ; RVO:61388963 Keywords : carborane * estradiol * metathesis * estrogen receptor * steroid ligand Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.302, year: 2013

  3. Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5α-androst-16-en-3α-ol

    International Nuclear Information System (INIS)

    Vincent, Jeremy; Shan, Li; Fan, Ming; Brunzelle, Joseph S.; Forman, Barry M.; Fernandez, Elias J.

    2004-01-01

    The purification and structure determination of the murine constitutive androstane receptor bound to its inverse agonist/antagonist androstenol is described. The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement

  4. Identification of cinnabarinic acid as a novel endogenous aryl hydrocarbon receptor ligand that drives IL-22 production.

    Directory of Open Access Journals (Sweden)

    Margaret M Lowe

    Full Text Available The aryl hydrocarbon receptor (AHR binds to environmental toxicants including synthetic halogenated aromatic hydrocarbons and is involved in a diverse array of biological processes. Recently, the AHR was shown to control host immunity by affecting the balance between inflammatory T cells that produce IL-17 (Th17 and IL-22 versus regulatory T cells (Treg involved in tolerance. While environmental AHR ligands can mediate this effect, endogenous ligands are likely to be more relevant in host immune responses. We investigated downstream metabolites of tryptophan as potential AHR ligands because (1 tryptophan metabolites have been implicated in regulating the balance between Th17 and Treg cells and (2 many of the AHR ligands identified thus far are derivatives of tryptophan. We characterized the ability of tryptophan metabolites to bind and activate the AHR and to increase IL-22 production in human T cells. We report that the tryptophan metabolite, cinnabarinic acid (CA, is an AHR ligand that stimulates the differentiation of human and mouse T cells producing IL-22. We compare the IL-22-stimulating activity of CA to that of other tryptophan metabolites and define stimulation conditions that lead to CA production from immune cells. Our findings link tryptophan metabolism to AHR activation and define a novel endogenous AHR agonist with potentially broad biological functions.

  5. Identification of cinnabarinic acid as a novel endogenous aryl hydrocarbon receptor ligand that drives IL-22 production.

    Science.gov (United States)

    Lowe, Margaret M; Mold, Jeff E; Kanwar, Bittoo; Huang, Yong; Louie, Alexander; Pollastri, Michael P; Wang, Cuihua; Patel, Gautam; Franks, Diana G; Schlezinger, Jennifer; Sherr, David H; Silverstone, Allen E; Hahn, Mark E; McCune, Joseph M

    2014-01-01

    The aryl hydrocarbon receptor (AHR) binds to environmental toxicants including synthetic halogenated aromatic hydrocarbons and is involved in a diverse array of biological processes. Recently, the AHR was shown to control host immunity by affecting the balance between inflammatory T cells that produce IL-17 (Th17) and IL-22 versus regulatory T cells (Treg) involved in tolerance. While environmental AHR ligands can mediate this effect, endogenous ligands are likely to be more relevant in host immune responses. We investigated downstream metabolites of tryptophan as potential AHR ligands because (1) tryptophan metabolites have been implicated in regulating the balance between Th17 and Treg cells and (2) many of the AHR ligands identified thus far are derivatives of tryptophan. We characterized the ability of tryptophan metabolites to bind and activate the AHR and to increase IL-22 production in human T cells. We report that the tryptophan metabolite, cinnabarinic acid (CA), is an AHR ligand that stimulates the differentiation of human and mouse T cells producing IL-22. We compare the IL-22-stimulating activity of CA to that of other tryptophan metabolites and define stimulation conditions that lead to CA production from immune cells. Our findings link tryptophan metabolism to AHR activation and define a novel endogenous AHR agonist with potentially broad biological functions.

  6. An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis

    Science.gov (United States)

    Quintana, Francisco J.; Murugaiyan, Gopal; Farez, Mauricio F.; Mitsdoerffer, Meike; Tukpah, Ann-Marcia; Burns, Evan J.; Weiner, Howard L.

    2010-01-01

    The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3+ Treg, Tr1 cells, and IL-17–producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3+ Treg compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3+ Treg in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3+ Treg in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3+ Treg that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3+ Treg differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders. PMID:21068375

  7. Using novel descriptor accounting for ligand-receptor interactions to define and visually explore biologically relevant chemical space.

    Science.gov (United States)

    Rabal, Obdulia; Oyarzabal, Julen

    2012-05-25

    The definition and pragmatic implementation of biologically relevant chemical space is critical in addressing navigation strategies in the overlapping regions where chemistry and therapeutically relevant targets reside and, therefore, also key to performing an efficient drug discovery project. Here, we describe the development and implementation of a simple and robust method for representing biologically relevant chemical space as a general reference according to current knowledge, independently of any reference space, and analyzing chemical structures accordingly. Underlying our method is the generation of a novel descriptor (LiRIf) that converts structural information into a one-dimensional string accounting for the plausible ligand-receptor interactions as well as for topological information. Capitalizing on ligand-receptor interactions as a descriptor enables the clustering, profiling, and comparison of libraries of compounds from a chemical biology and medicinal chemistry perspective. In addition, as a case study, R-groups analysis is performed to identify the most populated ligand-receptor interactions according to different target families (GPCR, kinases, etc.), as well as to evaluate the coverage of biologically relevant chemical space by structures annotated in different databases (ChEMBL, Glida, etc.).

  8. Soluble CD40 ligand contributes to blood-brain barrier breakdown and central nervous system inflammation in multiple sclerosis and neuromyelitis optica spectrum disorder.

    Science.gov (United States)

    Masuda, Hiroki; Mori, Masahiro; Uchida, Tomohiko; Uzawa, Akiyuki; Ohtani, Ryohei; Kuwabara, Satoshi

    2017-04-15

    Soluble CD40 ligand (sCD40L) is reported to disrupt the blood-brain barrier (BBB). Cerebrospinal fluid (CSF) and serum sCD40L levels were measured in 29 multiple sclerosis (MS), 29 neuromyelitis optica spectrum disorder (NMOSD), and 27 disease control (DC) patients. In MS, serum sCD40L levels were higher than in DCs and positively correlated with the CSF/serum albumin ratio (Qalb). In NMOSD, CSF sCD40L levels were significantly increased compared to DCs, and were correlated to Qalb, CSF cell counts, protein concentrations, and interleukin-6 levels. sCD40L could be involved in BBB disruption in MS, whereas it may contribute to CNS inflammation in NMOSD. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

    Directory of Open Access Journals (Sweden)

    Sylvie Faucher

    Full Text Available BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127 and common gamma (CD132 chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127 is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

  10. The relaxin family peptide receptors and their ligands: new developments and paradigms in the evolution from jawless fish to mammals.

    Science.gov (United States)

    Yegorov, Sergey; Bogerd, Jan; Good, Sara V

    2014-12-01

    Relaxin family peptide receptors (Rxfps) and their ligands, relaxin (Rln) and insulin-like (Insl) peptides, are broadly implicated in the regulation of reproductive and neuroendocrine processes in mammals. Most placental mammals harbour genes for four receptors, namely rxfp1, rxfp2, rxfp3 and rxfp4. The number and identity of rxfps in other vertebrates are immensely variable, which is probably attributable to intraspecific variation in reproductive and neuroendocrine regulation. Here, we highlight several interesting, but greatly overlooked, aspects of the rln/insl-rxfp evolutionary history: the ancient origin, recruitment of novel receptors, diverse roles of selection, differential retention and lineage-specific loss of genes over evolutionary time. The tremendous diversity of rln/insl and rxfp genes appears to have arisen from two divergent receptors and one ligand that were duplicated by whole genome duplications (WGD) in early vertebrate evolution, although several genes, notably relaxin in mammals, were also duplicated via small scale duplications. Duplication and loss of genes have varied across lineages: teleosts retained more WGD-derived genes, dominated by those thought to be involved in neuroendocrine regulation (rln3, insl5 and rxfp 3/4 genes), while eutherian mammals witnessed the diversification and rapid evolution of genes involved in reproduction (rln/insl3). Several genes that arose early in evolutionary history were lost in most mammals, but retained in teleosts and, to a lesser extent, in early diverging tetrapods. To elaborate on their evolutionary history, we provide updated phylogenies of the Rxfp1/2 and Rxfp3/4 receptors and their ligands, including new sequences from early diverging vertebrate taxa such as coelacanth, skate, spotted gar, and lamprey. We also summarize the recent progress made towards understanding the functional biology of Rxfps in non-mammalian taxa, providing a new conceptual framework for research on Rxfp signaling across

  11. [Bis(TrimethylsilylMethyl]Lithium and -Sodium: Solubility in Alkanes and Complexes with O- and N- Donor Ligands

    Directory of Open Access Journals (Sweden)

    Markus von Pilgrim

    2017-06-01

    Full Text Available In contrast to alkyl compounds of lithium, which play an important role in organometallic chemistry, the corresponding heavier alkali metal compounds are less investigated. These compounds are mostly insoluble in inert solvents or undergo solvolysis in coordinating solvents due to their high reactivity. An exception from this typical behavior is demonstrated by bis(trimethylsilylmethylsodium. This study examines alkane solutions of bis(trimethylsilylmethyllithium and -sodium by NMR spectroscopic and cryoscopic methods. In addition, structural studies by X-ray crystallography of the corresponding compounds coordinated by O- and N- ligands (tetrahydrofuran and tetramethylethylenediamine present possible structural motifs of the uncoordinated compounds in solution.

  12. A Macrocyclic Agouti-Related Protein/[Nle4, DPhe7]α-Melanocyte Stimulating Hormone Chimeric Scaffold Produces Sub-nanomolar Melanocortin Receptor Ligands

    OpenAIRE

    Ericson, Mark D.; Freeman, Katie T.; Schnell, Sathya M.; Haskell-Luevano, Carrie

    2017-01-01

    The melanocortin system consists of five receptor subtypes, endogenous agonists, and naturally occurring antagonists. These receptors and ligands have been implicated in numerous biological pathways including processes linked to obesity and food intake. Herein, a truncation structure-activity relationship study of chimeric agouti-related protein (AGRP)/[Nle4, DPhe7]α-Melanocyte Stimulating Hormone (NDP-MSH) ligands is reported. The tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp repl...

  13. Differential expression of ligands for NKG2D and DNAM-1 receptors by epithelial ovarian cancer-derived exosomes and its influence on NK cell cytotoxicity.

    Science.gov (United States)

    Labani-Motlagh, Alireza; Israelsson, Pernilla; Ottander, Ulrika; Lundin, Eva; Nagaev, Ivan; Nagaeva, Olga; Dehlin, Eva; Baranov, Vladimir; Mincheva-Nilsson, Lucia

    2016-04-01

    Cancers constitutively produce and secrete into the blood and other biofluids 30-150 nm-sized endosomal vehicles called exosomes. Cancer-derived exosomes exhibit powerful influence on a variety of biological mechanisms to the benefit of the tumors that produce them. We studied the immunosuppressive ability of epithelial ovarian cancer (EOC) exosomes on two cytotoxic pathways of importance for anticancer immunity-the NKG2D receptor-ligand pathway and the DNAM-1-PVR/nectin-2 pathway. Using exosomes, isolated from EOC tumor explant and EOC cell-line culture supernatants, and ascitic fluid from EOC patients, we studied the expression of NKG2D and DNAM-1 ligands on EOC exosomes and their ability to downregulate the cognate receptors. Our results show that EOC exosomes differentially and constitutively express NKG2D ligands from both MICA/B and ULBP families on their surface, while DNAM-1 ligands are more seldom expressed and not associated with the exosomal membrane surface. Consequently, the NKG2D ligand-bearing EOC exosomes significantly downregulated the NKG2D receptor expression on peripheral blood mononuclear cells (PBMC) while the DNAM-1 receptor was unaffected. The downregulation of NKG2D receptor expression was coupled to inhibition of NKG2D receptor-ligand-mediated degranulation and cytotoxicity measured in vitro with OVCAR-3 and K562 cells as targets. The EOC exosomes acted as a decoy impairing the NKG2D mediated cytotoxicity while the DNAM-1 receptor-ligand system remained unchanged. Taken together, our results support and explain the mechanism behind the recently reported finding that in EOC, NK-cell recognition and killing of tumor cells was mainly dependent on DNAM-1 signaling while the contribution of the NKG2D receptor-ligand pathway was complementary and uncertain.

  14. Generation of Affibody ligands binding interleukin-2 receptor alpha/CD25.

    Science.gov (United States)

    Grönwall, Caroline; Snelders, Eveline; Palm, Anna Jarelöv; Eriksson, Fredrik; Herne, Nina; Ståhl, Stefan

    2008-06-01

    Affibody molecules specific for human IL-2Ralpha, the IL-2 (interleukin-2) receptor alpha subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody molecules bound to CD4+ CD25+ PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

  15. A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

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    Han CY

    2013-04-01

    Full Text Available Cui-yan Han,1,2 Li-ling Yue,2 Ling-yu Tai,1 Li Zhou,2 Xue-yan Li,2 Gui-hua Xing,2 Xing-gang Yang,1 Ming-shuang Sun,1 Wei-san Pan1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 2Qiqihar Medical University, Qiqihar, People’s Republic of China Abstract: The epidermal growth factor receptor (EGFR serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy. Keywords: EGFR, small peptide, tumor targeting, lung cancer, NLC

  16. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

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    Tasnim S. Mohamed

    2015-11-01

    Full Text Available Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity.

  17. Receptor activator NFkappaB-ligand and osteoprotegerin protein expression in human periapical cysts and granulomas.

    Science.gov (United States)

    Menezes, Renato; Bramante, Clóvis Monteiro; da Silva Paiva, Katiúcia Batista; Letra, Ariadne; Carneiro, Everdan; Fernando Zambuzzi, Willian; Granjeiro, José Mauro

    2006-09-01

    The purpose of this study was to determine the expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG) associated with bone destruction in periapical cysts and granulomas. Forty human dental chronic periapical lesions were collected after periapical surgery. The lesions collected were fixed in 10% buffered formalin and histologically processed. At least 2 sections of each specimen were stained with hematoxylin and eosin for microscopic diagnosis. After that, 10 human periapical granulomas and 10 cysts were selected for immunohistochemical analysis for RANKL, OPG, and CD68+. Polymorphonuclear neutrophils, macrophages, endothelial cells, and lymphocytes were stained for RANKL and OPG in both lesions. Epithelial cells were also stained for RANKL and OPG in periapical cysts. Quantitative analysis was conducted and the results were expressed as a ratio of the number of immunostained cells over the total number of cells in the field (n = 100). The ratio of RANKL+/total cells was higher than OPG+/total cells in periapical granulomas (0.553 +/- 0.153 and 0.483 +/- 0.189, respectively; P cysts (0.519 +/- 0.09 and 0.339 +/- 0.117, respectively; P cysts. However, the ratio RANKL+/OPG+ in granulomas (1.336 +/- 0.723) and cysts (1.404 +/- 0.385) was not significantly different. The ratio of CD68+/total cells was significantly higher in granulomas (0.381 +/- 0.040) than in cysts (0.307 +/- 0.068) (P cysts and granulomas, strongly suggesting the involvement of these gene products in the development of periapical lesions.

  18. Copper, nickel and zinc complexes of a new water-soluble thiosemicarbazone ligand: Synthesis, characterization, stability and biological evaluation

    Czech Academy of Sciences Publication Activity Database

    Hosseini-Yazdi, S.A.; Mirzaahmadi, A.; Khandar, A.A.; Mahdavi, M.; Rahimian, A.; Eigner, Václav; Dušek, Michal; Zarrini, G.

    2017-01-01

    Roč. 248, Dec (2017), s. 658-667 ISSN 0167-7322 R&D Projects: GA ČR(CZ) GA15-12653S; GA MŠk(CZ) LO1603 EU Projects: European Commission(CZ) CZ.2.16/3.1.00/24510 Institutional support: RVO:68378271 Keywords : cytotoxicity * thiosemicarbazone * water-soluble Schiff base * stability * antioxidant * crystal structure Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 3.648, year: 2016

  19. Development of an image analysis screen for estrogen receptor alpha (ERα) ligands through measurement of nuclear translocation dynamics.

    Science.gov (United States)

    Dull, Angie; Goncharova, Ekaterina; Hager, Gordon; McMahon, James B

    2010-11-01

    We have developed a robust high-content assay to screen for novel estrogen receptor alpha (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen utilizes a green fluorescent protein tagged-glucocorticoid/estrogen receptor (GFP-GRER) chimera which consisted of the N-terminus of the glucocorticoid receptor fused to the human ER ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands, and translocated to the nucleus in response to stimulation with ERα agonists or antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. The assay was validated with known ERα agonists and antagonists, and the Library of Pharmacologically Active Compounds (LOPAC 1280). Additionally, screening of crude natural product extracts demonstrated the robustness of the assay, and the ability to quantitate the effects of toxicity on nuclear translocation dynamics. The GFP-GRER nuclear translocation assay was very robust, with z' values >0.7, CVs screening of natural product extracts. This assay has been developed for future primary screening of synthetic, pure natural products, and natural product extracts libraries available at the National Cancer Institute at Frederick. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

    Science.gov (United States)

    Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

    2016-04-01

    The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The role of MCP-1-CCR2 ligand-receptor axis in chondrocyte degradation and disease progress in knee osteoarthritis

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    Yuan-kun Xu

    Full Text Available BACKGROUND: Osteoarthritis (OA is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1 and receptor chemokine (C-C motif receptor 2 (CCR2 in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001 and the expression of MMP-13 also increased (P < 0.05. MCP-1 stimulation also induced (or enhanced the apoptosis of OA chondrocytes (P < 0.05. Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13 in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA model CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  3. Systemic factors related to soluble (prorenin receptor in plasma of patients with proliferative diabetic retinopathy.

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    Keitaro Hase

    Full Text Available (Prorenin receptor [(PRR], a new component of the tissue renin-angiotensin system (RAS, plays a crucial role in inflammation and angiogenesis in the eye, thus contributing to the development of proliferative diabetic retinopathy (PDR. In this study, we investigated systemic factors related to plasma levels of soluble form of (PRR [s(PRR] in patients with PDR. Twenty type II diabetic patients with PDR and 20 age-matched, non-diabetic patients with idiopathic macular diseases were enrolled, and plasma levels of various molecules were measured by enzyme-linked immunosorbent assays. Human retinal microvascular endothelial cells were stimulated with several diabetes-related conditions to evaluate changes in gene expression using real-time quantitative PCR. Of various systemic parameters examined, the PDR patients had significantly higher blood sugar and serum creatinine levels than non-diabetic controls. Protein levels of s(PRR, prorenin, tumor necrosis factor (TNF-α, complement factor D (CFD, and leucine-rich α-2-glycoprotein 1 (LRG1 significantly increased in the plasma of PDR subjects as compared to non-diabetes, with positive correlations detected between s(PRR and these inflammatory molecules but not prorenin. Estimated glomerular filtration rate and serum creatinine were also correlated with plasma s(PRR, but not prorenin, levels. Among the inflammatory molecules correlated with s(PRR in the plasma, TNF-α, but not CFD or LRG1, application to retinal endothelial cells upregulated the mRNA expression of (PRR but not prorenin, while stimulation with high glucose enhanced both (PRR and prorenin expression. These findings suggested close relationships between plasma s(PRR and diabetes-induced factors including chronic inflammation, renal dysfunction, and hyperglycemia in patients with PDR.

  4. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  5. Soluble tumor necrosis factor receptor-I in preterm infants with chorioamnionitis.

    Science.gov (United States)

    Sato, Miho; Nishimaki, Shigeru; An, Hiromi; Shima, Yoshio; Naruto, Takuya; Sugai, Toshiyuki; Iwasaki, Shiho; Seki, Kazuo; Imagawa, Tomoyuki; Mori, Masaaki; Yokota, Shumpei

    2009-04-01

    The aim of our study was (i) to determine whether chorioamnionitis (CAM) is associated with an elevated soluble tumor necrosis factor receptor I (sTNFR-I) level and (ii) to examine the time course of the concentration of sTNFR-I in preterm infants after birth. We measured sTNFR-I levels in the cord blood of 112 preterm infants (gestational age < or =34 weeks), and those in peripheral blood of 30 preterm infants on days 7, 14, 21 and 28. The median value for the sTNFR-I was significantly elevated in 33 infants with CAM at stage 3 (4618 pg/mL) compared with the 52 infants without CAM (2866 pg/mL), or the 13 infants with CAM at stage 1 (3638 pg/mL) and the 14 infants at stage 2 (3242 pg/mL). The severity of CAM is an independent factor for the elevation of cord blood sTNFR-I. The sTNFR-I level on day 0 was significantly higher in eight infants with CAM at stage 3 than in the 22 infants without CAM or with CAM at stage 1 and 2; however there were no significant differences on days 7, 14, 21 and 28. The serum level of sTNFR-I showed a significant gradual decline with time. We suggest that there is an association between elevated sTNFR-I levels in cord blood and maternal CAM, and this elevation may reflect the fetal inflammation. However the elevation of sTNFR-I could not persist postnatally for a long time.

  6. Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer

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    Kim Wun-Jae

    2011-04-01

    Full Text Available Abstract Background Most bladder cancer patients experience lymphatic metastasis in the course of disease progression, yet the relationship between lymphangiogenesis and lymphatic metastasis is not well known. The aim of this study is to elucidate underlying mechanisms of how expanded lymphatic vessels and tumor microenvironment interacts each other and to find effective therapeutic options to inhibit lymphatic metastasis. Results The orthotopic urinary bladder cancer (OUBC model was generated by intravesical injection of MBT-2 cell lines. We investigated the angiogenesis, lymphangiogenesis, and CD11b+/CD68+ tumor-associated macrophages (TAM by using immunofluorescence staining. OUBC displayed a profound lymphangiogenesis and massive infiltration of TAM in primary tumor and lymphatic metastasis in lymph nodes. TAM flocked near lymphatic vessels and express higher levels of VEGF-C/D than CD11b- cells. Because VEGFR-3 was highly expressed in lymphatic vascular endothelial cells, TAM could assist lymphangiogenesis by paracrine manner in bladder tumor. VEGFR-3 expressing adenovirus was administered to block VEGF-C/D signaling pathway and clodronate liposome was used to deplete TAM. The blockade of VEGF-C/D with soluble VEGF receptor-3 markedly inhibited lymphangiogenesis and lymphatic metastasis in OUBC. In addition, the depletion of TAM with clodronate liposome exerted similar effects on OUBC. Conclusion VEGF-C/D are the main factors of lymphangiogenesis and lymphatic metastasis in bladder cancer. Moreover, TAM plays an important role in these processes by producing VEGF-C/D. The inhibition of lymphangiogenesis could provide another therapeutic target to inhibit lymphatic metastasis and recurrence in patients with invasive bladder cancer.

  7. Soluble transferrin receptor as a marker of erythropoiesis in patients undergoing high-flux hemodialysis

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    Pei Yin

    2017-11-01

    Full Text Available Anemia is a common complication in chronic kidney disease (CKD patients receiving hemodialysis. The effect of high-flux dialysis (HFD on anemia remains unclear. This prospective study aimed to evaluate the effect of HFD on anemia, and the potential of soluble transferrin receptor (sTfR as a marker of iron status and erythropoiesis in CKD patients on hemodialysis. Forty patients, who switched from conventional low-flux dialysis to HFD for 12 months, were enrolled in this study. The levels of sTfR, hemoglobin (Hb, iron, and nutritional markers, as well as the dose of recombinant human erythropoietin (rhEPO and use of chalybeate were determined at 0, 2, 6, and 12 months after starting HFD. HFD significantly increased the hemoglobin level and reduced sTfR level in CKD patients (p < 0.05. In addition, significant decreasing linear trends were observed for rhEPO dosage and chalybeate use (p < 0.05. The level of sTfR was positively correlated with the percentage of reticulocytes (RET%, rhEPO dose, and chalybeate use, while it was negatively correlated with Hb levels and total iron-binding capacity results (all p < 0.05. A univariate generalized estimating equation (GEE model showed that the Hb level, RET%, rhEPO dose, and chalybeate use were the variables associated with sTfR levels. A multivariate GEE model showed that the time points when hemodialysis was performed were the variables associated significantly with sTfR levels. Overall, our findings suggest that HFD can effectively improve renal anemia in hemodialysis patients, and sTfR could be used as a marker of erythropoiesis in HFD patients.

  8. Bronchoalveolar lavage analysis, gallium-67 lung scanning and soluble interleukin-2 receptor levels in asbestos exposure

    International Nuclear Information System (INIS)

    Delclos, G.L.; Flitcraft, D.G.; Brousseau, K.P.; Windsor, N.T.; Nelson, D.L.; Wilson, R.K.; Lawrence, E.C.

    1989-01-01

    This study examined different markers of lung immunologic and inflammatory responses to previous asbestos exposure. We performed bronchoalveolar lavage (BAL) and gallium-67 ( 67 Ga) lung scans and measured serum and BAL soluble interleukin-2 receptor (IL-2R) and angiotensin-converting enzyme (SACE) levels in 32 subjects with a history of significant asbestos exposure, 14 without (EXP) and 18 with (ASB) radiographic evidence of asbestosis. BAL analysis revealed increases in neutrophils in both ASB and EXP when compared to controls (P less than 0.01), which persisted after adjustment for smoking category. Although significant abnormalities of macrophage and total lymphocyte profiles were not found in the study population, lymphocyte subpopulation analysis revealed elevation of BAL T4/T8 ratios in the entire study group (ASB + EXP) when compared to controls (P less than 0.05), independent of smoking category. 67 Ga lung scan activity was increased in 56% of ASB and in 36% of EXP: no correlations between positive scans and different radiological and functional parameters could be found. There was no significant elevation of mean SACE, serum, or BAL IL-2R levels in any of the study categories. These data suggest that asbestos exposure may be associated with parenchymal inflammation, even in the absence of clinical criteria for asbestosis. Abnormalities of gallium uptake and of BAL analysis reflect the clinically inapparent inflammation. The increased BAL T4/T8 ratios observed suggest that abnormal local pulmonary immunoregulation may play a role in the pathogenesis of asbestos-related lung diseases

  9. Prediction of response to medical therapy by serum soluble (pro)renin receptor levels in Graves' disease.

    Science.gov (United States)

    Mizuguchi, Yuki; Morimoto, Satoshi; Kimura, Shihori; Takano, Noriyoshi; Yamashita, Kaoru; Seki, Yasufumi; Bokuda, Kanako; Yatabe, Midori; Yatabe, Junichi; Watanabe, Daisuke; Ando, Takashi; Ichihara, Atsuhiro

    2018-01-01

    Antithyroid drugs are generally selected as the first-line treatment for Graves' Disease (GD); however, the existence of patients showing resistance or severe side effects to these drugs is an important issue to be solved. The (pro)renin receptor [(P)RR] is a multi-functional protein that activates the tissue renin-angiotensin system and is an essential constituent of vacuolar H+-ATPase, necessary for the autophagy-lysosome pathway. (P)RR is cleaved to soluble (s)(P)RR, which reflects the status of (P)RR expression. In this retrospective study, we aimed to investigate whether serum s(P)RR concentration can be used as a biomarker to predict the outcome of antithyroid drug treatment in GD patients. Serum s(P)RR levels were measured in 54 untreated GD patients and 47 control participants. Effects of medical treatment with antithyroid drugs on these levels were investigated in GD patients. Serum s(P)RR levels were significantly higher in patients with Graves' disease than in control subjects (PGraves' disease. High serum s(P)RR levels were associated with resistance to antithyroid drug treatment, suggesting that serum s(P)RR concentration can be used as a useful biomarker to predict the outcome of antithyroid drug treatment in these patients. Patients with Graves' disease with low body mass index showed higher levels of serum soluble (pro)renin receptor levels than those with high body mass index. In addition, in patients with Graves' disease, serum triglyceride levels were negatively correlated with serum soluble (pro)renin receptor levels. All these data indicated an association between low nutrient condition due to hyperthyroidism and increased (pro)renin receptor expression in these patients, suggesting that (pro)renin receptor expression could be increased in the process of stimulating intracellular energy production via activating autophagy function to compensate energy loss.

  10. Prediction of response to medical therapy by serum soluble (pro)renin receptor levels in Graves’ disease

    Science.gov (United States)

    Kimura, Shihori; Takano, Noriyoshi; Yamashita, Kaoru; Seki, Yasufumi; Bokuda, Kanako; Yatabe, Midori; Yatabe, Junichi; Watanabe, Daisuke; Ando, Takashi

    2018-01-01

    Antithyroid drugs are generally selected as the first-line treatment for Graves’ Disease (GD); however, the existence of patients showing resistance or severe side effects to these drugs is an important issue to be solved. The (pro)renin receptor [(P)RR] is a multi-functional protein that activates the tissue renin-angiotensin system and is an essential constituent of vacuolar H+-ATPase, necessary for the autophagy-lysosome pathway. (P)RR is cleaved to soluble (s)(P)RR, which reflects the status of (P)RR expression. In this retrospective study, we aimed to investigate whether serum s(P)RR concentration can be used as a biomarker to predict the outcome of antithyroid drug treatment in GD patients. Serum s(P)RR levels were measured in 54 untreated GD patients and 47 control participants. Effects of medical treatment with antithyroid drugs on these levels were investigated in GD patients. Serum s(P)RR levels were significantly higher in patients with Graves’ disease than in control subjects (Pantithyroid drug treatment, suggesting that serum s(P)RR concentration can be used as a useful biomarker to predict the outcome of antithyroid drug treatment in these patients. Patients with Graves’ disease with low body mass index showed higher levels of serum soluble (pro)renin receptor levels than those with high body mass index. In addition, in patients with Graves’ disease, serum triglyceride levels were negatively correlated with serum soluble (pro)renin receptor levels. All these data indicated an association between low nutrient condition due to hyperthyroidism and increased (pro)renin receptor expression in these patients, suggesting that (pro)renin receptor expression could be increased in the process of stimulating intracellular energy production via activating autophagy function to compensate energy loss. PMID:29621332

  11. Bibenzimidazole containing mixed ligand cobalt(III) complex as a selective receptor for iodide

    Digital Repository Service at National Institute of Oceanography (India)

    Indumathy, R.; Parameswarana, P.S.; Aiswarya, C.V.; Nair, B.U.

    Two new mixed ligand cobalt(III) complexes containing bibenzimidazole (bbenzimH2) ligand with composition [Co(phen)2bbenzimH2](ClO4)3 (1) and [Co(bpy)2bbenzimH2](ClO4...

  12. Association of soluble Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL with central adiposity and low-density lipoprotein cholesterol.

    Directory of Open Access Journals (Sweden)

    Gloria Brombo

    Full Text Available OBJECTIVE: Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL, in addition to having a prognostic value in patients with cardiovascular disease, seems to interact with adiposity, insulin resistance and other cardiovascular risk factors. However, the results of previous clinical studies, focused on the association of TRAIL with selected metabolic or anthropometric indices were inconclusive. The aim of this study was to further investigate how soluble TRAIL concentrations independently correlate with major cardiovascular risk factors, including lipid, glycemic and anthropometric features. MATERIALS/METHODS: We examined the associations between serum soluble TRAIL concentrations, measured by ELISA, and lipid, glycemic and anthropometric features in 199 subjects recruited at our Metabolic Outpatient Clinic. RESULTS: Soluble TRAIL concentrations had a significant and direct correlation with total cholesterol (p = 0.046, LDL-cholesterol (p = 0.032, triglycerides (p = 0.01, body mass index (p = 0.046, waist circumference (p = 0.008, fat mass (p = 0.056 and insulin (p = 0.046 and an inverse correlation with HDL-cholesterol (p = 0.02. In multivariable regression analyses adjusted for potential confounders (age, gender, C-reactive protein, HDL-cholesterol, triglycerides, waist circumference, and insulin, TRAIL levels continued to have an independent correlation with LDL-cholesterol and waist circumference (r(2 = 0.04. CONCLUSIONS: Serum TRAIL levels were weakly but significantly and independently associated with waist circumference, a marker of visceral adiposity, and with LDL-cholesterol. Further studies are needed to clarify the biological basis of these relationships.

  13. Association of soluble Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) with central adiposity and low-density lipoprotein cholesterol.

    Science.gov (United States)

    Brombo, Gloria; Volpato, Stefano; Secchiero, Paola; Passaro, Angelina; Bosi, Cristina; Zuliani, Giovanni; Zauli, Giorgio

    2013-01-01

    Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL), in addition to having a prognostic value in patients with cardiovascular disease, seems to interact with adiposity, insulin resistance and other cardiovascular risk factors. However, the results of previous clinical studies, focused on the association of TRAIL with selected metabolic or anthropometric indices were inconclusive. The aim of this study was to further investigate how soluble TRAIL concentrations independently correlate with major cardiovascular risk factors, including lipid, glycemic and anthropometric features. We examined the associations between serum soluble TRAIL concentrations, measured by ELISA, and lipid, glycemic and anthropometric features in 199 subjects recruited at our Metabolic Outpatient Clinic. Soluble TRAIL concentrations had a significant and direct correlation with total cholesterol (p = 0.046), LDL-cholesterol (p = 0.032), triglycerides (p = 0.01), body mass index (p = 0.046), waist circumference (p = 0.008), fat mass (p = 0.056) and insulin (p = 0.046) and an inverse correlation with HDL-cholesterol (p = 0.02). In multivariable regression analyses adjusted for potential confounders (age, gender, C-reactive protein, HDL-cholesterol, triglycerides, waist circumference, and insulin), TRAIL levels continued to have an independent correlation with LDL-cholesterol and waist circumference (r(2) = 0.04). Serum TRAIL levels were weakly but significantly and independently associated with waist circumference, a marker of visceral adiposity, and with LDL-cholesterol. Further studies are needed to clarify the biological basis of these relationships.

  14. EANM procedure guidelines for brain neurotransmission SPECT/PET using dopamine D2 receptor ligands, version 2

    DEFF Research Database (Denmark)

    Van Laere, Koen; Varrone, Andrea; Booij, Jan

    2010-01-01

    receptor SPECT or PET studies, and to achieve a high quality standard of dopamine D2 receptor imaging, which will increase the impact of this technique in neurological practice.The present document is an update of the first guidelines for SPECT using D2 receptor ligands labelled with (123)I [1......The guidelines summarize the current views of the European Association of Nuclear Medicine Neuroimaging Committee (ENC). The aims of the guidelines are to assist nuclear medicine practitioners in making recommendations, performing, interpreting and reporting the results of clinical dopamine D2......] and was guided by the views of the Society of Nuclear Medicine Brain Imaging Council [2], and the individual experience of experts in European countries. The guidelines intend to present information specifically adapted to European practice. The information provided should be taken in the context of local...

  15. Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

    Science.gov (United States)

    Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

    2003-03-14

    Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

  16. Steroid receptor coactivator 1 deficiency increases MMTV-neu mediated tumor latency and differentiation specific gene expression, decreases metastasis, and inhibits response to PPAR ligands

    International Nuclear Information System (INIS)

    Han, Ji Seung; Crowe, David L

    2010-01-01

    The peroxisome proliferator activated receptor (PPAR) subgroup of the nuclear hormone receptor superfamily is activated by a variety of natural and synthetic ligands. PPARs can heterodimerize with retinoid X receptors, which have homology to other members of the nuclear receptor superfamily. Ligand binding to PPAR/RXRs results in recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP). Both SRC-1 and CBP are histone acetyltransferases, which by modifying nucleosomal histones, produce more open chromatin structure and increase transcriptional activity. Nuclear hormone receptors can recruit limiting amounts of coactivators from other transcription factor binding sites such as AP-1, thereby inhibiting the activity of AP-1 target genes. PPAR and RXR ligands have been used in experimental breast cancer therapy. The role of coactivator expression in mammary tumorigenesis and response to drug therapy has been the subject of recent studies. We examined the effects of loss of SRC-1 on MMTV-neu mediated mammary tumorigenesis. SRC-1 null mutation in mammary tumor prone mice increased the tumor latency period, reduced tumor proliferation index and metastasis, inhibited response to PPAR and RXR ligands, and induced genes involved in mammary gland differentiation. We also examined human breast cancer cell lines overexpressing SRC-1 or CBP. Coactivator overexpression increased cellular proliferation with resistance to PPAR and RXR ligands and remodeled chromatin of the proximal epidermal growth factor receptor promoter. These results indicate that histone acetyltransferases play key roles in mammary tumorigenesis and response to anti-proliferative therapies

  17. Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands.

    Science.gov (United States)

    Korpis, Katharina; Weber, Frauke; Brune, Stefanie; Wünsch, Bernhard; Bednarski, Patrick J

    2014-01-01

    Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. The role of ligands on the equilibria between functional states of a G protein-coupled receptor.

    Science.gov (United States)

    Kim, Tae Hun; Chung, Ka Young; Manglik, Aashish; Hansen, Alexandar L; Dror, Ron O; Mildorf, Thomas J; Shaw, David E; Kobilka, Brian K; Prosser, R Scott

    2013-06-26

    G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed two distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Analysis of the spectra as a function of temperature revealed that for all ligands, the activation intermediate is entropically favored and enthalpically disfavored. β2AR enthalpy changes toward activation are notably lower than those observed with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR are weaker. Positive entropy changes toward activation likely reflect greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter analysis. Ligands greatly influence the overall changes in enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

  19. Androgen receptor regulates nuclear trafficking and nuclear domain residency of corepressor HDAC7 in a ligand-dependent fashion

    International Nuclear Information System (INIS)

    Karvonen, Ulla; Jaenne, Olli A.; Palvimo, Jorma J.

    2006-01-01

    In addition to chromosomal proteins, histone deacetylases (HDACs) target transcription factors in transcriptional repression. Here, we show that the class II HDAC family member HDAC7 is an efficient corepressor of the androgen receptor (AR). HDAC7 resided in the cytoplasm in the absence of AR or a cognate ligand, but hormone-occupancy of AR induced nuclear transfer of HDAC7. Nuclear colocalization pattern of AR and HDAC7 was dependent on the nature of the ligand. In the presence of testosterone, a portion of HDAC7 localized to pearl-like nuclear domains, whereas AR occupied with antagonistic ligands cyproterone acetate- or casodex (bicalutamide) recruited HDAC7 from these domains to colocalize with the receptor in speckles and nucleoplasm in a more complete fashion. Ectopic expression of PML-3 relieved the repressive effect of HDAC7 on AR function by sequestering HDAC7 to PML-3 domains. AR acetylation at Lys630/632/633 was not the target of HDAC7 repression, since repression of AR function was independent of these acetylation sites. Moreover, the deacetylase activity of HDAC7 was in part dispensable in the repression of AR function. In sum, our results identify HDAC7 as a novel AR corepressor whose subcellular and subnuclear compartmentalization can be regulated in an androgen-selective manner

  20. Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptorligand, rosiglitazone

    International Nuclear Information System (INIS)

    Wakui, Yuta; Inoue, Jun; Ueno, Yoshiyuki; Fukushima, Koji; Kondo, Yasuteru; Kakazu, Eiji; Obara, Noriyuki; Kimura, Osamu; Shimosegawa, Tooru

    2010-01-01

    Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-α, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPARα ligand, bezafibrate, and a PPARγ ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPARγ, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-α-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

  1. Oxytocin receptor ligand binding in embryonic tissue and postnatal brain development of the C57BL/6J mouse

    Directory of Open Access Journals (Sweden)

    Elizabeth eHammock

    2013-12-01

    Full Text Available Oxytocin (OXT has drawn increasing attention as a developmentally relevant neuropeptide given its role in the brain regulation of social behavior. It has been suggested that OXT plays an important role in the infant brain during caregiver attachment in nurturing familial contexts, but there is incomplete experimental evidence. Mouse models of OXT system genes have been particularly informative for the role of the OXT system in social behavior, however, the developing brain areas that could respond to ligand activation of the OXT receptor (OXTR have yet to be identified in this species. Here we report new data revealing dynamic ligand-binding distribution of OXTR in the developing mouse brain. Using male and female C57BL/6J mice at postnatal days (P 0, 7, 14, 21, 35, and 60 we quantified OXTR ligand binding in several brain areas which changed across development. Further, we describe OXTR ligand binding in select tissues of the near-term whole embryo at E18.5. Together, these data aid in the interpretation of findings in mouse models of the OXT system and generate new testable hypotheses for developmental roles for OXT in mammalian systems. We discuss our findings in the context of developmental disorders (including autism, attachment biology, and infant physiological regulation.

  2. Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptor-{gamma} ligand, rosiglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Wakui, Yuta; Inoue, Jun [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan); Ueno, Yoshiyuki, E-mail: yueno@mail.tains.tohoku.ac.jp [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan); Fukushima, Koji; Kondo, Yasuteru; Kakazu, Eiji; Obara, Noriyuki; Kimura, Osamu; Shimosegawa, Tooru [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan)

    2010-05-28

    Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-{alpha}, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPAR{alpha} ligand, bezafibrate, and a PPAR{gamma} ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPAR{gamma}, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-{alpha}-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

  3. Peroxisome Proliferator-Activated ReceptorLigands: Potential Pharmacological Agents for Targeting the Angiogenesis Signaling Cascade in Cancer

    Directory of Open Access Journals (Sweden)

    Costas Giaginis

    2008-01-01

    Full Text Available Peroxisome proliferator-activated receptor-γ (PPAR-γ has currently been considered as molecular target for the treatment of human metabolic disorders. Experimental data from in vitro cultures, animal models, and clinical trials have shown that PPAR-γ ligand activation regulates differentiation and induces cell growth arrest and apoptosis in a variety of cancer types. Tumor angiogenesis constitutes a multifaceted process implicated in complex downstream signaling pathways that triggers tumor growth, invasion, and metastasis. In this aspect, accumulating in vitro and in vivo studies have provided extensive evidence that PPAR-γ ligands can function as modulators of the angiogenic signaling cascade. In the current review, the crucial role of PPAR-γ ligands and the underlying mechanisms participating in tumor angiogenesis are summarized. Targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments with conventional chemotherapy; however, special attention should be taken as there is also substantial evidence to support that PPAR-γ ligands can enhance angiogenic phenotype in tumoral cells.

  4. Involvement of the Retinoid X Receptor Ligand in the Anti-Inflammatory Effect Induced by Peroxisome Proliferator-Activated Receptor Agonist In Vivo

    Directory of Open Access Journals (Sweden)

    Atsuki Yamamoto

    2011-01-01

    Full Text Available Peroxisome proliferator-activated receptor γ (PPARγ forms a heterodimeric DNA-binding complex with retinoid X receptors (RXRs. It has been reported that the effect of the PPAR agonist is reduced in hepatocyte RXR-deficient mice. Therefore, it is suggested that the endogenous RXR ligand is involved in the PPARγ agonist-induced anti-inflammatory effect. However, the participation of the RXR ligand in the PPARγ-induced anti-inflammatory effect is unknown. Here, we investigated the influence of RXR antagonist on the anti-inflammatory effect of PPARγ agonist pioglitazone in carrageenan test. In addition, we also examined the influence of PPAR antagonist on the anti-inflammatory effect induced by RXR agonist NEt-3IP. The RXR antagonist suppressed the antiedema effect of PPARγ agonist. In addition, the anti-inflammatory effect of RXR agonist was suppressed by PPARγ antagonist. PPARγ agonist-induced anti-inflammatory effects were reversed by the RXR antagonist. Thus, we showed that the endogenous RXR ligand might contribute to the PPARγ agonist-induced anti-inflammatory effect.

  5. New advances in pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors

    Science.gov (United States)

    Greig, Nigel H.; Reale, Marcella; Tata, Ada Maria

    2016-01-01

    The cholinergic system is expressed in neuronal and in non-neuronal tissues. Acetylcholine (ACh), synthesized in and out of the nervous system can locally contribute to modulation of various cell functions (e.g. survival, proliferation). Considering that the cholinergic system and its functions are impaired in a number of disorders, the identification of new pharmacological approaches to regulate cholinergic system components appears of great relevance. The present review focuses on recent pharmacological drugs able to modulate the activity of cholinergic receptors and thereby, cholinergic function, with an emphasis on the muscarinic receptor subtype, and additionally covers the cholinesterases, the main enzymes involved in ACh hydrolysis. The presence and function of muscarinic receptor subtypes both in neuronal and non-neuronal cells has been demonstrated using extensive pharmacological data emerging from studies on transgenic mice. The possible involvement of ACh in different pathologies has been proposed in recent years and is becoming an important area of study. Although the lack of selective muscarinic receptor ligands has for a long time limited the definition of therapeutic treatment based on muscarinic receptors as targets, some muscarinic ligands such as cevimeline (patents US4855290; US5571918) or xanomeline (patent, US5980933) have been developed and used in pre-clinical or in clinical studies for the treatment of nervous system diseases (Alzheimer’ and Sjogren’s diseases). The present review focuses on the potential implications of muscarinic receptors in different pathologies, including tumors. Moreover, the future use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that some muscarinic antagonists currently used in the treatment of genitourinary disease (e.g. darifenacin, patent, US5096890; US6106864) have also been demonstrated to arrest tumor progression in nude mice. The involvement of muscarinic

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

    Science.gov (United States)

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

    2018-02-01

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

  7. Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation

    Directory of Open Access Journals (Sweden)

    Miguel Angel Burguillos

    2015-03-01

    Full Text Available Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4 in microglia’s inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3 released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain.

  8. Use of plasma C-reactive protein, procalcitonin, neutrophils,macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble triggering receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Andersen, Ove; Kronborg, Gitte

    2007-01-01

    the diagnostic characteristics of novel and routinely used biomarkers of sepsis alone and in combination. Methods: This prospective cohort study included patients with systemic inflammatory response syndrome who were suspected of having community-acquired infections. It was conducted in a medical emergency...... department and department of infectious diseases at a university hospital. A multiplex immunoassay measuring soluble urokinase-type plasminogen activator (suPAR) and soluble triggering receptor expressed on myeloid cells (sTREM)-1 and macrophage migration inhibitory factor (MIF) was used in parallel...... with standard measurements of C-reactive protein (CRP), procalcitonin (PCT), and neutrophils. Two composite markers were constructed – one including a linear combination of the three best performing markers and another including all six – and the area under the receiver operating characteristic curve (AUC...

  9. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3

    DEFF Research Database (Denmark)

    Hudson, Brian D; Tikhonova, Irina G; Pandey, Sunil K

    2012-01-01

    Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C...

  10. Identification of novel peptide ligands for the cancer-specific receptor mutation EFGRvIII using a mixture-based synthetic combinatorial library

    DEFF Research Database (Denmark)

    Denholt, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2009-01-01

    We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in se...

  11. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

    Science.gov (United States)

    Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W.; Trudeau, Louis-Eric

    2014-01-01

    Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells. PMID:24022593

  12. Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors

    Directory of Open Access Journals (Sweden)

    Irene Veneziani

    2018-01-01

    Full Text Available Neuroblastoma (NB, the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR, triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted.

  13. Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors

    Science.gov (United States)

    Veneziani, Irene; Brandetti, Elisa; Ognibene, Marzia; Pezzolo, Annalisa; Pistoia, Vito

    2018-01-01

    Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted. PMID:29805983

  14. Dynamic ligand modulation of EPO receptor pools, and dysregulation by polycythemia-associated EPOR alleles.

    Directory of Open Access Journals (Sweden)

    Seema Singh

    Full Text Available Erythropoietin (EPO and its cell surface receptor (EPOR are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1 High- Mr EPOR forms become obviously expressed only when EPO is limited. 2 EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3 Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size. 4 In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products. Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects. New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via

  15. Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes.

    Directory of Open Access Journals (Sweden)

    Meral Tunc-Ozdemir

    Full Text Available Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1 modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22. These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy.The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants.A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex

  16. Dynamic Ligand Modulation of EPO Receptor Pools, and Dysregulation by Polycythemia-Associated EPOR Alleles

    Science.gov (United States)

    Singh, Seema; Verma, Rakesh; Pradeep, Anamika; Leu, Karen; Mortensen, R. Bruce; Young, Peter R.; Oyasu, Miho; Schatz, Peter J.; Green, Jennifer M.; Wojchowski, Don M.

    2012-01-01

    Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-