Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

PubMed Central

Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

2011-01-01

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

Optical methods in modeling nicotine effect on the surface water of cell membranes

NASA Astrophysics Data System (ADS)

Alexandrova, Tatyana V.; Rogacheva, Svetlana M.; Kuznetsov, Pavel E.; Gubina, Tamara I.

2005-06-01

Fluorescence and spectrophotometric methods have been used for investigation of nicotine action on the state and mobility of the surface water. The surfaces of membranes and proteins were simulated with the help of liposomes and ultradispersive diamonds consequently. Nicotine was shown to reduce the stability of liposomes and to change the aggregative ability of ultradispersive diamonds. The wave-like curves for the nicotine concentration dependences of the pointed features were observed. Such shape of responses was suggested to be due to the changing in structure and dynamics of water hydrogen bonds net near the surface of the model systems induced by nicotine molecules. The surface water phase was supposed to be one of signal elements ofthe ligand receptor recognition process.

Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

NASA Astrophysics Data System (ADS)

Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

2011-02-01

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.

PubMed

Heredia, Jeremiah D; Park, Jihye; Brubaker, Riley J; Szymanski, Steven K; Gill, Kevin S; Procko, Erik

2018-06-01

Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 2.45 and W161 4.50 ) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously. Copyright © 2018 by The American Association of Immunologists, Inc.

Unique Directional Motility of Influenza C Virus Controlled by Its Filamentous Morphology and Short-Range Motions.

PubMed

Sakai, Tatsuya; Takagi, Hiroaki; Muraki, Yasushi; Saito, Mineki

2018-01-15

Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the distances of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that like IAV, ICV also has a motility that is driven by the receptor exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology. IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as the motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has a unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase. Copyright © 2018 Sakai et al.

Surface potentials measure ion concentrations near lipid bilayers during rapid solution changes.

PubMed Central

Laver, D R; Curtis, B A

1996-01-01

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle. Images FIGURE 1 FIGURE 4 FIGURE 5 FIGURE 8 PMID:8842210

Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salaita, Khalid; Nair, Pradeep M; Petit, Rebecca S

2009-09-09

Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitativemore » analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.« less

Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor

PubMed Central

Padilla-Morales, Luis F.; Morales-Pérez, Claudio L.; De La Cruz-Rivera, Pamela C.; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A.

2011-01-01

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility. PMID:21922299

Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

PubMed

Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

2014-12-12

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study

NASA Astrophysics Data System (ADS)

Zhao, Yuejie; Singh, Arunima; Xu, Yongmei; Zong, Chengli; Zhang, Fuming; Boons, Geert-Jan; Liu, Jian; Linhardt, Robert J.; Woods, Robert J.; Amster, I. Jonathan

2017-01-01

Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed.

Neurotrophin responsiveness of sympathetic neurons is regulated by rapid mobilization of the p75 receptor to the cell surface through TrkA activation of Arf6.

PubMed

Edward Hickman, F; Stanley, Emily M; Carter, Bruce D

2018-05-22

The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is up regulated resulting in formation of TrkA-p75 complexes, which are high affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 GEFs. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth while the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system. SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface availability, may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs. Copyright © 2018 the authors.

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

PubMed

Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

2017-06-15

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lad, P.M.; Olson, C.V.; Grewal, I.S.

1986-03-05

Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components.more » Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.« less

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

PubMed Central

Jacquier, V.; Prummer, M.; Segura, J.-M.; Pick, H.; Vogel, H.

2006-01-01

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception. PMID:16980412

Receptors and aging: structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca(2+)-mobilization and gene-expression profiles.

PubMed

Faury, G; Molinari, J; Rusova, E; Mariko, B; Raveaud, S; Huber, P; Velebny, V; Robert, A M; Robert, L

2011-01-01

Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

PubMed

Sarkar, Mitul; Koland, John G

2016-01-01

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

Functional distribution of Ca2+-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells.

PubMed

Tomé, Angelo R; Castro, Enrique; Santos, Rosa M; Rosário, Luís M

2007-06-14

Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells. The [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30-50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells. Although neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretion.

NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition.

PubMed

Baez, María Verónica; Cercato, Magalí Cecilia; Jerusalinsky, Diana Alicia

2018-01-01

NMDA ionotropic glutamate receptors (NMDARs) are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Kainate Receptors Inhibit Glutamate Release Via Mobilization of Endocannabinoids in Striatal Direct Pathway Spiny Projection Neurons.

PubMed

Marshall, John J; Xu, Jian; Contractor, Anis

2018-04-18

Kainate receptors are members of the glutamate receptor family that function by both generating ionotropic currents through an integral ion channel pore and coupling to downstream metabotropic signaling pathways. They are highly expressed in the striatum, yet their roles in regulating striatal synapses are not known. Using mice of both sexes, we demonstrate that GluK2-containing kainate receptors expressed in direct pathway spiny projection neurons (dSPNs) inhibit glutamate release at corticostriatal synapses in the dorsolateral striatum. This inhibition requires postsynaptic kainate-receptor-mediated mobilization of a retrograde endocannabinoid (eCB) signal and activation of presynaptic CB1 receptors. This pathway can be activated during repetitive 25 Hz trains of synaptic stimulation, causing short-term depression of corticostriatal synapses. This is the first study to demonstrate a role for kainate receptors in regulating eCB-mediated plasticity at the corticostriatal synapse and demonstrates an important role for these receptors in regulating basal ganglia circuits. SIGNIFICANCE STATEMENT The GRIK2 gene, encoding the GluK2 subunit of the kainate receptor, has been linked to several neuropsychiatric and neurodevelopmental disorders including obsessive compulsive disorder (OCD). Perseverative behaviors associated with OCD are known to result from pathophysiological changes in the striatum and kainate receptor knock-out mice have striatal-dependent phenotypes. However, the role of kainate receptors in striatal synapses is not known. We demonstrate that GluK2-containing kainate receptors regulate corticostriatal synapses by mobilizing endocannabinoids from direct pathway spiny projection neurons. Synaptic activation of GluK2 receptors during trains of synaptic input causes short-term synaptic depression, demonstrating a novel role for these receptors in regulating striatal circuits. Copyright © 2018 the authors 0270-6474/18/383901-10$15.00/0.

Tetraspanin-3 is an organizer of the multi-subunit Nogo-A signaling complex.

PubMed

Thiede-Stan, Nina K; Tews, Björn; Albrecht, David; Ristic, Zorica; Ewers, Helge; Schwab, Martin E

2015-10-01

To ensure precision and specificity of ligand-receptor-induced signaling, co-receptors and modulatory factors play important roles. The membrane-bound ligand Nogo-A (an isoform encoded by RTN4) induces inhibition of neurite outgrowth, cell spreading, adhesion and migration through multi-subunit receptor complexes. Here, we identified the four-transmembrane-spanning protein tetraspanin-3 (TSPAN3) as a new modulatory co-receptor for the Nogo-A inhibitory domain Nogo-A-Δ20. Single-molecule tracking showed that TSPAN3 molecules in the cell membrane reacted to binding of Nogo-A with elevated mobility, which was followed by association with the signal-transducing Nogo-A receptor sphingosine-1-phosphate receptor 2 (S1PR2). Subsequently, TSPAN3 was co-internalized as part of the Nogo-A-ligand-receptor complex into early endosomes, where it subsequently separated from Nogo-A and S1PR2 to be recycled to the cell surface. The functional importance of the Nogo-A-TSPAN3 interaction is shown by the fact that knockdown of TSPAN3 strongly reduced the Nogo-A-induced S1PR2 clustering, RhoA activation, cell spreading and neurite outgrowth inhibition. In addition to the modulatory functions of TSPAN3 on Nogo-A-S1PR2 signaling, these results illustrate the very dynamic spatiotemporal reorganizations of membrane proteins during ligand-induced receptor complex organization. © 2015. Published by The Company of Biologists Ltd.

Enhancement of bradykinin and resensitization of its B2 receptor.

PubMed

Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

1999-03-01

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

The Four Rs of Environmental Dredging: Resuspension, Release, Residual, and Risk

DTIC Science & Technology

2008-02-01

sufficiently mobile to avoid the plume and potentially impact the health of less mobile aquatic vertebrates and invertebrates . Resettling of suspended...spatial dimension of both exposures and effects will vary among receptor groups. Exposures to sessile or relatively immobile receptors will vary more... reproduction ), but differ in regard to how they experience exposures associated with dredging in both the short and long term. For these reasons, risks

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.

PubMed

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-11-15

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

PubMed Central

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-01-01

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the “phosphate-sensor” of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins. PMID:22039220

Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2) and glutamatergic receptors.

PubMed

Oswald, Duane J; Lee, Albert; Trinidad, Monique; Chi, Cheryl; Ren, Ruiyi; Rich, Celeste B; Trinkaus-Randall, Vickery

2012-01-01

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

The 17,18-epoxyeicosatetraenoic acid-G protein-coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques.

PubMed

Nagatake, Takahiro; Shiogama, Yumiko; Inoue, Asuka; Kikuta, Junichi; Honda, Tetsuya; Tiwari, Prabha; Kishi, Takayuki; Yanagisawa, Atsushi; Isobe, Yosuke; Matsumoto, Naomi; Shimojou, Michiko; Morimoto, Sakiko; Suzuki, Hidehiko; Hirata, So-Ichiro; Steneberg, Pär; Edlund, Helena; Aoki, Junken; Arita, Makoto; Kiyono, Hiroshi; Yasutomi, Yasuhiro; Ishii, Masaru; Kabashima, Kenji; Kunisawa, Jun

2017-12-27

Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE) is a recently identified new class of antiallergic and anti-inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. We evaluated the effectiveness of 17,18-EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18-EpETE. Contact hypersensitivity was induced by topical application of 2,4-dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. Neutrophil mobility was examined by means of imaging analysis in vivo and neutrophil culture in vitro. The receptor for 17,18-EpETE was identified by using the TGF-α shedding assay, and the receptor's involvement in the anti-inflammatory effects of 17,18-EpETE was examined by using KO mice and specific inhibitor treatment. We found that preventive or therapeutic treatment with 17,18-EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18-EpETE was recognized by G protein-coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant-induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18-EpETE was abolished in the absence or inhibition of GPR40. 17,18-EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

PubMed

Fediuk, J; Sikarwar, A S; Lizotte, P P; Hinton, M; Nolette, N; Dakshinamurti, S

2015-02-01

Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Persistent Increase in Microglial RAGE Contributes to Chronic Stress-Induced Priming of Depressive-like Behavior.

PubMed

Franklin, Tina C; Wohleb, Eric S; Zhang, Yi; Fogaça, Manoela; Hare, Brendan; Duman, Ronald S

2018-01-01

Chronic stress-induced inflammatory responses occur in part via danger-associated molecular pattern (DAMP) molecules, such as high mobility group box 1 protein (HMGB1), but the receptor(s) underlying DAMP signaling have not been identified. Microglia morphology and DAMP signaling in enriched rat hippocampal microglia were examined during the development and expression of chronic unpredictable stress (CUS)-induced behavioral deficits, including long-term, persistent changes after CUS. The results show that CUS promotes significant morphological changes and causes robust upregulation of HMGB1 messenger RNA in enriched hippocampal microglia, an effect that persists for up to 6 weeks after CUS exposure. This coincides with robust and persistent upregulation of receptor for advanced glycation end products (RAGE) messenger RNA, but not toll-like receptor 4 in hippocampal microglia. CUS also increased surface expression of RAGE protein on hippocampal microglia as determined by flow cytometry and returned to basal levels 5 weeks after CUS. Importantly, exposure to short-term stress was sufficient to increase RAGE surface expression as well as anhedonic behavior, reflecting a primed state that results from a persistent increase in RAGE messenger RNA expression. Further evidence for DAMP signaling in behavioral responses is provided by evidence that HMGB1 infusion into the hippocampus was sufficient to cause anhedonic behavior and by evidence that RAGE knockout mice were resilient to stress-induced anhedonia. Together, the results provide evidence of persistent microglial HMGB1-RAGE expression that increases vulnerability to depressive-like behaviors long after chronic stress exposure. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Adaptor protein-3 is required in dendritic cells for optimal Toll-like receptor signaling from phagosomes and antigen presentation to CD4+ T cells

PubMed Central

Mantegazza, Adriana R.; Guttentag, Susan H.; El-Benna, Jamel; Sasai, Miwa; Iwasaki, Akiko; Shen, Hao; Laufer, Terri M.; Marks, Michael S.

2012-01-01

SUMMARY Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4+ T cell activation and Th1 effector function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores. PMID:22560444

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

PubMed

Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

2016-12-01

Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala 19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

PubMed Central

Cook, Brian L.; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P.; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

2009-01-01

Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. PMID:19581598

Large-scale production and study of a synthetic G protein-coupled receptor: human olfactory receptor 17-4.

PubMed

Cook, Brian L; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

2009-07-21

Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be approximately 50% alpha-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

PubMed

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

PubMed

Ding, K H; Husain, S; Akhtar, R A; Isales, C M; Abdel-Latif, A A

1997-09-01

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.

Live Imaging of Kv7.2/7.3 Cell Surface Dynamics at the Axon Initial Segment: High Steady-State Stability and Calpain-Dependent Excitotoxic Downregulation Revealed.

PubMed

Benned-Jensen, Tau; Christensen, Rasmus Kordt; Denti, Federico; Perrier, Jean-Francois; Rasmussen, Hanne Borger; Olesen, Søren-Peter

2016-02-17

The voltage-gated K(+) channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)-TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP-TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containing NMDA receptors, Ca(2+) influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABAA receptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily. The voltage-gated K(+) channels Kv7.2 and Kv7.3 exert strong control over action potential generation, but little is known about their cell surface dynamics. Using a novel phluorin-based approach, we here show that these channels are highly stable at steady state and different types of neuronal stimulation. However, at high glutamate loads, they undergo a rapid calpain-dependent endocytosis that likely represents an early response during excitotoxic states. Copyright © 2016 the authors 0270-6474/16/362261-06$15.00/0.

Endocannabinoid signaling mediates oxytocin-driven social reward.

PubMed

Wei, Don; Lee, DaYeon; Cox, Conor D; Karsten, Carley A; Peñagarikano, Olga; Geschwind, Daniel H; Gall, Christine M; Piomelli, Daniele

2015-11-10

Marijuana exerts profound effects on human social behavior, but the neural substrates underlying such effects are unknown. Here we report that social contact increases, whereas isolation decreases, the mobilization of the endogenous marijuana-like neurotransmitter, anandamide, in the mouse nucleus accumbens (NAc), a brain structure that regulates motivated behavior. Pharmacological and genetic experiments show that anandamide mobilization and consequent activation of CB1 cannabinoid receptors are necessary and sufficient to express the rewarding properties of social interactions, assessed using a socially conditioned place preference test. We further show that oxytocin, a neuropeptide that reinforces parental and social bonding, drives anandamide mobilization in the NAc. Pharmacological blockade of oxytocin receptors stops this response, whereas chemogenetic, site-selective activation of oxytocin neurons in the paraventricular nucleus of the hypothalamus stimulates it. Genetic or pharmacological interruption of anandamide degradation offsets the effects of oxytocin receptor blockade on both social place preference and cFos expression in the NAc. The results indicate that anandamide-mediated signaling at CB1 receptors, driven by oxytocin, controls social reward. Deficits in this signaling mechanism may contribute to social impairment in autism spectrum disorders and might offer an avenue to treat these conditions.

GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps

PubMed Central

Zeigerer, Anja; Lampson, Michael A.; Karylowski, Ola; Sabatini, David D.; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E.

2002-01-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level. PMID:12134080

GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps.

PubMed

Zeigerer, Anja; Lampson, Michael A; Karylowski, Ola; Sabatini, David D; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E

2002-07-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Pinoresinol-4,4'-di-O-beta-D-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts.

PubMed

Do, Kee Hun; Choi, Young Whan; Kim, Eun Kyoung; Yun, Sung Ji; Kim, Min Sung; Lee, Sun Young; Ha, Jung Min; Kim, Jae Ho; Kim, Chi Dae; Son, Beung Gu; Kang, Jum Soon; Khan, Ikhlas A; Bae, Sun Sik

2009-06-01

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4'-di-O-beta-D-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 microM PDG resulted in strong stimulation of MEF cell migration and the EC(50) was about 2 microM. Pretreatment with pertussis toxin (PTX), an inhibitor of G(i) protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G(i)-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10 microM), which is a selective antagonist for LPA(1) and LPA(3) receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.

Dynamic interactions of the asialoglycoprotein receptor subunits with coated pits. Enhanced interactions of H2 following association with H1.

PubMed

Katzir, Z; Nardi, N; Geffen, I; Fuhrer, C; Henis, Y I

1994-08-26

Lateral mobility studies comparing native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, can measure membrane protein-coated pit interactions in intact cells (Fire, E., Zwart, D., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). We applied this approach to study the interactions of the H1 and H2 human asialoglycoprotein receptor subunits with coated pits. The lateral mobilities of singly expressed and coexpressed H1 and H2B (the H2 species that reaches the cell surface) were measured by fluorescence photobleaching recovery. They were compared with mutant proteins, H1(5A) (Tyr-5 replaced by Ala) and H2(5A) (Phe-5 replaced by Ala). While the mobile fractions of H1, H2B, and their mutants were similar, the lateral diffusion rate (measured by D, the lateral diffusion coefficient) was significantly slower for H1, whether expressed alone or with H2B. Coexpression with H1 reduced D of H2B to that of H1. Disruption of the clathrin lattices by hypertonic medium elevated D of H1, H1(5A), H2B, and H2(5A) to the same final level, without affecting their mobile fractions. Cytosol acidification, which retains altered clathrin lattices attached to the membrane and prevents coated vesicle formation, immobilized part of the H1 molecules, reflecting stable entrapment in "frozen" coated pits. H1(5A), H2B, and H2(5A) were not affected; however, coexpression of H2B with H1 conferred the sensitivity to cytosol acidification on H2B. Our results suggest that H1 lateral mobility is inhibited by dynamic interactions with coated pits in which Tyr-5 is involved. H2B resembles H1(5A) rather than H1, and its interactions with coated pits are weaker; efficient interaction of H2B with coated pits depends on complex formation with H1.

Diarylureas as allosteric modulators of the cannabinoid CB1 receptor: structure-activity relationship studies on 1-(4-chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea (PSNCBAM-1).

PubMed

German, Nadezhda; Decker, Ann M; Gilmour, Brian P; Gay, Elaine A; Wiley, Jenny L; Thomas, Brian F; Zhang, Yanan

2014-09-25

The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [(3)H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.

CD22 ligation inhibits downstream B cell receptor signaling and Ca(2+) flux upon activation.

PubMed

Sieger, N; Fleischer, S J; Mei, H E; Reiter, K; Shock, A; Burmester, G R; Daridon, C; Dörner, T

2013-03-01

CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases. Copyright © 2013 by the American College of Rheumatology.

Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling

PubMed Central

Gur-Cohen, Shiri; Kollet, Orit; Graf, Claudine; Esmon, Charles T.; Ruf, Wolfram; Lapidot, Tsvee

2016-01-01

The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of several cell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs), endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR), which is used as a marker to isolate them. EPCR/PAR1 signaling in endothelial cells has anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflammation. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR+ LT-HSC BM retention and egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production, maintaining NOlow LT-HSC BM retention with increased VLA4 expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generation and activate different PAR1 signaling which overcomes BM EPCR+ LT-HSC retention, inducing their recruitment to the bloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulation of CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses new roles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1 signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production. PMID:26928241

Regulation of ghrelin secretion and action.

PubMed

Camiña, Jesus P; Carreira, Marcos C; Micic, Dragan; Pombo, Manuel; Kelestimur, Fahrettin; Dieguez, Carlos; Casanueva, Felipe F

2003-10-01

The pulsatile release of growth hormone (GH) from anterior pituitary gland is regulated by the interplay of at least two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. Furthermore, release of GH in vivo may also be controlled by a third type of receptor, the growth hormone secretagogue receptor, a G-protein-coupled receptor, called GHS receptor type 1a (GHSR1a), which was identified in the pituitary and the hypothalamus in humans using a nonpeptidyl growth hormone secretagogue (MK-0677). Ghrelin, the endogenous ligand for the GHS-R1a, is a 28-amino-acid peptide isolated from human stomach that is modified by a straight chain octanoyl group covalently linked to Ser3, which is essential for its endocrine activity. This hormone, predominantly expressed and secreted by the stomach, has a dual action on GH secretion and food intake, showing interdependency between these actions. The finding that fasting and food intake, respectively, increase and decrease the secretion of ghrelin suggests that this hormone may be the bridge connecting somatic growth and body composition with energy metabolism, and appears to play a role in the alteration of energy homeostasis and body weight in pathophysiological states such as hypothyroidism and hyperthyroidism. Despite this, little is known about the intracellular signaling through which ghrelin exerts its regulatory actions. Activation of intracellular calcium mobilization is one of the earliest known cellular signals elicited by ghrelin. In HEK- 293 cells expressing the GHS-R1a, ghrelin induces a biphasic cytosolic calcium elevation characterized by a spike phase of the response, which reflects Ins(1,4,5)P3- dependent calcium mobilization of intracellular stores, and a sustained phase of the response, which is due to calcium influx across the plasma membrane triggered by aperture of capacitative calcium channels (store-operated calcium channels). Upon repeated administration, ghrelin showed a marked suppression of ghrelin-mediated elevations of intracellular calcium. This homologous desensitization represents an important physiological mechanism that modulates receptor responsiveness and acts as an information filter for intracellular signaling system. The discovery of ghrelin adds a new component to the complex machinery responsible for regulation of GH secretion in connection with the regulation of appetite and energy homeostasis.

Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors.

PubMed

Duty, J Andrew; Szodoray, Peter; Zheng, Nai-Ying; Koelsch, Kristi A; Zhang, Qingzhao; Swiatkowski, Mike; Mathias, Melissa; Garman, Lori; Helms, Christina; Nakken, Britt; Smith, Kenneth; Farris, A Darise; Wilson, Patrick C

2009-01-16

Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

PubMed

Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fuller, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

2006-07-01

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression.

PubMed

Ramsay, Andrew J; Dong, Ying; Hunt, Melanie L; Linn, MayLa; Samaratunga, Hemamali; Clements, Judith A; Hooper, John D

2008-05-02

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.

β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

PubMed

Galaz-Montoya, Monica; Wright, Sara J; Rodriguez, Gustavo J; Lichtarge, Olivier; Wensel, Theodore G

2017-06-16

Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor

PubMed Central

Treanor, Bebhinn; Depoil, David; Gonzalez-Granja, Aitor; Barral, Patricia; Weber, Michele; Dushek, Omer; Bruckbauer, Andreas; Batista, Facundo D.

2010-01-01

Summary Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igβ as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival. PMID:20171124

Calcium Signalling through Ligand-Gated Ion Channels such as P2X1 Receptors in the Platelet and other Non-Excitable Cells.

PubMed

Mahaut-Smith, Martyn P; Taylor, Kirk A; Evans, Richard J

2016-01-01

Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca(2+) whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells. Much of the detailed information on the contribution of P2X1 to Ca(2+) signalling and downstream functional events has been derived from the platelet. The underlying primary P2X1-generated signalling event in non-excitable cells is principally due to Ca(2+) influx, although Na(+) entry will also occur along with membrane depolarization. P2X1 receptor stimulation can lead to additional Ca(2+) mobilization via a range of routes such as amplification of G-protein-coupled receptor-dependent Ca(2+) responses. This chapter also considers the mechanism by which cells generate extracellular ATP for autocrine or paracrine activation of P2X1 receptors. For example cytosolic ATP efflux can result from opening of pannexin anion-permeable channels or following damage to the cell membrane. Alternatively, ATP stored in specialised secretory vesicles can undergo quantal release via the process of exocytosis. Examples of physiological or pathophysiological roles of P2X1-dependent signalling in non-excitable cells are also discussed, such as thrombosis and immune responses.

SiGe derivatization by spontaneous reduction of aryl diazonium salts

NASA Astrophysics Data System (ADS)

Girard, A.; Geneste, F.; Coulon, N.; Cardinaud, C.; Mohammed-Brahim, T.

2013-10-01

Germanium semiconductors have interesting properties for FET-based biosensor applications since they possess high surface roughness allowing the immobilization of a high amount of receptors on a small surface area. Since SiGe combined low cost of Si and intrinsic properties of Ge with high mobility carriers, we focused the study on this particularly interesting material. The comparison of the efficiency of a functionalization process involving the spontaneous reduction of diazonium salts is studied on Si(1 0 0), SiGe and Ge semiconductors. XPS analysis of the functionalized surfaces reveals the presence of a covalent grafted layer on all the substrates that was confirmed by AFM. Interestingly, the modified Ge derivatives have still higher surface roughness after derivatization. To support the estimated thickness by XPS, a step measurement of the organic layers is done by AFM or by profilometer technique after a O2 plasma etching of the functionalized layer. This original method is well-adapted to measure the thickness of thin organic films on rough substrates such as germanium. The analyses show a higher chemical grafting on SiGe substrates compared with Si and Ge semiconductors.

Non-pharmacological treatment affects neuropeptide expression in neuropathic pain model.

PubMed

Santos, Fabio Martinez; Silva, Joyce Teixeira; Rocha, Igor Rafael Correia; Martins, Daniel Oliveira; Chacur, Marucia

2018-05-15

Chronic constriction injury (CCI) of the sciatic nerve elicits changes in neuropeptide expression on the dorsal root ganglia (DRG). The neural mobilization (NM) technique is a noninvasive method that has been proven clinically effective in reducing pain. The aim of this study was to analyze the expression of substance P, transient receptor potential vanilloid 1 (TRPV1) and opioid receptors in the DRG of rats with chronic constriction injury and to compare it to animals that received NM treatment. CCI was performed on adult male rats. Each animal was submitted to 10 sessions of neural mobilization every other day, starting 14 days after the CCI injury. At the end of the sessions, the DRG (L4-L6) were analyzed using Western blot assays for substance P, TRPV1 and opioid receptors (µ-opioid receptor, δ-opioid receptor and κ-opioid receptor). We observed a decreased substance P and TRPV1 expression (48% and 35%, respectively) and an important increase of µ-opioid receptor expression (200%) in the DRG after NM treatment compared to control animals. The data provide evidence that NM promotes substantial changes in neuropeptide expression in the DRG; these results may provide new options for treating neuropathic pain. Copyright © 2018 Elsevier B.V. All rights reserved.

Mobilization of endocrine-disrupting chemicals and estrogenic activity in simulated rainfall runoff from land-applied biosolids.

PubMed

Giudice, Ben D; Young, Thomas M

2011-10-01

Municipal biosolids are commonly applied to land as soil amendment or fertilizer as a form of beneficial reuse of what could otherwise be viewed as waste. Balanced against this benefit are potential risks to groundwater and surface water quality from constituents that may be mobilized during storm events. The objective of the present study was to characterize the mobilization of selected endocrine-disrupting compounds, heavy metals, and total estrogenic activity in rainfall runoff from land-applied biosolids. Rainfall simulations were conducted on soil plots amended with biosolids. Surface runoff and leachate was collected and analyzed for the endocrine-disrupting compounds bisphenol A, 17α-ethynylestradiol, triclocarban, triclosan, octylphenol, and nonylphenol; a suite of 16 metals; and estrogenic activity via the estrogen receptor-mediated chemical activated luciferase gene expression (ER-CALUX) bioassay. Triclocarban (2.3-17.3 ng/L), triclosan (<51-309 ng/L), and octylphenol (<4.9-203 ng/L) were commonly detected. Chromium (2.0-22 µg/L), Co (2.5-10 µg/L), Ni (28-235 µg/L), Cu (14-110 µg/L), As (1.2-2.7 µg/L), and Se (0.29-12 µg/L) were quantifiable over background levels. Triclosan, Ni, and Cu were detected at levels that might pose some risk to aquatic life, though levels of metals in the biosolids were well below the maximum allowable regulatory limits. The ER-CALUX results were mostly explained by background bisphenol A contamination and octylphenol in runoff, although unknown contributors or matrix effects were also found. Copyright © 2011 SETAC.

Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

PubMed Central

Paolini, R; Kinet, J P

1993-01-01

Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912

Expression of a truncated form of the endoplasmic reticulum chaperone protein, σ1 receptor, promotes mitochondrial energy depletion and apoptosis.

PubMed

Shioda, Norifumi; Ishikawa, Kiyoshi; Tagashira, Hideaki; Ishizuka, Toru; Yawo, Hiromu; Fukunaga, Kohji

2012-07-06

The σ1 receptor (σ(1)R) regulates endoplasmic reticulum (ER)/mitochondrial interorganellar Ca(2+) mobilization through the inositol 1,4,5-trisphosphate receptor (IP(3)R). Here, we observed that expression of a novel splice variant of σ(1)R, termed short form σ(1)R (σ(1)SR), has a detrimental effect on mitochondrial energy production and cell survival. σ(1)SR mRNA lacks 47 ribonucleotides encoding exon 2, resulting in a frameshift and formation of a truncated receptor. σ(1)SR localizes primarily in the ER at perinuclear regions and forms a complex with σ(1)R but not with IP(3)R in the mitochondrion-associated ER membrane. Overexpression of both σ(1)R and the truncated isoform promotes mitochondrial elongation with increased ER mitochondrial contact surface. σ(1)R overexpression increases the efficiency of mitochondrial Ca(2+) uptake in response to IP(3)R-driven stimuli, whereas σ(1)SR overexpression reduces it. Most importantly, σ(1)R promotes ATP production via increased mitochondrial Ca(2+) uptake, promoting cell survival in the presence of ER stress. By contrast, σ(1)SR suppresses ATP production following ER stress, enhancing cell death. Taken together, the newly identified σ(1)SR isoform interferes with σ(1)R function relevant to mitochondrial energy production under ER stress conditions, promoting cellular apoptosis.

Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

PubMed

Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

2014-04-25

The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Isolation frequency of Candida present on the surfaces of mobile phones and handsx.

PubMed

Kordecka, Anna; Krajewska-Kułak, Elżbieta; Łukaszuk, Cecylia; Kraszyńska, Bogumiła; Kułak, Wojciech

2016-06-01

It is known that mobile phones may play a role in microorganism transmission. The aim of this study was to analyze the relationship between the number of Candida genera/species isolated from samples collected from the surfaces of mobile phones and the hands of the staff as well as the preferred health-related behavior. The mycological evaluation included 175 mobile telephones and the hands of staff members at the University Hospital in Białystok, Poland. We used the Count-Tact(TM) applicator, with CandiSelect (Bio-Rad). Self-administered questionnaire was used to gather data on mobile phones disinfection practices. Assessment of the preferred health-related behavior was based on The Multidemensional Health Locus of Control Scale (MHLC). Out of 175 mobile phones, 131 (74.9 %) were colonized. Candida glabrata, C. albicans and C.krusei were isolated more frequently from the hand as well as phone surface. The mean number of Candida colonies was higher in samples collected from hand surfaces than mobile phone surfaces. No significant correlation was found between the preferred health-related behavior and the frequency of washing hands, the way of using a mobile phone, the number of colonies or the isolation frequency for the fungi collected from the surface of the phones and hands of their owners. Only 19.4 % of the participants cleaned the surface of their phones. The prevalence of mobile phone contamination by Candida is high in the University Hospital in Białystok, Poland. Candida albicans, C. glabrata, and C. krusei were the dominant species in the samples collected from mobile phones and hands. These results pose the need to develop guidelines for mobile phone disinfection.

Synaptic Bistability Due to Nucleation and Evaporation of Receptor Clusters

NASA Astrophysics Data System (ADS)

Burlakov, V. M.; Emptage, N.; Goriely, A.; Bressloff, P. C.

2012-01-01

We introduce a bistability mechanism for long-term synaptic plasticity based on switching between two metastable states that contain significantly different numbers of synaptic receptors. One state is characterized by a two-dimensional gas of mobile interacting receptors and is stabilized against clustering by a high nucleation barrier. The other state contains a receptor gas in equilibrium with a large cluster of immobile receptors, which is stabilized by the turnover rate of receptors into and out of the synapse. Transitions between the two states can be initiated by either an increase (potentiation) or a decrease (depotentiation) of the net receptor flux into the synapse. This changes the saturation level of the receptor gas and triggers nucleation or evaporation of receptor clusters.

Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.

PubMed

Zheng, Huimei; Wang, Xuexiang; Guo, Pengfei; Ge, Wanzhong; Yan, Qinfeng; Gao, Weiqiang; Xi, Yongmei; Yang, Xiaohang

2017-05-01

In Drosophila, fat-body remodeling accompanied with fat mobilization is an ecdysone-induced dynamic process that only occurs during metamorphosis. Here, we show that the activated Drosophila platelet-derived growth factor/VEGF receptor (PVR) is sufficient to induce shape changes in the fat body, from thin layers of tightly conjugated polygonal cells to clusters of disaggregated round-shaped cells. These morphologic changes are reminiscent of those seen during early pupation upon initiation of fat-body remodeling. Activation of PVR also triggers an early onset of lipolysis and mobilization of internal storage, as revealed by the appearance of small lipid droplets and up-regulated lipolysis-related genes. We found that PVR displays a dynamic expression pattern in the fat body and peaks at the larval-prepupal transition under the control of ecdysone signaling. Removal of PVR, although it does not prevent ecdysone-induced fat-body remodeling, causes ecdysone signaling to be up-regulated. Our data reveal that PVR is active in a dual-secured mechanism that involves an ecdysone-induced fat-body remodeling pathway and a reinforced PVR pathway for effective lipid mobilization. Ectopic expression of activated c-kit-the mouse homolog of PVR in the Drosophila fat body-also results in a similar phenotype. This may suggest a novel function of c-kit as it relates to lipid metabolism in mammals.-Zheng, H., Wang, X., Guo, P., Ge, W., Yan, Q., Gao, W., Xi, Y., Yang, X. Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila . © FASEB.

Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

PubMed Central

Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

2015-01-01

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse

PubMed Central

Hivroz, Claire; Saitakis, Michael

2016-01-01

T lymphocyte activation is a pivotal step of the adaptive immune response. It requires the recognition by T-cell receptors (TCR) of peptides presented in the context of major histocompatibility complex molecules (pMHC) present at the surface of antigen-presenting cells (APCs). T lymphocyte activation also involves engagement of costimulatory receptors and adhesion molecules recognizing ligands on the APC. Integration of these different signals requires the formation of a specialized dynamic structure: the immune synapse. While the biochemical and molecular aspects of this cell–cell communication have been extensively studied, its mechanical features have only recently been addressed. Yet, the immune synapse is also the place of exchange of mechanical signals. Receptors engaged on the T lymphocyte surface are submitted to many tensile and traction forces. These forces are generated by various phenomena: membrane undulation/protrusion/retraction, cell mobility or spreading, and dynamic remodeling of the actomyosin cytoskeleton inside the T lymphocyte. Moreover, the TCR can both induce force development, following triggering, and sense and convert forces into biochemical signals, as a bona fide mechanotransducer. Other costimulatory molecules, such as LFA-1, engaged during immune synapse formation, also display these features. Moreover, T lymphocytes themselves are mechanosensitive, since substrate stiffness can modulate their response. In this review, we will summarize recent studies from a biophysical perspective to explain how mechanical cues can affect T lymphocyte activation. We will particularly discuss how forces are generated during immune synapse formation; how these forces affect various aspects of T lymphocyte biology; and what are the key features of T lymphocyte response to stiffness. PMID:26913033

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

PubMed Central

Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

2006-01-01

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils

PubMed Central

Paulsson, J M; Moshfegh, A; Dadfar, E; Held, C; Jacobson, S H; Lundahl, J

2012-01-01

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL-1R1) following in-vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL-1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL-1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL-1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL-1R1 that could be mobilized by N-formyl-methionine-leucine-phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL-1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in-vivo extravasation. The present results demonstrate that IL-1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL-1R1 as demonstrated by the induction of chemokines following IL-1 stimulation. The results indicate a potential role for IL-1 in the activation of neutrophils at inflammatory sites. PMID:22385245

A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

PubMed Central

Boudreau, Amy C.; Milovanovic, Mike; Conrad, Kelly L.; Nelson, Christopher; Ferrario, Carrie R.; Wolf, Marina E.

2012-01-01

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then cell surface-expressed receptors are covalently crosslinked to nearby proteins using the membrane-impermeable, bifunctional crosslinker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after crosslinking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. PMID:22470150

Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

EPA Science Inventory

The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

Leptin inhibits neutrophil apoptosis in children via ERK/NF-κB-dependent pathways.

PubMed

Sun, Zhizhi; Dragon, Stéphane; Becker, Allan; Gounni, Abdelilah S

2013-01-01

Previous studies have shown that delayed neutrophil apoptosis is associated with chronic airway diseases. Leptin is an adipocyte-derived hormone that acts as a regulator of energy homeostasis and food intake. Emerging evidence suggests that leptin can regulate immune responses including the release of proinflammatory cytokines and protection of inflammatory cells from apoptosis. Serum leptin is increased during allergic reactions in the airways. However, the expression and function of leptin receptor in neutrophils isolated from children is not known. Flow cytometry was used to detect leptin receptor expression in neutrophils isolated from allergic asthmatic (n = 14), allergic non asthmatic (n = 21), non allergic asthmatic (n = 7) and healthy children (n = 23); confocal laser scanning microscopy combined with immunofluorescence was performed to detect intracellular pool of leptin receptor; Annexin-V/PI staining and caspase 3 activity was used to determine neutrophil survival. Pharmacological inhibitors were utilized to understand the role of MAPK and NF-κB pathway in leptin-induced neutrophil survival. A heterogeneous leptin receptor expression was observed on neutrophils isolated from children. Neutrophils isolated from healthy children expressed more leptin receptor than those from allergic asthmatic (P<0.05) but not allergic non-asthmatic (P>0.05) or non-allergic asthmatic children (n = 7, P>0.05). Neutrophils isolated from children express an intracellular pool of leptin receptor that was mobilized to the cell surface upon GM-CSF stimulation. Finally, leptin exhibited anti-apoptotic properties on neutrophils via NF-κB and MEK1/2 MAPK pathway. Collectively, our data suggest that leptin may enhance airway inflammation by promoting neutrophil survival.

Leptin Inhibits Neutrophil Apoptosis in Children via ERK/NF-κB-Dependent Pathways

PubMed Central

Sun, Zhizhi; Dragon, Stéphane; Becker, Allan; Gounni, Abdelilah S.

2013-01-01

Introduction and Rationale Previous studies have shown that delayed neutrophil apoptosis is associated with chronic airway diseases. Leptin is an adipocyte-derived hormone that acts as a regulator of energy homeostasis and food intake. Emerging evidence suggests that leptin can regulate immune responses including the release of proinflammatory cytokines and protection of inflammatory cells from apoptosis. Serum leptin is increased during allergic reactions in the airways. However, the expression and function of leptin receptor in neutrophils isolated from children is not known. Methods Flow cytometry was used to detect leptin receptor expression in neutrophils isolated from allergic asthmatic (n = 14), allergic non asthmatic (n = 21), non allergic asthmatic (n = 7) and healthy children (n = 23); confocal laser scanning microscopy combined with immunofluorescence was performed to detect intracellular pool of leptin receptor; Annexin-V/PI staining and caspase 3 activity was used to determine neutrophil survival. Pharmacological inhibitors were utilized to understand the role of MAPK and NF-κB pathway in leptin-induced neutrophil survival. Results and Conclusion A heterogeneous leptin receptor expression was observed on neutrophils isolated from children. Neutrophils isolated from healthy children expressed more leptin receptor than those from allergic asthmatic (P<0.05) but not allergic non-asthmatic (P>0.05) or non-allergic asthmatic children (n = 7, P>0.05). Neutrophils isolated from children express an intracellular pool of leptin receptor that was mobilized to the cell surface upon GM-CSF stimulation. Finally, leptin exhibited anti-apoptotic properties on neutrophils via NF-κB and MEK1/2 MAPK pathway. Collectively, our data suggest that leptin may enhance airway inflammation by promoting neutrophil survival. PMID:23383125

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

NASA Technical Reports Server (NTRS)

Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1995-01-01

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

Effect of surface mobility on the particle sliding along a bubble or a solid sphere.

PubMed

Wang, Weixing; Zhou, Zhiang; Nandakumar, K; Xu, Zhenghe; Masliyah, Jacob H

2003-03-01

The sliding velocity of glass beads on a spherical surface, made either of an air bubble or of a glass sphere held stationary, is measured to investigate the effect of surface mobility on the particle sliding velocity. The sliding process is recorded with a digital camera and analyzed frame by frame. The sliding glass bead was found to accelerate with increasing angular position on the collector's surface. It reaches a maximum velocity at an angular position of about 100 degrees and then, under certain conditions, the glass bead leaves the surface of the collector. The sliding velocity of the glass bead depends strongly on the surface mobility of a bubble, decreasing with decreasing surface mobility. By a mobile surface we mean one which cannot set up resistive forces to an applied stress on the surface. The sliding velocity on a rigid surface, such as a glass sphere, is much lower than that on a mobile bubble surface. The sliding velocity can be described through a modified Stokes equation. A numerical factor in the modified Stokes equation is determined by fitting the experimental data and is found to increase with decreasing surface mobility. Hydrophobic glass beads sliding on a hydrophobic glass sphere were found to stick at the point of impact without sliding if the initial angular position of the impact is less than some specific angle, which is defined as the critical sticking angle. The sticking of the glass beads can be attributed to the capillary contracting force created by the formation of a cavity due to spontaneous receding of the nonwetting liquid from the contact zone. The relationship between the critical sticking angle and the particle size is established based on the Yushchenko [J. Colloid Interface Sci. 96 (1983) 307] analysis.

Heterogeneity of chemokine cell-surface receptor expression in triple-negative breast cancer

PubMed Central

Norton, Kerri-Ann; Popel, Aleksander S; Pandey, Niranjan B

2015-01-01

Introduction: Tumor heterogeneity is a well-established concept in cancer research. In this paper, we examine an additional type of tumor cell heterogeneity - tumor cell-surface receptor heterogeneity. Methods: We use flow cytometry to measure the frequency and numbers of cell-surface receptors on triple negative breast cancer cell lines. Results: We find two distinct populations of human triple-negative breast cancer cells MDA-MB-231 when they are grown in culture, one with low surface levels of various chemokine receptors and a second with much higher levels. The population with high surface levels of these receptors is increased in the more metastatic MDA-MB-231-luc-d3h2ln cell line. Conclusion: We hypothesize that this high cell-surface receptor population is involved in metastasis. We find that the receptor high populations can be modulated by tumor conditioned media and IL6 treatment indicating that the tumor microenvironment is important for the maintenance and sizes of these populations. PMID:26101698

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition.

PubMed

Sheokand, Navdeep; Kumar, Santosh; Malhotra, Himanshu; Tillu, Vikas; Raje, Chaaya Iyengar; Raje, Manoj

2013-06-01

The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron. These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays. This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized. Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis. Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.

PubMed

Elhaji, Youssef A; Stoica, Ileana; Dennis, Sheldon; Purisima, Enrico O; Lumbroso, Rose; Beitel, Lenore K; Trifiro, Mark A

2006-03-15

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.

Notch2 blockade enhances hematopoietic stem cell mobilization and homing.

PubMed

Wang, Weihuan; Yu, Shuiliang; Myers, Jay; Wang, Yiwei; Xin, William W; Albakri, Marwah; Xin, Alison W; Li, Ming; Huang, Alex Y; Xin, Wei; Siebel, Christian W; Lazarus, Hillard M; Zhou, Lan

2017-10-01

Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT. Copyright© 2017 Ferrata Storti Foundation.

Electron mobility on the surface of liquid Helium: influence of surface level atoms and depopulation of lowest subbands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grigoriev, P. D., E-mail: grigorev@itp.ac.ru; Dyugaev, A. M.; Lebedeva, E. V.

2008-02-15

The temperature dependence of electron mobility is examined. We calculate the contribution to the electron scattering rate from the surface level atoms (SLAs), proposed in [10]. This contribution is substantial at low temperatures T < 0.5, when the He vapor concentration is exponentially small. We also study the effect of depopulation of the lowest energy subband, which leads to an increase in the electron mobility at high temperature. The results explain certain long-standing discrepancies between the existing theory and experiment on electron mobility on the surface of liquid helium.

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

PubMed

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

PubMed Central

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

Asymmetrical Macromolecular Complex Formation of Lysophosphatidic Acid Receptor 2 (LPA2) Mediates Gradient Sensing in Fibroblasts*

PubMed Central

Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P.

2014-01-01

Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca2+ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca2+ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts. PMID:25542932

Single-cell analyses reveal that KISS1R-expressing cells undergo sustained kisspeptin-induced signaling that is dependent upon an influx of extracellular Ca2+.

PubMed

Babwah, Andy V; Pampillo, Macarena; Min, Le; Kaiser, Ursula B; Bhattacharya, Moshmi

2012-12-01

The kisspeptin receptor (KISS1R) is a Gα(q/11)-coupled seven-transmembrane receptor activated by a group of peptides referred to as kisspeptins (Kps). The Kp/KISS1R signaling system is a powerful regulator of GnRH secretion, and inactivating mutations in this system are associated with hypogonadotropic hypogonadism. A recent study revealed that Kp triggers prolonged signaling; not from the inability of the receptor to undergo rapid desensitization, but instead from the maintenance of a dynamic and active pool of KISS1R at the cell surface. To investigate this further, we hypothesized that if a dynamic pool of receptor is maintained at the cell surface for a protracted period, chronic Kp-10 treatment would trigger the sustained activation of Gα(q/11) as evidenced through the prolonged activation of phospholipase C, protein kinase C, and prolonged mobilization of intracellular Ca(2+). Through single-cell analyses, we tested our hypothesis in human embryonic kidney (HEK) 293 cells and found that was indeed the case. We subsequently determined that prolonged KISS1R signaling was not a phenomenon specific to HEK 293 cells but is likely a conserved property of KISS1R-expressing cells because evidence of sustained KISS1R signaling was also observed in the GT1-7 GnRH neuronal and Chinese hamster ovary cell lines. While exploring the regulation of prolonged KISS1R signaling, we identified a critical role for extracellular Ca(2+). We found that although free intracellular Ca(2+), primarily derived from intracellular stores, was sufficient to trigger the acute activation of a major KISS1R secondary effector, protein kinase C, it was insufficient to sustain chronic KISS1R signaling; instead extracellular Ca(2+) was absolutely required for this.

Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

PubMed

Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

2016-05-01

G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Colloidal mobilization of arsenic from mining-affected soils by surface runoff.

PubMed

Gomez-Gonzalez, Miguel Angel; Voegelin, Andreas; Garcia-Guinea, Javier; Bolea, Eduardo; Laborda, Francisco; Garrido, Fernando

2016-02-01

Scorodite-rich wastes left as a legacy of mining and smelting operations pose a threat to environmental health. Colloids formed by the weathering of processing wastes may control the release of arsenic (As) into surface waters. At a former mine site in Madrid (Spain), we investigated the mobilization of colloidal As by surface runoff from weathered processing wastes and from sediments in the bed of a draining creek and a downstream sedimentation-pond. Colloids mobilized by surface runoff during simulated rain events were characterized for their composition, structure and mode of As uptake using asymmetric flow field-flow fractionation coupled to inductively plasma mass spectrometry (AF4-ICP-MS) and X-ray absorption spectroscopy (XAS) at the As and Fe K-edges. Colloidal scorodite mobilized in surface runoff from the waste pile is acting as a mobile As carrier. In surface runoff from the river bed and the sedimentation pond, ferrihydrite was identified as the dominant As-bearing colloidal phase. The results from this study suggest that mobilization of As-bearing colloids by surface runoff may play an important role in the dispersion of As from metallurgical wastes deposited above ground and needs to be considered in risk assessment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins

NASA Astrophysics Data System (ADS)

Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard

1991-06-01

Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

PubMed

Driesbaugh, Kathryn H; Buzza, Marguerite S; Martin, Erik W; Conway, Gregory D; Kao, Joseph P Y; Antalis, Toni M

2015-02-06

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of two test methods to ensure accurate surface firmness and stability measurements for accessibility.

PubMed

Axelson, Peter W; Hurley, Seanna L

2018-05-01

The firmness and stability of indoor and outdoor surfacing are critical to the accessibility and safety of all environments for people with mobility impairments and/or who use mobility devices. ASTM F1951 laboratory test procedures include pass/fail criteria for determining playground surface accessibility by comparing the work to propel up a 1:14 (7.1%) grade ramp to that of the test surface in a wheelchair. A portable instrumented surface indenter (ISI) was developed to validate that accessibility results obtained in the laboratory are maintained in the field where the surface is installed and used. Accessibility measurements have been made on indoor and outdoor surfaces tested in the laboratory using both the ASTM F1951 and the ISI over 13 years. Correlations between these two methods were calculated. A strong correlation has been demonstrated for the sum of the ISI firmness and stability results compared to the sum of the ASTM F1951 straight propulsion and turning results (R 2 =0.9006). The portable ISI can be used to verify that the firmness and stability of an installed surface in the field correlates to the accessibility results of the surface tested in the laboratory concurrently according to ASTM F1951 and the ISI. Implications for Rehabilitation The Instrumented Surface Indenter (ISI) allows for surfaces in all environments to be tested for firmness and stability, which is critical for wheelchair user safety, especially during rehabilitation when learning to use a wheelchair. The ISI allows for surfaces in all environments to be tested for firmness and stability, which increases access to all indoor and outdoor surfaces, thereby improving the quality of life for people who have mobility impairments and/or use mobility devices, such as canes, crutches, walkers, and wheelchairs. Using the ISI to test the firmness and stability of installed playground surfaces increases access to playgrounds for children with mobility impairments, facilitating developmentally critical peer-play opportunities for children who use mobility devices. Using the ISI to test the firmness and stability of installed playground surfaces increases access to playgrounds for people with mobility impairments, allowing adults who use a mobility device to supervise and play with children in their lives.

Effect of surface roughness of trench sidewalls on electrical properties in 4H-SiC trench MOSFETs

NASA Astrophysics Data System (ADS)

Kutsuki, Katsuhiro; Murakami, Yuki; Watanabe, Yukihiko; Onishi, Toru; Yamamoto, Kensaku; Fujiwara, Hirokazu; Ito, Takahiro

2018-04-01

The effects of the surface roughness of trench sidewalls on electrical properties have been investigated in 4H-SiC trench MOSFETs. The surface roughness of trench sidewalls was well controlled and evaluated by atomic force microscopy. The effective channel mobility at each measurement temperature was analyzed on the basis of the mobility model including optical phonon scattering. The results revealed that surface roughness scattering had a small contribution to channel mobility, and at the arithmetic average roughness in the range of 0.4-1.4 nm, there was no correlation between the experimental surface roughness and the surface roughness scattering mobility. On the other hand, the characteristics of the gate leakage current and constant current stress time-dependent dielectric breakdown tests demonstrated that surface morphology had great impact on the long-term reliability of gate oxides.

Surface receptors on human haematopoietic cell lines.

PubMed Central

Huber, C; Sundström, C; Nilsson, K; Wigzell, H

1976-01-01

The expression of complement receptors, of Fc receptors, of SRBC receptors and of S-Ig was investigated on human haematopoietic cell lines of proved malignant derivation. According to their origin and to a panel of phenotypic markers these lines have been classified into lymphoma lines, myeloma lines and leukemia lines. Results were compared with those obtained on non-malignant EBV carrying lymphoblastoid cell lines (LCL). Among the lymphoid cell lines the LCL showed a pattern of B-lymphocyte surface markers, i.e. surface immunoglobulins, C3 receptors but low density of Fc receptors. The non-Burkitt lymphoma lines bore in varying degree these B-lymphocyte markers. The lines U-698 M and DG-75 were exceptional in having only surface immunoglobulin. The Burkitt lymphoma lines had all B-lymphocyte markers. The myeloma lines differed from the lymphoid lines in lacking C3 and Fc receptors and showed only trace amounts of surface immunoglobulins. In contrast to lymphoid and myeloma lines, the leukaemia lines were completely lacking surface immunoglobulins, but showed C3 and Fc receptors in variable densities. On line, the ALL derived line MOLT-3 showed the capacity to spontaneous rosette formation with SRBC. The findings that LCL presented a homogeneous pattern of B-lymphocyte surface markers may be of value in order to discriminate between these lines and lines derived from haematopoietic malignancies other than Burkitt lymphomas. PMID:963908

The varying stability of benthic homes: hydrologic regime and sediment supply control the timing and intensity of bed mobility

NASA Astrophysics Data System (ADS)

Pfeiffer, A.; Finnegan, N. J.

2017-12-01

Gravel river beds provide an ephemeral architecture for the benthic inhabitants of river ecosystems. Periphyton and benthic macroinvertebrates that live on or within the gravel are subject to catastrophic disruption upon mobilization of the surface gravel during floods. Because sediment supply varies by orders of magnitude across North America, and rivers have adjusted to convey their imposed loads, river bed surface mobility varies enormously. Climate also varies widely across the continent, yielding a range of flood timing, duration, and intermittency. Together, the differences in sediment supply and hydrologic patterns result in diverse regimes of benthic habitat stability. To quantitatively characterize these regimes, we calculate decades-scale time series of estimated bed surface mobility using sediment transport equations (Wilcock and Crowe, 2003). The method requires measurements of the bed surface grainsize distribution, channel slope, and standard USGS stream gauging records. We calculate the fraction of the bed surface grain size distribution that is mobile at any given flow, as well as the intensity of transport. We use the time series of bed mobility to compare between rivers and regions. In many snowmelt-dominated rivers in Idaho, a period of moderate bed mobility (W* > 0.002) generally occurs during the annual melt, and can last for days. In rivers draining the central and northern Appalachians, bed mobility is comparatively rare and occurs during short duration floods. Rivers on the tectonically active West Coast tend to experience bed mobility during most winter storms, with brief (hours long) periods of high transport rates (W* > 0.02) during storm peaks. The timing and intensity of bed mobility varies with hydrologic regime and sediment supply; these contrasts in bed mobility lead to diverse structural templates for river ecosystems.

Bradykinin Induces TRPV1 Exocytotic Recruitment in Peptidergic Nociceptors

PubMed Central

Mathivanan, Sakthikumar; Devesa, Isabel; Changeux, Jean-Pierre; Ferrer-Montiel, Antonio

2016-01-01

Transient receptor potential vanilloid I (TRPV1) sensitization in peripheral nociceptors is a prominent phenomenon that occurs in inflammatory pain conditions. Pro-algesic agents can potentiate TRPV1 activity in nociceptors through both stimulation of its channel gating and mobilization of channels to the neuronal surface in a context dependent manner. A recent study reported that ATP-induced TRPV1 sensitization in peptidergic nociceptors involves the exocytotic release of channels trafficked by large dense core vesicles (LDCVs) that cargo alpha-calcitonin gene related peptide alpha (αCGRP). We hypothesized that, similar to ATP, bradykinin may also use different mechanisms to sensitize TRPV1 channels in peptidergic and non-peptidergic nociceptors. We found that bradykinin notably enhances the excitability of peptidergic nociceptors, and sensitizes TRPV1, primarily through the bradykinin receptor 2 pathway. Notably, bradykinin sensitization of TRPV1 in peptidergic nociceptors was significantly blocked by inhibiting Ca2+-dependent neuronal exocytosis. In addition, silencing αCGRP gene expression, but not substance P, drastically reduced bradykinin-induced TRPV1 sensitization in peptidergic nociceptors. Taken together, these findings indicate that bradykinin-induced sensitization of TRPV1 in peptidergic nociceptors is partially mediated by the exocytotic mobilization of new channels trafficked by αCGRP-loaded LDCVs to the neuronal membrane. Our findings further imply a central role of αCGRP peptidergic nociceptors in peripheral algesic sensitization, and substantiate that inhibition of LDCVs exocytosis is a valuable therapeutic strategy to treat pain, as it concurrently reduces the release of pro-inflammatory peptides and the membrane recruitment of thermoTRP channels. PMID:27445816

Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R)

PubMed Central

Chernova, Irene; Lai, Jian-Ping; Li, Haiying; Schwartz, Lynnae; Tuluc, Florin; Korchak, Helen M.; Douglas, Steven D.; Kilpatrick, Laurie E.

2009-01-01

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP-preferring receptor neurokinin-1 receptor (NK1R) has two forms: a full-length NK1R (NK1R-F) isoform and a truncated NK1R (NK1R-T) isoform, which lacks the terminal cytoplasmic 96-aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R-T but not NK1R-F. SP interactions with NK1R-T did not mobilize calcium (Ca2+), but SP mobilized Ca2+ when the NK1R-F was transfected into monocytes. However, the NK1R-T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5-elicited Ca2+ mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R-T also enhanced CCL5-mediated chemotaxis, which was ERK1/2-dependent. NK1R-T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R-T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R-T is functional in human monocytes and activates select signaling pathways, and the NK1R-T-mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain. PMID:18835883

Mobile Lunar Base Concepts

NASA Astrophysics Data System (ADS)

Cohen, Marc M.

2004-02-01

This paper describes three innovative concepts for a mobile lunar base. These concept combine design research for habitat architecture, mobility systems, habitability, radiation protection, human factors, and living and working environments on the lunar surface. The mobile lunar base presents several key advantages over conventional static base notions. These advantages concern landing zone safety, the requirement to move modules over the lunar surface, and the ability to stage mobile reconnaissance with effective systemic redundancy. All of these concerns lead to the consideration of a mobile walking habitat module and base design. The key issues involve landing zone safety, the ability to transport habitat modules across the surface, and providing reliability and redundancy to exploration traverses in pressurized vehicles. With self-ambulating lunar base modules, it will be feasible to have each module separate itself from its retro-rocket thruster unit, and walk five to ten km away from the LZ to a pre-selected site. These mobile modules can operate in an autonomous or teleoperated mode to navigate the lunar surface. At the site of the base, the mobile modules can combine together; make pressure port connections among themselves, to create a multi-module pressurized lunar base.

The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element.

PubMed

Romine, L E; Wood, J R; Lamia, L A; Prendergast, P; Edwards, D P; Nardulli, A M

1998-05-01

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.

Receptor model comparisons and wind direction analyses of volatile organic compounds and submicrometer particles in an arid, binational, urban air shed.

PubMed

Mukerjee, Shaibal; Norris, Gary A; Smith, Luther A; Noble, Christopher A; Neas, Lucas M; Ozkaynak, A Halûk; Gonzales, Melissa

2004-04-15

The relationship between continuous measurements of volatile organic compounds sources and particle number was evaluated at a Photochemical Assessment Monitoring Station Network (PAMS) site located near the U.S.-Mexico Border in central El Paso, TX. Sources of volatile organic compounds (VOCs) were investigated using the multivariate receptor model UNMIX and the effective variance least squares receptor model known as Chemical Mass Balance (CMB, Version 8.0). As expected from PAMS measurements, overall findings from data screening as well as both receptor models confirmed that mobile sources were the major source of VOCs. Comparison of hourly source contribution estimates (SCEs) from the two receptor models revealed significant differences in motor vehicle exhaust and evaporative gasoline contributions. However, the motor vehicle exhaust contributions were highly correlated with each other. Motor vehicle exhaust was also correlated with the ultrafine and accumulation mode particle count, which suggests that motor vehicle exhaust is a source of these particles at the measurement site. Wind sector analyses were performed using the SCE and pollutant data to assess source location of VOCs, particle count, and criteria pollutants. Results from this study have application to source apportionment studies and mobile source emission control strategies that are ongoing in this air shed.

Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cornett, L.E.; Norris, J.S.

1987-11-01

In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation ofmore » unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.« less

Mobile colloid generation induced by a cementitious plume: mineral surface-charge controls on mobilization.

PubMed

Li, Dien; Kaplan, Daniel I; Roberts, Kimberly A; Seaman, John C

2012-03-06

Cementitious materials are increasingly used as engineered barriers and waste forms for radiological waste disposal. Yet their potential effect on mobile colloid generation is not well-known, especially as it may influence colloid-facilitated contaminant transport. Whereas previous papers have studied the introduction of cement colloids into sediments, this study examined the influence of cement leachate chemistry on the mobilization of colloids from a subsurface sediment collected from the Savannah River Site, USA. A sharp mobile colloid plume formed with the introduction of a cement leachate simulant. Colloid concentrations decreased to background concentrations even though the aqueous chemical conditions (pH and ionic strength) remained unchanged. Mobile colloids were mainly goethite and to a lesser extent kaolinite. The released colloids had negative surface charges and the mean particle sizes ranged primarily from 200 to 470 nm. Inherent mineralogical electrostatic forces appeared to be the controlling colloid removal mechanism in this system. In the background pH of ~6.0, goethite had a positive surface charge, whereas quartz (the dominant mineral in the immobile sediment) and kaolinite had negative surface charges. Goethite acted as a cementing agent, holding kaolinite and itself onto the quartz surfaces due to the electrostatic attraction. Once the pH of the system was elevated, as in the cementitious high pH plume front, the goethite reversed to a negative charge, along with quartz and kaolinite, then goethite and kaolinite colloids were mobilized and a sharp spike in turbidity was observed. Simulating conditions away from the cementitious source, essentially no colloids were mobilized at 1:1000 dilution of the cement leachate or when the leachate pH was ≤ 8. Extreme alkaline pH environments of cementitious leachate may change mineral surface charges, temporarily promoting the formation of mobile colloids.

SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

PubMed Central

Lightfoot, Yaíma L; Selle, Kurt; Yang, Tao; Goh, Yong Jun; Sahay, Bikash; Zadeh, Mojgan; Owen, Jennifer L; Colliou, Natacha; Li, Eric; Johannssen, Timo; Lepenies, Bernd; Klaenhammer, Todd R; Mohamadzadeh, Mansour

2015-01-01

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD. PMID:25666591

SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis.

PubMed

Lightfoot, Yaíma L; Selle, Kurt; Yang, Tao; Goh, Yong Jun; Sahay, Bikash; Zadeh, Mojgan; Owen, Jennifer L; Colliou, Natacha; Li, Eric; Johannssen, Timo; Lepenies, Bernd; Klaenhammer, Todd R; Mohamadzadeh, Mansour

2015-04-01

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD. © 2015 The Authors.

Regional Variation in Gravel Riverbed Mobility, Controlled by Hydrologic Regime and Sediment Supply

NASA Astrophysics Data System (ADS)

Pfeiffer, Allison M.; Finnegan, Noah J.

2018-04-01

The frequency and intensity of riverbed mobility are of paramount importance to the inhabitants of river ecosystems as well as to the evolution of bed surface structure. Because sediment supply varies by orders of magnitude across North America, the intensity of bedload transport varies by over an order of magnitude. Climate also varies widely across the continent, yielding a range of flood timing, duration, and intermittency. Together, the differences in sediment supply and hydroclimate result in diverse regimes of bed surface stability. To quantitatively characterize this regional variation, we calculate multidecadal time series of estimated bed surface mobility for 29 rivers using sediment transport equations. We use these data to compare predicted bed mobility between rivers and regions. There are statistically significant regional differences in the (a) exceedance probability of bed-mobilizing flows (W* > 0.002), (b) maximum bed mobility, and (c) number of discrete bed-mobilizing events in a year.

Composition-dependent metallic glass alloys correlate atomic mobility with collective glass surface dynamics.

PubMed

Nguyen, Duc; Zhu, Zhi-Guang; Pringle, Brian; Lyding, Joseph; Wang, Wei-Hua; Gruebele, Martin

2016-06-22

Glassy metallic alloys are richly tunable model systems for surface glassy dynamics. Here we study the correlation between atomic mobility, and the hopping rate of surface regions (clusters) that rearrange collectively on a minute to hour time scale. Increasing the proportion of low-mobility copper atoms in La-Ni-Al-Cu alloys reduces the cluster hopping rate, thus establishing a microscopic connection between atomic mobility and dynamics of collective rearrangements at a glass surface made from freshly exposed bulk glass. One composition, La60Ni15Al15Cu10, has a surface resistant to re-crystallization after three heating cycles. When thermally cycled, surface clusters grow in size from about 5 glass-forming units to about 8 glass-forming units, evidence of surface aging without crystal formation, although its bulk clearly forms larger crystalline domains. Such kinetically stable glass surfaces may be of use in applications where glassy coatings stable against heating are needed.

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

PubMed

Ries, Jonas; Yu, Shuizi Rachel; Burkhardt, Markus; Brand, Michael; Schwille, Petra

2009-09-01

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

Distinct mobilization of leukocytes and hematopoietic stem cells by CXCR4 peptide antagonist LY2510924 and monoclonal antibody LY2624587

PubMed Central

Peng, Sheng-Bin; Van Horn, Robert D.; Yin, Tinggui; Brown, Robin M.; Roell, William C.; Obungu, Victor H.; Ruegg, Charles; Wroblewski, Victor J.; Raddad, Eyas; Stille, John R.

2017-01-01

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy. PMID:29212254

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Molecular Insight into the Slipperiness of Ice.

PubMed

Weber, Bart; Nagata, Yuki; Ketzetzi, Stefania; Tang, Fujie; Smit, Wilbert J; Bakker, Huib J; Backus, Ellen H G; Bonn, Mischa; Bonn, Daniel

2018-05-16

Measurements of the friction coefficient of steel-on-ice over a large temperature range reveal very high friction at low temperatures (-100 °C) and a steep decrease in the friction coefficient with increasing temperature. Very low friction is only found over the limited temperature range typical for ice skating. The strong decrease in the friction coefficient with increasing temperature exhibits Arrhenius behavior with an activation energy of E a ≈ 11.5 kJ mol -1 . Remarkably, molecular dynamics simulations of the ice-air interface reveal a very similar activation energy for the mobility of surface molecules. Weakly hydrogen-bonded surface molecules diffuse over the surface in a rolling motion, their number and mobility increasing with increasing temperature. This correlation between macroscopic friction and microscopic molecular mobility indicates that slippery ice arises from the high mobility of its surface molecules, making the ice surface smooth and the shearing of the weakly bonded surface molecules easy.

Real space mapping of ionic diffusion and electrochemical activity in energy storage and conversion materials

DOEpatents

Kalinin, Sergei V; Balke, Nina; Kumar, Amit; Dudney, Nancy J; Jesse, Stephen

2014-05-06

A method and system for probing mobile ion diffusivity and electrochemical reactivity on a nanometer length scale of a free electrochemically active surface includes a control module that biases the surface of the material. An electrical excitation signal is applied to the material and induces the movement of mobile ions. An SPM probe in contact with the surface of the material detects the displacement of mobile ions at the surface of the material. A detector measures an electromechanical strain response at the surface of the material based on the movement and reactions of the mobile ions. The use of an SPM tip to detect local deformations allows highly reproducible measurements in an ambient environment without visible changes in surface structure. The measurements illustrate effective spatial resolution comparable with defect spacing and well below characteristic grain sizes of the material.

Electron mobility enhancement in metalorganic-vapor-phase-epitaxy-grown InAlN high-electron-mobility transistors by control of surface morphology of spacer layer

NASA Astrophysics Data System (ADS)

Yamada, Atsushi; Ishiguro, Tetsuro; Kotani, Junji; Nakamura, Norikazu

2018-01-01

We demonstrated low-sheet-resistance metalorganic-vapor-phase-epitaxy-grown InAlN high-electron-mobility transistors using AlGaN spacers with excellent surface morphology. We systematically investigated the effects of AlGaN spacer growth conditions on surface morphology and electron mobility. We found that the surface morphology of InAlN barriers depends on that of AlGaN spacers. Ga desorption from AlGaN spacers was suppressed by increasing the trimethylaluminum (TMA) supply rate, resulting in the small surface roughnesses of InAlN barriers and AlGaN spacers. Moreover, we found that an increase in the NH3 supply rate also improved the surface morphologies of InAlN barriers and AlGaN spacers as long as the TMA supply rate was high enough to suppress the degradation of GaN channels. Finally, we realized a low sheet resistance of 185.5 Ω/sq with a high electron mobility of 1210 cm2 V-1 s-1 by improving the surface morphologies of AlGaN spacers and InAlN barriers.

Cocaine-Induced Endocannabinoid Mobilization in the Ventral Tegmental Area.

PubMed

Wang, Huikun; Treadway, Tyler; Covey, Daniel P; Cheer, Joseph F; Lupica, Carl R

2015-09-29

Cocaine is a highly addictive drug that acts upon the brain's reward circuitry via the inhibition of monoamine uptake. Endogenous cannabinoids (eCB) are lipid molecules released from midbrain dopamine (DA) neurons that modulate cocaine's effects through poorly understood mechanisms. We find that cocaine stimulates release of the eCB, 2-arachidonoylglycerol (2-AG), in the rat ventral midbrain to suppress GABAergic inhibition of DA neurons, through activation of presynaptic cannabinoid CB1 receptors. Cocaine mobilizes 2-AG via inhibition of norepinephrine uptake and promotion of a cooperative interaction between Gq/11-coupled type-1 metabotropic glutamate and α1-adrenergic receptors to stimulate internal calcium stores and activate phospholipase C. The disinhibition of DA neurons by cocaine-mobilized 2-AG is also functionally relevant because it augments DA release in the nucleus accumbens in vivo. Our results identify a mechanism through which the eCB system can regulate the rewarding and addictive properties of cocaine. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamics of Multiple Trafficking Behaviors of Individual Synaptic Vesicles Revealed by Quantum-Dot Based Presynaptic Probe

PubMed Central

Lee, Suho; Jung, Kyung Jin; Jung, Hyun Suk; Chang, Sunghoe

2012-01-01

Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity. PMID:22666444

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells.

PubMed

Ding, K H; Latimer, A J; Abdel-Latif, A A

1999-01-01

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.

Curb Mounting, Vertical Mobility, and Inverted Mobility on Rough Surfaces Using Microspine-Enabled Robots

NASA Technical Reports Server (NTRS)

Parness, Aaron

2012-01-01

Three robots that extend microspine technology to enable advanced mobility are presented. First, the Durable Reconnaissance and Observation Platform (DROP) and the ReconRobotics Scout platform use a new rotary configuration of microspines to provide improved soldier-portable reconnaissance by moving rapidly over curbs and obstacles, transitioning from horizontal to vertical surfaces, climbing rough walls and surviving impacts. Next, the four-legged LEMUR robot uses new configurations of opposed microspines to anchor to both manmade and natural rough surfaces. Using these anchors as feet enables mobility in unstructured environments, from urban disaster areas to deserts and caves.

Influence of carboxymethyl cellulose and sodium alginate on sweetness intensity of Aspartame.

PubMed

Han, Xue; Xu, Shu-Zhen; Dong, Wen-Rui; Wu, Zhai; Wang, Ren-Hai; Chen, Zhong-Xiu

2014-12-01

Sensory evaluation of Aspartame in the presence of sodium carboxymethyl cellulose (CMC-L) and sodium alginate (SA) revealed that only CMC-L showed a suppression effect, while SA did not. By using an artificial taste receptor model, we found that the presence of SA or CMC-L resulted in a decrease in association constants. Further investigation of CMC-L solution revealed that the decrease in water mobility and diffusion also contribute to the suppression effect. In the case of SA, the decreased viscosity and comparatively higher amount of free water facilitated the diffusion of sweetener, which might compensate for the decreased binding constant between Aspartame and receptor. This may suppress the impact of SA on sweetness intensity. The results suggest that exploring the binding affinity of taste molecules with the receptor, along with water mobility and diffusion in hydrocolloidal structures, provide sufficient information for understanding the mechanism behind the effect of macromolecular hydrocolloids on taste. Copyright © 2014 Elsevier Ltd. All rights reserved.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4*

PubMed Central

Dolezal, Olan; Cao, Benjamin; See, Heng B.; Pfleger, Kevin D. G.; Gorry, Paul R.; Pow, Andrew; Viduka, Katerina; Lim, Kevin; Lu, Bernadine G. C.; Chang, Denison H. C.; Murray-Rust, Thomas; Dogovski, Con; Doerflinger, Marcel; Zhang, Yuan; Parisi, Kathy; Casey, Joanne L.; Nuttall, Stewart D.; Foley, Michael

2016-01-01

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an “i-body,” the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor. PMID:27036939

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perron, Amelie; Sharif, Nadder; Gendron, Louis

2006-05-12

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less

Chronic production of angiotensin IV in the brain leads to hypertension that is reversible with an angiotensin II AT1 receptor antagonist.

PubMed

Lochard, Nadheige; Thibault, Gaétan; Silversides, David W; Touyz, Rhian M; Reudelhuber, Timothy L

2004-06-11

Angiotensin IV (Ang IV) is a metabolite of the potent vasoconstrictor angiotensin II (Ang II). Because specific binding sites for this peptide have been reported in numerous tissues including the brain, it has been suggested that a specific Ang IV receptor (AT4) might exist. Bolus injection of Ang IV in brain ventricles has been implicated in learning, memory, and localized vasodilatation. However, the functions of Ang IV in a physiological context are still unknown. In this study, we generated a transgenic (TG) mouse model that chronically releases Ang IV peptide specifically in the brain. TG mice were found to be hypertensive by the tail-cuff method as compared with control littermates. Treatment with the angiotensin-converting enzyme inhibitor captopril had no effect on blood pressure, but surprisingly treatment with the Ang II AT1 receptor antagonist candesartan normalized the blood pressure despite the fact that the levels of Ang IV in the brains of TG mice were only 4-fold elevated over the normal endogenous level of Ang peptides. Calcium mobilization assays performed on cultured CHO cells chronically transfected with the AT1 receptor confirm that low-dose Ang IV can mobilize calcium via the AT1 receptor only in the presence of Ang II, consistent with an allosteric mechanism. These results suggest that chronic elevation of Ang IV in the brain can induce hypertension that can be treated with angiotensin II AT1 receptor antagonists.

Acetylcholine is released from taste cells, enhancing taste signalling

PubMed Central

Dando, Robin; Roper, Stephen D

2012-01-01

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

Monoclonal TCR-redirected tumor cell killing.

PubMed

Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

2012-06-01

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Structural Origin of Enhanced Dynamics at the Surface of a Glassy Alloy

NASA Astrophysics Data System (ADS)

Sun, Gang; Saw, Shibu; Douglass, Ian; Harrowell, Peter

2017-12-01

The enhancement of mobility at the surface of an amorphous alloy is studied using a combination of molecular dynamic simulations and normal mode analysis of the nonuniform distribution of Debye-Waller factors. The increased mobility at the surface is found to be associated with the appearance of Arrhenius temperature dependence. We show that the transverse Debye-Waller factor exhibits a peak at the surface. Over the accessible temperature range, we find that the bulk and surface diffusion coefficients obey the same empirical relationship with the respective Debye-Waller factors. Extrapolating this relationship to lower T , we argue that the observed decrease in the constraint at the surface is sufficient to account for the experimentally observed surface enhancement of mobility.

Proteins at the Biomaterial Electrolyte Interface

NASA Astrophysics Data System (ADS)

Tengvall, Pentti

2005-03-01

Proteins adsorb rapidly onto solid and polymeric surfaces because the association process is in the vast majority of cases energetically favourable, i.e. exothermic. The most common exceptions to this rule are hydrophilic interfaces with low net charge and high mobility, e.g. immobilized PEGs. Current research in the research area tries to understand and control unwanted and wanted adsorption by studying the adsorption kinetics, protein surface binding specificity, protein exchange at interfaces, and surface protein repulsion mechanisms. In blood plasma model systems humoral cascade reactions such as surface mediated coagulation and immune complement raise considerable interest due to the immediate association to blood compatibility, and in tissue applications the binding between surfaces and membrane receptors in cells and tissues. Thus, the understanding of interfacial events at the protein level is of large importance in applications such as blood and tissue contacting biomaterials, in vitro medical and biological diagnostics, food industry and in marine anti-fouling technology. Well described consequences of adsorption are a lowered system energy, increased system entropy, irreversible binding, conformational changes, specific surface/protein interactions, and in biomedical materials applications surface opsonization followed by cell-surface interactions and a host tissue response. This lecture will deal with some mechanisms known to be of importance for the adsorption processes, such as the influence of surface chemistry and surface energy, the composition of the protein solution, the Vroman effect, and residence time. Examples will be shown from ellipsometric experiments using different model surfaces in single/few protein solutions, and specific attention be given to blood serum and plasma experiments on coagulation and immune complement at interfaces.

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

PubMed

Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

2015-02-03

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

PubMed Central

Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

2015-01-01

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Laboratory investigation of surface processes on airless bodies due to electrostatic dust mobilization

NASA Astrophysics Data System (ADS)

Wang, X.; Hood, N.; Schwan, J.; Hsu, H. W.; Horanyi, M.

2017-12-01

Electrostatic dust mobilization on the surfaces of airless bodies due to direct exposure to solar wind and solar ultraviolet (UV) radiation has been suggested from a number of unusual planetary observations and supported by our recent laboratory experiments. This electrostatic process may have a significant contribution in the evolution of these surfaces in addition to other surface processes, e.g., thermal fragmentation. The critical questions are how this process changes the surface physical characteristics and how efficient this process can be. We report new laboratory experiments that record dust activities as function of the incoming fluxes of photons or energetic electrons over a long exposure time under Earth gravity. Dust is observed to hop and move on the surface, causing the significant change in surface morphology and becoming smoother over time. Our results indicate that the dynamics of dust mobilization may be complicated by temporal charging effect as dust moves. Various sizes and types of dust are examined, showing large effects on dust mobilization. These laboratory data will help us to predict the electrostatic surface processes and estimate their timescales in space conditions.

Aquifer modification: an approach to improve the mobility of nanoscale zero-valent iron particles used for in situ groundwater remediation

NASA Astrophysics Data System (ADS)

MicicBatka, Vesna; Schmid, Doris; Marko, Florian; Velimirovic, Milica; Wagner, Stephan; von der Kammer, Frank; Hofmann, Thilo

2015-04-01

Successful emplacement of nanoscale zero-valent iron (nZVI) within the contaminated source zone is a prerequisite for the use of nZVI technology in groundwater remediation. Emplacement of nZVI is influenced i.e., by the injection technique and the injection velocity applied, as well as by the mobility of nZVI in the subsurface. Whereas processes linked to the injection can be controlled by the remediation practitioners, the mobility of nZVI in the subsurface remains limited. Even though mobility of nZVI is somewhat improved by surface coating with polyelectrolytes, it is still greatly affected by the groundwater composition and physical and chemical heterogeneities of aquifer grains. In order to promote mobility of nZVI it is needed to alter the surface charge heterogeneities of aquifer grains. Modifying the aquifer grain's surfaces by means of polyelectrolyte coating is an approach proposed to increase the overall negative surface charge of the aquifer grain surfaces, hinder deposition of nZVI onto aquifer grains, and finally promote nZVI mobility. In this study the effect of different polyelectrolytes on the nZVI mobility is tested in natural sands deriving from real brownfield sites that are proposed to be remediated using the nZVI technology. Sands collected from brownfield sites were characterized in terms of grain size distribution, mineralogical and chemical composition, and organic carbon content. Furthermore, surface charge of these sands was determined in both, low- and high ionic strength background solutions. Finally, changes of the sand's surface charges were examined after addition of the proposed aquifer modifiers, lignin sulfonate and humic acid. Surface charge of brownfield sands in low ionic strength background solution is more negative compared to that in high ionic strength background solution. An increase in negative surface potential of brownfield sand was recorded when aquifer modifiers were applied in a background solution with low ionic strength, indicating their potential to improve nZVI mobility under comparable environmental conditions. In contrast, no significant change of the surface potential of brownfield sand was observed when aquifer modifiers were applied in a background solution with high ionic strength. The potential of the aquifer modifiers to promote the mobility of nZVI was furthermore tested in flow-through columns, starting with the one filled with natural quartz sand with rough surface, low ionic strength background solutions and pre-injecting lignin sulfonate in concentration of 50 mg/L. The preliminary results showed that the pre-injection of lignin sulfonate does increase mobility of nZVI under this experimental condition. Further mobility tests will be carried out in order to elucidate the potential of the aquifer modifiers to promote the mobility of nZVI in sands with a complex mineralogy and in the background solutions with varying ionic strength, in order to account for the condition that resemble those at polluted sites. This research receives funding from the European Union's Seventh Framework Programme FP7/2007-2013 under grant agreement n°309517.

Phosphorylated Epidermal Growth Factor Receptor Expression Is Associated With Clinicopathologic Parameters and Patient Survival in Mobile Tongue Squamous Cell Carcinoma.

PubMed

Theocharis, Stamatios; Giaginis, Constantinos; Dana, Eugene; Thymara, Irene; Rodriguez, Jose; Patsouris, Efstratios; Klijanienko, Jerzy

2017-03-01

Phosphorylated epidermal growth factor receptor (pEGFR) activates several signaling pathways, resulting in tumor-promoting cellular activities, and has been implicated in malignant transformation and disease progression. The present study evaluated the clinical significance of pEGFR protein expression in mobile tongue squamous cell carcinoma (SCC). The present cohort study included 48 patients with mobile tongue SCC. We evaluated whether pEGFR immunohistochemical protein expression is associated with clinical variables and patient outcome. Of the 48 patients included in the present cohort study, 25 were men and 23 were women. The median patient age was 60 years (interquartile range 53 to 72). pEGFR protein expression was significantly increased in well-differentiated tumors compared with poorly differentiated tumors (P = .001). Elevated pEGFR protein expression was significantly more frequently observed in mobile tongue SCC cases with a well-defined tumor shape and an earlier disease stage (P = .010 and P = .019, respectively). Patients with mobile tongue SCC presenting with elevated pEGFR expression had longer overall and disease-free survival times compared with those with low pEGFR expression (P = .015 and P = .006, respectively; log-rank test). On multivariate analysis, pEGFR expression proved to be an independent prognostic factor of both overall and disease-free survival (P = .008 and P = .044, respectively; Cox regression analysis). The results of the present study support evidence that the pEGFR signaling pathway might be implicated in the malignant transformation of mobile tongue SCC. Additional studies are recommended to validate whether pEGFR could be used as a potential biomarker and therapeutic target in mobile tongue SCC. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

PubMed

Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

2005-05-01

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

PubMed Central

Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

2013-01-01

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

PubMed

He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen-induced pulmonary inflammation in mice.

PubMed

Frei, R B; Luschnig, P; Parzmair, G P; Peinhaupt, M; Schranz, S; Fauland, A; Wheelock, C E; Heinemann, A; Sturm, E M

2016-07-01

Accumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) has been proposed to elicit eosinophil migration in a CB2 receptor/Gi/o -dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of 2-AG into eicosanoids. Here, we explored the direct contribution of specific CB2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo. In vitro studies including CB2 expression, adhesion and migratory responsiveness, respiratory burst, degranulation, and calcium mobilization were conducted in human peripheral blood eosinophils and mouse bone marrow-derived eosinophils. Allergic airway inflammation was assessed in mouse models of acute OVA-induced asthma and directed eosinophil migration. CB2 expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB2 receptor agonist JWH-133 induced a moderate migratory response in eosinophils. However, short-term exposure to JWH-133 potently enhanced chemoattractant-induced eosinophil shape change, chemotaxis, CD11b surface expression, and adhesion as well as production of reactive oxygen species. Receptor specificity of the observed effects was confirmed in eosinophils from CB2 knockout mice and by using the selective CB2 antagonist SR144528. Of note, systemic application of JWH-133 clearly primed eosinophil-directed migration in vivo and aggravated both AHR and eosinophil influx into the airways in a CB2 -specific manner. This effect was completely absent in eosinophil-deficient ∆dblGATA mice. Our data indicate that CB2 may directly contribute to the pathogenesis of eosinophil-driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB2 -mediated priming of eosinophils. Hence, antagonism of CB2 receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Dynamic changes of surface soil organic carbon and light-fraction organic carbon after mobile dune afforestation with Mongolian pine in Horqin Sandy Land].

PubMed

Shang, Wen; Li, Yu-qiang; Wang, Shao-kun; Feng, Jing; Su, Na

2011-08-01

This paper studied the dynamic changes of surface (0-15 cm) soil organic carbon (SOC) and light-fraction organic carbon (LFOC) in 25- and 35-year-old sand-fixing Mongolian pine (Pinus sylvestris var. mongolica) plantations in Horqin Sandy Land, with a mobile dune as a comparison site. After the afforestation on mobile dune, the content of coarse sand in soil decreased, while that of fine sand and clay-silt increased significantly. The SOC and LFOC contents also increased significantly, but tended to decrease with increasing soil depth. Afforestation increased the storages of SOC and LFOC in surface soil, and the increment increased with plantation age. In the two plantations, the increment of surface soil LFOC storage was much higher than that of SOC storage, suggesting that mobile dune afforestation had a larger effect on surface soil LFOC than on SOC.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius

PubMed Central

Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.

1999-01-01

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907

Surface code—biophysical signals for apoptotic cell clearance

NASA Astrophysics Data System (ADS)

Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, Luis E.

2013-12-01

Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

Norepinephrine reuptake inhibition promotes mobilization in mice: potential impact to rescue low stem cell yields

PubMed Central

Lucas, Daniel; Bruns, Ingmar; Battista, Michela; Mendez-Ferrer, Simon; Magnon, Claire; Kunisaki, Yuya

2012-01-01

The mechanisms mediating hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF are complex. We have found previously that G-CSF–enforced mobilization is controlled by peripheral sympathetic nerves via norepinephrine (NE) signaling. In the present study, we show that G-CSF likely alters sympathetic tone directly and that methods to increase adrenergic activity in the BM microenvironment enhance progenitor mobilization. Peripheral sympathetic nerve neurons express the G-CSF receptor and ex vivo stimulation of peripheral sympathetic nerve neurons with G-CSF reduced NE reuptake significantly, suggesting that G-CSF potentiates the sympathetic tone by increasing NE availability. Based on these data, we investigated the NE reuptake inhibitor desipramine in HSPC mobilization. Whereas desipramine did not by itself elicit circulating HSPCs, it increased G-CSF–triggered mobilization efficiency significantly and rescued mobilization in a model mimicking “poor mobilizers.” Therefore, these data suggest that blockade of NE reuptake may be a novel therapeutic target to increase stem cell yield in patients. PMID:22422821

NCI-H295R, a human adrenal cortex-derived cell line, expresses purinergic receptors linked to Ca²⁺-mobilization/influx and cortisol secretion.

PubMed

Nishi, Haruhisa; Arai, Hirokazu; Momiyama, Toshihiko

2013-01-01

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A(2A) and A(2B)), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10-1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1-1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10-100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.

Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

PubMed Central

Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

2015-01-01

ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

Immunocytochemical localization of muscarinic, adrenergic and AT1 receptors.

PubMed

Schulze, W; Fu, M L

1996-01-01

By indirect immunofluorescence and post-embedding EM gold technique, the localization of alpha 1-adrenergic, M2-muscarinic and angiotensin II receptor-I (AT1) were determinated. With antipeptide antibodies directed against the second extracellular loops of all three receptors, these receptors were found to be localized at the sarcolemma of adult rat cardiomyocytes and at the surface membranes of cultivated neonatal heart cells. Additionally, M2 receptors were localized along T-tubule membranes of both rat and human adult cardiomyocytes. alpha 1-Adrenergic receptors were found intracellular near the surface of atrial granules (ANF-granules). By using M2 and alpha 1-adrenergic receptor antibodies the strongest fluorescence was found in the right atrium of the rat. Besides the localization in cardiomyocytes, AT1 receptors were also localized in outer plasma membranes and the endoplasmic reticulum of fibroblasts, and the surface of smooth muscle cells of the major arteries and veins. Likewise, the muscarinic M2 receptors were found along the outer membranes of endothelial cells from capillaries and endocardium.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

Adenovirus receptors and their implications in gene delivery

PubMed Central

Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

2010-01-01

Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

Peripheral and spinal activation of cannabinoid receptors by joint mobilization alleviates postoperative pain in mice.

PubMed

Martins, D F; Mazzardo-Martins, L; Cidral-Filho, F J; Gadotti, V M; Santos, A R S

2013-01-01

The present study was undertaken to investigate the relative contribution of cannabinoid receptors (CBRs) subtypes and to analyze cannabimimetic mechanisms involved in the inhibition of anandamide (AEA) and 2-arachidonoyl glycerol degradation on the antihyperalgesic effect of ankle joint mobilization (AJM). Mice (25-35g) were subjected to plantar incision (PI) and 24h after surgery animals received the following treatments, AJM for 9min, AEA (10mg/kg, intraperitoneal [i.p.]), WIN 55,212-2 (1.5mg/kg, i.p.), URB937 (0.01-1mg/kg, i.p.; a fatty acid amide hydrolase [FAAH] inhibitor) or JZL184 (0.016-16mg/kg, i.p.; a monoacylglycerol lipase [MAGL] inhibitor). Withdrawal frequency to mechanical stimuli was assessed 24h after PI and at different time intervals after treatments. Receptor specificity was investigated using selective CB1R (AM281) and CB2R (AM630) antagonists. In addition, the effect of the FAAH and MAGL inhibitors on the antihyperalgesic action of AJM was investigated. AJM, AEA, WIN 55,212-2, URB937 and JZL184 decreased mechanical hyperalgesia induced by PI. The antihyperalgesic effect of AJM was reversed by pretreatment with AM281 given by intraperitoneal and intrathecal routes, but not intraplantarly. Additionally, intraperitoneal and intraplantar, but not intrathecal administration of AM630 blocked AJM-induced antihyperalgesia. Interestingly, in mice pretreated with FAAH or the MAGL inhibitor the antihyperalgesic effect of AJM was significantly longer. This article presents data addressing the CBR mechanisms underlying the antihyperalgesic activity of joint mobilization as well as of the endocannabinoid catabolic enzyme inhibitors in the mouse postoperative pain model. Joint mobilization and these enzymes offer potential targets to treat postoperative pain. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

PubMed Central

Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E.; Schief, William R.; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D.

2009-01-01

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded β-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate—and structurally plastic—layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated β-sandwich and providing for conformational diversity used in immune evasion. A “layered” gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a β-sandwich clamp maintains gp120–gp41 interaction and regulates gp41 transitions. PMID:20080564

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility.

PubMed

Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Kwon, Young Do; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E; Schief, William R; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D

2010-01-19

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

An Automaton Rover for Extreme Environments: Rethinking an Approach to Surface Mobility

NASA Astrophysics Data System (ADS)

Sauder, J.; Hilgemman, E.; Stack, K.; Kawata, J.; Parness, A.; Johnson, M.

2017-11-01

An Automaton Rover for Extreme Environments (AREE) enables long duration in-situ mobility on the surface of Venus through a simplified design and robust mechanisms. The goal is to design a rover capable of operating for months on the surface of Venus.

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

PubMed

Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

2015-12-01

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

The neural mobilization technique modulates the expression of endogenous opioids in the periaqueductal gray and improves muscle strength and mobility in rats with neuropathic pain

PubMed Central

2014-01-01

Background The neural mobilization (NM) technique is a noninvasive method that has been proven to be clinically effective in reducing pain; however, the molecular mechanisms involved remain poorly understood. The aim of this study was to analyze whether NM alters the expression of the mu-opioid receptor (MOR), the delta-opioid receptor (DOR) and the Kappa-opioid receptor (KOR) in the periaqueductal gray (PAG) and improves locomotion and muscle force after chronic constriction injury (CCI) in rats. Methods The CCI was imposed on adult male rats followed by 10 sessions of NM every other day, starting 14 days after the CCI injury. At the end of the sessions, the PAG was analyzed using Western blot assays for opioid receptors. Locomotion was analyzed by the Sciatic functional index (SFI), and muscle force was analyzed by the BIOPAC system. Results An improvement in locomotion was observed in animals treated with NM compared with injured animals. Animals treated with NM showed an increase in maximal tetanic force of the tibialis anterior muscle of 172% (p < 0.001) compared with the CCI group. We also observed a decrease of 53% (p < 0.001) and 23% (p < 0.05) in DOR and KOR levels, respectively, after CCI injury compared to those from naive animals and an increase of 17% (p < 0.05) in KOR expression only after NM treatment compared to naive animals. There were no significant changes in MOR expression in the PAG. Conclusion These data provide evidence that a non-pharmacological NM technique facilitates pain relief by endogenous analgesic modulation. PMID:24884961

Postconditioning: "Toll-erating" mesenteric ischemia-reperfusion injury?

PubMed

Rosero, Olivér; Ónody, Péter; Kovács, Tibor; Molnár, Dávid; Fülöp, András; Lotz, Gábor; Harsányi, László; Szijártó, Attila

2017-04-01

Postconditioning may prove to be a suitable method to decrease ischemia-reperfusion injury of intestine after mesenteric arterial occlusion. Toll-like-receptor-4 is involved in the pathophysiology of organ damage after ischemia-reperfusion; therefore, the aim of our study was to investigate the effect of postconditioning on the mucosal expression of toll-like-receptor-4. Male Wistar rats (n = 10/group) underwent 60 minutes of superior mesenteric artery occlusion followed by 6 hours of reperfusion in 3 groups: sham-operated, ischemia-reperfusion, and a postconditioned group. Postconditioning was performed by 6 alternating cycles of 10 seconds of reperfusion/reocclusion. Blood and tissue samples were collected at the end of reperfusion. Intestinal histopathologic changes and immunohistochemical expression of mucosal caspase-3, antioxidant status, and protein levels of high-mobility group box-1 and toll-like-receptor-4 were assessed. Immunofluorescent labeling and confocal microscopic analysis of toll-like-receptor-4 were performed. Mucosal and serum levels of interleukin-6 and tumor necrosis factor-α protein were measured. Histologic alterations in the postconditioned group were associated with decreased caspase-3 positivity, less toll-like-receptor-4 mRNA, and less protein expression of high-mobility group box-1 and toll-like-receptor-4 in the intestinal villi compared with the ischemia-reperfusion group. Furthermore, a significantly improved antioxidant state of the intestinal mucosa and less mucosal and serum protein levels of interleukin-6 and tumor necrosis factor-α were detected in the postconditioned group. Small intestinal ischemia-reperfusion injury in male Wistar rats caused by the occlusion of the superior mesenteric artery was ameliorated by the use of postconditioning, showing a more favorable inflammatory response, which may be attributed to the decreased mucosal expression of toll-like-receptor-4. Copyright © 2016 Elsevier Inc. All rights reserved.

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

PubMed Central

Tzafriri, A. Rami; Edelman, Elazer R.

2006-01-01

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered. PMID:17117924

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling.

PubMed

Takekura, H; Takeshima, H; Nishimura, S; Takahashi, M; Tanabe, T; Flockerzi, V; Hofmann, F; Franzini-Armstrong, C

1995-10-01

Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Yamada, M; Yamada, M; Lombet, A; Forgez, P; Rostène, W

1998-01-01

Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp; Sekiguchi, Toshio; Nagata, Sayaka

2016-02-19

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding andmore » AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.« less

Magnetically-refreshable receptor platform structures for reusable nano-biosensor chips

NASA Astrophysics Data System (ADS)

Yoo, Haneul; Lee, Dong Jun; Cho, Dong-guk; Park, Juhun; Nam, Ki Wan; Tak Cho, Young; Park, Jae Yeol; Chen, Xing; Hong, Seunghun

2016-01-01

We developed a magnetically-refreshable receptor platform structure which can be integrated with quite versatile nano-biosensor structures to build reusable nano-biosensor chips. This structure allows one to easily remove used receptor molecules from a biosensor surface and reuse the biosensor for repeated sensing operations. Using this structure, we demonstrated reusable immunofluorescence biosensors. Significantly, since our method allows one to place receptor molecules very close to a nano-biosensor surface, it can be utilized to build reusable carbon nanotube transistor-based biosensors which require receptor molecules within a Debye length from the sensor surface. Furthermore, we also show that a single sensor chip can be utilized to detect two different target molecules simply by replacing receptor molecules using our method. Since this method does not rely on any chemical reaction to refresh sensor chips, it can be utilized for versatile biosensor structures and virtually-general receptor molecular species.

The mapping of yeast's G-protein coupled receptor with an atomic force microscope

NASA Astrophysics Data System (ADS)

Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

2015-03-01

An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

PubMed Central

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-01-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

NASA Astrophysics Data System (ADS)

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-06-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment of Vasculogenic and Hematopoietic Stem Cells

PubMed Central

Hattori, Koichi; Dias, Sergio; Heissig, Beate; Hackett, Neil R.; Lyden, David; Tateno, Masatoshi; Hicklin, Daniel J.; Zhu, Zhenping; Witte, Larry; Crystal, Ronald G.; Moore, Malcolm A.S.; Rafii, Shahin

2001-01-01

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF165, matrix-bound VEGF189, or Ang-1 into mice. VEGF165, but not VEGF189, induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2+ circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF165 was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF165, but not Ang-1–induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis. PMID:11342585

In vitro evaluation of biodegradable microspheres with surface-bound ligands.

PubMed

Keegan, Mark E; Royce, Sara M; Fahmy, Tarek; Saltzman, W Mark

2006-02-21

Protein ligands were conjugated to the surface of biodegradable microspheres. These microsphere-ligand conjugates were then used in two in vitro model systems to evaluate the effect of conjugated ligands on microsphere behavior. Microsphere retention in agarose columns was increased by ligands on the microsphere surface specific for receptors on the agarose matrix. In another experiment, conjugating the lectin Ulex europaeus agglutinin 1 to the microsphere surface increased microsphere adhesion to Caco-2 monolayers compared to control microspheres. This increase in microsphere adhesion was negated by co-administration of l-fucose, indicating that the increase in adhesion is due to specific interaction of the ligand with carbohydrate receptors on the cell surface. These results demonstrate that the ligands conjugated to the microspheres maintain their receptor binding activity and are present on the microsphere surface at a density sufficient to target the microspheres to both monolayers and three-dimensional matrices bearing complementary receptors.

Engineering of PDMS Surfaces for use in Microsystems for Capture and Isolation of Complex and Biomedically Important Proteins: Epidermal Growth Factor Receptor as a Model System

PubMed Central

Lowe, Aaron M.; Ozer, Byram H.; Wiepz, Gregory J.; Bertics, Paul J.; Abbott, Nicholas L.

2009-01-01

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was 32P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (H11 and 111.6) and one phosphospecific EGF receptor antibody (pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82:1, exceeding the signal-to-background measured on the ELISA plate (<48:1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein from the PDMS surface was found to be 75–81%. However, the transfer of non-specifically bound protein was substantially less than 75%, thus leading to the important finding that mechanical transfer of the EGF receptor leads to an approximately four-fold increase in signal-to-background from 20:1 to 88:1. The signal-to-background obtained following mechanical transfer is also better than that obtained using ELISA plates and stripping buffer (<48:1). The EGF receptor is a clinically important protein and the target of numerous anticancer agents and thus these results, when combined, provide guidance for the design of PDMS-based microanalytical systems for the capture and isolation of complex and clinically important transmembrane proteins. PMID:18651079

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

PubMed

Lowe, Aaron M; Ozer, Byram H; Wiepz, Gregory J; Bertics, Paul J; Abbott, Nicholas L

2008-08-01

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein from the PDMS surface was found to be 75-81%. However, the transfer of non-specifically bound protein was substantially less than 75%, thus leading to the important finding that mechanical transfer of the EGF receptor leads to an approximately four-fold increase in signal-to-background from 20 : 1 to 88 : 1. The signal-to-background obtained following mechanical transfer is also better than that obtained using ELISA plates and stripping buffer (<48 : 1). The EGF receptor is a clinically important protein and the target of numerous anticancer agents and thus these results, when combined, provide guidance for the design of PDMS-based microanalytical systems for the capture and isolation of complex and clinically important transmembrane proteins.

Arsenic Mobilization Through Microbial Bioreduction of Ferrihydrite Nanoparticles

NASA Astrophysics Data System (ADS)

Tadanier, C. J.; Roller, J.; Schreiber, M. E.

2004-12-01

Under anaerobic conditions Fe(III)-reducing microorganisms can couple the reduction of solid phase Fe(III) (hydr)oxides with the oxidation of organic carbon. Nutrients and trace metals, such as arsenic, associated with Fe(III) hydroxides may be mobilized through microbially-mediated surface reduction. Although arsenic mobilization has been attributed to mineral surface reduction in a variety of pristine and contaminated environments, minimal information exists on the mechanisms causing this arsenic mobilization. Understanding of the fundamental biochemical and physicochemical processes involved in these mobilization mechanisms is still limited, and has been complicated by the often contradictory and interchangeable terminology used in the literature to describe them. We studied arsenic mobilization mechanisms using a series of controlled microcosm experiments containing aggregated arsenic-bearing ferrihydrite nanoparticles and an Fe(III)-reducing microorganism, Geobacter metallireducens. The phase distribution of iron and arsenic was determined through filtration and ultracentrifugation techniques. Experimental results showed that in the biotic trials, approximately 10 percent of the Fe(III) was reduced to Fe(II) by microbial activity, which remained associated with ferrihydrite surfaces. Biotic activity resulted in changes in nanoparticle surface potential and caused deflocculation of nanoparticle aggregates. Deflocculated nanoparticles were able to pass through a 0.2 micron filter and could only be removed from solution by ultracentrifugation. Arsenic mobilized over time in the biotic trials was found to be exclusively associated with the nanoparticles; 98 percent of arsenic that passed through a 0.2 micron filter was removed from solution by ultracentrifugation. None of these changes were observed in abiotic controls. Because arsenic contamination of natural waters due to mobilization from mineral surfaces is a significant route of human arsenic exposure worldwide, improved understanding of the biologically-mediated mechanisms that partition arsenic between solid and solution phases is required for development of effective treatment and remediation strategies.

The influence of surfaces on the transient terahertz conductivity and electron mobility of GaAs nanowires

NASA Astrophysics Data System (ADS)

Joyce, Hannah J.; Baig, Sarwat A.; Parkinson, Patrick; Davies, Christopher L.; Boland, Jessica L.; Tan, H. Hoe; Jagadish, Chennupati; Herz, Laura M.; Johnston, Michael B.

2017-06-01

Bare unpassivated GaAs nanowires feature relatively high electron mobilities (400-2100 cm2 V-1 s-1) and ultrashort charge carrier lifetimes (1-5 ps) at room temperature. These two properties are highly desirable for high speed optoelectronic devices, including photoreceivers, modulators and switches operating at microwave and terahertz frequencies. When engineering these GaAs nanowire-based devices, it is important to have a quantitative understanding of how the charge carrier mobility and lifetime can be tuned. Here we use optical-pump-terahertz-probe spectroscopy to quantify how mobility and lifetime depend on the nanowire surfaces and on carrier density in unpassivated GaAs nanowires. We also present two alternative frameworks for the analysis of nanowire photoconductivity: one based on plasmon resonance and the other based on Maxwell-Garnett effective medium theory with the nanowires modelled as prolate ellipsoids. We find the electron mobility decreases significantly with decreasing nanowire diameter, as charge carriers experience increased scattering at nanowire surfaces. Reducing the diameter from 50 nm to 30 nm degrades the electron mobility by up to 47%. Photoconductivity dynamics were dominated by trapping at saturable states existing at the nanowire surface, and the trapping rate was highest for the nanowires of narrowest diameter. The maximum surface recombination velocity, which occurs in the limit of all traps being empty, was calculated as 1.3  ×  106 cm s-1. We note that when selecting the optimum nanowire diameter for an ultrafast device, there is a trade-off between achieving a short lifetime and a high carrier mobility. To achieve high speed GaAs nanowire devices featuring the highest charge carrier mobilities and shortest lifetimes, we recommend operating the devices at low charge carrier densities.

Cellular Membrane Phospholipids Act as a Depository for Quaternary Amine containing Drugs thus competing with the Acetylcholine / Nicotinic Receptor

PubMed Central

Barbacci, Damon; Jackson, Shelley N.; Muller, Ludovic; Egan, Thomas; Lewis, Ernest K.; Schultz, J. Albert; Woods, Amina S.

2014-01-01

We previously demonstrated that ammonium- or guanidinium- phosphate interactions are key to forming non-covalent complexes (NCXs) through salt bridge formation with G-protein coupled receptors (GPCR), which are immersed in the cell membrane's lipids. The present work highlights MALDI ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI IM oTOF MS) as a method to determine qualitative and relative quantitative affinity of drugs to form NCXs with targeted GPCRs' epitopes in a model system using, bis-quaternary amine based drugs, α- and β- subunit epitopes of the nicotinic acetylcholine receptor' (nAChR) and phospholipids. Bis-quaternary amines proved to have a strong affinity for all nAChR epitopes and negatively charged phospholipids, even in the presence of the physiological neurotransmitter acetylcholine. Ion mobility baseline separated isobaric phosphatidyl ethanolamine and a matrix cluster, providing an accurate estimate for phospholipid counts. Overall this technique is a powerful method for screening drugs' interactions with targeted lipids and protein respectively containing quaternary amines and guanidinium moieties. PMID:22506649

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Recognition and Classification of Road Condition on the Basis of Friction Force by Using a Mobile Robot

NASA Astrophysics Data System (ADS)

Watanabe, Tatsuhito; Katsura, Seiichiro

A person operating a mobile robot in a remote environment receives realistic visual feedback about the condition of the road on which the robot is moving. The categorization of the road condition is necessary to evaluate the conditions for safe and comfortable driving. For this purpose, the mobile robot should be capable of recognizing and classifying the condition of the road surfaces. This paper proposes a method for recognizing the type of road surfaces on the basis of the friction between the mobile robot and the road surfaces. This friction is estimated by a disturbance observer, and a support vector machine is used to classify the surfaces. The support vector machine identifies the type of the road surface using feature vector, which is determined using the arithmetic average and variance derived from the torque values. Further, these feature vectors are mapped onto a higher dimensional space by using a kernel function. The validity of the proposed method is confirmed by experimental results.

Significant Transient Mobility of Platinum Clusters via a Hot Precursor State on the Alumina Surface.

PubMed

Beniya, Atsushi; Hirata, Hirohito; Watanabe, Yoshihide

2016-11-17

Relaxation dynamics of hot metal clusters on oxide surfaces play a crucial role in a variety of physical and chemical processes. However, their transient mobility has not been investigated as much as other systems such as atoms and molecules on metal surfaces due to experimental difficulties. To study the role of the transient mobility of clusters on the oxide surface, we investigated the initial adsorption process of size-selected Pt clusters on a thin Al 2 O 3 film. Soft-landing the size-selected clusters while suppressing the thermal migration resulted in the transient migration controlling the initial adsorption states as an isolated and aggregated cluster, as revealed using scanning tunneling microscopy. We demonstrate that transient migration significantly contributes to the initial cluster adsorption process; the cross section for aggregation is seven times larger than the expected value from geometrical considerations, indicating that metal clusters are highly mobile during a energy dissipation process on the oxide surface.

Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties

PubMed Central

Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.

2004-01-01

A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854

Fast Modulation of μ-Opioid Receptor (MOR) Recycling Is Mediated by Receptor Agonists*

PubMed Central

Roman-Vendrell, Cristina; Yu, Y. Joy; Yudowski, Guillermo Ariel

2012-01-01

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca2+-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance. PMID:22378794

Does the low hole transport mass in <110> and <111> Si nanowires lead to mobility enhancements at high field and stress: A self-consistent tight-binding study

NASA Astrophysics Data System (ADS)

Kotlyar, R.; Linton, T. D.; Rios, R.; Giles, M. D.; Cea, S. M.; Kuhn, K. J.; Povolotskyi, Michael; Kubis, Tillmann; Klimeck, Gerhard

2012-06-01

The hole surface roughness and phonon limited mobility in the silicon <100>, <110>, and <111> square nanowires under the technologically important conditions of applied gate bias and stress are studied with the self-consistent Poisson-sp3d5s*-SO tight-binding bandstructure method. Under an applied gate field, the hole carriers in a wire undergo a volume to surface inversion transition diminishing the positive effects of the high <110> and <111> valence band nonparabolicities, which are known to lead to the large gains of the phonon limited mobility at a zero field in narrow wires. Nonetheless, the hole mobility in the unstressed wires down to the 5 nm size remains competitive or shows an enhancement at high gate field over the large wire limit. Down to the studied 3 nm sizes, the hole mobility is degraded by strong surface roughness scattering in <100> and <110> wires. The <111> channels are shown to experience less surface scattering degradation. The physics of the surface roughness scattering dependence on wafer and channel orientations in a wire is discussed. The calculated uniaxial compressive channel stress gains of the hole mobility are found to reduce in the narrow wires and at the high field. This exacerbates the stressed mobility degradation with size. Nonetheless, stress gains of a factor of 2 are obtained for <110> wires down to 3 nm size at a 5×1012 cm-2 hole inversion density per gate area.

Ballbot-type motion of N-heterocyclic carbenes on gold surfaces

NASA Astrophysics Data System (ADS)

Wang, Gaoqiang; Rühling, Andreas; Amirjalayer, Saeed; Knor, Marek; Ernst, Johannes Bruno; Richter, Christian; Gao, Hong-Jun; Timmer, Alexander; Gao, Hong-Ying; Doltsinis, Nikos L.; Glorius, Frank; Fuchs, Harald

2017-02-01

Recently, N-heterocyclic carbenes (NHCs) were introduced as alternative anchors for surface modifications and so offered many attractive features, which might render them superior to thiol-based systems. However, little effort has been made to investigate the self-organization process of NHCs on surfaces, an important aspect for the formation of self-assembled monolayers (SAMs), which requires molecular mobility. Based on investigations with scanning tunnelling microscopy and first-principles calculations, we provide an understanding of the microscopic mechanism behind the high mobility observed for NHCs. These NHCs extract a gold atom from the surface, which leads to the formation of an NHC-gold adatom complex that displays a high surface mobility by a ballbot-type motion. Together with their high desorption barrier this enables the formation of ordered and strongly bound SAMs. In addition, this mechanism allows a complementary surface-assisted synthesis of dimeric and hitherto unknown trimeric NHC gold complexes on the surface.

Ballbot-type motion of N-heterocyclic carbenes on gold surfaces.

PubMed

Wang, Gaoqiang; Rühling, Andreas; Amirjalayer, Saeed; Knor, Marek; Ernst, Johannes Bruno; Richter, Christian; Gao, Hong-Jun; Timmer, Alexander; Gao, Hong-Ying; Doltsinis, Nikos L; Glorius, Frank; Fuchs, Harald

2017-02-01

Recently, N-heterocyclic carbenes (NHCs) were introduced as alternative anchors for surface modifications and so offered many attractive features, which might render them superior to thiol-based systems. However, little effort has been made to investigate the self-organization process of NHCs on surfaces, an important aspect for the formation of self-assembled monolayers (SAMs), which requires molecular mobility. Based on investigations with scanning tunnelling microscopy and first-principles calculations, we provide an understanding of the microscopic mechanism behind the high mobility observed for NHCs. These NHCs extract a gold atom from the surface, which leads to the formation of an NHC-gold adatom complex that displays a high surface mobility by a ballbot-type motion. Together with their high desorption barrier this enables the formation of ordered and strongly bound SAMs. In addition, this mechanism allows a complementary surface-assisted synthesis of dimeric and hitherto unknown trimeric NHC gold complexes on the surface.

An In Silico Model of Endotoxic Shock Mediators (Briefing Charts)

DTIC Science & Technology

2012-03-12

complex probably because receptor activates multiple signaling pathways choline P O acetyl...calcium mobilization phospholipase C phospholipase D phospholipase A2 eicosanoids inositol metabolism protein kinase C COOH NH2 PAF

Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function.

PubMed

Xing, Juan; Petranka, John G; Davis, Felicity M; Desai, Pooja N; Putney, James W; Bird, Gary S

2014-03-01

Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases.

PubMed

Field, K A; Apgar, J R; Hong-Geller, E; Siraganian, R P; Baird, B; Holowka, D

2000-10-01

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.

Simulated molecular-scale interaction of supercritical fluid mobile and stationary phases.

PubMed

Siders, Paul D

2017-12-08

In supercritical fluid chromatography, molecules from the mobile phase adsorb on the stationary phase. Stationary-phase alkylsilane-terminated silica surfaces might adsorb molecules at the silica, among the silanes, on a silane layer, or in pore space between surfaces. Mobile phases of carbon dioxide, pure and modified with methanol, and stationary phases were simulated at the molecular scale. Classical atomistic force fields were used in Gibbs-ensemble hybrid Monte Carlo calculations. Excess adsorption of pure carbon dioxide mobile phase peaked at fluid densities of 0.002-0.003Å -3 . Mobile phase adsorption from 7% methanol in carbon dioxide peaked at lower fluid density. Methanol was preferentially adsorbed from the mixed fluid. Surface silanes prevented direct interaction of fluid-phase molecules with silica. Some adsorbed molecules mixed with tails of bonded silanes; some formed layers above the silanes. Much adsorption occurred by filling the space between surfaces in the stationary-phase model. The distribution in the stationary phase of methanol molecules from a modified fluid phase varied with pressure. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic mobility applications, program evaluation : national-level impacts and costs estimation : final report.

DOT National Transportation Integrated Search

2016-07-01

The vision of the Dynamic Mobility Applications (DMA) program is to expedite the development, testing, and deployment of innovative mobility applications that maximize system productivity and enhance mobility of individuals within the surface transpo...

Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

PubMed

Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P

2013-03-01

Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.

Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes.

PubMed Central

Bussell, S J; Koch, D L; Hammer, D A

1995-01-01

Tracer diffusion coefficients of integral membrane proteins (IMPs) in intact plasma membranes are often much lower than those found in blebbed, organelle, and reconstituted membranes. We calculate the contribution of hydrodynamic interactions to the tracer, gradient, and rotational diffusion of IMPs in plasma membranes. Because of the presence of immobile IMPs, Brinkman's equation governs the hydrodynamics in plasma membranes. Solutions of Brinkman's equation enable the calculation of short-time diffusion coefficients of IMPs. There is a large reduction in particle mobilities when a fraction of them is immobile, and as the fraction increases, the mobilities of the mobile particles continue to decrease. Combination of the hydrodynamic mobilities with Monte Carlo simulation results, which incorporate excluded area effects, enable the calculation of long-time diffusion coefficients. We use our calculations to analyze results for tracer diffusivities in several different systems. In erythrocytes, we find that the hydrodynamic theory, when combined with excluded area effects, closes the gap between existing theory and experiment for the mobility of band 3, with the remaining discrepancy likely due to direct obstruction of band 3 lateral mobility by the spectrin network. In lymphocytes, the combined hydrodynamic-excluded area theory provides a plausible explanation for the reduced mobility of sIg molecules induced by binding concanavalin A-coated platelets. However, the theory does not explain all reported cases of "anchorage modulation" in all cell types in which receptor mobilities are reduced after binding by concanavalin A-coated platelets. The hydrodynamic theory provides an explanation of why protein lateral mobilities are restricted in plasma membranes and why, in many systems, deletion of the cytoplasmic tail of a receptor has little effect on diffusion rates. However, much more data are needed to test the theory definitively. We also predict that gradient and tracer diffusivities are the same to leading order. Finally, we have calculated rotational diffusion coefficients in plasma membranes. They decrease less rapidly than translational diffusion coefficients with increasing protein immobilization, and the results agree qualitatively with the limited experimental data available. PMID:7612825

Single Particle Tracking reveals two distinct environments for CD4 receptors at the surface of living T lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mascalchi, Patrice; Lamort, Anne Sophie; Salome, Laurence

2012-01-06

Highlights: Black-Right-Pointing-Pointer We studied the diffusion of single CD4 receptors on living lymphocytes. Black-Right-Pointing-Pointer This study reveals that CD4 receptors have either a random or confined diffusion. Black-Right-Pointing-Pointer The dynamics of unconfined CD4 receptors was accelerated by a temperature raise. Black-Right-Pointing-Pointer The dynamics of confined CD4 receptors was unchanged by a temperature raise. Black-Right-Pointing-Pointer Our results suggest the existence of two different environments for CD4 receptors. -- Abstract: We investigated the lateral diffusion of the HIV receptor CD4 at the surface of T lymphocytes at 20 Degree-Sign C and 37 Degree-Sign C by Single Particle Tracking using Quantum Dots. Wemore » found that the receptors presented two major distinct behaviors that were not equally affected by temperature changes. About half of the receptors showed a random diffusion with a diffusion coefficient increasing upon raising the temperature. The other half of the receptors was permanently or transiently confined with unchanged dynamics on raising the temperature. These observations suggest that two distinct subpopulations of CD4 receptors with different environments are present at the surface of living T lymphocytes.« less

Slippery Liquid-Infused Porous Surfaces and Droplet Transportation by Surface Acoustic Waves

NASA Astrophysics Data System (ADS)

Luo, J. T.; Geraldi, N. R.; Guan, J. H.; McHale, G.; Wells, G. G.; Fu, Y. Q.

2017-01-01

On a solid surface, a droplet of liquid will stick due to the capillary adhesion, and this causes low droplet mobility. To reduce contact line pinning, surface chemistry can be coupled to micro- and/or nanostructures to create superhydrophobic surfaces on which a droplet balls up into an almost spherical shape, thus, minimizing the contact area. Recent progress in soft matter has now led to alternative lubricant-impregnated surfaces capable of almost zero contact line pinning and high droplet mobility without causing droplets to ball up and minimize the contact area. Here we report an approach to surface-acoustic-wave- (SAW) actuated droplet transportation enabled using such a surface. These surfaces maintain the contact area required for efficient energy and momentum transfer of the wave energy into the droplet while achieving high droplet mobility and a large footprint, therefore, reducing the threshold power required to induce droplet motion. In our approach, we use a slippery layer of lubricating oil infused into a self-assembled porous hydrophobic layer, which is significantly thinner than the SAW wavelength, and avoid damping of the wave. We find a significant reduction (up to 85%) in the threshold power for droplet transportation compared to that using a conventional surface-treatment method. Moreover, unlike droplets on superhydrophobic surfaces, where interaction with the SAW induces a transition from a Cassie-Baxter state to a Wenzel state, the droplets on our liquid-impregnated surfaces remain in a mobile state after interaction with the SAW.

Small Body Hopper Mobility Concepts

NASA Technical Reports Server (NTRS)

Howe, A. Scott; Gernhardt, Michael L.; Lee, Dave E.; Crues, E. Zack; Dexter, Dan E.; Abercromby, Andrew F. J.; Chappell, Steve P.; Nguyen, Hung T.

2015-01-01

A propellant-saving hopper mobility system was studied that could help facilitate the exploration of small bodies such as Phobos for long-duration human missions. The NASA Evolvable Mars Campaign (EMC) has proposed a mission to the moons of Mars as a transitional step for eventual Mars surface exploration. While a Mars transit habitat would be parked in High-Mars Orbit (HMO), crew members would visit the surface of Phobos multiple times for up to 14 days duration (up to 50 days at a time with logistics support). This paper describes a small body surface mobility concept that is capable of transporting a small, two-person Pressurized Exploration Vehicle (PEV) cabin to various sites of interest in the low-gravity environment. Using stored kinetic energy between bounces, a propellant-saving hopper mobility system can release the energy to vector the vehicle away from the surface in a specified direction. Alternatively, the stored energy can be retained for later use while the vehicle is stationary in respect to the surface. The hopper actuation was modeled using a variety of launch velocities, and the hopper mobility was evaluated using NASA Exploration Systems Simulations (NExSyS) for transit between surface sites of interest. A hopper system with linear electromagnetic motors and mechanical spring actuators coupled with Control Moment Gyroscope (CMG) for attitude control will use renewable electrical power, resulting in a significant propellant savings.

Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

PubMed

VandenBussche, C J; Mulrooney, T J; Frazier, W R; Dakshanamurthy, S; Hurley, C K

2009-03-01

Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

Infrared thermography based studies on mobile phone induced heating

NASA Astrophysics Data System (ADS)

Lahiri, B. B.; Bagavathiappan, S.; Soumya, C.; Jayakumar, T.; Philip, John

2015-07-01

Here, we report the skin temperature rise due to the absorption of radio frequency (RF) energy from three handheld mobile phones using infrared thermography technique. Experiments are performed under two different conditions, viz. when the mobile phones are placed in soft touch with the skin surface and away from the skin surface. Additionally, the temperature rise of mobile phones during charging, operation and simultaneous charging and talking are monitored under different exposure conditions. It is observed that the temperature of the cheek and ear regions monotonically increased with time during the usage of mobile phones and the magnitude of the temperature rise is higher for the mobile phone with higher specific absorption rate. The increase in skin temperature is higher when the mobile phones are in contact with the skin surface due to the combined effect of absorption of RF electromagnetic power and conductive heat transfer. The increase in the skin temperature in non-contact mode is found to be within the safety limit of 1 °C. The measured temperature rise is in good agreement with theoretical predictions. The empirical equation obtained from the temperature rise on the cheek region of the subjects correlates well with the specific absorption rate of the mobile phones. Our study suggests that the use of mobile phones in non-contact mode can significantly lower the skin temperature rise during its use and hence, is safer compared to the contact mode.

Differential mobilization of terrestrial carbon pools in Eurasian Arctic river basins.

PubMed

Feng, Xiaojuan; Vonk, Jorien E; van Dongen, Bart E; Gustafsson, Örjan; Semiletov, Igor P; Dudarev, Oleg V; Wang, Zhiheng; Montluçon, Daniel B; Wacker, Lukas; Eglinton, Timothy I

2013-08-27

Mobilization of Arctic permafrost carbon is expected to increase with warming-induced thawing. However, this effect is challenging to assess due to the diverse processes controlling the release of various organic carbon (OC) pools from heterogeneous Arctic landscapes. Here, by radiocarbon dating various terrestrial OC components in fluvially and coastally integrated estuarine sediments, we present a unique framework for deconvoluting the contrasting mobilization mechanisms of surface vs. deep (permafrost) carbon pools across the climosequence of the Eurasian Arctic. Vascular plant-derived lignin phenol (14)C contents reveal significant inputs of young carbon from surface sources whose delivery is dominantly controlled by river runoff. In contrast, plant wax lipids predominantly trace ancient (permafrost) OC that is preferentially mobilized from discontinuous permafrost regions, where hydrological conduits penetrate deeper into soils and thermokarst erosion occurs more frequently. Because river runoff has significantly increased across the Eurasian Arctic in recent decades, we estimate from an isotopic mixing model that, in tandem with an increased transfer of young surface carbon, the proportion of mobilized terrestrial OC accounted for by ancient carbon has increased by 3-6% between 1985 and 2004. These findings suggest that although partly masked by surface carbon export, climate change-induced mobilization of old permafrost carbon is well underway in the Arctic.

30 CFR 57.12027 - Grounding mobile equipment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Grounding mobile equipment. 57.12027 Section 57... Surface and Underground § 57.12027 Grounding mobile equipment. Frame grounding or equivalent protection shall be provided for mobile equipment powered through trailing cables. ...

High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor

PubMed Central

Suhr, Steven T.; Gil, Elad B.; Senut, Marie-Claude; Gage, Fred H.

1998-01-01

Our studies of the Bombyx mori ecdysone receptor (BE) revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the ultraspiracle gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this “gain of function” to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate “reporter” viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells. PMID:9653129

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

PubMed Central

Van Zoelen, E J; Peters, P H; Afink, G B; Van Genesen, S; De Roos, D G; Van Rotterdam, W; Theuvenet, A P

1994-01-01

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells. Images Figure 5 PMID:8135739

Structure-activity relationships of seco-prezizaane and picrotoxane/picrodendrane terpenoids by Quasar receptor-surface modeling.

PubMed

Schmidt, Thomas J; Gurrath, Marion; Ozoe, Yoshihisa

2004-08-01

The seco-prezizaane-type sesquiterpenes pseudoanisatin and parviflorolide from Illicium are noncompetitive antagonists at housefly (Musca domestica) gamma-aminobutyric acid (GABA) receptors. They show selectivity toward the insect receptor and thus represent new leads toward selective insecticides. Based on the binding data for 13 seco-prezizaane terpenoids and 17 picrotoxane and picrodendrane-type terpenoids to housefly and rat GABA receptors, a QSAR study was conducted by quasi-atomistic receptor-surface modeling (Quasar). The resulting models provide insight into the structural basis of selectivity and properties of the binding sites at GABA receptor-coupled chloride channels of insects and mammals.

Intrapulmonary receptors in the Tegu lizard: II. Functional characteristics and localization;.

PubMed

Scheid, P; Kuhlmann, W D; Fedde, M R

1977-02-01

Intrapulmonary receptors identified in the Tegu lizard by single-unit vagal recording (Fedde et al., 1977) were subjected to a number of stimuli and localized within the lung. Some carbon dioxide receptors could follow periodic changes in intrapulmonary CO2 concentrations as rapidly as 1.3 Hz; No oxygen sensitivity was observed with this receptor type, and halothane markedly depressed the discharge frequency. In response to intravenously injected acetazolamide they increased their discharge frequency and became almost totally insensitive to CO2, suggesting molecular per se is not the direct controller of receptor discharge; These receptors show many of the functional characteristics described for those in the avian lung. Afferent activity from both CO2 and mechanoreceptors could be elicited by electrically stimulating the lung surface. The CO2 receptors appeared to be organized in a receptive field covering more than 1 cm2 of lung surface, multiple receptors being innervated by a single afferent fiber. Activity in afferent fibers from mechanoreceptors could be evoked from only one distinct spot on the lung surface. Conduction velocities of afferent fibers from CO2 receptors ranged from 1 to 3 m-sec-1; from mechanoreceptors, from 1.9 to 5.2 m-sec-1.

Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

PubMed

Sabri, F; Tresoldi, E; Di Stefano, M; Polo, S; Monaco, M C; Verani, A; Fiore, J R; Lusso, P; Major, E; Chiodi, F; Scarlatti, G

1999-11-25

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages. Copyright 1999 Academic Press.

The pharmacology of TUG-891, a potent and selective agonist of the free fatty acid receptor 4 (FFA4/GPR120), demonstrates both potential opportunity and possible challenges to therapeutic agonism.

PubMed

Hudson, Brian D; Shimpukade, Bharat; Mackenzie, Amanda E; Butcher, Adrian J; Pediani, John D; Christiansen, Elisabeth; Heathcote, Helen; Tobin, Andrew B; Ulven, Trond; Milligan, Graeme

2013-11-01

TUG-891 [3-(4-((4-fluoro-4'-methyl-[1,1'-biphenyl]-2-yl)methoxy)phenyl)propanoic acid] was recently described as a potent and selective agonist for the long chain free fatty acid (LCFA) receptor 4 (FFA4; previously G protein-coupled receptor 120, or GPR120). Herein, we have used TUG-891 to further define the function of FFA4 and used this compound in proof of principle studies to indicate the therapeutic potential of this receptor. TUG-891 displayed similar signaling properties to the LCFA α-linolenic acid at human FFA4 across various assay end points, including stimulation of Ca²⁺ mobilization, β-arrestin-1 and β-arrestin-2 recruitment, and extracellular signal-regulated kinase phosphorylation. Activation of human FFA4 by TUG-891 also resulted in rapid phosphorylation and internalization of the receptor. While these latter events were associated with desensitization of the FFA4 signaling response, removal of TUG-891 allowed both rapid recycling of FFA4 back to the cell surface and resensitization of the FFA4 Ca²⁺ signaling response. TUG-891 was also a potent agonist of mouse FFA4, but it showed only limited selectivity over mouse FFA1, complicating its use in vivo in this species. Pharmacologic dissection of responses to TUG-891 in model murine cell systems indicated that activation of FFA4 was able to mimic many potentially beneficial therapeutic properties previously reported for LCFAs, including stimulating glucagon-like peptide-1 secretion from enteroendocrine cells, enhancing glucose uptake in 3T3-L1 adipocytes, and inhibiting release of proinflammatory mediators from RAW264.7 macrophages, which suggests promise for FFA4 as a therapeutic target for type 2 diabetes and obesity. Together, these results demonstrate both potential but also significant challenges that still need to be overcome to therapeutically target FFA4.

[Stimuli sensitive changes in electrical surface properties of soft membranes: from a synthesized polymer to a biological system].

PubMed

Makino, K

1997-01-01

The electrical surface properties of biological cells have been studied, which provided us with the fundamental knowledge about the cell surface. The change in shape or biological functions of cells may affect the surface properties and can be detected by electrokinetic measurements. Biological cell surfaces are covered with polysaccharide chains, some are charged and some are not. Some polysaccharides produce a hydrogel matrixes under a proper condition. We thus consider it reasonable that cell surface is approximated by a hydrogel surface. Electrophoretic mobility measurements are useful for studying the surface properties of biological cells suspended as colloidal particles in an electrolyte solution. The electro-osmotic velocity measurements on the other hand are advantageous to the study of the surface properties of slab-shaped biological systems such as membranes. This work was started with a hydrogel, as a model material. As a hydrogel, poly(N-isopropylacrylamide) poly(NIPAAm), abbreviated as hereafter, was chosen, because this hydrogel changes its volume depending on temperature. The dependence of the electrophoretic mobility of latex particles covered with poly(NIPAAm) hydrogel layer or of the electro-osmotic mobility on poly(NIPAAm) plate upon temperature and ionic strength of the dispersing medium was well explained with an electrophoretic mobility formula for "soft particles" developed by Ohshima. The electrokinetic measurements and the explanation of data with an electrophoretic mobility formula for "soft particles" give us information about the surface charge density and the "softness" of soft surfaces. On the basis of the findings with hydrogels, we have discussed the relationship between the changes in shape or function of the biological cells and the change in physicochemical surface properties using these measurements. To study the change in physicochemical properties of the cell surface caused by apoptosis, we have measured the electrophoretic mobilities of intact and apoptotic human promyelocytic leukemia cell lines, HL-60RG cells. We have also studied the differences observed in surface properties of malignant lymphosarcoma cell line, RAW117-P, and its variant, RAW117-H10, with a high metastatic property to the liver. In both cases, the cell surfaces became softer by the changes of biological functions. We have applied electrophoresis and electro-osmosis measurements to the study of the electrokinetic surface properties of rat basophilic leukemia cells, RBL cells. It was also found that the surface of Human umbilical vein endothelial cells, HUVEC, is considerably soft as compared with those of other biological cells we have studied before.

Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

PubMed Central

Tremblay, Tammy-Lynn; Hill, Jennifer J.

2017-01-01

Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct. PMID:28422167

Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

PubMed Central

Stevenson, M; Zhang, X H; Volsky, D J

1987-01-01

Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

PubMed Central

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

Reciprocal and activity-dependent regulation of surface AMPA and NMDA receptors in cultured neurons

PubMed Central

Li, Guo Hua; Jackson, Michael F; Orser, Beverley A; MacDonald, John F

2010-01-01

Activation of NMDA receptors (NMDARs) can modulate excitatory synaptic transmission in the central nervous system by dynamically altering the number of synaptic AMPA receptors (AMPARs). The surface expression of NMDARs themselves is also subject to modulation in an activity-dependent manner. In addition to NMDAR-induced changes in AMPAR expression, AMPARs have also been found to regulate their own surface expression, independently of NMDARs. However, whether or not AMPARs and NMDARs might reciprocally regulate their surface expression has not previously been systematically explored. We utilized surface biotinylation assays and stimulation protocols intended to selectively stimulate various glutamate receptor subpopulations (e.g. AMPARs vs NMDARs; synaptic vs extrasynaptic). We reveal that activation of synaptic NMDARs increases the surface expression of both NMDAR and AMPAR subunits, while activation of extrasynaptic NMDAR produces the opposite effect. Surprisingly, we find that selective activation of AMPARs reduces the surface expression of not only AMPARs but also of NMDARs. These results suggest that both AMPARs and NMDARs at synaptic sites are subject to modulation by multiple signalling pathways in an activity-dependent way. PMID:21383896

A leptin-regulated circuit controls glucose mobilization during noxious stimuli.

PubMed

Flak, Jonathan N; Arble, Deanna; Pan, Warren; Patterson, Christa; Lanigan, Thomas; Goforth, Paulette B; Sacksner, Jamie; Joosten, Maja; Morgan, Donald A; Allison, Margaret B; Hayes, John; Feldman, Eva; Seeley, Randy J; Olson, David P; Rahmouni, Kamal; Myers, Martin G

2017-08-01

Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor-expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.

Microscopic visualization of metabotropic glutamate receptors on the surface of living cells using bifunctional magnetic resonance imaging probes.

PubMed

Mishra, Anurag; Mishra, Ritu; Gottschalk, Sven; Pal, Robert; Sim, Neil; Engelmann, Joern; Goldberg, Martin; Parker, David

2014-02-19

A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualize mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.

Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

DTIC Science & Technology

2014-05-01

in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors

PubMed Central

Budi, Erine H.; Muthusamy, Baby Periyanayaki; Derynck, Rik

2015-01-01

Increased activity of transforming growth factor β (TGF-β), which binds to and stimulates cell surface receptors, contributes to cancer progression and fibrosis by driving epithelial cells toward a migratory mesenchymal phenotype and increasing the abundance of extracellular matrix proteins. The abundance of TGF-β receptors at the cell surface determines cellular responsiveness to TGF-β, which is often produced by the same cells that have the receptors, and thus serves as an autocrine signal. We found that Akt-mediated phosphorylation of AS160, a RabGAP [guanosine triphosphatase (GTPase)-activating protein] promoted the translocation of TGF-β receptors from intracellular stores to the plasma membrane of mouse embryonic fibroblasts (MEFs) and NMuMG epithelial cells. Consequently, insulin, which is commonly used to treat hyperglycemia and activates Akt signaling, increased the amount of TGF-β receptors at the cell surface, thereby enhancing TGF-β responsiveness. This insulin-induced increase in autocrine TGF-β signaling contributed to insulin-induced gene expression responses, attenuated the epithelial phenotype, and promoted the migration of NMuMG cells. Furthermore, the enhanced delivery of TGF-β receptors at the cell surface enabled insulin to increase TGF-β-induced gene responses. The enhancement of TGF-β responsiveness in response to Akt activation may help to explain the biological effects of insulin, the progression of cancers in which Akt is activated, and the increased incidence of fibroses in diabetes. PMID:26420907

Mobile autonomous robotic apparatus for radiologic characterization

DOEpatents

Dudar, Aed M.; Ward, Clyde R.; Jones, Joel D.; Mallet, William R.; Harpring, Larry J.; Collins, Montenius X.; Anderson, Erin K.

1999-01-01

A mobile robotic system that conducts radiological surveys to map alpha, beta, and gamma radiation on surfaces in relatively level open areas or areas containing obstacles such as stored containers or hallways, equipment, walls and support columns. The invention incorporates improved radiation monitoring methods using multiple scintillation detectors, the use of laser scanners for maneuvering in open areas, ultrasound pulse generators and receptors for collision avoidance in limited space areas or hallways, methods to trigger visible alarms when radiation is detected, and methods to transmit location data for real-time reporting and mapping of radiation locations on computer monitors at a host station. A multitude of high performance scintillation detectors detect radiation while the on-board system controls the direction and speed of the robot due to pre-programmed paths. The operators may revise the preselected movements of the robotic system by ethernet communications to remonitor areas of radiation or to avoid walls, columns, equipment, or containers. The robotic system is capable of floor survey speeds of from 1/2-inch per second up to about 30 inches per second, while the on-board processor collects, stores, and transmits information for real-time mapping of radiation intensity and the locations of the radiation for real-time display on computer monitors at a central command console.

Mobile autonomous robotic apparatus for radiologic characterization

DOEpatents

Dudar, A.M.; Ward, C.R.; Jones, J.D.; Mallet, W.R.; Harpring, L.J.; Collins, M.X.; Anderson, E.K.

1999-08-10

A mobile robotic system is described that conducts radiological surveys to map alpha, beta, and gamma radiation on surfaces in relatively level open areas or areas containing obstacles such as stored containers or hallways, equipment, walls and support columns. The invention incorporates improved radiation monitoring methods using multiple scintillation detectors, the use of laser scanners for maneuvering in open areas, ultrasound pulse generators and receptors for collision avoidance in limited space areas or hallways, methods to trigger visible alarms when radiation is detected, and methods to transmit location data for real-time reporting and mapping of radiation locations on computer monitors at a host station. A multitude of high performance scintillation detectors detect radiation while the on-board system controls the direction and speed of the robot due to pre-programmed paths. The operators may revise the preselected movements of the robotic system by ethernet communications to remonitor areas of radiation or to avoid walls, columns, equipment, or containers. The robotic system is capable of floor survey speeds of from 1/2-inch per second up to about 30 inches per second, while the on-board processor collects, stores, and transmits information for real-time mapping of radiation intensity and the locations of the radiation for real-time display on computer monitors at a central command console. 4 figs.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

PubMed

Cantini, Marco; Gomide, Karina; Moulisova, Vladimira; González-García, Cristina; Salmerón-Sánchez, Manuel

2017-09-01

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

From Human Megakaryocytes to Platelets: Effects of Aspirin on High-Mobility Group Box 1/Receptor for Advanced Glycation End Products Axis

PubMed Central

Mardente, Stefania; Mari, Emanuela; Massimi, Isabella; Tafani, Marco; Guerriero, Raffaella; Morsilli, Ornella; Pulcinelli, Fabio M.; Bianchi, Marco E.; Zicari, Alessandra

2018-01-01

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin “in vivo” and “in vitro” not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1. PMID:29375570

A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

PubMed Central

Shepard, Blythe D.; Natarajan, Niranjana; Protzko, Ryan J.; Acres, Omar W.; Pluznick, Jennifer L.

2013-01-01

Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. PMID:23840901

The majority of ACTH receptor (MC2R) mutations found in Familial Glucocorticoid Deficiency type 1 lead to defective trafficking of the receptor to the cell surface

PubMed Central

TT, Chung; TR, Webb; LF, Chan; SN, Cooray; LA, Metherell; PJ, King; JP, Chapple; AJL, Clark

2008-01-01

Context: There are at least twenty-four missense, non-conservative mutations found in the ACTH receptor (Melanocortin 2 receptor, MC2R) which have been associated with the autosomal recessive disease Familial Glucocorticoid Deficiency (FGD) type 1. The characterization of these mutations has been hindered by difficulties in establishing a functional heterologous cell transfection system for MC2R. Recently the melanocortin 2 receptor accessory protein (MRAP) was identified as essential for trafficking of MC2R to the cell surface; therefore a functional characterization of MC2R mutations is now possible. Objective: To elucidate the molecular mechanisms responsible for defective MC2R function in FGD. Methods: Stable cell lines expressing human MRAPα were established and transiently transfected with wild-type or mutant MC2R. Functional characterization of mutant MC2R was performed using a cell surface expression assay, a cAMP reporter assay, confocal microscopy and co-immunoprecipitation of MRAPα. Results: Two thirds of all MC2R mutations had a significant reduction in cell surface trafficking even though MRAPα interacted with all mutants. Analysis of those mutant receptors that reached the cell surface indicated that 4/6 failed to signal, following stimulation with ACTH. Conclusion: The majority of MC2R mutations found in FGD fail to function because they fail to traffic to the cell surface. PMID:18840636

Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

PubMed

Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

2015-11-15

Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

Lunar surface exploration using mobile robots

NASA Astrophysics Data System (ADS)

Nishida, Shin-Ichiro; Wakabayashi, Sachiko

2012-06-01

A lunar exploration architecture study is being carried out by space agencies. JAXA is carrying out research and development of a mobile robot (rover) to be deployed on the lunar surface for exploration and outpost construction. The main target areas for outpost construction and lunar exploration are mountainous zones. The moon's surface is covered by regolith. Achieving a steady traversal of such irregular terrain constitutes the major technical problem for rovers. A newly developed lightweight crawler mechanism can effectively traverse such irregular terrain because of its low contact force with the ground. This fact was determined on the basis of the mass and expected payload of the rover. This paper describes a plan for Japanese lunar surface exploration using mobile robots, and presents the results of testing and analysis needed in their development. This paper also gives an overview of the lunar exploration robot to be deployed in the SELENE follow-on mission, and the composition of its mobility, navigation, and control systems.

Novel method for minority-carrier mobility measurement using photoconductance decay with chemically passivated and plasma damaged surfaces

NASA Astrophysics Data System (ADS)

Stephens, A. W.; Green, M. A.

1996-10-01

A method for measuring minority-carrier mobility using microwave-detected photoconductance decay without requiring bulk lifetime, estimates is presented. Three different measurements on a single sample yield values for surface recombination velocity, bulk lifetime, and diffusivity. For each measurement the surface conditions of the sample are changed, allowing extraction of different parameters. The usefulness of 0.08 molar ethanol/iodine solution as a means of achieving such good surface passivation is demonstrated. The following procedure was used to achieve high surface recombination. A CF4 plasma surface etch was shown to achieve the same level of surface damage as mechanical abrasion. The advantage of the new method is that it completely eliminates the chance of breaking samples during the abrasion process, which is of particular advantage for thin samples. The new experimental method for minority-carrier mobility measurement is evaluated using carrier lifetime measurements made on a commercially available Leo Giken ``Wafer-τ'' lifetime tester.

Ras-Directed N-Glycoproteins are Novel Early Biomarkers for Tumorigenesis and Malignant Transformation, and Therapeutic Targets of Neurofibromatosis Type I

DTIC Science & Technology

2012-09-01

translocation of receptors from the cytoplasm to the cell surface and retained receptors in the ER and Golgi apparatus , but had no effect on normal...glucose (2-DG) as two potential glycosylation inhibitors. Because proteins travelling to the Golgi apparatus for the consequent steps of...inhibited the transportation of receptors from the cytoplasm to the cell surface and retained receptors in the ER and Golgi apparatus (Fig 3). To

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed Central

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation. PMID:9405282

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation.

High-Mobility Group Box 1 Inhibits Gastric Ulcer Healing through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products

PubMed Central

Nadatani, Yuji; Watanabe, Toshio; Tanigawa, Tetsuya; Ohkawa, Fumikazu; Takeda, Shogo; Higashimori, Akira; Sogawa, Mitsue; Yamagami, Hirokazu; Shiba, Masatsugu; Watanabe, Kenji; Tominaga, Kazunari; Fujiwara, Yasuhiro; Takeuchi, Koji; Arakawa, Tetsuo

2013-01-01

High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced glycation end products (RAGE), leading to tissue injury. Although HMGB1’s ability to induce inflammation has been well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1’s effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα, whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses. PMID:24244627

High-mobility group box 1 inhibits gastric ulcer healing through Toll-like receptor 4 and receptor for advanced glycation end products.

PubMed

Nadatani, Yuji; Watanabe, Toshio; Tanigawa, Tetsuya; Ohkawa, Fumikazu; Takeda, Shogo; Higashimori, Akira; Sogawa, Mitsue; Yamagami, Hirokazu; Shiba, Masatsugu; Watanabe, Kenji; Tominaga, Kazunari; Fujiwara, Yasuhiro; Takeuchi, Koji; Arakawa, Tetsuo

2013-01-01

High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced glycation end products (RAGE), leading to tissue injury. Although HMGB1's ability to induce inflammation has been well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1's effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα, whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses.

Friction-Controlled Traction Force in Cell Adhesion

PubMed Central

Pompe, Tilo; Kaufmann, Martin; Kasimir, Maria; Johne, Stephanie; Glorius, Stefan; Renner, Lars; Bobeth, Manfred; Pompe, Wolfgang; Werner, Carsten

2011-01-01

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo. PMID:22004739

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Experimental evaluation of a new morphological approximation of the articular surfaces of the ankle joint.

PubMed

Belvedere, Claudio; Siegler, Sorin; Ensini, Andrea; Toy, Jason; Caravaggi, Paolo; Namani, Ramya; Giannini, Giulia; Durante, Stefano; Leardini, Alberto

2017-02-28

The mechanical characteristics of the ankle such as its kinematics and load transfer properties are influenced by the geometry of the articulating surfaces. A recent, image-based study found that these surfaces can be approximated by a saddle-shaped, skewed, truncated cone with its apex oriented laterally. The goal of this study was to establish a reliable experimental technique to study the relationship between the geometry of the articular surfaces of the ankle and its mobility and stability characteristics and to use this technique to determine if morphological approximations of the ankle surfaces based on recent discoveries, produce close to normal behavior. The study was performed on ten cadavers. For each specimen, a process based on medical imaging, modeling and 3D printing was used to produce two subject specific artificial implantable sets of the ankle surfaces. One set was a replica of the natural surfaces. The second approximated the ankle surfaces as an original saddle-shaped truncated cone with apex oriented laterally. Testing under cyclic loading conditions was then performed on each specimen following a previously established technique to determine its mobility and stability characteristics under three different conditions: natural surfaces; artificial surfaces replicating the natural surface morphology; and artificial approximation based on the saddle-shaped truncated cone concept. A repeated measure analysis of variance was then used to compare between the three conditions. The results show that (1): the artificial surfaces replicating natural morphology produce close to natural mobility and stability behavior thus establishing the reliability of the technique; and (2): the approximated surfaces based on saddle-shaped truncated cone concept produce mobility and stability behavior close to the ankle with natural surfaces. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mobilizing stem cells from normal donors: is it possible to improve upon G-CSF?

PubMed

Cashen, A F; Lazarus, H M; Devine, S M

2007-05-01

Currently, granulocyte colony stimulating factor (G-CSF) remains the standard mobilizing agent for peripheral blood stem cell (PBSC) donors, allowing the safe collection of adequate PBSCs from the vast majority of donors. However, G-CSF mobilization can be associated with some significant side effects and requires a multi-day dosing regimen. The other cytokine approved for stem cell mobilization, granulocyte-macrophage colony stimulating factor (GM-CSF), alters graft composition and may reduce the development of graft-versus-host disease, but a significant minority of donors fails to provide sufficient CD34+ cells with GM-CSF and some experience unacceptable toxicity. AMD3100 is a promising new mobilizing agent, which may have several advantages over G-CSF for donor mobilization. As it is a direct antagonist of the interaction between the chemokine stromal-derived factor-1 and its receptor CXCR4, AMD3100 mobilizes PBSCs within hours rather than days. It is also well tolerated, with no significant side effects reported in any of the clinical trials to date. Studies of autologous and allogeneic transplantation of AMD3100 mobilized grafts have demonstrated prompt and stable engraftment. Here, we review the current state of stem cell mobilization in normal donors and discuss novel strategies for donor stem cell mobilization.

Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

DNA-based probes for flow cytometry analysis of endocytosis and recycling.

PubMed

Dumont, Claire; Czuba, Ewa; Chen, Moore; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

2017-04-01

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients' survival.

PubMed

Theocharis, Stamatios; Giaginis, Constantinos; Alexandrou, Paraskevi; Rodriguez, Jose; Tasoulas, Jason; Danas, Eugene; Patsouris, Efstratios; Klijanienko, Jerzy

2016-03-01

Cannabinoid receptors (CB1R and CB2R) constitute essential members of the endocannabinoid system (ECS) which participates in many different functions indispensable to homeostatic regulation in several tissues, exerting also antitumorigenic effects. The present study aimed to assess the clinical significance of CB1R and CB2R protein expression in mobile tongue squamous cell carcinoma (SCC). CB1R and CB2R expression was assessed immunohistochemically on 28 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics and overall and disease-free patients' survival. CB1R, CB2R, and concomitant CB1R/CB2R expression was significantly increased in older compared to younger mobile tongue SCC patients (p = 0.0243, p = 0.0079, and p = 0.0366, respectively). Enhanced CB2R and concomitant CB1R/CB2R expression was significantly more frequently observed in female compared to male mobile tongue SCC patients (p = 0.0025 and p = 0.0016, respectively). Elevated CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well-defined tumor shape compared to those with diffuse (p = 0.0430). Mobile tongue SCC patients presenting enhanced CB1R, CB2R, or concomitant CB1R/CB2R expression showed significantly longer overall (log-rank test, p = 0.004, p = 0.011, p = 0.018, respectively) and disease-free (log-rank test, p = 0.003, p = 0.007, p = 0.027, respectively) survival times compared to those with low expression. In multivariate analysis, CB1R was identified as an independent prognostic factor for disease-free patients' survival (Cox-regression analysis, p = 0.032). The present study provides evidence that CB1R and CB2R may play a role in the pathophysiological aspects of the mobile tongue SCC and even each molecule may constitute a potential target for the development of novel anti-cancer drugs for this type of malignancy.

Inhibition of the mobility of mouse lymphocyte surface immunoglobulins by locally bound concanavalin A.

PubMed Central

Henis, Y I; Elson, E L

1981-01-01

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed. PMID:6940124

Enhancement of field effect mobility of poly(3-hexylthiophene) thin film transistors by soft-lithographical nanopatterning on the gate-dielectric surface

NASA Astrophysics Data System (ADS)

Park, Jeong-Ho; Kang, Seok-Ju; Park, Jeong-Woo; Lim, Bogyu; Kim, Dong-Yu

2007-11-01

The submicroscaled octadecyltrichlorosilane (OTS) line patterns on gate-dielectric surfaces were introduced into the fabrication of organic field effect transistors (OFETs). These spin-cast regioregular poly(3-hexylthiophene) films on soft-lithographically patterned SiO2 surfaces yielded a higher hole mobility (˜0.072cm2/Vs ) than those of unpatterned (˜0.015cm2/Vs) and untreated (˜5×10-3cm2/Vs) OFETs. The effect of mobility enhancement as a function of the patterned line pitch was investigated in structural and geometric characteristics. The resulting improved mobility is likely attributed to the formation of efficient π-π stacking as a result of guide-assisted, local self-organization-involved molecular interactions between the poly(3-hexylthiophene) polymer and the geometrical OTS patterns.

Inhibitory mechanism of an extract of Althaea officinalis L. on endothelin-1-induced melanocyte activation.

PubMed

Kobayashi, Akemi; Hachiya, Akira; Ohuchi, Atsushi; Kitahara, Takashi; Takema, Yoshinori

2002-02-01

It is known that expression of endothelin-1 (ET-1) increases in the epidermis after UVB irradiation, and that this plays an important role during the induction of pigmentation both as a mitogen and as a melanogen for normal human melanocytes (NHMC). When ET-1 acts on NHMC via the endothelin B receptor (ET(B)R) on their cell surface, mobilization of intracellular calcium is induced, which is followed by activation of Raf-1 located upstream of mitogen activated protein kinase (MAPK). We have continued the search for new agent which inhibit this calcium mobilization and we have found that an extract of Althaea officinalis L. has such an action. In this study, we investigated the precise inhibitory mechanism of this botanical extract on the ET-1-induced activation of melanocytes. Treatment of NHMC with this extract abrogated the stimulatory effect of ET-1 on proliferation and also on activation of MAPK in the intracellular signal transduction pathway, but did not affect the binding of ET-1 to the ET(B)R or the production of Inositol 1,4,5-Trisphosphate (IP3). Further, when this extract was used to treat normal human keratinocytes (NHKC), secretion of ET-1 by those cells was reduced. Taken together, these findings indicate that an extract of A. officinalis inhibits both the secretion of ET-1 from NHKC and the action of ET-1 on NHMC mainly by suppressing the ET-1-induced calcium mobilization without the modification of IP3 production, which in turn suggests that this extract is a useful ingredient for a whitening agent.

Rapid Steroid Hormone Actions Initiated at the Cell Surface and the Receptors that Mediate Them with an Emphasis on Recent Progress in Fish Models

PubMed Central

Thomas, Peter

2011-01-01

In addition to the classic genomic mechanism of steroid action mediated by activation of intracellular nuclear receptors, there is now extensive evidence that steroids also activate receptors on the cell surface to initiate rapid intracellular signaling and biological responses that are often nongenomic. Recent progress in our understanding of rapid, cell surface-initiated actions of estrogens, progestins, androgens and corticosteroids and the identities of the membrane receptors that act as their intermediaries is briefly reviewed with a special emphasis on studies in teleost fish. Two recently discovered novel proteins with seven-transmembrane domains, G protein-coupled receptor 30 (GPR30), and membrane progestin receptors (mPRs) have the ligand binding and signaling characteristics of estrogen and progestin membrane receptors, respectively, but their functional significance is disputed by some researchers. GPR30 is expressed on the cell surface of fish oocytes and mediates estrogen inhibition of oocyte maturation. mPRα is also expressed on the oocyte cell surface and is the intermediary in progestin induction of oocyte maturation in fish. Recent results suggest there is cross-talk between these two hormonal pathways and that there is reciprocal down-regulation of GPR30 and mPRα expression by estrogens and progestins at different phases of oocyte development to regulate the onset of oocyte maturation. There is also evidence in fish that mPRs are involved in progestin induction of sperm hypermotility and anti-apoptotic actions in ovarian follicle cells. Nonclassical androgen and corticosteroid actions have also been described in fish models but the membrane receptors mediating these actions have not been identified. PMID:22154643

Brain region-selective cellular redistribution of mGlu5 but not GABA(B) receptors following methamphetamine-induced associative learning.

PubMed

Herrold, Amy A; Voigt, Robin M; Napier, T Celeste

2011-12-01

Alterations in receptor expression and distribution between cell surface and cytoplasm are means by which psychostimulants regulate neurotransmission. Metabotropic glutamate receptor group I, subtype 5 (mGluR5) and GABA(B) receptors (GABA(B) R) are critically involved in the development and expression of stimulant-induced behaviors, including conditioned place preference (CPP), an index of drug-seeking. However, it is not known if psychostimulant-induced CPP alters the trafficking of these receptors. To fill this gap, this study used methamphetamine (Meth)-induced CPP in rats to ascertain if receptor changes occur in limbic brain regions that regulate drug-seeking, the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP). To do so, ex vivo tissue was assessed for changes in expression and surface vs. intracellular distribution of mGluR5 and GABA(B) Rs. There was a decrease in the surface to intracellular ratio of mGluR5 in the mPFC in Meth-conditioned rats, commensurate with an increase in intracellular levels. mGluR5 levels in the NAc or the VP were unaltered. There were no changes for GABA(B) R in any brain region assayed. This ex vivo snapshot of metabotropic glutamate and GABA receptor cellular distribution following induction of Meth-induced CPP is the first report to determine if these receptors are differentially altered after Meth-induced CPP. The results suggest that this Meth treatment paradigm likely induced a compensatory change in mGluR5 surface to intracellular ratio such that the surface remains unaltered while an increase in intracellular protein occurred. Copyright © 2011 Wiley-Liss, Inc.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes

PubMed Central

Santhosh, KT; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, AJ; Dakshinamurti, S

2011-01-01

BACKGROUND AND PURPOSE Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor–mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS TP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. PMID:21385177

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes.

PubMed

Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

2011-04-01

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes

PubMed Central

Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

2011-01-01

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time–courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gαi protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. PMID:21255008

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus.

PubMed

Naylor, David E; Liu, Hantao; Niquet, Jerome; Wasterlain, Claude G

2013-06-01

After 1h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE. Copyright © 2013 Elsevier Inc. All rights reserved.

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus

PubMed Central

Naylor, David E.; Liu, Hantao; Niquet, Jerome; Wasterlain, Claude G.

2017-01-01

After 1 h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE. PMID:23313318

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Identification and quantification of the rat hepatocyte asialoglycoprotein receptor.

PubMed Central

Schwartz, A L; Marshak-Rothstein, A; Rup, D; Lodish, H F

1981-01-01

The asialoglycoprotein receptor from rat liver was purified by solubilization and affinity chromatography on asialoorosomucoid-Sepharose. The preparation yielded four distinct polypeptides of Mr 40,000-120,000. We prepared a monoclonal antibody that both immunoprecipitates solubilized receptor activity and blocks the binding of galactose-terminal glycoproteins to immobilized receptor. The monoclonal antibody and a rabbit antireceptor antiserum immunoprecipitated all four polypeptide species. Peptide analysis by two-dimensional chromatography of the individual 125I-labeled species showed nearly identical patterns, which also suggested that the four polypeptides have a similar primary structure. To identify and quantitate the asialoglycoprotein receptor on the hepatocyte cell surface, intact cells were iodinated with lactoperoxidase, and the solubilized membranes were treated with antireceptor antibody. The Mr 55,000 and Mr 65,000 species were the major species found. Our results suggest that the Mr of the surface receptor is at least 55,000 and that it comprises between 1-2% of the iodinated hepatocyte surface protein. Images PMID:6267585

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation

PubMed Central

Sivori, Simona; Falco, Michela; Marcenaro, Emanuela; Parolini, Silvia; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro

2002-01-01

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor. PMID:11917118

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

30 CFR 57.12005 - Protection of power conductors from mobile equipment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Protection of power conductors from mobile... NONMETAL MINES Electricity Surface and Underground § 57.12005 Protection of power conductors from mobile equipment. Mobile equipment shall not run over power conductors, nor shall loads be dragged over power...

30 CFR 57.12005 - Protection of power conductors from mobile equipment.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Protection of power conductors from mobile... NONMETAL MINES Electricity Surface and Underground § 57.12005 Protection of power conductors from mobile equipment. Mobile equipment shall not run over power conductors, nor shall loads be dragged over power...

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

PubMed Central

Schweitzer-Stenner, R; Tamir, I; Pecht, I

1997-01-01

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response. PMID:9168023

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

PubMed

Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma

2018-04-30

Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Surveying GPCR solubilisation conditions using surface plasmon resonance.

PubMed

Navratilova, Iva Hopkins; Aristotelous, Tonia; Bird, Louise E; Hopkins, Andrew L

2018-06-15

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Role of σ1 Receptors in Learning and Memory and Alzheimer's Disease-Type Dementia.

PubMed

Maurice, Tangui; Goguadze, Nino

2017-01-01

The present chapter will review the role of σ 1 receptor in learning and memory and neuroprotection , against Alzheimer's type dementia. σ 1 Receptor agonists have been tested in a variety of pharmacological and pathological models of learning impairments in rodents these last past 20 years. Their anti-amnesic effects have been explained by the wide-range modulatory role of σ 1 receptors on Ca 2+ mobilizations, neurotransmitter responses, and particularly glutamate and acetylcholine systems, and neurotrophic factors. Recent observations from genetic and pharmacological studies have shown that σ 1 receptor can also be targeted in neurodegenerative diseases, and particularly Alzheimer's disease . Several compounds, acting partly through the σ 1 receptor, have showed effective neuroprotection in transgenic mouse models of Alzheimer's disease . We will review the data and discuss the possible mechanisms of action, particularly focusing on oxidative stress and mitochondrial integrity, trophic factors and a novel hypothesis suggesting a functional interaction between the σ 1 receptor and α 7 nicotinic acetylcholine receptor. Finally, we will discuss the pharmacological peculiarities of non-selective σ 1 receptor ligands, now developed as neuroprotectants in Alzheimer's disease , and positive modulators, recently described and that showed efficacy against learning and memory deficits.

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Quantitative Characterization of Magnetic Mobility of Nanoparticle in Solution-Based Condition.

PubMed

Rodoplu, Didem; Boyaci, Ismail H; Bozkurt, Akif G; Eksi, Haslet; Zengin, Adem; Tamer, Ugur; Aydogan, Nihal; Ozcan, Sadan; Tugcu-Demiröz, Fatmanur

2015-01-01

Magnetic nanoparticles are considered as the ideal substrate to selectively isolate target molecules or organisms from sample solutions in a wide variety of applications including bioassays, bioimaging and environmental chemistry. The broad array of these applications in fields requires the accurate magnetic characterization of nanoparticles for a variety of solution based-conditions. Because the freshly synthesized magnetic nanoparticles demonstrated a perfect magnetization value in solid form, they exhibited a different magnetic behavior in solution. Here, we present simple quantitative method for the measurement of magnetic mobility of nanoparticles in solution-based condition. Magnetic mobility of the nanoparticles was quantified with initial mobility of the particles using UV-vis absorbance spectroscopy in water, ethanol and MES buffer. We demonstrated the efficacy of this method through a systematic characterization of four different core-shell structures magnetic nanoparticles over three different surface modifications. The solid nanoparticles were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD) and saturation magnetization (Ms). The surfaces of the nanoparticles were functionalized with 11-mercaptoundecanoic acid and bovine serum albumin BSA was selected as biomaterial. The effect of the surface modification and solution media on the stability of the nanoparticles was monitored by zeta potentials and hydrodynamic diameters of the nanoparticles. Results obtained from the mobility experiments indicate that the initial mobility was altered with solution media, surface functionalization, size and shape of the magnetic nanoparticle. The proposed method easily determines the interactions between the magnetic nanoparticles and their surrounding biological media, the magnetophoretic responsiveness of nanoparticles and the initial mobilities of the nanoparticles.

Understanding mobility degeneration mechanism in organic thin-film transistors (OTFT)

NASA Astrophysics Data System (ADS)

Wang, Wei; Wang, Long; Xu, Guangwei; Gao, Nan; Wang, Lingfei; Ji, Zhuoyu; Lu, Congyan; Lu, Nianduan; Li, Ling; Liu, Miwng

2017-08-01

Mobility degradation at high gate bias is often observed in organic thin film transistors. We propose a mechanism for this confusing phenomenon, based on the percolation theory with the presence of disordered energy landscape with an exponential density of states. Within a simple model we show how the surface states at insulator/organic interface trap a portion of channel carriers, and result in decrease of mobility as well as source/drain current with gate voltage. Depending on the competition between the carrier accumulation and surface trapping effect, two different carrier density dependences of mobility are obtained, in excellent agreement with experiment data.

Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata

PubMed Central

Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin

2016-01-01

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata. Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation. PMID:27421895

Particle compositions with a pre-selected cell internalization mode

NASA Technical Reports Server (NTRS)

Ferrari, Mauro (Inventor); Decuzzi, Paolo (Inventor)

2012-01-01

A method of formulating a particle composition having a pre-selected cell internalization mode involves selecting a target cell having surface receptors and obtaining particles that have i) surface moieties, that have an affinity for or are capable of binding to the surface receptors of the cell and ii) a preselected shape, where a surface distribution of the surface moieties on the particles and the shape of the particles are effective for the pre-selected cell internalization mode.

Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling & paired-agent principles from nuclear medicine and optical imaging

PubMed Central

Tichauer, Kenneth M.; Wang, Yu; Pogue, Brian W.; Liu, Jonathan T. C.

2015-01-01

The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche. PMID:26134619

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

PubMed

Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

1999-10-01

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction.

PubMed

Stenmark, Pål; Dupuy, Jérôme; Imamura, Akihiro; Kiso, Makoto; Stevens, Raymond C

2008-08-15

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

Molecular-dynamics analysis of mobile helium cluster reactions near surfaces of plasma-exposed tungsten

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Lin; Maroudas, Dimitrios, E-mail: maroudas@ecs.umass.edu; Hammond, Karl D.

We report the results of a systematic atomic-scale analysis of the reactions of small mobile helium clusters (He{sub n}, 4 ≤ n ≤ 7) near low-Miller-index tungsten (W) surfaces, aiming at a fundamental understanding of the near-surface dynamics of helium-carrying species in plasma-exposed tungsten. These small mobile helium clusters are attracted to the surface and migrate to the surface by Fickian diffusion and drift due to the thermodynamic driving force for surface segregation. As the clusters migrate toward the surface, trap mutation (TM) and cluster dissociation reactions are activated at rates higher than in the bulk. TM produces W adatoms and immobile complexes ofmore » helium clusters surrounding W vacancies located within the lattice planes at a short distance from the surface. These reactions are identified and characterized in detail based on the analysis of a large number of molecular-dynamics trajectories for each such mobile cluster near W(100), W(110), and W(111) surfaces. TM is found to be the dominant cluster reaction for all cluster and surface combinations, except for the He{sub 4} and He{sub 5} clusters near W(100) where cluster partial dissociation following TM dominates. We find that there exists a critical cluster size, n = 4 near W(100) and W(111) and n = 5 near W(110), beyond which the formation of multiple W adatoms and vacancies in the TM reactions is observed. The identified cluster reactions are responsible for important structural, morphological, and compositional features in the plasma-exposed tungsten, including surface adatom populations, near-surface immobile helium-vacancy complexes, and retained helium content, which are expected to influence the amount of hydrogen re-cycling and tritium retention in fusion tokamaks.« less

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

PubMed Central

Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F.; Rudel, Thomas

2015-01-01

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance. PMID:25906164

EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis.

PubMed

Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F; Rudel, Thomas

2015-04-01

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia

Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less

Immunomodulatory intervention with Gamma interferon in mice with sepsis.

PubMed

Wang, Yu; Kong, Bing-Bing; Yang, Wen-Ping; Zhao, Xin; Zhang, Rong

2017-09-15

Sepsis-triggered immune paralysis including T-cell dysfunction increase susceptibility to infection. Gamma interferon (IFNg) exert beneficial effects in patients with sepsis. Herein, we speculated that IFNg may attenuate T-cell dysfunction induced by sepsis, although the mechanisms remain elusive. To test this hypothesis, we used a model based on cecal ligation and puncture (CLP) to induce sepsis in mice. Male C57BL/6 mice were pretreated with recombinant human IFNg (0.01μg/g of body weight) before CLP. The immunophenotyping of cell surface receptor expression, and regulatory T cells (CD4+CD25+Foxp3+) were quantified by flow cytometry. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. Formation of IFNg and interleukin 4 (IL-4) in the spleen and plasma levels of TNF-α, IL-6, high-mobility group box 1 (HMGB1) were determined using enzyme-linked immunosorbent assay. IFNg markedly inhibited the reduction in cytokine secretion from lipopolysaccharide (LPS)-stimulated splenocytes. IFNg-treated mices had significantly decreased percentages of programmed cell death 1 (PD-1) receptors, increased the percentages of positive costimulatory receptor CD28 on CD4 T cells expressing. IFNg markedly reduced T-cell apoptosis through upregulating the expression of Bcl-2. CLP-induced formation of regulatory T cells in the spleen was abolished in IFNg -treated mices. Moreover, IFNg treatment reduced plasma levels of TNF-α, IL-6, HMGB1. IFNg can be a powerful regulator of immune function under sepsis conditions. Therefore, targeted immune-enhancement with IFNg may be a valid therapeutic approach in sepsis. Copyright © 2017 Elsevier Inc. All rights reserved.

Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.

PubMed

Lam, E W; Glassford, J; van der Sman, J; Banerji, L; Pizzey, A R; Shaun, N; Thomas, B; Klaus, G G

1999-10-01

Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein/E2F complexes in mouse B lymphocytes following stimulation by anti-IgM, anti-CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two "free" E2F complexes consisting of E2F4/DP1 and E2F1-3/DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB/E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.

Parenteral medium-chain triglyceride-induced neutrophil activation is not mediated by a Pertussis Toxin sensitive receptor.

PubMed

Versleijen, Michelle W J; van Esterik, Joantine C J; Roelofs, Hennie M J; van Emst-de Vries, Sjenet E; Willems, Peter H G M; Wanten, Geert J A

2009-02-01

Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5-1 microg/mL, 1.5 h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5 mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca(2+) mobilization and on LCT/MCT (5 mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca(2+) concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas

PubMed Central

Alexander, Jodi L.; Oliphant, Andrew; Wilcockson, David C.; Audsley, Neil; Down, Rachel E.; Lafont, Rene; Webster, Simon G.

2018-01-01

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides. PMID:29379412

A Rapid Screen Technique for Estimating Nanoparticle Transport in Porous Media

EPA Science Inventory

Quantifying the mobility of engineered nanoparticles in hydrologic pathways from point of release to human or ecological receptors is essential for assessing environmental exposures. Column transport experiments are a widely used technique to estimate the transport parameters of ...

Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein*

PubMed Central

Mori, Yasunori; Fukuda, Mitsunori; Henley, Jeremy M.

2014-01-01

Glutamate receptors are fundamental for control synaptic transmission, synaptic plasticity, and neuronal excitability. However, many of the molecular mechanisms underlying their trafficking remain elusive. We previously demonstrated that the small GTPase Rab17 regulates dendritic trafficking in hippocampal neurons. Here, we investigated the role(s) of Rab17 in AMPA receptor (AMPAR) and kainate receptor (KAR) trafficking. Although Rab17 knockdown did not affect surface expression of the AMPAR subunit GluA1 under basal or chemically induced long term potentiation conditions, it significantly reduced surface expression of the KAR subunit GluK2. Rab17 co-localizes with Syntaxin-4 in the soma, dendritic shaft, the tips of developing hippocampal neurons, and in spines. Rab17 knockdown caused Syntaxin-4 redistribution away from dendrites and into axons in developing hippocampal neurons. Syntaxin-4 knockdown reduced GluK2 but had no effect on GluA1 surface expression. Moreover, overexpression of constitutively active Rab17 promoted dendritic surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. These data suggest that Rab17 mediates the dendritic trafficking of Syntaxin-4 to selectively regulate dendritic surface insertion of GluK2-containing KARs in rat hippocampal neurons. PMID:24895134

The Host Cell Receptors for Measles Virus and Their Interaction with the Viral Hemagglutinin (H) Protein

PubMed Central

Lin, Liang-Tzung; Richardson, Christopher D.

2016-01-01

The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles “blind” to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces. PMID:27657109

The Host Cell Receptors for Measles Virus and Their Interaction with the Viral Hemagglutinin (H) Protein.

PubMed

Lin, Liang-Tzung; Richardson, Christopher D

2016-09-20

The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles "blind" to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces.

Laser-stimulated desorption of organic molecules from surfaces, as a method of increasing the efficiency of ion mobility spectrometry analysis.

PubMed

Akmalov, Artem E; Chistyakov, Alexander A; Kotkovskii, Gennadii E

2017-08-01

Application of laser-induced desorption was investigated as a method of increasing the efficiency of gas phase analyzers on principles of field asymmetric ion mobility spectrometry. Mass spectrometric data of investigations of laser desorption of pentaerythritoltetranitrate molecules and cyclotetramethylenetetranitramine molecules from quartz substrate under vacuum were obtained. Laser sources a Nd 3+ :YAG with nanosecond pulse duration (λ = 532 nm) and a continuous wave diode laser (λ = 440 nm) were used. It was shown that both laser sources have different desorption abilities. This is expressed in various time of appearance of desorbed products that is caused by different heating mechanisms of surface layer. The desorbed quantity under action of both laser sources exceeds the detection threshold for all modern gas phase analyzers. It should be noted that despite the presence of surface dissociation of explosives under laser radiation, the quantity of nondissociated molecules is large enough for detection by ion mobility and field asymmetric ion mobility spectrometers. The optimal parameters of laser radiation for effective removal (evaporation) molecules of low-volatile compounds from surfaces are defined. The conclusion about preferable use of a Nd 3+ :YAG laser for increasing the detection ability of detectors based on ion mobility spectrometry was made.

Capturing the sensitivity of land-use regression models to short-term mobile monitoring campaigns using air pollution micro-sensors.

PubMed

Minet, L; Gehr, R; Hatzopoulou, M

2017-11-01

The development of reliable measures of exposure to traffic-related air pollution is crucial for the evaluation of the health effects of transportation. Land-use regression (LUR) techniques have been widely used for the development of exposure surfaces, however these surfaces are often highly sensitive to the data collected. With the rise of inexpensive air pollution sensors paired with GPS devices, we witness the emergence of mobile data collection protocols. For the same urban area, can we achieve a 'universal' model irrespective of the number of locations and sampling visits? Can we trade the temporal representation of fixed-point sampling for a larger spatial extent afforded by mobile monitoring? This study highlights the challenges of short-term mobile sampling campaigns in terms of the resulting exposure surfaces. A mobile monitoring campaign was conducted in 2015 in Montreal; nitrogen dioxide (NO 2 ) levels at 1395 road segments were measured under repeated visits. We developed LUR models based on sub-segments, categorized in terms of the number of visits per road segment. We observe that LUR models were highly sensitive to the number of road segments and to the number of visits per road segment. The associated exposure surfaces were also highly dissimilar. Copyright © 2017 Elsevier Ltd. All rights reserved.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

PubMed

Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

2011-07-01

Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Retrograde transport of the transmembrane estrogen receptor, G-protein-coupled-receptor-30 (GPR30/GPER) from the plasma membrane towards the nucleus.

PubMed

Cheng, Shi-Bin; Graeber, Carl T; Quinn, Jeffrey A; Filardo, Edward J

2011-08-01

G-protein-coupled receptor 30 (GPR30/GPER) belongs to the seven transmembrane receptor (7TMR) superfamily, the most common class of surface receptor with approximately 800 known members. GPER promotes estrogen binding and rapid signaling via membrane-associated enzymes resulting in increased cAMP and release of heparan bound epidermal growth factor (proHB-EGF) from breast cancer cells. However, GPER is predominately localized intracellularly in breast cancer cells with minor amounts of receptor on the cell surface, an observation that has caused some controversy regarding its potential role as a plasma membrane estrogen receptor. Using the widely employed approach of tracking recombinant 7TMRs by surface labeling live cells, we have begun to characterize and compare the endocytic fate of GPER to other similarly labeled 7TMRs. Upon ectopic expression in human embryonic kidney HEK-293 cells, functional GPER is generated as these cells acquire the capacity to stimulate cAMP and activate cyclic AMP responsive binding protein in response to estradiol-17 beta stimulation. GPER is detectable on the cell surface by immunofluorescent analysis using HA-specific antibodies, albeit the bulk of the receptor is located intracellularly. Like β1AR (beta 1 adrenergic receptor) and CXCR4 (C-X-C chemokine receptor 4), GPER exits the plasma membrane via clathrin-coated pits and enters early endosomes. Interestingly, GPER has a destination that is uncommon among 7TMRs, as it accumulates in a perinuclear compartment. Like many 7TMRs (approximately one-third), GPER trafficking from the plasma membrane is constitutive (occurs in the absence of agonist). However, its route of intracellular trafficking is highly unusual, as 7TMRs typically recycle to the plasma membrane (e.g. β1AR) or are degraded in lysosomes (e.g. CXCR4). The accumulation of GPER in the perinuclear space and its possible significance for attenuating estrogen action via this newly recognized membrane estrogen receptor is discussed herein. Published by Elsevier Inc.

Diffusion Barriers, Mechanical Forces, and the Biophysics of Phagocytosis.

PubMed

Ostrowski, Philip P; Grinstein, Sergio; Freeman, Spencer A

2016-07-25

Phagocytes recognize and eliminate pathogens, alert other tissues of impending threats, and provide a link between innate and adaptive immunity. They also maintain tissue homeostasis, consuming dead cells without causing alarm. The receptor engagement, signal transduction, and cytoskeletal rearrangements underlying phagocytosis are paradigmatic of other immune responses and bear similarities to macropinocytosis and cell migration. We discuss how the glycocalyx restricts access to phagocytic receptors, the processes that enable receptor engagement and clustering, and the remodeling of the actin cytoskeleton that controls the mobility of membrane proteins and lipids and provides the mechanical force propelling the phagocyte membrane toward and around the phagocytic prey. Copyright © 2016 Elsevier Inc. All rights reserved.

The impact of mobile phone use on where we look and how we walk when negotiating floor based obstacles.

PubMed

Timmis, Matthew A; Bijl, Herre; Turner, Kieran; Basevitch, Itay; Taylor, Matthew J D; van Paridon, Kjell N

2017-01-01

Pedestrians regularly engage with their mobile phone whilst walking. The current study investigated how mobile phone use affects where people look (visual search behaviour) and how they negotiate a floor based hazard placed along the walking path. Whilst wearing a mobile eye tracker and motion analysis sensors, participants walked up to and negotiated a surface height change whilst writing a text, reading a text, talking on the phone, or without a phone. Differences in gait and visual search behaviour were found when using a mobile phone compared to when not using a phone. Using a phone resulted in looking less frequently and for less time at the surface height change, which led to adaptations in gait by negotiating it in a manner consistent with adopting an increasingly cautious stepping strategy. When using a mobile phone, writing a text whilst walking resulted in the greatest adaptions in gait and visual search behaviour compared to reading a text and talking on a mobile phone. Findings indicate that mobile phone users were able to adapt their visual search behaviour and gait to incorporate mobile phone use in a safe manner when negotiating floor based obstacles.

The impact of mobile phone use on where we look and how we walk when negotiating floor based obstacles

PubMed Central

Bijl, Herre; Turner, Kieran; Basevitch, Itay; Taylor, Matthew J. D.; van Paridon, Kjell N.

2017-01-01

Pedestrians regularly engage with their mobile phone whilst walking. The current study investigated how mobile phone use affects where people look (visual search behaviour) and how they negotiate a floor based hazard placed along the walking path. Whilst wearing a mobile eye tracker and motion analysis sensors, participants walked up to and negotiated a surface height change whilst writing a text, reading a text, talking on the phone, or without a phone. Differences in gait and visual search behaviour were found when using a mobile phone compared to when not using a phone. Using a phone resulted in looking less frequently and for less time at the surface height change, which led to adaptations in gait by negotiating it in a manner consistent with adopting an increasingly cautious stepping strategy. When using a mobile phone, writing a text whilst walking resulted in the greatest adaptions in gait and visual search behaviour compared to reading a text and talking on a mobile phone. Findings indicate that mobile phone users were able to adapt their visual search behaviour and gait to incorporate mobile phone use in a safe manner when negotiating floor based obstacles. PMID:28665942

Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion

PubMed Central

Liu, Li; Li, Chang-jun; Lu, Yun; Zong, Xian-gang; Luo, Chao; Sun, Jun; Guo, Lian-jun

2015-01-01

GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy, and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3β and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit, and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage. PMID:26412641

Influence of traditional agricultural practices on mobilization of arsenic from sediments to groundwater in Bengal delta.

PubMed

Farooq, S H; Chandrasekharam, D; Berner, Z; Norra, S; Stüben, D

2010-11-01

In the wake of the idea that surface derived dissolved organic carbon (DOC) plays an important role in the mobilization of arsenic (As) from sediments to groundwater and may provide a vital tool in understanding the mechanism of As contamination (mobilization/fixation) in Bengal delta; a study has been carried out. Agricultural fields that mainly cultivate rice (paddy fields) leave significantly large quantities of organic matter/organic carbon on the surface of Bengal delta which during monsoon starts decomposing and produces DOC. The DOC thus produced percolates down with rain water and mobilizes As from the sediments. Investigations on sediment samples collected from a paddy field clearly indicate that As coming on to the surface along with the irrigation water accumulates itself in the top few meters of sediment profile. The column experiments carried out on a 9 m deep sediment profile demonstrates that DOC has a strong potential to mobilize As from the paddy fields and the water recharging the aquifer through such agricultural fields contain As well above the WHO limit thus contaminating the shallow groundwater. Experiment also demonstrates that decay of organic matter induces reducing condition in the sediments. Progressively increasing reducing conditions not only prevent the adsorption of As on mineral surfaces but also cause mobilization of previously sorbed arsenic. There seems to be a cyclic pattern where As from deeper levels comes to the surface with irrigational water, accumulates itself in the sediments, and ultimately moves down to the shallow groundwater. The extensive and continual exploitation of intermediate/deep groundwater accelerates this cyclic process and helps in the movement of shallow contaminated groundwater to the deeper levels. Copyright © 2010 Elsevier Ltd. All rights reserved.

Regulation of P2Y1 receptor traffic by sorting Nexin 1 is retromer independent.

PubMed

Nisar, Shaista; Kelly, Eamonn; Cullen, Pete J; Mundell, Stuart J

2010-04-01

The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

30 CFR 77.403-1 - Mobile equipment; rollover protective structures (ROPS).

Code of Federal Regulations, 2010 CFR

2010-07-01

... WORK AREAS OF UNDERGROUND COAL MINES Safeguards for Mechanical Equipment § 77.403-1 Mobile equipment... surface coal mines or the surface work areas of underground coal mines shall be provided with rollover... complying with paragraph (d) (1) (iii) (A) of this section. Stresses shall not exceed the ultimate strength...

30 CFR 77.403-1 - Mobile equipment; rollover protective structures (ROPS).

Code of Federal Regulations, 2011 CFR

2011-07-01

... WORK AREAS OF UNDERGROUND COAL MINES Safeguards for Mechanical Equipment § 77.403-1 Mobile equipment... surface coal mines or the surface work areas of underground coal mines shall be provided with rollover... complying with paragraph (d) (1) (iii) (A) of this section. Stresses shall not exceed the ultimate strength...

KENNEDY SPACE CENTER, FLA. - A worker sandblasts the surface behind the Mobile Launcher Platform on Launch Pad 39A . Routine maintenance includes sandblasting and repainting as preventive means to minimize corrosion.

NASA Image and Video Library

2003-09-12

KENNEDY SPACE CENTER, FLA. - A worker sandblasts the surface behind the Mobile Launcher Platform on Launch Pad 39A . Routine maintenance includes sandblasting and repainting as preventive means to minimize corrosion.

Lateral diffusion and signaling of receptor for advanced glycation end-products (RAGE): a receptor involved in chronic inflammation.

PubMed

Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

2018-01-01

Membrane diffusion is one of the key mechanisms in the cellular function of receptors. The signaling of receptors for advanced glycation end-products (RAGE) has been extensively studied in the context of several pathological conditions, however, very little is known about RAGE diffusion. To fill this gap, RAGE lateral diffusion is probed in native, cholesterol-depleted, and cytoskeleton-altered cellular conditions. In native GM07373 cellular conditions, RAGE has a 90% mobile fraction and an average diffusion coefficient of 0.3 μm 2 /s. When depolymerization of the actin cytoskeleton is inhibited with the small molecule jasplakinolide (Jsp), the RAGE mobile fraction and diffusion coefficient decrease by 22 and 37%, respectively. In contrast, depolymerizing the filamentous actin cytoskeleton using the small molecule cytochalasin D (CD) does not alter the RAGE diffusion properties. There is a 70 and 50% decrease in phosphorylation of extracellular signal-regulated kinase (p-ERK) when the actin cytoskeleton is disrupted by CD or Jsp, respectively, in RAGE-expressing GM07373 cells. Disrupting the actin cytoskeleton in GM07373 cells that do not express detectable amounts of RAGE results in no change in p-ERK. Cholesterol depletion results in no statistically significant change in the diffusion properties of RAGE or p-ERK. This work presents a strong link between the actin cytoskeleton and RAGE diffusion and downstream signaling, and serves to further our understanding of the factors influencing RAGE lateral diffusion.

Stress Proteins and Initiation of Immune Response: Chaperokine activity of Hsp72

PubMed Central

Asea, Alexzander

2006-01-01

From its original description as solely an intracellular molecular chaperone, heat shock proteins have now been shown to function as initiators of the host's immune response. Although the exact mechanism by which intracellular heat shock proteins leave cells is still incompletely understood, recent work from several labs suggest that heat shock proteins are released by both passive (necrotic) and active (physiological) mechanisms. Binding to specific surface receptors is a prerequisite for the initiation of an immune response. To date, several cell surface proteins have been described as the receptor for seventy kilo-Dalton heat shock protein (Hsp70) including Toll-like receptors 2 and 4 with their cofactor CD14, the scavenger receptor CD36, the low-density lipoprotein receptor-related protein CD91, the C-type lectin receptor LOX-1, and another member of the scavenger super-family SR-A plus the co-stimulatory molecule, CD40. Binding of Hsp70 to these surface receptors specifically activates intracellular signaling cascades, which in turn exert immunoregulatory effector functions; a process known as the chaperokine activity of Hsp70. This review will highlight recent advances in understanding the mechanism by which Hsp70 initiates the host's immune response. PMID:16385842

Stress proteins and initiation of immune response: chaperokine activity of hsp72.

PubMed

Asea, Alexzander

2005-01-01

From its original description as solely an intracellular molecular chaperone, heat shock proteins have now been shown to function as initiators of the host's immune response. Although the exact mechanism by which intracellular heat shock proteins leave cells is still incompletely understood, recent work from several labs suggest that heat shock proteins are released by both passive (necrotic) and active (physiological) mechanisms. Binding to specific surface receptors is a prerequisite for the initiation of an immune response. To date, several cell surface proteins have been described as the receptor for seventy kilo-Dalton heat shock protein (Hsp70) including Toll-like receptors 2 and 4 with their cofactor CD14, the scavenger receptor CD36, the low-density lipoprotein receptor-related protein CD91, the C-type lectin receptor LOX-1, and another member of the scavenger super-family SR-A plus the co-stimulatory molecule, CD40. Binding of Hsp70 to these surface receptors specifically activates intracellular signaling cascades, which in turn exert immunoregulatory effector functions; a process known as the chaperokine activity of Hsp70. This review will highlight recent advances in understanding the mechanism by which Hsp70 initiates the host's immune response.

Observation of Wigner crystal phase and ripplon-limited mobility behavior in monolayer CVD MoS2 with grain boundary.

PubMed

Chen, Jyun-Hong; Zhong, Yuan-Liang; Li, Lain-Jong; Chen, Chii-Dong

2018-06-01

Two-dimensional electron gas (2DEG) is crucial in condensed matter physics and is present on the surface of liquid helium and at the interface of semiconductors. Monolayer MoS 2 of 2D materials also contains 2DEG in an atomic layer as a field effect transistor (FET) ultrathin channel. In this study, we synthesized double triangular MoS 2 through a chemical vapor deposition method to obtain grain boundaries for forming a ripple structure in the FET channel. When the temperature was higher than approximately 175 K, the temperature dependence of the electron mobility μ was consistent with those in previous experiments and theoretical predictions. When the temperature was lower than approximately 175 K, the mobility behavior decreased with the temperature; this finding was also consistent with that of the previous experiments. We are the first research group to explain the decreasing mobility behavior by using the Wigner crystal phase and to discover the temperature independence of ripplon-limited mobility behavior at lower temperatures. Although these mobility behaviors have been studied on the surface of liquid helium through theories and experiments, they have not been previously analyzed in 2D materials and semiconductors. We are the first research group to report the similar temperature-dependent mobility behavior of the surface of liquid helium and the monolayer MoS 2 .

Observation of Wigner crystal phase and ripplon-limited mobility behavior in monolayer CVD MoS2 with grain boundary

NASA Astrophysics Data System (ADS)

Chen, Jyun-Hong; Zhong, Yuan-Liang; Li, Lain-Jong; Chen, Chii-Dong

2018-06-01

Two-dimensional electron gas (2DEG) is crucial in condensed matter physics and is present on the surface of liquid helium and at the interface of semiconductors. Monolayer MoS2 of 2D materials also contains 2DEG in an atomic layer as a field effect transistor (FET) ultrathin channel. In this study, we synthesized double triangular MoS2 through a chemical vapor deposition method to obtain grain boundaries for forming a ripple structure in the FET channel. When the temperature was higher than approximately 175 K, the temperature dependence of the electron mobility μ was consistent with those in previous experiments and theoretical predictions. When the temperature was lower than approximately 175 K, the mobility behavior decreased with the temperature; this finding was also consistent with that of the previous experiments. We are the first research group to explain the decreasing mobility behavior by using the Wigner crystal phase and to discover the temperature independence of ripplon-limited mobility behavior at lower temperatures. Although these mobility behaviors have been studied on the surface of liquid helium through theories and experiments, they have not been previously analyzed in 2D materials and semiconductors. We are the first research group to report the similar temperature-dependent mobility behavior of the surface of liquid helium and the monolayer MoS2.

Surface Soil Preparetion for Leguminous Plants Growing in Degraded Areas by Mining Located in Amazon Forest-Brazil

NASA Astrophysics Data System (ADS)

Irio Ribeiro, Admilson; Hashimoto Fengler, Felipe; Araújo de Medeiros, Gerson; Márcia Longo, Regina; Frederici de Mello, Giovanna; José de Melo, Wanderley

2015-04-01

The revegetation of areas degraded by mining usually requires adequate mobilization of surface soil for the development of the species to be implemented. Unlike the traditional tillage, which has periodicity, the mobilization of degraded areas for revegetation can only occur at the beginning of the recovery stage. In this sense, the process of revegetation has as purpose the establishment of local native vegetation with least possible use of inputs and superficial tillage in order to catalyze the process of natural ecological succession, promoting the reintegration of areas and minimizing the negative impacts of mining activities in environmental. In this context, this work describes part of a study of land reclamation by tin exploitation in the Amazon ecosystem in the National Forest Jamari- Rondonia Brazil. So, studied the influence of surface soil mobilization in pit mine areas and tailings a view to the implementation of legumes. The results show that the surface has areas of mobilizing a significant effect on the growth of leguminous plants, areas for both mining and to tailings and pit mine areas.

Truncated Lévy flights and agenda-based mobility are useful for the assessment of personal human exposure.

PubMed

Schlink, Uwe; Ragas, Ad M J

2011-01-01

Receptor-oriented approaches can assess the individual-specific exposure to air pollution. In such an individual-based model we analyse the impact of human mobility to the personal exposure that is perceived by individuals simulated in an exemplified urban area. The mobility models comprise random walk (reference point mobility, RPM), truncated Lévy flights (TLF), and agenda-based walk (RPMA). We describe and review the general concepts and provide an inter-comparison of these concepts. Stationary and ergodic behaviour are explained and applied as well as performance criteria for a comparative evaluation of the investigated algorithms. We find that none of the studied algorithm results in purely random trajectories. TLF and RPMA prove to be suitable for human mobility modelling, because they provide conditions for very individual-specific trajectories and exposure. Suggesting these models we demonstrate the plausibility of their results for exposure to air-borne benzene and the combined exposure to benzene and nonane. Copyright © 2011 Elsevier Ltd. All rights reserved.

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Flow-through nanohole array based sensor implemented on analogue smartphone components

NASA Astrophysics Data System (ADS)

Gomez-Cruz, Juan; Nair, Srijit; Ascanio, Gabriel; Escobedo, Carlos

2017-08-01

Mobile communications have massively populated the consumer electronics market over the past few years and it is now ubiquitous, providing a timeless opportunity for the development of smartphone-based technologies as point-of-care (POC) diagnosis tools1 . The expectation for a fully integrated smartphone-based sensor that enables applications such as environmental monitoring, explosive detection and biomedical analysis has increased among the scientific community in the past few years2,3. The commercialization forecast for smartphone-based sensing technologies is very promising, but reliable, miniature and cost-effective sensing platforms that can adapt to portable electronics in still under development. In this work, we present an integrated sensing platform based on flow-through metallic nanohole arrays. The nanohole arrays are 260 nm in diameter and 520 nm in pitch, fabricated using Focused Ion Beam (FIB) lithography. A white LED resembling a smartphone flash LED serves as light source to excite surface plasmons and the signal is recorded via a Complementary Metal-Oxide-Semiconductor (CMOS) module. The sensing abilities of the integrated sensing platform is demonstrated for the detection of (i) changes in bulk refractive index (RI), (ii) real-time monitoring of surface modification by receptor-analyte system of streptavidin-biotin.

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Yuting; Zhou, Lubing; Gunnet, Joseph W.

2006-06-23

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A{sub 2} (PLA{sub 2})/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeuticmore » effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca{sup 2+}-mobilization and enhanced bradykinin-promoted Ca{sup 2+}-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPAR{gamma} agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.« less

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, T.; Ui, M.

1985-06-25

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

Multi-Purpose Avionic Architecture for Vision Based Navigation Systems for EDL and Surface Mobility Scenarios

NASA Astrophysics Data System (ADS)

Tramutola, A.; Paltro, D.; Cabalo Perucha, M. P.; Paar, G.; Steiner, J.; Barrio, A. M.

2015-09-01

Vision Based Navigation (VBNAV) has been identified as a valid technology to support space exploration because it can improve autonomy and safety of space missions. Several mission scenarios can benefit from the VBNAV: Rendezvous & Docking, Fly-Bys, Interplanetary cruise, Entry Descent and Landing (EDL) and Planetary Surface exploration. For some of them VBNAV can improve the accuracy in state estimation as additional relative navigation sensor or as absolute navigation sensor. For some others, like surface mobility and terrain exploration for path identification and planning, VBNAV is mandatory. This paper presents the general avionic architecture of a Vision Based System as defined in the frame of the ESA R&T study “Multi-purpose Vision-based Navigation System Engineering Model - part 1 (VisNav-EM-1)” with special focus on the surface mobility application.

Co-Requirement of PICK1 Binding and PKC Phosphorylation for Stable Surface Expression of the Metabotropic Glutamate Receptor mGluR7

PubMed Central

Suh, Young Ho; Pelkey, Kenneth A.; Lavezzari, Gabriela; Roche, Paul A.; Huganir, Richard L.; McBain, Chris J.; Roche, Katherine W.

2008-01-01

SUMMARY The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release, and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a novel form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo. PMID:18549785

Rearrangement and expression of the human {Psi}C{lambda}6 gene segment results in a surface Ig receptor with a truncated light chain constant region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiernholm, N.B.J.; Verkoczy, L.K.; Berinstein, N.L.

1995-05-01

The constant region of the human Ig{lambda} locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C{lambda}1, J-C{lambda}2, J-C{lambda}3, and J-C{lambda}7 gene segments are functional, and code for the four distinct Ig{lambda} isotypes found in human serum, the J-C{lambda}4, J-C{lambda}5, and J-C{lambda}6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C{lambda}6 gene segment has been documented, in the majority of cases, J-C{lambda}6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C{lambda}6more » gene segment do occur, and that such a rearrangement encodes an Ig{lambda} protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg{triangle}CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig{lambda} L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg{triangle}CL receptors, suggesting that the latter could possibly function as an Ag receptor. 35 refs., 7 figs.« less

The influence of hydrodynamic slip on the electrophoretic mobility of a spherical colloidal particle

NASA Astrophysics Data System (ADS)

Khair, Aditya S.; Squires, Todd M.

2009-04-01

Recent theoretical studies have suggested a significant enhancement in electro-osmotic flows over hydrodynamically slipping surfaces, and experiments have indeed measured O(1) enhancements. In this paper, we investigate whether an equivalent effect occurs in the electrophoretic motion of a colloidal particle whose surface exhibits hydrodynamic slip. To this end, we compute the electrophoretic mobility of a uniformly charged spherical particle with slip length λ as a function of the zeta (or surface) potential of the particle ζ and diffuse-layer thickness κ-1. In the case of a thick diffuse layer, κa ≪1 (where a is the particle size), simple arguments show that slip does lead to an O(1) enhancement in the mobility, owing to the reduced viscous drag on the particle. On the other hand, for a thin-diffuse layer κa ≫1, the situation is more complicated. A detailed asymptotic analysis, following the method of O'Brien [J. Colloid Interface Sci. 92, 204 (1983)], reveals that an O(κλ) increase in the mobility occurs at low-to-moderate zeta potentials (with ζ measured on the scale of thermal voltage kBT /e≈25 mV). However, as ζ is further increased, the mobility decreases and ultimately becomes independent of the slip length—the enhancement is lost—which is due to the importance of nonuniform surface conduction within the thin-diffuse layer, at large ζ and large, but finite, κa. Our asymptotic calculations for thick and thin-diffuse layers are corroborated and bridged by computation of the mobility from the numerical solution of the full electrokinetic equations (using the method of O'Brien and White [J. Chem. Soc., Faraday Trans. 2 74, 1607 (1978)]). In summary, then, we demonstrate that hydrodynamic slip can indeed produce an enhancement in the electrophoretic mobility; however, such enhancements will not be as dramatic as the previously studied κa →∞ limit would suggest. Importantly, this conclusion applies not only to electrophoresis but also to electro-osmosis over highly charged surfaces, wherein any inhomogeneities (e.g., due to curvature, roughness, charge patterning, or a variation in slip length) will drive nonuniform surface conduction, which prevents the significant slip-driven flow enhancements predicted for a uniform highly charged surface.

Chemical Modification of the Olfactory Receptor Epithelium of Vertebrate Species

DTIC Science & Technology

1990-06-28

Pre-column Derivatization Procedure: 1.0 mL of the Jeffamine solution was mixed with 1.0 mL of NaCN, 5.0 mL of phosphate buffer pH 9.5 followed by 1.0...running buffer. All the unprotonated components elute at the same time because their rate of elution is controlled only by the rate of electroosmotic ...elecarosomotic mobility under our experimental conditions. Using an average elution time of 22.2 min the measured electroosmotic mobility is 1.3 x 10-4 cm2

Insulin release: the receptor hypothesis.

PubMed

Malaisse, Willy J

2014-07-01

It is currently believed that the stimulation of insulin release by nutrient secretagogues reflects their capacity to act as fuel in pancreatic islet beta cells. In this review, it is proposed that such a fuel concept is not incompatible with a receptor hypothesis postulating the participation of cell-surface receptors in the recognition of selected nutrients as insulinotropic agents. Pursuant to this, attention is drawn to such matters as the anomeric specificity of the beta cell secretory response to D-glucose and its perturbation in diabetes mellitus, the insulinotropic action of artificial sweeteners, the possible role of bitter taste receptors in the stimulation of insulin secretion by L-glucose pentaacetate, the recently documented presence of cell-surface sweet taste receptors in insulin-producing cells, the multimodal signalling process resulting from the activation of these latter receptors, and the presence in beta cells of a sweet taste receptor mediating the fructose-induced potentiation of glucose-stimulated insulin secretion.

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenmark, P.; Dupuy, J.; Inamura, A.

2009-05-26

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in themore » toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez-Miranda, Elizabeth; Ibarra-Sanchez, Alfredo; Gonzalez-Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx

IgE-antigen-dependent crosslinking of the high affinity IgE receptor (Fc{epsilon}RI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca{sup 2+}) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls Fc{epsilon}RI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed Fc{epsilon}RI-dependent Ca{sup 2+} mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn -/- knock out mice. Fyn -/- BMMCs showed a markedmore » defect in extracellular Ca{sup 2+} influx after Fc{epsilon}RI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd{sup 3+}) partially blocked Fc{epsilon}RI-induced Ca{sup 2+} influx in WT cells but, in contrast, completely inhibited Ca{sup 2+} mobilization in Fyn -/- cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca{sup 2+} channels (2-aminoethoxyphenyl-borane, 2-APB) blocked Fc{epsilon}RI-induced maximal Ca{sup 2+} rise in WT but not in Fyn -/- cells. Ca{sup 2+} entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in Fc{epsilon}RI-stimulated mast cells.« less

Suppression of receptor-mediated Ca2+ mobilization and functional leukocyte responses by hyperforin.

PubMed

Feisst, Christian; Werz, Oliver

2004-04-15

We have recently identified hyperforin, a lipophilic constituent of the herb Hypericum perforatum (St. John's wort), as a dual inhibitor of the proinflammatory enzymes cyclooxygenase-1 and 5-lipoxygenase. The aim of the present study was to further elucidate antiinflammatory properties and respective targets of hyperforin. We found that hyperforin inhibited the generation of reactive oxygen species (ROS) as well as the release of leukocyte elastase (degranulation) in human isolated polymorphonuclear leukocytes (PMNL), challenged by the G protein-coupled receptor (GPCR) ligand N-formyl-methionyl-leucyl-phenylalanine (fMLP) with an IC 50 approximately equal 0.3 microM. When PMNL were stimulated with phorbol-12-myristate-13-acetate (PMA) or ionomycin, hyperforin (up to 10 microM) failed to inhibit ROS production and elastase release, respectively. Moreover, hyperforin blocked receptor-mediated Ca(2+) mobilization ( IC 50 approximately equal 0.4 and 4 microM, respectively) in PMNL and monocytic cells, and caused a rapid decline of the intracellular Ca(2+) concentration in resting cells. In contrast, the Ca(2+) influx induced by ionomycin or thapsigargin was not suppressed. Comparative studies with the specific phospholipase C inhibitor U-73122 and hyperforin revealed similarities between both compounds. Thus, U-73122 and hyperforin blocked fMLP- and PAF-induced Ca(2+) mobilization, ROS formation, and elastase release, but failed to suppress these responses when cells were stimulated by PMA or ionomycin. Also, both compounds rapidly decreased basal Ca(2+) levels in resting cells and led to a rapid decline of the Ca(2+) elevations evoked by fMLP or PAF. Our data suggest that hyperforin targets component(s) within G protein signaling cascades that regulate Ca(2+) homeostasis, coupled to proinflammatory leukocyte functions.

Cloning and Characterization of the Mouse Hepatitis Virus Receptor

DTIC Science & Technology

1991-02-11

materials. Viruses may also adhere to cell surfaces non-specifically through electrostatic interactions (Tardieu et al., 1982). Virus particles might be... viruses can utilize more than one type of receptor and that specific virus receptors may be present in low numbers on the cell surface or may be labile...known example of this type of interaction is the enhancement of virus infection by antibodies, which has been demonstrated for several viruses

Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

PubMed Central

Monine, Michael I.; Posner, Richard G.; Savage, Paul B.; Faeder, James R.; Hlavacek, William S.

2010-01-01

Abstract We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-FcεRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important. PMID:20085718

Arsenic mobilization in shallow aquifers due to CO 2 intrusion from storage reservoirs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Ting; Dai, Zhenxue; Viswanathan, Hari S.

We developed an integrated framework of combined batch experiments and reactive transport simulations to quantify water-rock-CO 2 interactions and arsenic (As) mobilization responses to CO 2 and/or saline water leakage into USDWs. Experimental and simulation results suggest that when CO 2 is introduced, pH drops immediately that initiates release of As from clay minerals. Calcite dissolution can increase pH slightly and cause As re-adsorption. Thus, the mineralogy of the USDW is ultimately a determining factor of arsenic fate and transport. Salient results suggest that: (1) As desorption/adsorption from/onto clay minerals is the major reaction controlling its mobilization, and clay mineralsmore » could mitigate As mobilization with surface complexation reactions; (2) dissolution of available calcite plays a critical role in buffering pH; (3) high salinity in general hinders As release from minerals; and (4) the magnitude and quantitative uncertainty of As mobilization are predicated on the values of reaction rates and surface area of calcite, adsorption surface areas and equilibrium constants of clay minerals, and cation exchange capacity. Results of this study are intended to improve ability to quantify risks associated with potential leakage of reservoir fluids into shallow aquifers, in particular the possible environmental impacts of As mobilization at carbon sequestration sites.« less

A dragline-forming mobile robot inspired by spiders.

PubMed

Wang, Liyu; Culha, Utku; Iida, Fumiya

2014-03-01

Mobility of wheeled or legged machines can be significantly increased if they are able to move from a solid surface into a three-dimensional space. Although that may be achieved by addition of flying mechanisms, the payload fraction will be the limiting factor in such hybrid mobile machines for many applications. Inspired by spiders producing draglines to assist locomotion, the paper proposes an alternative mobile technology where a robot achieves locomotion from a solid surface into a free space. The technology resembles the dragline production pathway in spiders to a technically feasible degree and enables robots to move with thermoplastic spinning of draglines. As an implementation, a mobile robot has been prototyped with thermoplastic adhesives as source material of the draglines. Experimental results show that a dragline diameter range of 1.17-5.27 mm was achievable by the 185 g mobile robot in descending locomotion from the solid surface of a hanging structure with a power consumption of 4.8 W and an average speed of 5.13 cm min(-1). With an open-loop controller consisting of sequences of discrete events, the robot has demonstrated repeatable dragline formation with a relative deviation within -4% and a length close to the metre scale.

Arsenic mobilization in shallow aquifers due to CO 2 intrusion from storage reservoirs

DOE PAGES

Xiao, Ting; Dai, Zhenxue; Viswanathan, Hari S.; ...

2017-06-05

We developed an integrated framework of combined batch experiments and reactive transport simulations to quantify water-rock-CO 2 interactions and arsenic (As) mobilization responses to CO 2 and/or saline water leakage into USDWs. Experimental and simulation results suggest that when CO 2 is introduced, pH drops immediately that initiates release of As from clay minerals. Calcite dissolution can increase pH slightly and cause As re-adsorption. Thus, the mineralogy of the USDW is ultimately a determining factor of arsenic fate and transport. Salient results suggest that: (1) As desorption/adsorption from/onto clay minerals is the major reaction controlling its mobilization, and clay mineralsmore » could mitigate As mobilization with surface complexation reactions; (2) dissolution of available calcite plays a critical role in buffering pH; (3) high salinity in general hinders As release from minerals; and (4) the magnitude and quantitative uncertainty of As mobilization are predicated on the values of reaction rates and surface area of calcite, adsorption surface areas and equilibrium constants of clay minerals, and cation exchange capacity. Results of this study are intended to improve ability to quantify risks associated with potential leakage of reservoir fluids into shallow aquifers, in particular the possible environmental impacts of As mobilization at carbon sequestration sites.« less

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

PubMed Central

Tsukaguchi, H; Matsubara, H; Taketani, S; Mori, Y; Seido, T; Inada, M

1995-01-01

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder. Images PMID:7560098

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

P17, an Original Host Defense Peptide from Ant Venom, Promotes Antifungal Activities of Macrophages through the Induction of C-Type Lectin Receptors Dependent on LTB4-Mediated PPARγ Activation.

PubMed

Benmoussa, Khaddouj; Authier, Hélène; Prat, Mélissa; AlaEddine, Mohammad; Lefèvre, Lise; Rahabi, Mouna Chirine; Bernad, José; Aubouy, Agnès; Bonnafé, Elsa; Leprince, Jérome; Pipy, Bernard; Treilhou, Michel; Coste, Agnès

2017-01-01

Despite the growing knowledge with regard to the immunomodulatory properties of host defense peptides, their impact on macrophage differentiation and on its associated microbicidal functions is still poorly understood. Here, we demonstrated that the P17, a new cationic antimicrobial peptide from ant venom, induces an alternative phenotype of human monocyte-derived macrophages (h-MDMs). This phenotype is characterized by a C-type lectin receptors (CLRs) signature composed of mannose receptor (MR) and Dectin-1 expression. Concomitantly, this activation is associated to an inflammatory profile characterized by reactive oxygen species (ROS), interleukin (IL)-1β, and TNF-α release. P17-activated h-MDMs exhibit an improved capacity to recognize and to engulf Candida albicans through the overexpression both of MR and Dectin-1. This upregulation requires arachidonic acid (AA) mobilization and the activation of peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor through the leukotriene B4 (LTB4) production. AA/LTB4/PPARγ/Dectin-1-MR signaling pathway is crucial for P17-mediated anti-fungal activity of h-MDMs, as indicated by the fact that the activation of this axis by P17 triggered ROS production and inflammasome-dependent IL-1β release. Moreover, we showed that the increased anti-fungal immune response of h-MDMs by P17 was dependent on intracellular calcium mobilization triggered by the interaction of P17 with pertussis toxin-sensitive G-protein-coupled receptors on h-MDMs. Finally, we also demonstrated that P17-treated mice infected with C. albicans develop less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf Candida , to produce ROS and IL-1β and to kill the yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts upstream PPARγ/CLRs axis and offer new immunomodulatory therapeutic perspectives in the field of infectious diseases.

The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells.

PubMed

Ariazi, Eric A; Brailoiu, Eugen; Yerrum, Smitha; Shupp, Heather A; Slifker, Michael J; Cunliffe, Heather E; Black, Michael A; Donato, Anne L; Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R; Dun, Nae J; Jordan, V Craig

2010-02-01

The G protein-coupled receptor GPR30 binds 17beta-estradiol (E(2)) yet differs from classic estrogen receptors (ERalpha and ERbeta). GPR30 can mediate E(2)-induced nongenomic signaling, but its role in ERalpha-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERalpha-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E(2) and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca(2+) mobilization studies, GPR30, but not ERalpha, mediated E(2)-induced Ca(2+) responses because E(2), 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca(2+) increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E(2)-induced and G-1-induced Ca(2+) mobilization, but ERalpha depletion did not. Interestingly, GPR30-coupled Ca(2+) responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1) phase. Thus, GPR30 antagonizes growth of ERalpha-positive breast cancer and may represent a new target to combat this disease.

P17, an Original Host Defense Peptide from Ant Venom, Promotes Antifungal Activities of Macrophages through the Induction of C-Type Lectin Receptors Dependent on LTB4-Mediated PPARγ Activation

PubMed Central

Benmoussa, Khaddouj; Authier, Hélène; Prat, Mélissa; AlaEddine, Mohammad; Lefèvre, Lise; Rahabi, Mouna Chirine; Bernad, José; Aubouy, Agnès; Bonnafé, Elsa; Leprince, Jérome; Pipy, Bernard; Treilhou, Michel; Coste, Agnès

2017-01-01

Despite the growing knowledge with regard to the immunomodulatory properties of host defense peptides, their impact on macrophage differentiation and on its associated microbicidal functions is still poorly understood. Here, we demonstrated that the P17, a new cationic antimicrobial peptide from ant venom, induces an alternative phenotype of human monocyte-derived macrophages (h-MDMs). This phenotype is characterized by a C-type lectin receptors (CLRs) signature composed of mannose receptor (MR) and Dectin-1 expression. Concomitantly, this activation is associated to an inflammatory profile characterized by reactive oxygen species (ROS), interleukin (IL)-1β, and TNF-α release. P17-activated h-MDMs exhibit an improved capacity to recognize and to engulf Candida albicans through the overexpression both of MR and Dectin-1. This upregulation requires arachidonic acid (AA) mobilization and the activation of peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor through the leukotriene B4 (LTB4) production. AA/LTB4/PPARγ/Dectin-1-MR signaling pathway is crucial for P17-mediated anti-fungal activity of h-MDMs, as indicated by the fact that the activation of this axis by P17 triggered ROS production and inflammasome-dependent IL-1β release. Moreover, we showed that the increased anti-fungal immune response of h-MDMs by P17 was dependent on intracellular calcium mobilization triggered by the interaction of P17 with pertussis toxin-sensitive G-protein-coupled receptors on h-MDMs. Finally, we also demonstrated that P17-treated mice infected with C. albicans develop less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf Candida, to produce ROS and IL-1β and to kill the yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts upstream PPARγ/CLRs axis and offer new immunomodulatory therapeutic perspectives in the field of infectious diseases. PMID:29250064

MobileFusion: real-time volumetric surface reconstruction and dense tracking on mobile phones.

PubMed

Ondrúška, Peter; Kohli, Pushmeet; Izadi, Shahram

2015-11-01

We present the first pipeline for real-time volumetric surface reconstruction and dense 6DoF camera tracking running purely on standard, off-the-shelf mobile phones. Using only the embedded RGB camera, our system allows users to scan objects of varying shape, size, and appearance in seconds, with real-time feedback during the capture process. Unlike existing state of the art methods, which produce only point-based 3D models on the phone, or require cloud-based processing, our hybrid GPU/CPU pipeline is unique in that it creates a connected 3D surface model directly on the device at 25Hz. In each frame, we perform dense 6DoF tracking, which continuously registers the RGB input to the incrementally built 3D model, minimizing a noise aware photoconsistency error metric. This is followed by efficient key-frame selection, and dense per-frame stereo matching. These depth maps are fused volumetrically using a method akin to KinectFusion, producing compelling surface models. For each frame, the implicit surface is extracted for live user feedback and pose estimation. We demonstrate scans of a variety of objects, and compare to a Kinect-based baseline, showing on average ∼ 1.5cm error. We qualitatively compare to a state of the art point-based mobile phone method, demonstrating an order of magnitude faster scanning times, and fully connected surface models.

Olfactory receptor Olfr544 responding to azelaic acid regulates glucagon secretion in α-cells of mouse pancreatic islets.

PubMed

Kang, NaNa; Bahk, Young Yil; Lee, NaHye; Jae, YoonGyu; Cho, Yoon Hee; Ku, Cheol Ryong; Byun, Youngjoo; Lee, Eun Jig; Kim, Min-Soo; Koo, JaeHyung

2015-05-08

Olfactory receptors (ORs) are extensively expressed in olfactory as well as non-olfactory tissues. Although many OR transcripts are expressed in non-olfactory tissues, only a few studies demonstrate the functional role of ORs. Here, we verified that mouse pancreatic α-cells express potential OR-mediated downstream effectors. Moreover, high levels of mRNA for the olfactory receptors Olfr543, Olfr544, Olfr545, and Olfr1349 were expressed in α-cells as assessed using RNA-sequencing, microarray, and quantitative real-time RT-PCR analyses. Treatment with dicarboxylic acids (azelaic acid and sebacic acid) increased intracellular Ca(2+) mobilization in pancreatic α-cells. The azelaic acid-induced Ca(2+) response as well as glucagon secretion was concentration- and time-dependent manner. Olfr544 was expressed in α-cells, and the EC50 value of azelaic acid to Olfr544 was 19.97 μM, whereas Olfr545 did not respond to azelaic acid. Our findings demonstrate that Olfr544 responds to azelaic acid to regulate glucagon secretion through Ca(2+) mobilization in α-cells of the mouse pancreatic islets, suggesting that Olfr544 may be an important therapeutic target for metabolic diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Anti-high mobility group box-1 antibody therapy for traumatic brain injury.

PubMed

Okuma, Yu; Liu, Keyue; Wake, Hidenori; Zhang, Jiyong; Maruo, Tomoko; Date, Isao; Yoshino, Tadashi; Ohtsuka, Aiji; Otani, Naoki; Tomura, Satoshi; Shima, Katsuji; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Takahashi, Hideo K; Mori, Shuji; Nishibori, Masahiro

2012-09-01

High mobility group box-1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti-HMGB1 monoclonal antibody (mAb) to attenuate brain injury. Traumatic brain injury was induced in rats or mice by fluid percussion. Anti-HMGB1 mAb or control mAb was administered intravenously after TBI. Anti-HMGB1 mAb remarkably inhibited fluid percussion-induced brain edema in rats, as detected by T2-weighted magnetic resonance imaging; this was associated with inhibition of HMGB1 translocation, protection of blood-brain barrier (BBB) integrity, suppression of inflammatory molecule expression, and improvement of motor function. In contrast, intravenous injection of recombinant HMGB1 dose-dependently produced the opposite effects. Experiments using receptor for advanced glycation end product (RAGE)(-/-) , toll-like receptor-4 (TLR4)(-/-) , and TLR2(-/-) mice suggested the involvement of RAGE as the predominant receptor for HMGB1. Anti-HMGB1 mAb may provide a novel and effective therapy for TBI by protecting against BBB disruption and reducing the inflammatory responses induced by HMGB1. Copyright © 2012 American Neurological Association.

Autoinflammation Around AES Total Ankle Replacement Implants.

PubMed

Koivu, Helka; Takakubo, Yuya; Mackiewicz, Zygmunt; Al-Samadi, Ahmed; Soininen, Antti; Peled, Nitai; Kukis, Modestas; Trokovic, Nina; Konttinen, Yrjö T

2015-12-01

Failure of total ankle replacement (TAR) can be characterized by early peri-implant osteolysis even in the presence of very modest numbers of wear particles. The hypothesis of the study was that this reaction is in part mediated by autoinflammatory responses mediated via damage-associated molecular patterns (DAMPs, danger signals) and pattern-recognizing danger signal receptors (PRRs). Peri-implant tissue and control samples from 10 patients with AES implants were immunostained for hypoxia inducible factor-1α (HIF-1α), activated caspase-3, high-mobility group box 1 (HMGB1), receptor for advanced glycation end product (RAGE), and toll-like receptors TLR2 and TLR4. Results were evaluated on a 0 to 4 scale (from 0% to >50% stained area). Peri-implant tissue around failed TAR implants had a relatively high mean HIF-1α score of 3 on a scale, which however was similar in control samples. HMGB1 (a DAMP) was seen to be mobilized from nuclei to cellular cytoplasm, and the active caspase-3(+) cells were increased. All PRRs were increased in revision samples. Increased expression of HMGB1 and other danger signals together with increased PRR-dependent responsiveness could contribute to autoinflammatory peri-implantitis, multilocular cyst formation, and osteolysis in failed TAR implants. Level IV, case series. © The Author(s) 2015.

Record surface state mobility and quantum Hall effect in topological insulator thin films via interface engineering

DOE PAGES

Koirala, Nikesh; Han, Myung -Geun; Brahlek, Matthew; ...

2015-11-19

Material defects remain as the main bottleneck to the progress of topological insulators (TIs). In particular, efforts to achieve thin TI samples with dominant surface transport have always led to increased defects and degraded mobilities, thus making it difficult to probe the quantum regime of the topological surface states. Here, by utilizing a novel buffer layer scheme composed of an In 2Se 3/(Bi 0.5In 0.5) 2Se 3 heterostructure, we introduce a quantum generation of Bi 2Se 3 films with an order of magnitude enhanced mobilities than before. Furthermore, this scheme has led to the first observation of the quantum Hallmore » effect in Bi 2Se 3.« less

High Stability Pentacene Transistors Using Polymeric Dielectric Surface Modifier.

PubMed

Wang, Xiaohong; Lin, Guangqing; Li, Peng; Lv, Guoqiang; Qiu, Longzhen; Ding, Yunsheng

2015-08-01

1,6-bis(trichlorosilyl)hexane (C6Cl), polystyrene (PS), and cross-linked polystyrene (CPS) were investigated as gate dielectric modified layers for high performance organic transistors. The influence of the surface energy, roughness and morphology on the charge transport of the organic thin-film transistors (OTFTs) was investigated. The surface energy and roughness both affect the grain size of the pentacene films which will control the charge carrier mobility of the devices. Pentacene thin-film transistors fabricated on the CPS modified dielectric layers exhibited charge carrier mobility as high as 1.11 cm2 V-1 s-1. The bias stress stability for the CPS devices shows that the drain current only decays 1% after 1530 s and the mobility never decreases until 13530 s.

Cumulative effects of electrode and dielectric surface modifications on pentacene-based transistors

NASA Astrophysics Data System (ADS)

Devynck, Mélanie; Tardy, Pascal; Wantz, Guillaume; Nicolas, Yohann; Vellutini, Luc; Labrugère, Christine; Hirsch, Lionel

2012-01-01

Surface modifications of the dielectric and the metal of pentacene-based field effect transistors using self-assembled monolayer (SAM) were studied. First, a low interfacial trap density and pentacene 2D-growth were favored by the nonpolar and low surface energy of octadecyltrichlorosilane-based SAM. This treatment leaded to increased mobility up to 0.4 cm2 V-1 s-1 and no observable hysteresis on transfer curves. Second, reduced hole injection barrier and contact resistance were achieved by fluorinated thiols deposited on gold contacts resulting in an increased mobility up to 0.6 cm2 V-1 s-1. Finally, a high mobility of 2.6 cm2 V-1 s-1 was achieved by cumulative effects of both treatments.

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

PubMed Central

Ng, Wy Ching; Liong, Stella; Tate, Michelle D.; Irimura, Tatsuro; Denda-Nagai, Kaori; Brooks, Andrew G.; Londrigan, Sarah L.

2014-01-01

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca2+-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca2+-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV. PMID:24257596

Lithium-ion battery electrolyte mobility at nano-confined graphene interfaces

PubMed Central

Moeremans, Boaz; Cheng, Hsiu-Wei; Hu, Qingyun; Garces, Hector F.; Padture, Nitin P.; Renner, Frank Uwe; Valtiner, Markus

2016-01-01

Interfaces are essential in electrochemical processes, providing a critical nanoscopic design feature for composite electrodes used in Li-ion batteries. Understanding the structure, wetting and mobility at nano-confined interfaces is important for improving the efficiency and lifetime of electrochemical devices. Here we use a Surface Forces Apparatus to quantify the initial wetting of nanometre-confined graphene, gold and mica surfaces by Li-ion battery electrolytes. Our results indicate preferential wetting of confined graphene in comparison with gold or mica surfaces because of specific interactions of the electrolyte with the graphene surface. In addition, wetting of a confined pore proceeds via a profoundly different mechanism compared with wetting of a macroscopic surface. We further reveal the existence of molecularly layered structures of the confined electrolyte. Nanoscopic confinement of less than 4–5 nm and the presence of water decrease the mobility of the electrolyte. These results suggest a lower limit for the pore diameter in nanostructured electrodes. PMID:27562148

SnO2 promoted by alkali metal oxides for soot combustion: The effects of surface oxygen mobility and abundance on the activity

NASA Astrophysics Data System (ADS)

Rao, Cheng; Shen, Jiating; Wang, Fumin; Peng, Honggen; Xu, Xianglan; Zhan, Hangping; Fang, Xiuzhong; Liu, Jianjun; Liu, Wenming; Wang, Xiang

2018-03-01

In this study, SnO2-based catalysts promoted by different alkali metal oxides with a Sn/M (M = Li, Na, K, Cs) molar ratio of 9/1 have been prepared for soot combustion. In comparison with the un-modified SnO2 support, the activity of the modified catalysts has been evidently enhanced, following the sequence of CsSn1-9 > KSn1-9 > NaSn1-9 > LiSn1-9 > SnO2. As testified by Raman, H2-TPR, soot-TPR-MS, XPS and O2-TPD results, the incorporation of various alkali metal oxides can induce the formation of more abundant and mobile oxygen species on the surface of the catalysts. Moreover, quantified results have proved that the amount of the surface active oxygen species is nearly proportional to the activity of the catalysts. CsSn1-9, the catalyst promoted by cesium oxide, owns the largest amount of surface mobile oxygen species, thus having the highest activity among all the studied catalysts. It is concluded that the amount of surface active and mobile oxygen species is the major factor determining the activity of the catalysts for soot combustion.

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

PubMed

Buijs; Hlady

1997-06-01

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

Revealing Surface States in In-Doped SnTe Nanoplates with Low Bulk Mobility.

PubMed

Shen, Jie; Xie, Yujun; Cha, Judy J

2015-06-10

Indium (In) doping in topological crystalline insulator SnTe induces superconductivity, making In-doped SnTe a candidate for a topological superconductor. SnTe nanostructures offer well-defined nanoscale morphology and high surface-to-volume ratios to enhance surface effects. Here, we study In-doped SnTe nanoplates, In(x)Sn(1-x)Te, with x ranging from 0 to 0.1 and show they superconduct. More importantly, we show that In doping reduces the bulk mobility of In(x)Sn(1-x)Te such that the surface states are revealed in magnetotransport despite the high bulk carrier density. This is manifested by two-dimensional linear magnetoresistance in high magnetic fields, which is independent of temperature up to 10 K. Aging experiments show that the linear magnetoresistance is sensitive to ambient conditions, further confirming its surface origin. We also show that the weak antilocalization observed in In(x)Sn(1-x)Te nanoplates is a bulk effect. Thus, we show that nanostructures and reducing the bulk mobility are effective strategies to reveal the surface states and test for topological superconductors.

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

PubMed

Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

2016-03-29

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides.

PubMed

Kasai, Taishi; Hamaguchi, Tasuku; Miyata, Makoto

2015-09-01

The binding and gliding of Mycoplasma mobile on a plastic plate covered by 53 uniform oligosaccharides were analyzed. Mycoplasmas bound to and glided on only 21 of the fixed sialylated oligosaccharides (SOs), showing that sialic acid is essential as the binding target. The affinities were mostly consistent with our previous results on the inhibitory effects of free SOs and suggested that M. mobile recognizes SOs from the nonreducing end with four continuous sites as follows. (i and ii) A sialic acid at the nonreducing end is tightly recognized by tandemly connected two sites. (iii) The third site is recognized by a loose groove that may be affected by branches. (iv) The fourth site is recognized by a large groove that may be enhanced by branches, especially those with a negative charge. The cells glided on uniform SOs in manners apparently similar to those of the gliding on mixed SOs. The gliding speed was related inversely to the mycoplasma's affinity for SO, suggesting that the detaching step may be one of the speed determinants. The cells glided faster and with smaller fluctuations on the uniform SOs than on the mixtures, suggesting that the drag caused by the variation in SOs influences gliding behaviors. Mycoplasma is a group of bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide in the direction of the protrusion. These procedures are essential for parasitism. Usually, mycoplasmas glide on mixed sialylated oligosaccharides (SOs) derived from glycoprotein and glycolipid. Since gliding motility on uniform oligosaccharides has never been observed, this study gives critical information about recognition and interaction between receptors and SOs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Physical Biology in Cancer. 3. The role of cell glycocalyx in vascular transport of circulating tumor cells

PubMed Central

Mitchell, Michael J.

2013-01-01

Circulating tumor cells (CTCs) in blood are known to adhere to the luminal surface of the microvasculature via receptor-mediated adhesion, which contributes to the spread of cancer metastasis to anatomically distant organs. Such interactions between ligands on CTCs and endothelial cell-bound surface receptors are sensitive to receptor-ligand distances at the nanoscale. The sugar-rich coating expressed on the surface of CTCs and endothelial cells, known as the glycocalyx, serves as a physical structure that can control the spacing and, thus, the availability of such receptor-ligand interactions. The cancer cell glycocalyx can also regulate the ability of therapeutic ligands to bind to CTCs in the bloodstream. Here, we review the role of cell glycocalyx on the adhesion and therapeutic treatment of CTCs in the bloodstream. PMID:24133067

RNA Aptamer-Based Functional Ligands of the Neurotrophin Receptor, TrkB

PubMed Central

Huang, Yang Zhong; Hernandez, Frank J.; Gu, Bin; Stockdale, Katie R.; Nanapaneni, Kishore; Scheetz, Todd E.; Behlke, Mark A.; Peek, Andrew S.; Bair, Thomas; Giangrande, Paloma H.

2012-01-01

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (KD ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors. PMID:22752556

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

PubMed Central

Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

2013-01-01

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

PubMed Central

Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Bianchi, Marco E.; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

1998-01-01

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors. PMID:9671457

UVA phototransduction drives early melanin synthesis in human melanocytes.

PubMed

Wicks, Nadine L; Chan, Jason W; Najera, Julia A; Ciriello, Jonathan M; Oancea, Elena

2011-11-22

Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% ultraviolet A (UVA) and ~5% ultraviolet B (UVB) at the Earth's surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions [6] that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days [7]. In contrast, UVA causes primarily oxidative damage [8] and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or phospholipase C (PLC) inhibitors or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin [11] is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to 5-fold after 24 hr. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis and may underlie the mechanism of IPD in human skin. Copyright © 2011 Elsevier Ltd. All rights reserved.

A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

PubMed Central

Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I

1989-01-01

Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522

Flow condensation on copper-based nanotextured superhydrophobic surfaces.

PubMed

Torresin, Daniele; Tiwari, Manish K; Del Col, Davide; Poulikakos, Dimos

2013-01-15

Superhydrophobic surfaces have shown excellent ability to promote dropwise condensation with high droplet mobility, leading to enhanced surface thermal transport. To date, however, it is unclear how superhydrophobic surfaces would perform under the stringent flow condensation conditions of saturated vapor at high temperature, which can affect superhydrophobicity. Here, we investigate this issue employing "all-copper" superhydrophobic surfaces with controlled nanostructuring for minimal thermal resistance. Flow condensation tests performed with saturated vapor at a high temperature (110 °C) showed the condensing drops penetrate the surface texture (i.e., attain the Wenzel state with lower droplet mobility). At the same time, the vapor shear helped ameliorate the mobility and enhanced the thermal transport. At the high end of the examined vapor velocity range, a heat flux of ~600 kW m(-2) was measured at 10 K subcooling and 18 m s(-1) vapor velocity. This clearly highlights the excellent potential of a nanostructured superhydrophobic surface in flow condensation applications. The surfaces sustained dropwise condensation and vapor shear for five days, following which mechanical degradation caused a transition to filmwise condensation. Overall, our results underscore the need to investigate superhydrophobic surfaces under stringent and realistic flow condensation conditions before drawing conclusions regarding their performance in practically relevant condensation applications.

CD94/NKG2C is a killer effector molecule in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis.

PubMed

Morel, Esther; Escamochero, Salvador; Cabañas, Rosario; Díaz, Rosa; Fiandor, Ana; Bellón, Teresa

2010-03-01

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, bullous cutaneous diseases with uncertain pathogenesis, although cytotoxic T cells seem to be involved. Natural killer (NK)-like activity has been found in blister infiltrates. Cytotoxic T lymphocytes (CTLs) with NK-like activity (NK-CTLs) have been shown to express T-cell receptors restricted by the HLA-Ib molecule HLA-E. Alternatively, the HLA-E-specific activating receptor CD94/NKG2C can trigger T-cell receptor-independent cytotoxicity in CTLs. Our aim was to test whether HLA-E expression sensitizes keratinocytes to killing by CTLs with NK-like activity and to explore the expression of activating receptors specific for HLA-E in blister cytotoxic lymphocytes. We used flow cytometry and immunohistochemistry to analyze HLA-E expression in keratinocytes from affected skin in patients with SJS, TEN, and other less severe drug-induced exanthemas. The expression of CD94/NKG2C was analyzed by means of flow cytometry in PBMCs and blister cells from patients. PBMCs and blister cells were analyzed for their ability to kill HLA-E-expressing cells. Involvement of CD94/NKG2C in triggering degranulation of cytolytic cells was explored by means of CD107a mobilization assays and standard cytotoxicity chromium release assays. We found that keratinocytes from affected skin expressed HLA-E and that cell-surface HLA-E sensitizes keratinocytes to killing by CD94/NKG2C(+) CTLs. Frequencies of CD94/NKG2C(+) peripheral blood T and NK cells were increased in patients with SJS and TEN during the acute phase. Moreover, activated blister T and NK lymphocytes expressed CD94/NKG2C and were able to degranulate in response to HLA-E(+) cells in an NKG2C-dependent manner. CD94/NKG2C might be involved in triggering cytotoxic lymphocytes in patients with SJS and TEN.

Apollo 15 time and motion study

NASA Technical Reports Server (NTRS)

Kubis, J. F.; Elrod, J. T.; Rusnak, R.; Barnes, J. E.

1972-01-01

A time and motion study of Apollo 15 lunar surface activity led to examination of four distinct areas of crewmen activity. These areas are: an analysis of lunar mobility, a comparative analysis of tasks performed in 1-g training and lunar EVA, an analysis of the metabolic cost of two activities that are performed in several EVAs, and a fall/near-fall analysis. An analysis of mobility showed that the crewmen used three basic mobility patterns (modified walk, hop, side step) while on the lunar surface. These mobility patterns were utilized as adaptive modes to compensate for the uneven terrain and varied soil conditions that the crewmen encountered. A comparison of the time required to perform tasks at the final 1-g lunar EVA training sessions and the time required to perform the same task on the lunar surface indicates that, in almost all cases, it took significantly more time (on the order of 40%) to perform tasks on the moon. This increased time was observed even after extraneous factors (e.g., hardware difficulties) were factored out.

Measuring the lateral charge-carrier mobility in metal-insulator-semiconductor capacitors via Kelvin-probe.

PubMed

Milotti, Valeria; Pietsch, Manuel; Strunk, Karl-Philipp; Melzer, Christian

2018-01-01

We report a Kelvin-probe method to investigate the lateral charge-transport properties of semiconductors, most notably the charge-carrier mobility. The method is based on successive charging and discharging of a pre-biased metal-insulator-semiconductor stack by an alternating voltage applied to one edge of a laterally confined semiconductor layer. The charge carriers spreading along the insulator-semiconductor interface are directly measured by a Kelvin-probe, following the time evolution of the surface potential. A model is presented, describing the device response for arbitrary applied biases allowing the extraction of the lateral charge-carrier mobility from experimentally measured surface potentials. The method is tested using the organic semiconductor poly(3-hexylthiophene), and the extracted mobilities are validated through current voltage measurements on respective field-effect transistors. Our widely applicable approach enables robust measurements of the lateral charge-carrier mobility in semiconductors with weak impact from the utilized contact materials.

Measuring the lateral charge-carrier mobility in metal-insulator-semiconductor capacitors via Kelvin-probe

NASA Astrophysics Data System (ADS)

Milotti, Valeria; Pietsch, Manuel; Strunk, Karl-Philipp; Melzer, Christian

2018-01-01

We report a Kelvin-probe method to investigate the lateral charge-transport properties of semiconductors, most notably the charge-carrier mobility. The method is based on successive charging and discharging of a pre-biased metal-insulator-semiconductor stack by an alternating voltage applied to one edge of a laterally confined semiconductor layer. The charge carriers spreading along the insulator-semiconductor interface are directly measured by a Kelvin-probe, following the time evolution of the surface potential. A model is presented, describing the device response for arbitrary applied biases allowing the extraction of the lateral charge-carrier mobility from experimentally measured surface potentials. The method is tested using the organic semiconductor poly(3-hexylthiophene), and the extracted mobilities are validated through current voltage measurements on respective field-effect transistors. Our widely applicable approach enables robust measurements of the lateral charge-carrier mobility in semiconductors with weak impact from the utilized contact materials.

Phase-field modeling of void anisotropic growth behavior in irradiated zirconium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, G. M.; Wang, H.; Lin, De-Ye

2017-06-01

A three-dimensional (3D) phase field model was developed to study the effects of surface energy and diffusivity anisotropy on void growth behavior in irradiated Zr. The gamma surface energy function, which is used in the phase field model, was developed with the surface energy anisotropy calculated from the molecular dynamics (MD) simulations. It is assumed that vacancies have much larger mobility in c-axis than a- and b- axes while interstitials have much larger mobility in basal plane then that in c-axis. With the model, the equilibrium void morphology and the effect of defect concentrations and defect mobility anisotropy on voidmore » growth behavior were simulated. The simulations demonstrated that 1) The developed phase-field model can correctly reproduce the faceted void morphology predicted by the Wullf construction. 2) With isotropic diffusivity the void prefers to grow on the basal plane. 3) When the vacancy has large mobility along c-axis and interstitial has a large mobility on the basal plane of hexagonal closed packed (hcp) Zr alloys a platelet void grows in c-direction and shrinks on the basal plane, which is in agreement with the experimental observation of void growth behavior in irradiated Zr.« less

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

PubMed

Cai, Haogang; Muller, James; Depoil, David; Mayya, Viveka; Sheetz, Michael P; Dustin, Michael L; Wind, Shalom J

2018-04-30

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

Sliding tethered ligands add topological interactions to the toolbox of ligand–receptor design

PubMed Central

Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

2015-01-01

Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand–receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand–receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering. PMID:26350224

Identification of insulin as a novel retinoic acid receptor-related orphan receptor α target gene.

PubMed

Kuang, Jiangying; Hou, Xiaoming; Zhang, Jinlong; Chen, Yulong; Su, Zhiguang

2014-03-18

Insulin plays an important role in regulation of lipid and glucose metabolism. Retinoic acid receptor-related orphan receptor α (RORα) modulates physiopathological processes such as dyslipidemia and diabetes. In this study, we found overexpression of RORα in INS1 cells resulted in increased expression and secretion of insulin. Suppression of endogenous RORα caused a decrease of insulin expression. Luciferase and electrophoretic mobility shift assay (EMSA) assays demonstrated that RORα activated insulin transcription via direct binding to its promoter. RORα was also observed to regulate BETA2 expression, which is one of the insulin active transfactors. In vivo analyses showed that the insulin transcription is increased by the synthetic RORα agonist SR1078. These findings identify RORα as a transcriptional activator of insulin and suggest novel therapeutic opportunities for management of the disease. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Granulocyte colony-stimulating factor receptor signaling in severe congenital neutropenia, chronic neutrophilic leukemia, and related malignancies.

PubMed

Dwivedi, Pankaj; Greis, Kenneth D

2017-02-01

Granulocyte colony-stimulating factor is a hematopoietic cytokine that stimulates neutrophil production and hematopoietic stem cell mobilization by initiating the dimerization of homodimeric granulocyte colony-stimulating factor receptor. Different mutations of CSF3R have been linked to a unique spectrum of myeloid disorders and related malignancies. Myeloid disorders caused by the CSF3R mutations include severe congenital neutropenia, chronic neutrophilic leukemia, and atypical chronic myeloid leukemia. In this review, we provide an analysis of granulocyte colony-stimulating factor receptor, various mutations, and their roles in the severe congenital neutropenia, chronic neutrophilic leukemia, and malignant transformation, as well as the clinical implications and some perspective on approaches that could expand our knowledge with respect to the normal signaling mechanisms and those associated with mutations in the receptor. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

ELECTROKINETIC PHENOMENA. II : THE FACTOR OF PROPORTIONALITY FOR CATAPHORETIC AND ELECTROENDOSMOTIC MOBILITIES.

PubMed

Abramson, H A

1930-07-20

Two theories which predict different values for the ratio of V(E), the electroendosmotic velocity of a liquid past a surface, to V(p), the electric mobility of a particle of the same surface through the same liquid are discussed. The theory demanding that See PDF for Equation was supported by certain data of van der Grinten for a glass surface. Re-calculation of van der Grinten's data reveals that the ratio varies between 2.1 and 2.8. These results are in accord with previous data of Abramson. It is pointed out that glass is unsuitable for the investigation. The ratio See PDF for Equation is here determined for a flat surface and particles when both are covered by the same proteins. Under these conditions See PDF for Equation The theory is similarly tested for a round surface using a micro-cataphoresis cell. It is shown that See PDF for Equation for a round surface is approximately 1.00. These findings are confirmatory of previous data supporting the view that cataphoretic mobility is independent of the size and shape of the particles when all particles compared have similar surface constitutions.

Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

USDA-ARS?s Scientific Manuscript database

Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface. Copyright © 2017 Elsevier Inc. All rights reserved.

Reconfigurable Robust Routing for Mobile Outreach Network

NASA Technical Reports Server (NTRS)

Lin, Ching-Fang

2010-01-01

The Reconfigurable Robust Routing for Mobile Outreach Network (R3MOO N) provides advanced communications networking technologies suitable for the lunar surface environment and applications. The R3MOON techn ology is based on a detailed concept of operations tailored for luna r surface networks, and includes intelligent routing algorithms and wireless mesh network implementation on AGNC's Coremicro Robots. The product's features include an integrated communication solution inco rporating energy efficiency and disruption-tolerance in a mobile ad h oc network, and a real-time control module to provide researchers an d engineers a convenient tool for reconfiguration, investigation, an d management.

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.

PubMed

Vishnivetskiy, Sergey A; Gimenez, Luis E; Francis, Derek J; Hanson, Susan M; Hubbell, Wayne L; Klug, Candice S; Gurevich, Vsevolod V

2011-07-08

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.

Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins*

PubMed Central

Vishnivetskiy, Sergey A.; Gimenez, Luis E.; Francis, Derek J.; Hanson, Susan M.; Hubbell, Wayne L.; Klug, Candice S.; Gurevich, Vsevolod V.

2011-01-01

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. PMID:21471193

The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

PubMed

Galson, D L; Tsuchiya, T; Tendler, D S; Huang, L E; Ren, Y; Ogura, T; Bunn, H F

1995-04-01

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

The cell surface environment for pathogen recognition and entry.

PubMed

Stow, Jennifer L; Condon, Nicholas D

2016-04-01

The surface of mammalian cells offers an interface between the cell interior and its surrounding milieu. As part of the innate immune system, macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens, debris or dead cells. Their highly dynamic and constantly moving cell surface has extensions such as lamellipodia, filopodia and dorsal ruffles that help detect pathogens. Dorsal ruffles give rise to macropinosomes for rapid, high volume non-selective fluid sampling, receptor internalisation and plasma membrane turnover. Ruffles can also generate phagocytic cups for the receptor-mediated uptake of pathogens or particles. The membrane lipids, actin cytoskeleton, receptors and signalling proteins that constitute these cell surface domains are discussed. Although the cell surface is designed to counteract pathogens, many bacteria, viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival. Nevertheless, these features offer important potential for developing vaccines, drugs and preventative measures to help fight infection.

Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

PubMed Central

Els-Heindl, Sylvia; Chollet, Constance; Scheidt, Holger A.; Beck-Sickinger, Annette G.; Meiler, Jens; Huster, Daniel

2015-01-01

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide’s secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide’s positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8–17 form an α-helix, while residues 21–23 and 26–27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane. PMID:25803439

A gain-of-function screen for genes that influence axon guidance identifies the NF-kappaB protein dorsal and reveals a requirement for the kinase Pelle in Drosophila photoreceptor axon targeting.

PubMed

Mindorff, Elizabeth N; O'Keefe, David D; Labbé, Alain; Yang, Jennie Ping; Ou, Yimiao; Yoshikawa, Shingo; van Meyel, Donald J

2007-08-01

To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We mobilized the Gene Search (GS) P element and identified 42 lines with insertions in unique loci, including leak/roundabout2, which encodes an axon guidance receptor and confirms the utility of our screen. The genes we identified encode proteins of diverse classes, some acting near the cell surface and others in the cytoplasm or nucleus. We found that one GS line drove misexpression of the NF-kappaB transcription factor Dorsal, causing motor axons to bypass their correct termination sites. In the developing visual system, Dorsal misexpression also caused photoreceptor axons to reach incorrect positions within the optic lobe. This mistargeting occurred without observable changes of cell fate and correlated with localization of ectopic Dorsal in distal axons. We found that Dorsal and its inhibitor Cactus are expressed in photoreceptors, though neither was required for axon targeting. However, mutation analyses of genes known to act upstream of Dorsal revealed a requirement for the interleukin receptor-associated kinase family kinase Pelle for layer-specific targeting of photoreceptor axons, validating our screen as a means to identify new molecular determinants of nervous system development in vivo.

Tissue damage negatively regulates LPS-induced macrophage necroptosis.

PubMed

Li, Z; Scott, M J; Fan, E K; Li, Y; Liu, J; Xiao, G; Li, S; Billiar, T R; Wilson, M A; Jiang, Y; Fan, J

2016-09-01

Infection is a common clinical complication following tissue damage resulting from surgery and severe trauma. Studies have suggested that cell pre-activation by antecedent trauma/tissue damage profoundly impacts the response of innate immune cells to a secondary infectious stimulus. Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that control cell release of inflammatory mediators from important innate immune executive cells such as macrophages (Mφ), which critically regulate the progress of inflammation. In this study, we investigated the mechanism and role of trauma/tissue damage in the regulation of LPS-induced Mφ necroptosis using a mouse model simulating long-bone fracture. We demonstrate that LPS acting through Toll-like receptor (TLR) 4 promotes Mφ necroptosis. However, necroptosis is ameliorated by high-mobility group box 1 (HMGB1) release from damaged tissue. We show that HMGB1 acting through cell surface receptor for advanced glycation end products (RAGE) upregulates caveolin-1 expression, which in turn induces caveolae-mediated TLR4 internalization and desensitization to decrease Mφ necroptosis. We further show that RAGE-MyD88 activation of Cdc42 and subsequent activation of transcription factor Sp1 serves as a mechanism underlying caveolin-1 transcriptional upregulation. These results reveal a previous unidentified protective role of damage-associated molecular pattern (DAMP) molecules in restricting inflammation in response to exogenous pathogen-associated molecular pattern molecules.

Tissue damage negatively regulates LPS-induced macrophage necroptosis

PubMed Central

Li, Z; Scott, M J; Fan, E K; Li, Y; Liu, J; Xiao, G; Li, S; Billiar, T R; Wilson, M A; Jiang, Y; Fan, J

2016-01-01

Infection is a common clinical complication following tissue damage resulting from surgery and severe trauma. Studies have suggested that cell pre-activation by antecedent trauma/tissue damage profoundly impacts the response of innate immune cells to a secondary infectious stimulus. Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that control cell release of inflammatory mediators from important innate immune executive cells such as macrophages (Mφ), which critically regulate the progress of inflammation. In this study, we investigated the mechanism and role of trauma/tissue damage in the regulation of LPS-induced Mφ necroptosis using a mouse model simulating long-bone fracture. We demonstrate that LPS acting through Toll-like receptor (TLR) 4 promotes Mφ necroptosis. However, necroptosis is ameliorated by high-mobility group box 1 (HMGB1) release from damaged tissue. We show that HMGB1 acting through cell surface receptor for advanced glycation end products (RAGE) upregulates caveolin-1 expression, which in turn induces caveolae-mediated TLR4 internalization and desensitization to decrease Mφ necroptosis. We further show that RAGE-MyD88 activation of Cdc42 and subsequent activation of transcription factor Sp1 serves as a mechanism underlying caveolin-1 transcriptional upregulation. These results reveal a previous unidentified protective role of damage-associated molecular pattern (DAMP) molecules in restricting inflammation in response to exogenous pathogen-associated molecular pattern molecules. PMID:26943325

Anomalous mobility of highly charged particles in pores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Yinghua; Yang, Crystal; Hinkle, Preston

2015-07-16

Single micropores in resistive-pulse technique were used to understand a complex dependence of particle mobility on its surface charge density. We show that the mobility of highly charged carboxylated particles decreases with the increase of the solution pH due to an interplay of three effects: (i) ion condensation, (ii) formation of an asymmetric electrical double layer around the particle, and (iii) electroosmotic flow induced by the charges on the pore walls and the particle surfaces. The results are important for applying resistive-pulse technique to determine surface charge density and zeta potential of the particles. As a result, the experiments alsomore » indicate the presence of condensed ions, which contribute to the measured current if a sufficiently high electric field is applied across the pore.« less

Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression*

PubMed Central

Coke, Christopher J.; Scarlett, Kisha A.; Chetram, Mahandranauth A.; Jones, Kia J.; Sandifer, Brittney J.; Davis, Ahriea S.; Marcus, Adam I.

2016-01-01

The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. PMID:26841863

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

PubMed

Speranskiy, Kirill; Kurnikova, Maria

2005-08-30

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

Lubricant-infused micro/nano-structured surfaces with tunable dynamic omniphobicity at high temperatures

DOE PAGES

Daniel, Daniel; Mankin, Max N.; Belisle, Rebecca A.; ...

2013-06-10

Omniphobic surfaces that can repel fluids at temperatures higher than 100 °C are rare. Most state-of- the-art liquid-repellent materials are based on the lotus effect, where a thin air layer is maintained throughout micro/nanotextures leading to high mobility of liquids. However, such behavior eventually fails at elevated temperatures when the surface tension of test liquids decreases significantly. Here, we demonstrate a class of lubricant-infused structured surfaces that can maintain a robust omniphobic state even for low-surface-tension liquids at temperatures up to at least 200 °C. We also demonstrate how liquid mobility on such surfaces can be tuned by a factormore » of 1000.« less

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Active radar guides missile to its target: receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems.

PubMed

Yan, Jing-Jun; Liao, Jia-Zhi; Lin, Ju-Sheng; He, Xing-Xing

2015-01-01

Patients with hepatocellular carcinoma (HCC) usually present at advanced stages and do not benefit from surgical resection, so drug therapy should deserve a prominent place in unresectable HCC treatment. But chemotherapy agents, such as doxorubicin, cisplatin, and paclitaxel, frequently encounter important problems such as low specificity and non-selective biodistribution. Recently, the development of nanotechnology led to significant breakthroughs to overcome these problems. Decorating the surfaces of nanoparticulate-based drug carriers with homing devices has demonstrated its potential in concentrating chemotherapy agents specifically to HCC cells. In this paper, we reviewed the current status of active targeting strategies for nanoparticulate systems based on various receptors such as asialoglycoprotein receptor, transferrin receptor, epidermal growth factor receptor, folate receptor, integrin, and CD44, which are abundantly expressed on the surfaces of hepatocytes or liver cancer cells. Furthermore, we pointed out their merits and defects and provided theoretical references for further research.

Peripheral blood stem cell mobilization: the CXCR2 ligand GRObeta rapidly mobilizes hematopoietic stem cells with enhanced engraftment properties.

PubMed

Pelus, Louis M; Fukuda, Seiji

2006-08-01

Chemokines direct the movement of leukocytes, including hematopoietic stem and progenitor cells, and can mobilize hematopoietic cells from marrow to peripheral blood where they can be used for transplantation. In this review, we will discuss the stem cell mobilizing activities and mechanisms of action of GRObeta, a CXC chemokine ligand for the CXCR2 receptor. GRObeta rapidly mobilizes short- and long-term repopulating cells in mice and/or monkeys and synergistically enhances mobilization responses when combined with the widely used clinical mobilizer, granulocyte colony-stimulating factor (G-CSF). The hematopoietic graft mobilized by GRObeta contains significantly more CD34(neg), Sca-1+, c-kit+, lineage(neg) (SKL) cells than the graft mobilized by G-CSF. In mice, stem cells mobilized by GRObeta demonstrate a competitive advantage upon long-term repopulation analysis and restore neutrophil and platelet counts significantly faster than cells mobilized by G-CSF. Even greater advantage in repopulation and restoration of hematopoiesis are observed with stem cells mobilized by the combination of GRObeta and G-CSF. GRObeta-mobilized SKL cells demonstrate enhanced adherence to vascular cell adhesion molecule-1 and VCAM(pos) endothelial cells and home more efficiently to bone marrow in vivo. The marrow homing ability of GRObeta-mobilized cells is less dependent on the CXCR4/SDF-1 axis than cells mobilized by G-CSF. The mechanism of mobilization by GRObeta requires active matrix metalloproteinase-9 (MMP-9), which results from release of pro-MMP-9 from peripheral blood, and marrow neutrophils, which alters the stoichiometry between pro-MMP-9 and its inhibitor tissue inhibitor of metalloproteinase-1, resulting in MMP-9 activation. The efficacy and rapid action of GRObeta and lack of proinflammatory activity make it an attractive agent to supplement mobilization by G-CSF. In addition, GRObeta may also have clinical mobilizing efficacy on its own, reducing the overall time and costs associated with peripheral blood stem cell transplantation.

Interaction of tachykinins with phospholipid membranes: A neutron diffraction study

NASA Astrophysics Data System (ADS)

Darkes, Malcolm J. M.; Davies, Sarah M. A.; Bradshaw, Jeremy P.

Tachykinins are a group of peptides which bind to G-protein-coupled receptors. Receptor affinity appears to depend on different secondary structures of tachykinin which share the same hydrophobic carboxy-terminal sequence, FXGLM. Receptor activation is thought to be due to the carboxy-terminal submerging into the bilayer and the amino-terminal binding on the surface. Binding of tachykinins to phospholipid bilayers may take place both on the aqueous membrane surface and in the hydrophobic region. The two-state equilibrium appears to depend on the surface charge of the membrane. Deuterating substance P and neurokinin A at their carboxy-terminals, our results show two populations of label for each peptide. One is very close to the water-hydrocarbon interface, the other some 13 Å deeper. We report that the bilayer location of the two tachykinins is remarkably similar, thereby inferring that receptor specifity must be controlled by finer levels of structure.

THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS1

PubMed Central

Purdy, Amanda K.; Alvarez-Arias, Diana A.; Oshinsky, Jennifer; James, Ashley M.; Serebriiskii, Ilya; Campbell, Kerry S.

2014-01-01

Stable surface expression of human inhibitory killer cell immunoglobulin-like receptors (KIR) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC-I+ cells. Our recent experiments, however, have found that antibody-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon antibody engagement with the receptor, as compared to untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIR though their ITIM domains. Based upon our results, we propose a model in which non-engaged KIR are internalized by this mechanism, whereas engagement with MHC-I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors. PMID:25238755

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemčovičová, Ivana; Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava; Zajonc, Dirk M., E-mail: dzajonc@liai.org

2014-03-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155more » as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions are evolutionary conserved.« less

Conceptual site models as a tool in evaluating ecological health: the case of the Department of Energy's Amchitka Island nuclear test site.

PubMed

Burger, Joanna; Mayer, Henry J; Greenberg, Michael; Powers, Charles W; Volz, Conrad D; Gochfeld, Michael

2006-07-01

Managers of contaminated sites are faced with options ranging from monitoring natural attenuation to complete removal of contaminants to meet residential health standards. Conceptual site models (CSMs) are one tool used by the U.S. Department of Energy (DOE) and other environmental managers to understand, track, help with decisions, and communicate with the public about the risk from contamination. CSMs are simplified graphical representations of the sources, releases, transport and exposure pathways, and receptors, along with possible barriers to interdict pathways and reduce exposure. In this article, three CSMs are created using Amchitka Island, where the remaining contamination is from underground nuclear test shot cavities containing large quantities of numerous radionuclides in various physical and chemical forms: (1) a typical underground nuclear test shot CSM (modeled after other sites), (2) an expanded CSM with more complex receptors, and (3) a regional CSM that takes into account contaminant pathways from sources other than Amchitka. The objective was to expand the CSM used by DOE to be more responsive to different types of receptors. Amchitka Island differs from other DOE test shot sites because it is surrounded by a marine environment that is highly productive and has a high biodiversity, and the source of contamination is underground, not on the surface. The surrounding waters of the Bering Sea and North Pacific Ocean are heavily exploited by commercial fisheries and provide the United States and other countries with a significant proportion of its seafood. It is proposed that the CSMs on Amchitka Island should focus more on the pathways of exposure and critical receptors, rather than sources and blocks. Further, CSMs should be incorporated within a larger regional model because of the potentially rapid transport within ocean ecosystems. The large number of migratory or highly mobile species that pass by Amchitka provide the potential for a direct pathway to the local human population, known as Aleut, and commercial fisheries, which are remote from the island itself. The exposure matrix for receptors requires expansion for the Amchitka Island ecosystem because of the valuable marine and seafood resources in the region. CSMs with an expanded exposure/receptor matrix can be used effectively to clarify the conceptualization of the problem for scientists, regulators, and the general public.

Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34+ cells mobilized by G-CSF and plerixafor.

PubMed

Uchida, Naoya; Bonifacino, Aylin; Krouse, Allen E; Metzger, Mark E; Csako, Gyorgy; Lee-Stroka, Agnes; Fasano, Ross M; Leitman, Susan F; Mattapallil, Joseph J; Hsieh, Matthew M; Tisdale, John F; Donahue, Robert E

2011-07-01

Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor produces significant mobilization of CD34(+) cells in rhesus macaques. We sought to evaluate whether these CD34(+) cells can stably reconstitute blood cells with lentiviral gene marking. We performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34(+) cells transduced with a lentiviral vector, and these data were compared with those of G-CSF and stem cell factor mobilization. G-CSF and plerixafor mobilization resulted in CD34(+) cell yields that were twofold higher than yields with G-CSF and stem cell factor. CD123 (interleukin-3 receptor) expression was greater in G-CSF and plerixafor-mobilized CD34(+) cells when compared to G-CSF alone. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages, similar to animals who received G-CSF and stem cell factor-mobilized grafts. Lymphocyte engraftment was accelerated in animals receiving the G-CSF and plerixafor-mobilized CD34(+) cells. One animal in the G-CSF and plerixafor group developed cold agglutinin-associated skin rash during the first 3 months of rapid lymphocyte recovery. One year after transplantation, all animals had 2% to 10% transgene expression in all blood cell lineages. G-CSF and plerixafor-mobilized CD34(+) cells accelerate lymphocyte engraftment and contain hematopoietic stem cell capable of reconstituting multilineage blood cells. These findings indicate important differences to consider in plerixafor-based hematopoietic stem cell mobilization protocols in rhesus macaques. Published by Elsevier Inc.

Combined effect of polarity and pH on the chromatographic behavior of some angiotensin II receptor antagonists and optimization of their determination in pharmaceutical dosage forms.

PubMed

Demiralay, Ebru Cubuk; Cubuk, Burcu; Ozkan, Sibel A; Alsancak, Guleren

2010-11-02

In the present study, the combined effect of mobile phase polarity and pH on retention behavior of some ARA-IIs (irbesartan, losartan, valsartan and telmisartan) is investigated. The linear relationships established between retention factors of the species and the polarity parameter of the mobile phase has proved to predict accurately retention in LC as a function of the acetonitrile content (50%, 55%, 60%, v/v). The suggested model uses the pH value in the acetonitrile-water mixture as mobile phase instead of pH value in water and takes into account the effect of activity coefficients. Moreover, correlation between retention and the mobile phase pH can be established allowing prediction of the retention behavior as a function of the mobile phase pH. The model can be used to estimate the pKa in an acetonitrile percentage between 50% and 60%, at 30 degrees C. The developed method was successfully applied to both the simultaneous separation of these drug-active compounds and individual determination in their commercial pharmaceutical dosage forms.

Size, weight and position: ion mobility spectrometry and imaging MS combined.

PubMed

Kiss, András; Heeren, Ron M A

2011-03-01

Size, weight and position are three of the most important parameters that describe a molecule in a biological system. Ion mobility spectrometry is capable of separating molecules on the basis of their size or shape, whereas imaging mass spectrometry is an effective tool to measure the molecular weight and spatial distribution of molecules. Recent developments in both fields enabled the combination of the two technologies. As a result, ion-mobility-based imaging mass spectrometry is gaining more and more popularity as a (bio-)analytical tool enabling the determination of the size, weight and position of several molecules simultaneously on biological surfaces. This paper reviews the evolution of ion-mobility-based imaging mass spectrometry and provides examples of its application in analytical studies of biological surfaces.

Thermodynamics of cell adhesion. II. Freely mobile repellers.

PubMed Central

Torney, D C; Dembo, M; Bell, G I

1986-01-01

The equilibrium adhesion of a cell or vesicle to a substrate is analyzed in a theoretical model in which two types of mobile molecules in the cell membrane are of interest: receptors that can form bonds with fixed ligands in the substrate and repellers that repel the substrate. If the repulsion between the repeller molecule and substrate is greater than kT, there is substantial redistribution of the repellers from the contact area. Coexisting equilibrium states are observed having comparable free energies (a) with unstretched bonds and repeller redistribution and (b) with stretched bonds and partial redistribution. PMID:3955182

Use pattern of pesticides and their predicted mobility into shallow groundwater and surface water bodies of paddy lands in Mahaweli river basin in Sri Lanka.

PubMed

Aravinna, Piyal; Priyantha, Namal; Pitawala, Amarasooriya; Yatigammana, Sudharma K

2017-01-02

Pesticides applied on agricultural lands reach groundwater by leaching, and move to offsite water bodies by direct runoff, erosion and spray drift. Therefore, an assessment of the mobility of pesticides in water resources is important to safeguard such resources. Mobility of pesticides on agricultural lands of Mahaweli river basin in Sri Lanka has not been reported to date. In this context, the mobility potential of 32 pesticides on surface water and groundwater was assessed by widely used pesticide risk indicators, such as Attenuation Factor (AF) index and the Pesticide Impact Rating Index (PIRI) with some modifications. Four surface water bodies having greater than 20% land use of the catchment under agriculture, and shallow groundwater table at 3.0 m depth were selected for the risk assessment. According to AF, carbofuran, quinclorac and thiamethoxam are three most leachable pesticides having AF values 1.44 × 10 -2 , 1.87 × 10 -3 and 5.70 × 10 -4 , respectively. Using PIRI, offsite movement of pesticides by direct runoff was found to be greater than with the erosion of soil particles for the study area. Carbofuran and quinclorac are most mobile pesticides by direct runoff with runoff fractions of 0.01 and 0.08, respectively, at the studied area. Thiamethoxam and novaluron are the most mobile pesticides by erosion with erosion factions of 1.02 × 10 -4 and 1.05 × 10 -4 , respectively. Expected pesticide residue levels in both surface and groundwater were predicted to remain below the USEPA health advisory levels, except for carbofuran, indicating that pesticide pollution is unlikely to exceed the available health guidelines in the Mahaweli river basin in Sri Lanka.

Electron Tomography Imaging of Surface Glycoproteins on Human Parainfluenza Virus 3: Association of Receptor Binding and Fusion Proteins before Receptor Engagement

PubMed Central

Gui, Long; Jurgens, Eric M.; Ebner, Jamie L.

2015-01-01

ABSTRACT In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a “heads-down” configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended “heads-up” form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN’s globular head does HN transmit its activating signal to F. PMID:25691596

Surface Expression of NMDA Receptor Changes during Memory Consolidation in the Crab "Neohelice granulata"

ERIC Educational Resources Information Center

Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin; Pedreira, Maria Eugenia; Freudenthal, Ramiro

2016-01-01

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab "Neohelice granulata". Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of…

Nanomechanical mapping of first binding steps of a virus to animal cells

NASA Astrophysics Data System (ADS)

Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

2017-02-01

Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). We present theoretical approaches to contour the free-energy landscape of early binding events between an engineered virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising "Hot-Spot" Lysine Residues.

PubMed

van den Biggelaar, Maartje; Madsen, Jesper J; Faber, Johan H; Zuurveld, Marleen G; van der Zwaan, Carmen; Olsen, Ole H; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

2015-07-03

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

PubMed

Kumeta, Masahiro; Panina, Yulia; Yamazaki, Hiroya; Takeyasu, Kunio; Yoshimura, Shige H

2018-06-13

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

The Reovirus Sigmal Aspartic Acid Sandwich: A Trimerization Motif Poised for Conformational Change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schelling,P.; Guglielml, K.; Kirchner, E.

2007-01-01

Reovirus attachment protein {sigma}1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The {sigma}1 protein is a filamentous, trimeric molecule with a globular {beta}-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the {sigma}1 subunit interface. A 1.75 {angstrom} structure of the {sigma}1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate thatmore » these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the {sigma}1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.« less

Dynamic conformational switching in the chemokine ligand is essential for G-protein-coupled receptor activation

PubMed Central

Joseph, Prem Raj B.; Sawant, Kirti V.; Isley, Angela; Pedroza, Mesias; Garofalo, Roberto P.; Richardson, Ricardo M.; Rajarathnam, Krishna

2014-01-01

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. Mutations in the GP motif caused various differences from native-like function to complete loss of activity that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity. PMID:24032673

Modelling and assessment of the electric field strength caused by mobile phone to the human head.

PubMed

Buckus, Raimondas; Strukcinskiene, Birute; Raistenskis, Juozas; Stukas, Rimantas

2016-06-01

Electromagnetic field exposure is the one of the most important physical agents that actively affects live organisms and environment. Active use of mobile phones influences the increase of electromagnetic field radiation. The aim of the study was to measure and assess the electric field strength caused by mobile phones to the human head. In this paper the software "COMSOL Multiphysics" was used to establish the electric field strength created by mobile phones around the head. The second generation (2G) Global System for Mobile (GSM) phones that operate in the frequency band of 900 MHz and reach the power of 2 W have a stronger electric field than (2G) GSM mobile phones that operate in the higher frequency band of 1,800 MHz and reach the power up to 1 W during conversation. The third generation of (3G) UMTS smart phones that effectively use high (2,100 MHz) radio frequency band emit the smallest electric field strength values during conversation. The highest electric field strength created by mobile phones is around the ear, i.e. the mobile phone location. The strength of mobile phone electric field on the phantom head decreases exponentially while moving sidewards from the center of the effect zone (the ear), and constitutes 1-12% of the artificial head's surface. The highest electric field strength values of mobile phones are associated with their higher power, bigger specific energy absorption rate (SAR) and lower frequency of mobile phone. The stronger electric field emitted by the more powerful mobile phones takes a higher percentage of the head surface. The highest electric field strength created by mobile phones is distributed over the user's ear.

Toward the Understanding of MNEI Sweetness from Hydration Map Surfaces

PubMed Central

De Simone, Alfonso; Spadaccini, Roberta; Temussi, Piero A.; Fraternali, Franca

2006-01-01

The binding mechanism of sweet proteins to their receptor, a G-protein-coupled receptor, is not supported by direct structural information. In principle, the key groups responsible for biological activity (glucophores) can be localized on a small structural unit (sweet finger) or spread on a larger surface area. A recently proposed model, called “wedge model”, implies a large surface of interaction with the receptor. To explore this model in greater detail, it is necessary to examine the physicochemical features of the surfaces of sweet proteins, since their interaction with the receptor, with respect to that of small sweeteners, is more dependent on general physicochemical properties of the interface, such as electrostatic potential and hydration. In this study, we performed exhaustive molecular dynamics simulations in explicit water of the sweet protein MNEI and of its structural mutant G-16A, whose sweetness is one order of magnitude lower than that of MNEI. Solvent density and self-diffusion calculated from molecular dynamics simulations suggest a likely area of interaction delimited by four stretches arranged as a tetrahedron whose shape is complementary to that of a cavity on the surface of the receptor, in agreement with the wedge model. The suggested area of interaction is amazingly consistent with known mutagenesis data. In addition, the asymmetric hydration of the only helix in both proteins hints at a specific role for this secondary structure element in orienting the protein during the binding process. PMID:16461400

World Language Students' Ethnographic Investigations of Culture through Mobile Devices

ERIC Educational Resources Information Center

Tuttle, Harry G.; Tuttle, Lori A.

2017-01-01

World language teachers can transform how their students learn culture through the use of mobile devices. When world language students use their mobile devices to access authentic current culture, they go from being passive receivers of culture to active cultural investigators. These students go from learning thin surface culture to exploring…

Noise Stress Induces an Epidermal Growth Factor Receptor/Xeroderma Pigmentosum-A Response in the Auditory Nerve.

PubMed

Guthrie, O'neil W

2017-03-01

In response to toxic stressors, cancer cells defend themselves by mobilizing one or more epidermal growth factor receptor (EGFR) cascades that employ xeroderma pigmentosum-A (XPA) to repair damaged genes. Recent experiments discovered that neurons within the auditory nerve exhibit basal levels of EGFR+XPA co-expression. This finding implied that auditory neurons in particular or neurons in general have the capacity to mobilize an EGFR+XPA defense. Therefore, the current study tested the hypothesis that noise stress would alter the expression pattern of EGFR/XPA within the auditory nerve. Design-based stereology was used to quantify the proportion of neurons that expressed EGFR, XPA, and EGFR+XPA with and without noise stress. The results revealed an intricate neuronal response that is suggestive of alterations to both co-expression and individual expression of EGFR and XPA. In both the apical and middle cochlear coils, the noise stress depleted EGFR+XPA expression. Furthermore, there was a reduction in the proportion of neurons that expressed XPA-alone in the middle coils. However, the noise stress caused a significant increase in the proportion of neurons that expressed EGFR-alone in the middle coils. The basal cochlear coils failed to mobilize a significant response to the noise stress. These results suggest that EGFR and XPA might be part of the molecular defense repertoire of the auditory nerve.

Noise Stress Induces an Epidermal Growth Factor Receptor/Xeroderma Pigmentosum–A Response in the Auditory Nerve

PubMed Central

Guthrie, O’neil W.

2017-01-01

In response to toxic stressors, cancer cells defend themselves by mobilizing one or more epidermal growth factor receptor (EGFR) cascades that employ xeroderma pigmentosum–A (XPA) to repair damaged genes. Recent experiments discovered that neurons within the auditory nerve exhibit basal levels of EGFR+XPA co-expression. This finding implied that auditory neurons in particular or neurons in general have the capacity to mobilize an EGFR+XPA defense. Therefore, the current study tested the hypothesis that noise stress would alter the expression pattern of EGFR/XPA within the auditory nerve. Design-based stereology was used to quantify the proportion of neurons that expressed EGFR, XPA, and EGFR+XPA with and without noise stress. The results revealed an intricate neuronal response that is suggestive of alterations to both co-expression and individual expression of EGFR and XPA. In both the apical and middle cochlear coils, the noise stress depleted EGFR+XPA expression. Furthermore, there was a reduction in the proportion of neurons that expressed XPA-alone in the middle coils. However, the noise stress caused a significant increase in the proportion of neurons that expressed EGFR-alone in the middle coils. The basal cochlear coils failed to mobilize a significant response to the noise stress. These results suggest that EGFR and XPA might be part of the molecular defense repertoire of the auditory nerve. PMID:28056182

Comprehensive Reactive Receiver Modeling for Diffusive Molecular Communication Systems: Reversible Binding, Molecule Degradation, and Finite Number of Receptors.

PubMed

Ahmadzadeh, Arman; Arjmandi, Hamidreza; Burkovski, Andreas; Schober, Robert

2016-10-01

This paper studies the problem of receiver modeling in molecular communication systems. We consider the diffusive molecular communication channel between a transmitter nano-machine and a receiver nano-machine in a fluid environment. The information molecules released by the transmitter nano-machine into the environment can degrade in the channel via a first-order degradation reaction and those that reach the receiver nano-machine can participate in a reversible bimolecular reaction with receiver receptor proteins. Thereby, we distinguish between two scenarios. In the first scenario, we assume that the entire surface of the receiver is covered by receptor molecules. We derive a closed-form analytical expression for the expected received signal at the receiver, i.e., the expected number of activated receptors on the surface of the receiver. Then, in the second scenario, we consider the case where the number of receptor molecules is finite and the uniformly distributed receptor molecules cover the receiver surface only partially. We show that the expected received signal for this scenario can be accurately approximated by the expected received signal for the first scenario after appropriately modifying the forward reaction rate constant. The accuracy of the derived analytical results is verified by Brownian motion particle-based simulations of the considered environment, where we also show the impact of the effect of receptor occupancy on the derived analytical results.

Measurement and Visualization of Tight Rock Exposed to CO2 Using NMR Relaxometry and MRI

PubMed Central

Wang, Haitao; Lun, Zengmin; Lv, Chengyuan; Lang, Dongjiang; Ji, Bingyu; Luo, Ming; Pan, Weiyi; Wang, Rui; Gong, Kai

2017-01-01

Understanding mechanisms of oil mobilization of tight matrix during CO2 injection is crucial for CO2 enhanced oil recovery (EOR) and sequestration engineering design. In this study exposure behavior between CO2 and tight rock of the Ordos Basin has been studied experimentally by using nuclear magnetic resonance transverse relaxation time (NMR T2) spectrum and magnetic resonance imaging (MRI) under the reservoir pressure and temperature. Quantitative analysis of recovery at the pore scale and visualization of oil mobilization are achieved. Effects of CO2 injection, exposure times and pressure on recovery performance have been investigated. The experimental results indicate that oil in all pores can be gradually mobilized to the surface of rock by CO2 injection. Oil mobilization in tight rock is time-consuming while oil on the surface of tight rock can be mobilized easily. CO2 injection can effectively mobilize oil in all pores of tight rock, especially big size pores. This understanding of process of matrix exposed to CO2 could support the CO2 EOR in tight reservoirs. PMID:28281697

Benthic foraminiferal census data from Mobile Bay, Alabama--counts of surface samples and box cores

USGS Publications Warehouse

Richwine, Kathryn A.; Osterman, Lisa E.

2012-01-01

A study was undertaken in order to understand recent environmental change in Mobile Bay, Alabama. For this study a series of surface sediment and box core samples was collected. The surface benthic foraminiferal data provide the modern baseline conditions of the bay and can be used as a reference for changing paleoenvironmental parameters recorded in the box cores. The 14 sampling locations were chosen in the bay to cover the wide diversity of fluvial and marine-influenced environments on both sides of the shipping channel.

Highly mobile oxygen holes in magnesium oxide

NASA Technical Reports Server (NTRS)

Freund, Minoru M.; Freund, Friedemann; Batllo, Francois

1989-01-01

High-purity MgO exhibits an unexpected giant anomaly of the apparent static dielectric constant and a positive surface charge of the order of 5 x 10 to the 21st/cu cm in the top 15 nm. It is postulated that the MgO matrix contains traces of peroxy defects, O2(2-), associated with Mg(2+) vacancies. Above approximately 400 C the O2(2-) dissociates to vacancy bound O(-) and highly mobile O(-) states, which diffuse to the surface, giving rise to a high surface conductivity.

Dielectric Modulation of Ion Transport near Interfaces

NASA Astrophysics Data System (ADS)

Antila, Hanne S.; Luijten, Erik

2018-03-01

Ion mobility and ionic conductance in nanodevices are known to deviate from bulk behavior, a phenomenon often attributed to surface effects. We demonstrate that dielectric mismatch between the electrolyte and the surface can qualitatively alter ionic transport in a counterintuitive manner. Instead of following the polarization-induced modulation of the concentration profile, mobility is enhanced or reduced by changes in the ionic atmosphere near the interface and affected by a polarization force parallel to the surface. In addition to revealing this mechanism, we explore the effect of salt concentration and electrostatic coupling.

Effects of self-assembled monolayer structural order, surface homogeneity and surface energy on pentacene morphology and thin film transistor device performance.

PubMed

Hutchins, Daniel Orrin; Weidner, Tobias; Baio, Joe; Polishak, Brent; Acton, Orb; Cernetic, Nathan; Ma, Hong; Jen, Alex K-Y

2013-01-04

A systematic study of six phosphonic acid (PA) self-assembled monolayers (SAMs) with tailored molecular structures is performed to evaluate their effectiveness as dielectric modifying layers in organic field-effect transistors (OFETs) and determine the relationship between SAM structural order, surface homogeneity, and surface energy in dictating device performance. SAM structures and surface properties are examined by near edge X-ray absorption fine structure (NEXAFS) spectroscopy, contact angle goniometry, and atomic force microscopy (AFM). Top-contact pentacene OFET devices are fabricated on SAM modified Si with a thermally grown oxide layer as a dielectric. For less ordered methyl- and phenyl-terminated alkyl ~(CH 2 ) 12 PA SAMs of varying surface energies, pentacene OFETs show high charge carrier mobilities up to 4.1 cm 2 V -1 s -1 . It is hypothesized that for these SAMs, mitigation of molecular scale roughness and subsequent control of surface homogeneity allow for large pentacene grain growth leading to high performance pentacene OFET devices. PA SAMs that contain bulky terminal groups or are highly crystalline in nature do not allow for a homogenous surface at a molecular level and result in charge carrier mobilities of 1.3 cm 2 V -1 s -1 or less. For all molecules used in this study, no causal relationship between SAM surface energy and charge carrier mobility in pentacene FET devices is observed.

Effects of self-assembled monolayer structural order, surface homogeneity and surface energy on pentacene morphology and thin film transistor device performance

PubMed Central

Hutchins, Daniel Orrin; Weidner, Tobias; Baio, Joe; Polishak, Brent; Acton, Orb; Cernetic, Nathan; Ma, Hong; Jen, Alex K.-Y.

2013-01-01

A systematic study of six phosphonic acid (PA) self-assembled monolayers (SAMs) with tailored molecular structures is performed to evaluate their effectiveness as dielectric modifying layers in organic field-effect transistors (OFETs) and determine the relationship between SAM structural order, surface homogeneity, and surface energy in dictating device performance. SAM structures and surface properties are examined by near edge X-ray absorption fine structure (NEXAFS) spectroscopy, contact angle goniometry, and atomic force microscopy (AFM). Top-contact pentacene OFET devices are fabricated on SAM modified Si with a thermally grown oxide layer as a dielectric. For less ordered methyl- and phenyl-terminated alkyl ~(CH2)12 PA SAMs of varying surface energies, pentacene OFETs show high charge carrier mobilities up to 4.1 cm2 V−1 s−1. It is hypothesized that for these SAMs, mitigation of molecular scale roughness and subsequent control of surface homogeneity allow for large pentacene grain growth leading to high performance pentacene OFET devices. PA SAMs that contain bulky terminal groups or are highly crystalline in nature do not allow for a homogenous surface at a molecular level and result in charge carrier mobilities of 1.3 cm2 V−1 s−1 or less. For all molecules used in this study, no causal relationship between SAM surface energy and charge carrier mobility in pentacene FET devices is observed. PMID:24086795

Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

PubMed Central

Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching

2013-01-01

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443

Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dwyer, B.P.

1991-04-23

The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digestsmore » of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.« less

Colossal positive magnetoresistance in surface-passivated oxygen-deficient strontium titanite.

PubMed

David, Adrian; Tian, Yufeng; Yang, Ping; Gao, Xingyu; Lin, Weinan; Shah, Amish B; Zuo, Jian-Min; Prellier, Wilfrid; Wu, Tom

2015-05-15

Modulation of resistance by an external magnetic field, i.e. magnetoresistance effect, has been a long-lived theme of research due to both fundamental science and device applications. Here we report colossal positive magnetoresistance (CPMR) (>30,000% at a temperature of 2 K and a magnetic field of 9 T) discovered in degenerate semiconducting strontium titanite (SrTiO3) single crystals capped with ultrathin SrTiO3/LaAlO3 bilayers. The low-pressure high-temperature homoepitaxial growth of several unit cells of SrTiO3 introduces oxygen vacancies and high-mobility carriers in the bulk SrTiO3, and the three-unit-cell LaAlO3 capping layer passivates the surface and improves carrier mobility by suppressing surface-defect-related scattering. The coexistence of multiple types of carriers and inhomogeneous transport lead to the emergence of CPMR. This unit-cell-level surface engineering approach is promising to be generalized to others oxides, and to realize devices with high-mobility carriers and interesting magnetoelectronic properties.

Bulk contribution to magnetotransport properties of low-defect-density Bi2Te3 topological insulator thin films

NASA Astrophysics Data System (ADS)

Ngabonziza, P.; Wang, Y.; Brinkman, A.

2018-04-01

An important challenge in the field of topological materials is to carefully disentangle the electronic transport contribution of the topological surface states from that of the bulk. For Bi2Te3 topological insulator samples, bulk single crystals and thin films exposed to air during fabrication processes are known to be bulk conducting, with the chemical potential in the bulk conduction band. For Bi2Te3 thin films grown by molecular beam epitaxy, we combine structural characterization (transmission electron microscopy), chemical surface analysis as function of time (x-ray photoelectron spectroscopy) and magnetotransport analysis to understand the low defect density and record high bulk electron mobility once charge is doped into the bulk by surface degradation. Carrier densities and electronic mobilities extracted from the Hall effect and the quantum oscillations are consistent and reveal a large bulk carrier mobility. Because of the cylindrical shape of the bulk Fermi surface, the angle dependence of the bulk magnetoresistance oscillations is two dimensional in nature.

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

PubMed

Barrera, G J; Tortolero, G Sanchez

2016-01-01

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokines and promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions among TFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulation of PAR-2 by siRNA resulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensins expression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

PubMed Central

Fakih, M G; Murphy, T F; Pattoli, M A; Berenson, C S

1997-01-01

Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties. PMID:9125549

Overexpression of Receptor for Advanced Glycation End Products and High-Mobility Group Box 1 in Human Dental Pulp Inflammation

PubMed Central

Tancharoen, Salunya; Tengrungsun, Tassanee; Suddhasthira, Theeralaksna; Kikuchi, Kiyoshi; Vechvongvan, Nuttavun; Maruyama, Ikuro

2014-01-01

High mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is released into the extracellular space and promotes inflammation. HMGB1 binds to related cell signaling transduction receptors, including receptor for advanced glycation end products (RAGE), which actively participate in vascular and inflammatory diseases. The aim of this study was to examine whether RAGE and HMGB1 are involved in the pathogenesis of pulpitis and investigate the effect of Prevotella intermedia (P. intermedia) lipopolysaccharide (LPS) on RAGE and HMGB1 expression in odontoblast-like cells (OLC-1). RAGE and HMGB1 expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Upregulated expression of RAGE was observed in odontoblasts, stromal pulp fibroblasts-like cells, and endothelial-like cell lining human pulpitis tissue. Strong cytoplasmic HMGB1 immunoreactivity was noted in odontoblasts, whereas nuclear HMGB1 immunoreactivity was seen in stromal pulp fibroblasts-like cells in human pulpitis tissue. LPS stimulated OLC-1 cells produced HMGB1 in a dose-dependent manner through RAGE. HMGB1 translocation towards the cytoplasm and secretion from OLC-1 in response to LPS was inhibited by TPCA-1, an inhibitor of NF-κB activation. These findings suggest that RAGE and HMGB1 play an important role in the pulpal immune response to oral bacterial infection. PMID:25114379

Protein Mobilization in Germinating Mung Bean Seeds Involves Vacuolar Sorting Receptors and Multivesicular Bodies1[W][OA

PubMed Central

Wang, Junqi; Li, Yubing; Lo, Sze Wan; Hillmer, Stefan; Sun, Samuel S.M.; Robinson, David G.; Jiang, Liwen

2007-01-01

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination. PMID:17322331

Low temperature mobility in hafnium-oxide gated germanium p-channel metal-oxide-semiconductor field-effect transistors

NASA Astrophysics Data System (ADS)

Beer, Chris; Whall, Terry; Parker, Evan; Leadley, David; De Jaeger, Brice; Nicholas, Gareth; Zimmerman, Paul; Meuris, Marc; Szostak, Slawomir; Gluszko, Grzegorz; Lukasiak, Lidia

2007-12-01

Effective mobility measurements have been made at 4.2K on high performance high-k gated germanium p-type metal-oxide-semiconductor field effect transistors with a range of Ge/gate dielectric interface state densities. The mobility is successfully modelled by assuming surface roughness and interface charge scattering at the SiO2 interlayer/Ge interface. The deduced interface charge density is approximately equal to the values obtained from the threshold voltage and subthreshold slope measurements on each device. A hydrogen anneal reduces both the interface state density and the surface root mean square roughness by 20%.

In situ investigation of the mobility of small gold clusters on cleaved MgO surfaces

NASA Technical Reports Server (NTRS)

Metois, J. J.; Heinemann, K.; Poppa, H.

1976-01-01

The mobility of small clusters of gold (about 10 A in diameter) on electron-beam-cleaved MgO surfaces was studied by in situ transmission electron microscopy under controlled vacuum and temperature conditions. During the first 10 min following a deposition at room temperature, over 10 per cent of the crystallites moved over short distances (about 20 A) discontinuously, with a velocity greater than 150 A/sec. Eighty per cent of the mobility events were characterized by the avoidance of proximity of other crystallites, and this was tentatively explained as the result of repulsive elastic forces between the interacting crystallites.

Mobile computing device configured to compute irradiance, glint, and glare of the sun

DOEpatents

Gupta, Vipin P; Ho, Clifford K; Khalsa, Siri Sahib

2014-03-11

Described herein are technologies pertaining to computing the solar irradiance distribution on a surface of a receiver in a concentrating solar power system or glint/glare emitted from a reflective entity. A mobile computing device includes at least one camera that captures images of the Sun and the entity of interest, wherein the images have pluralities of pixels having respective pluralities of intensity values. Based upon the intensity values of the pixels in the respective images, the solar irradiance distribution on the surface of the entity or glint/glare corresponding to the entity is computed by the mobile computing device.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

PubMed Central

2012-01-01

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

PubMed

Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

2012-03-02

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

A Methylene Group on C-2 of 24,24-Difluoro-19-nor-1α,25-Dihydroxyvitamin D3 Markedly Increases Bone Calcium Mobilization in vivo

PubMed Central

Flores, Agnieszka; Massarelli, Ilaria; Thoden, James B.; Plum, Lori A.; DeLuca, Hector F.

2015-01-01

Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig-Horner reaction as a key step. Structures and absolute configurations of analogues 3 and 5 were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D3 (1). Most important is that all of the 20S-configured derivatives (4 and 6) had high bone mobilizing activity in vivo. However, in the 20R series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20R molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D. PMID:26630444

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

PubMed Central

Axelrod, D; Koppel, D E; Schlessinger, J; Elson, E; Webb, W W

1976-01-01

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G. PMID:786399

Estimation of coefficient of rolling friction by the evolvent pendulum method

NASA Astrophysics Data System (ADS)

Alaci, S.; Ciornei, F. C.; Ciogole, A.; Ciornei, M. C.

2017-05-01

The paper presents a method for finding the coefficient of rolling friction using an evolvent pendulum. The pendulum consists in a fixed cylindrical body and a mobile body presenting a plane surface in contact with a cylindrical surface. The mobile body is placed over the fixed one in an equilibrium state; after applying a small impulse, the mobile body oscillates. The motion of the body is video recorded and afterwards the movie is analyzed by frames and the decrease with time of angular amplitude of the pendulum is found. The equation of motion is established for oscillations of the mobile body. The equation of motion, differential nonlinear, is integrated by Runge-Kutta method. Imposing the same damping both to model’s solution and to theoretical model, the value of coefficient of rolling friction is obtained. The last part of the paper presents results for actual pairs of materials. The main advantage of the method is the fact that the dimensions of contact regions are small, of order a few millimeters, and thus is substantially reduced the possibility of variation of mechanical characteristic for the two surfaces.

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2.

PubMed

Stokes, Alexander J; Wakano, Clay; Del Carmen, Kimberly A; Koblan-Huberson, Murielle; Turner, Helen

2005-03-01

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

Expression and nutritional regulation of the (pro)renin receptor in rat visceral adipose tissue.

PubMed

Achard, V; Tassistro, V; Boullu-Ciocca, S; Grino, M

2011-12-01

Early life nutritional environment plays an important role in the development of visceral adipose tissue and interacts with nutritional regulations in adulthood, leading to metabolic dysregulations. We hypothesized that the renin-angiotensin system may play a role in the programming-induced development of visceral adipose tissue. We studied, using a model of programming of overweight and glucose intolerance, obtained by post-natal overfeeding with consecutive highfat diet, the status of plasma renin activity and mesenteric adipose renin-angiotensin system, including the recently identified (pro)renin receptor, in adult rats. Post-natal overfeeding or high-fat feeding lead to overweight with increased visceral fat mass and adipocytes surface. When both paradigms were associated, adipocytes surface showed a disproportionate increase. A strong immunoreactivity for (pro)renin receptor was found in stromal cells. Plasma renin activity increased in programmed animals whereas (pro)renin receptor expressing cells density was stimulated by high-fat diet. There was a positive, linear relationship between plasma renin activity and (pro)renin receptor expressing cells density and adipocytes surface. Our experiments demonstrate that association of post-natal overfeeding and high-fat diet increased plasma renin activity and adipose (pro)renin receptor expression. Such phenomenon could explain, at least in part, the associated disproportionate adipocyte hypertrophy and its accompanying increased glucose intolerance.

Intracellular GPCRs Play Key Roles in Synaptic Plasticity.

PubMed

Jong, Yuh-Jiin I; Harmon, Steven K; O'Malley, Karen L

2018-02-16

The trillions of synaptic connections within the human brain are shaped by experience and neuronal activity, both of which underlie synaptic plasticity and ultimately learning and memory. G protein-coupled receptors (GPCRs) play key roles in synaptic plasticity by strengthening or weakening synapses and/or shaping dendritic spines. While most studies of synaptic plasticity have focused on cell surface receptors and their downstream signaling partners, emerging data point to a critical new role for the very same receptors to signal from inside the cell. Intracellular receptors have been localized to the nucleus, endoplasmic reticulum, lysosome, and mitochondria. From these intracellular positions, such receptors may couple to different signaling systems, display unique desensitization patterns, and/or show distinct patterns of subcellular distribution. Intracellular GPCRs can be activated at the cell surface, endocytosed, and transported to an intracellular site or simply activated in situ by de novo ligand synthesis, diffusion of permeable ligands, or active transport of non-permeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in synaptic plasticity and learning and memory. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools.

Nanolubrication: patterned lubricating films using ultraviolet (UV) irradiation on hard disks.

PubMed

Zhang, J; Hsu, S M; Liew, Y F

2007-01-01

Nanolubrication is emerging to be the key technical barrier in many devices. One of the key attributes for successful device lubrication is self-sustainability using only several molecular layers. For single molecular species lubrication, one desires bonding strength and molecular mobility to repair the contact by diffusing back to the contact. One way to achieve this is the use of mask to shield the surface with a patterned surface texture, put a monolayer on the surface and induce bonding. Then re-deposit mobile molecules on the surface to bring the thickness back to the desired thickness. This paper describes the use of long wavelength UV irradiation (320-390 nm) to induce bonding of a perfluoropolyether (PFPE) on CN(x) disks for magnetic hard disk application. This allows the use of irradiation to control the degree of bonding on CN(x) coatings. The effect of induced bonding based on this wavelength was studied by comparing 100% mobile PFPE, 100% bonded PFPE, and a mixture of mobile and bonded PFPE in a series of laboratory tests. Using a lateral force microscope, a diamond-tipped atomic force microscope, and a ball-on-inclined plane apparatus, the friction and wear characteristics of these three cases were obtained. Results suggested that the mixed PFPE has the highest shear rupture strength.

Role of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion.

PubMed

Mainali, Dipak; Syed, Aleem; Arora, Neha; Smith, Emily A

2014-12-01

Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24% increase in the mobile integrin population, (2) 14% of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45% increase in the diameter of the confined zone, and (4) there was a 29% increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.

Multiphase mean curvature flows with high mobility contrasts: A phase-field approach, with applications to nanowires

NASA Astrophysics Data System (ADS)

Bretin, Elie; Danescu, Alexandre; Penuelas, José; Masnou, Simon

2018-07-01

The structure of many multiphase systems is governed by an energy that penalizes the area of interfaces between phases weighted by surface tension coefficients. However, interface evolution laws depend also on interface mobility coefficients. Having in mind some applications where highly contrasted or even degenerate mobilities are involved, for which classical phase field models are inapplicable, we propose a new effective phase field approach to approximate multiphase mean curvature flows with mobilities. The key aspect of our model is to incorporate the mobilities not in the phase field energy (which is conventionally the case) but in the metric which determines the gradient flow. We show the consistency of such an approach by a formal analysis of the sharp interface limit. We also propose an efficient numerical scheme which allows us to illustrate the advantages of the model on various examples, as the wetting of droplets on solid surfaces or the simulation of nanowires growth generated by the so-called vapor-liquid-solid method.

Origin of the relatively low transport mobility of graphene grown through chemical vapor deposition

PubMed Central

Song, H. S.; Li, S. L.; Miyazaki, H.; Sato, S.; Hayashi, K.; Yamada, A.; Yokoyama, N.; Tsukagoshi, K.

2012-01-01

The reasons for the relatively low transport mobility of graphene grown through chemical vapor deposition (CVD-G), which include point defect, surface contamination, and line defect, were analyzed in the current study. A series of control experiments demonstrated that the determinant factor for the low transport mobility of CVD-G did not arise from point defects or surface contaminations, but stemmed from line defects induced by grain boundaries. Electron microscopies characterized the presence of grain boundaries and indicated the polycrystalline nature of the CVD-G. Field-effect transistors based on CVD-G without the grain boundary obtained a transport mobility comparative to that of Kish graphene, which directly indicated the detrimental effect of grain boundaries. The effect of grain boundary on transport mobility was qualitatively explained using a potential barrier model. Furthermore, the conduction mechanism of CVD-G was also investigated using the temperature dependence measurements. This study can help understand the intrinsic transport features of CVD-G. PMID:22468224

Spatial heterogeneity of mobilization processes and input pathways of herbicides into a brook in a small agricultural catchment

NASA Astrophysics Data System (ADS)

Doppler, Tobias; Lück, Alfred; Popow, Gabriel; Strahm, Ivo; Winiger, Luca; Gaj, Marcel; Singer, Heinz; Stamm, Christian

2010-05-01

Soil applied herbicides can be transported from their point of application (agricultural field) to surface waters during rain events. There they can have harmful effects on aquatic species. Since the spatial distribution of mobilization and transport processes is very heterogeneous, the contributions of different fields to the total load in a surface water body may differ considerably. The localization of especially critical areas (contributing areas) can help to efficiently minimize herbicide inputs to surface waters. An agricultural field becomes a contributing area when three conditions are met: 1) herbicides are applied, 2) herbicides are mobilized on the field and 3) the mobilized herbicides are transported rapidly to the surface water. In spring 2009, a controlled herbicide application was performed on corn fields in a small (ca 1 km2) catchment with intensive crop production in the Swiss plateau. Subsequently water samples were taken at different locations in the catchment with a high temporal resolution during rain events. We observed both saturation excess and hortonian overland flow during the field campaign. Both can be important mobilization processes depending on the intensity and quantity of the rain. This can lead to different contributing areas during different types of rain events. We will show data on the spatial distribution of herbicide loads during different types of rain events. Also the connectivity of the fields with the brook is spatially heterogeneous. Most of the fields are disconnected from the brook by internal sinks in the catchment, which prevents surface runoff from entering the brook directly. Surface runoff from these disconnected areas can only enter the brook rapidly via macropore-flow into tile drains beneath the internal sinks or via direct shortcuts to the drainage system (maintenance manholes, farmyard or road drains). We will show spatially distributed data on herbicide concentration in purely subsurface systems which shows how important such input pathways can be.

[The Impact of Electroacupuncture Intervention on Expression of 5-HTR 1 B/2 C Genes in Mice under Radiation Stimulation from Mobile Phone].

PubMed

Dai, Jian-yu; Chen, Yi-guo; Zhang, Xiao-qing

2015-08-01

To observe the effect of electroacupuncture (EA) stimulation of "Yifen" (TE 17), "Shenshu" (BL 23) on the expression of 5-hydroxytryptamine receptor 1 B (5-HTR 1 B) mRNA and 5-hydroxytryptamine receptor 2 C (5-HTR 2 C) mRNA in the cochlear nucleus tissue in mice experiencing radiation from mobile phone, so as to explore its mechanisms underlying improvement of tinnitus. Thirty Kunming mice were randomly divided into control group (n = 6) and modeling group (n = 24). The tinnitus model was established by giving the mice with mobile phone-radiation for 1 h in the morning and 1 h in the afternoon, continuously for 40 days. EA stimulation was applied to "Yifeng" (TE 17) group (n = 6) and "Shenshu" (BL 23) group (n = 6) for 20 min, once a day for 7 days. The expression of 5-THR 1 B/2 C mRNA in the cochlear nucleus was assayed by fluorescence quantitative polymerase chain reaction (real time-PCR). The expression level of 5-HTR 1 B was significantly lower in the model group than in the control group (P < 0.05), while that of 5-HTR 2 C mRNA significantly increased (P < 0.01). TE 17 group received a significant acupoint intervention effect (P < 0.01). Compared with TE 17 group, BL 23 group received a weaker effect (P < 0.05). EA of TE 17 can up-regulate expression level of 5-HTR 1 B and down-regulate expression level of 5-HTR 2 C in the cochlear nucleus in mice experiencing mobile-phone radiation.

Sensory-Targeted Ankle Rehabilitation Strategies for Chronic Ankle Instability.

PubMed

McKeon, Patrick O; Wikstrom, Erik A

2016-05-01

Deficient sensory input from damaged ankle ligament receptors is thought to contribute to sensorimotor deficits in those with chronic ankle instability (CAI). Targeting other viable sensory receptors may then enhance sensorimotor control in these patients. The purpose of this randomized controlled trial was to evaluate the effects of 2 wk of sensory-targeted ankle rehabilitation strategies (STARS) on patient- and clinician-oriented outcomes in those with CAI. Eighty patients with self-reported CAI participated. All patients completed patient-oriented questionnaires capturing self-reported function as well as the weight-bearing lunge test and an eyes-closed single-limb balance test. After baseline testing, patients were randomly allocated to four STARS groups: joint mobilization, plantar massage, triceps surae stretching, or control. Each patient in the intervention groups received six 5-min treatments of their respective STARS over 2 wk. All subjects were reassessed on patient- and clinician-oriented measures immediately after the intervention and completed a 1-month follow-up that consisted of patient-oriented measures. Change scores of the three STARS groups were compared with the control using independent t-tests and Hedges' g effect sizes with 95% confidence intervals. The joint mobilization group had the greatest weight-bearing lunge test improvement. Plantar massage had the most meaningful single-limb balance improvement. All STARS groups improved patient-oriented outcomes with joint mobilization having the most meaningful effect immediately after the intervention and plantar massage at the 1-month follow-up. Each STARS treatment offers unique contributions to the patient- and clinician-oriented rehabilitation outcomes of those with CAI. Both joint mobilization and plantar massage appear to demonstrate the greatest potential to improve sensorimotor function in those with CAI.

Lysophosphatidylcholine-induced cytotoxicity in osteoblast-like MG-63 cells: involvement of transient receptor potential vanilloid 2 (TRPV2) channels.

PubMed

Fallah, Abdallah; Pierre, Rachel; Abed, Elie; Moreau, Robert

2013-01-01

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 μM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.

Inhibition Of Call-Cell Binding By Kipid Assemblies

DOEpatents

Nagy, Jon O. , Bargatze, Robert F.

2003-12-16

This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

Inhibition of cell-cell binding by lipid assemblies

DOEpatents

Nagy, Jon O.; Bargatze, Robert F.

2001-05-22

This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor.

PubMed

Kim, Sun-Gyun; Lee, Bora; Kim, Dae-Hwan; Kim, Juhee; Lee, Seunghee; Lee, Soo-Kyung; Lee, Jae W

2013-10-01

Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

Structural Characterization of Vapor-deposited Organic Glasses

NASA Astrophysics Data System (ADS)

Gujral, Ankit

Physical vapor deposition, a common route of thin film fabrication for organic electronic devices, has recently been shown to produce organic glassy films with enhanced kinetic stability and anisotropic structure. Anisotropic structures are of interest in the organic electronics community as it has been shown that certain structures lead to enhanced device performance, such as higher carrier mobility and better light outcoupling. A mechanism proposed to explain the origin of the stability and anisotropy of vapor-deposited glasses relies on two parameters: 1) enhanced molecular mobility at the free surface (vacuum interface) of a glass, and 2) anisotropic molecular packing at the free surface of the supercooled liquid of the glass-forming system. By vapor-depositing onto a substrate maintained at Tsubstrate < Tg (where Tg is the glass transition temperature), the enhanced molecular mobility at the free surface allows every molecule that lands on the surface to at least partially equilibrate to the preferred anisotropic molecular packing motifs before being buried by further deposition. The extent of equilibration depends on the mobility at the surface, controlled by Tsubstrate, and the residence time on the free surface, controlled by the rate of deposition. This body of work deals with the optimization of deposition conditions and system chemistry to prepare and characterize films with functional anisotropic structures. Here, we show that structural anisotropy can be attained for a variety of molecular systems including a rod-shaped non-mesogen, TPD, a rod-shaped smectic mesogen, itraconazole, two discotic mesogens, phenanthroperylene-ester and triphenylene-ester, and a disc-shaped non-mesogen, m-MTDATA. Experimental evidence is also provided of the anisotropic molecular packing at the free surface (vacuum interface) for the disc-shaped systems that are consistent with the expectations of the proposed mechanism and the final bulk state of the vapor-deposited glasses.

Surface modification of ZnO nanorods with Hamilton receptors.

PubMed

Zeininger, Lukas; Klaumünzer, Martin; Peukert, Wolfgang; Hirsch, Andreas

2015-04-13

A new prototype of a Hamilton receptor suitable for the functionalization of inorganic nanoparticles was synthesized and characterized. The hydrogen bonding receptor was coupled to a catechol moiety, which served as anchor group for the functionalization of metal oxides, in particular zinc oxide. Synthesized zinc oxide nanorods [ZnO] were used for surface functionalization. The wet-chemical functionalization procedure towards monolayer-grafted particles [ZnO-HR] is described and a detailed characterization study is presented. In addition, the detection of specific cyanurate molecules is demonstrated. The hybrid structures [ZnO-HR-CA] were stable towards agglomeration and exhibited enhanced dispersability in apolar solvents. This observation, in combination with several spectroscopic experiments gave evidence of the highly directional supramolecular recognition at the surface of nanoparticles.

Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors.

PubMed

Penn, A C; Zhang, C L; Georges, F; Royer, L; Breillat, C; Hosy, E; Petersen, J D; Humeau, Y; Choquet, D

2017-09-21

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (less than 1 h) had initially been explained either by presynaptic increases in glutamate release or by direct modification of postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional postsynaptic AMPA receptors (AMPARs), sourced either from an intracellular reserve pool by exocytosis or from nearby extra-synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during early LTP remains unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Here, using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion markedly impairs synaptic potentiation of Schaffer collaterals and commissural inputs to the CA1 area of the mouse hippocampus in cultured slices, acute slices and in vivo. Our data also identify distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus inhibited fear conditioning, indicating that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein-protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning.

Transportation Planning and ITS: Putting the Pieces Together

DOT National Transportation Integrated Search

2013-11-01

Both the Dynamic Mobility Applications (DMA) and Active Transportation and Demand Management (ATDM) Programs have similar overarching goals to improve surface transportation system efficiency and individual traveler mobility. However, each program ha...

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Microbial contamination of mobile phones in a health care setting in Alexandria, Egypt.

PubMed

Selim, Heba Sayed; Abaza, Amani Farouk

2015-01-01

This study aimed at investigating the microbial contamination of mobile phones in a hospital setting. Swab samples were collected from 40 mobile phones of patients and health care workers at the Alexandria University Students' Hospital. They were tested for their bacterial contamination at the microbiology laboratory of the High Institute of Public Health. Quantification of bacteria was performed using both surface spread and pour plate methods. Isolated bacterial agents were identified using standard microbiological methods. Methicillin-resistant Staphylococcus aureus was identified by disk diffusion method described by Bauer and Kirby. Isolated Gram-negative bacilli were tested for being extended spectrum beta lactamase producers using the double disk diffusion method according to the Clinical and Laboratory Standards Institute recommendations. All of the tested mobile phones (100%) were contaminated with either single or mixed bacterial agents. The most prevalent bacterial contaminants were methicillin-resistant S. aureus and coagulase-negative staphylococci representing 53% and 50%, respectively. The mean bacterial count was 357 CFU/ml, while the median was 13 CFU/ml using the pour plate method. The corresponding figures were 2,192 and 1,720 organisms/phone using the surface spread method. Mobile phones usage in hospital settings poses a risk of transmission of a variety of bacterial agents including multidrug-resistant pathogens as methicillin-resistant S. aureus. The surface spread method is an easy and useful tool for detection and estimation of bacterial contamination of mobile phones.

Microbial contamination of mobile phones in a health care setting in Alexandria, Egypt

PubMed Central

Selim, Heba Sayed; Abaza, Amani Farouk

2015-01-01

Aim: This study aimed at investigating the microbial contamination of mobile phones in a hospital setting. Methods: Swab samples were collected from 40 mobile phones of patients and health care workers at the Alexandria University Students’ Hospital. They were tested for their bacterial contamination at the microbiology laboratory of the High Institute of Public Health. Quantification of bacteria was performed using both surface spread and pour plate methods. Isolated bacterial agents were identified using standard microbiological methods. Methicillin-resistant Staphylococcus aureus was identified by disk diffusion method described by Bauer and Kirby. Isolated Gram-negative bacilli were tested for being extended spectrum beta lactamase producers using the double disk diffusion method according to the Clinical and Laboratory Standards Institute recommendations. Results: All of the tested mobile phones (100%) were contaminated with either single or mixed bacterial agents. The most prevalent bacterial contaminants were methicillin-resistant S. aureus and coagulase-negative staphylococci representing 53% and 50%, respectively. The mean bacterial count was 357 CFU/ml, while the median was 13 CFU/ml using the pour plate method. The corresponding figures were 2,192 and 1,720 organisms/phone using the surface spread method. Conclusions: Mobile phones usage in hospital settings poses a risk of transmission of a variety of bacterial agents including multidrug-resistant pathogens as methicillin-resistant S. aureus. The surface spread method is an easy and useful tool for detection and estimation of bacterial contamination of mobile phones. PMID:25699226

Enhancement of the p27Kip1-mediated antiproliferative effect of trastuzumab (Herceptin) on HER2-overexpressing tumor cells.

PubMed

Marches, Radu; Uhr, Jonathan W

2004-11-10

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27Kip1-mediated G1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of p27Kip1 upregulation, inhibition of Cdk2 activity and magnitude of G1 arrest induced by the humanized Ab trastuzumab (Herceptin, HCT) on BT474 and SKBr3 HER2-overexpressing breast cancer cells correlates with the level of cell surface receptor. Thus, continuous exposure of cells to HCT for 72 hr results in downregulation of the cell surface receptor and a concurrent increase in the level of p27Kip1 protein. Discontinuation of Ab exposure after the first 8 hr results in failure to upregulate p27Kip1 and arrest of cell cycle progression. We show that the lysosomotropic amine chloroquine (CQ) augments receptor internalization in HER2-overexpressing cells either pretreated or continuously treated with HCT and leads to an increased and sustained inhibitory effect. The enhanced CQ-dependent loss of functional HER2 from the cell surface resulted in sustained inactivation of the serine/threonine kinase Akt, upregulation of p27Kip1 protein and inhibition of cyclin E/Cdk2 activity. Potentiation of the inhibitory effect of HCT by CQ was directly related to loss of HER2 from the plasma membrane since prevention of Ab-mediated receptor endocytosis by engagement of the receptor with immobilized HCT abrogated the effect of CQ.

Dysregulated cellular functions and cell stress pathways provide critical cues for activating and targeting natural killer cells to transformed and infected cells.

PubMed

Raulet, David H; Marcus, Assaf; Coscoy, Laurent

2017-11-01

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DAMPs, ageing, and cancer: The 'DAMP Hypothesis'.

PubMed

Huang, Jin; Xie, Yangchun; Sun, Xiaofang; Zeh, Herbert J; Kang, Rui; Lotze, Michael T; Tang, Daolin

2015-11-01

Ageing is a complex and multifactorial process characterized by the accumulation of many forms of damage at the molecular, cellular, and tissue level with advancing age. Ageing increases the risk of the onset of chronic inflammation-associated diseases such as cancer, diabetes, stroke, and neurodegenerative disease. In particular, ageing and cancer share some common origins and hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury, reprogrammed metabolism, and degradation system impairment (including within the ubiquitin-proteasome system and the autophagic machinery). Recent advances indicate that damage-associated molecular pattern molecules (DAMPs) such as high mobility group box 1, histones, S100, and heat shock proteins play location-dependent roles inside and outside the cell. These provide interaction platforms at molecular levels linked to common hallmarks of ageing and cancer. They can act as inducers, sensors, and mediators of stress through individual plasma membrane receptors, intracellular recognition receptors (e.g., advanced glycosylation end product-specific receptors, AIM2-like receptors, RIG-I-like receptors, and NOD1-like receptors, and toll-like receptors), or following endocytic uptake. Thus, the DAMP Hypothesis is novel and complements other theories that explain the features of ageing. DAMPs represent ideal biomarkers of ageing and provide an attractive target for interventions in ageing and age-associated diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Site-specific colloidal crystal nucleation by template-enhanced particle transport

NASA Astrophysics Data System (ADS)

Mishra, Chandan K.; Sood, A. K.; Ganapathy, Rajesh

2016-10-01

The monomer surface mobility is the single most important parameter that decides the nucleation density and morphology of islands during thin-film growth. During template-assisted surface growth in particular, low surface mobilities can prevent monomers from reaching target sites and this results in a partial to complete loss of nucleation control. Whereas in atomic systems a broad range of surface mobilities can be readily accessed, for colloids, owing to their large size, this window is substantially narrow and therefore imposes severe restrictions in extending template-assisted growth techniques to steer their self-assembly. Here, we circumvented this fundamental limitation by designing templates with spatially varying feature sizes, in this case moiré patterns, which in the presence of short-range depletion attraction presented surface energy gradients for the diffusing colloids. The templates serve a dual purpose: first, directing the particles to target sites by enhancing their surface mean-free paths and second, dictating the size and symmetry of the growing crystallites. Using optical microscopy, we directly followed the nucleation and growth kinetics of colloidal islands on these surfaces at the single-particle level. We demonstrate nucleation control, with high fidelity, in a regime that has remained unaccessed in theoretical, numerical, and experimental studies on atoms and molecules as well. Our findings pave the way for fabricating nontrivial surface architectures composed of complex colloids and nanoparticles as well.

Dietary acai fruit improves cognition and mobility in aged rats

USDA-ARS?s Scientific Manuscript database

Açai is a black-purple fruit (genus Euterpe) cultivated in the Amazon delta and in Brazil (Euterpe oleracea Mart. -EO), as well as Bolivia (Euterpe precatoria Mart. - EP), and it is known to be rich in polyphenolics that may affect cell-to-cell signaling, receptor sensitivity, inflammatory enzyme ac...

Metabotropic glutamate receptor 5 upregulates surface NMDA receptor expression in striatal neurons via CaMKII.

PubMed

Jin, Dao-Zhong; Xue, Bing; Mao, Li-Min; Wang, John Q

2015-10-22

Metabotropic and ionotropic glutamate receptors are closely clustered in postsynaptic membranes and are believed to interact actively with each other to control excitatory synaptic transmission. Metabotropic glutamate receptor 5 (mGluR5), for example, has been well documented to potentiate ionotropic NMDA receptor activity, although underlying mechanisms are poorly understood. In this study, we investigated the role of mGluR5 in regulating trafficking and subcellular distribution of NMDA receptors in adult rat striatal neurons. We found that the mGluR1/5 agonist DHPG concentration-dependently increased NMDA receptor GluN1 and GluN2B subunit expression in the surface membrane. Meanwhile, DHPG reduced GluN1 and GluN2B levels in the intracellular compartment. The effect of DHPG was blocked by an mGluR5 selective antagonist MTEP but not by an mGluR1 selective antagonist 3-MATIDA. Pretreatment with an inhibitor or a specific inhibitory peptide for synapse-enriched Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) also blocked the DHPG-stimulated redistribution of GluN1 and GluN2B. In addition, DHPG enhanced CaMKIIα activity and elevated GluN2B phosphorylation at a CaMKII-sensitive site (serine 1303). These results demonstrate that mGluR5 regulates trafficking of NMDA receptors in striatal neurons. Activation of mGluR5 appears to induce rapid trafficking of GluN1 and GluN2B to surface membranes through a signaling pathway involving CaMKII. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

NASA Astrophysics Data System (ADS)

Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

2015-03-01

Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression.

PubMed

Nelson, Christopher D; Kim, Myung Jong; Hsin, Honor; Chen, Yelin; Sheng, Morgan

2013-07-17

Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β. In organotypic rat hippocampal slices, we find that a nonphosphorylatable PSD-95 mutant (T19A) tagged with photoactivatable green fluorescent protein (PAGFP) shows enhanced stability in dendritic spines versus wild-type PSD-95, whereas the phosphomimetic mutant (PSD-95-T19D) is more readily dispersed. Further, overexpression of PSD-95-T19A, but not WT-PSD-95, impairs AMPA receptor internalization and the induction of LTD. These data indicate that phosphorylation on T19 by GSK-3β destabilizes PSD-95 within the PSD and is a critical step for AMPA receptor mobilization and LTD.

Ergot Alkaloids (Re)generate New Leads as Antiparasitics

PubMed Central

Chan, John D.; Agbedanu, Prince N.; Grab, Thomas; Zamanian, Mostafa; Dosa, Peter I.; Day, Timothy A.; Marchant, Jonathan S.

2015-01-01

Abstract Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors. PMID:26367744

A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

USDA-ARS?s Scientific Manuscript database

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

Ultrastructure studies on the papillae and the nonciliated sensory receptors of adult Spirometra erinacei (Cestoda, Pseudophyllidea).

PubMed

Okino, T; Hatsushika, R

1994-01-01

The small numerous papillae on the ventral surface of the gravid proglottid of adult Spirometra erinacei were studied by scanning electron microscopy. The arrangement of clumps of papillae was recognized on the surface of the central portion around the genital atrium, with lateral clumps being located above a pair of longitudinal nerve cords and marginal ones, on both sides of the proglottid. By transmission electron microscopy, two types of nonciliated sensory receptors were observed within the papillae. The type I, single receptor was embedded within a papilla. This dome-like sensory receptor contained two electron-dense collars and four rootlets surrounded by numerous thin filaments. The type II receptor was found arranged in groups in the area between the papillae, and the apical end was exposed to the external environment. This simple, club-like sensory receptor contained electron-lucent vesicles and microtubules. We believe that the papillae play an important role in cross-insemination.

Receptor-mediated cell mechanosensing

PubMed Central

Chen, Yunfeng; Ju, Lining; Rushdi, Muaz; Ge, Chenghao; Zhu, Cheng

2017-01-01

Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components of force, stress, and strain but also substrate rigidity, topology, and adhesiveness. This ability is crucial for the cell to respond to the surrounding mechanical cues and adapt to the changing environment. Examples of responses and adaptation include (de)activation, proliferation/apoptosis, and (de)differentiation. Receptor-mediated cell mechanosensing is a multistep process that is initiated by binding of cell surface receptors to their ligands on the extracellular matrix or the surface of adjacent cells. Mechanical cues are presented by the ligand and received by the receptor at the binding interface; but their transmission over space and time and their conversion into biochemical signals may involve other domains and additional molecules. In this review, a four-step model is described for the receptor-mediated cell mechanosensing process. Platelet glycoprotein Ib, T-cell receptor, and integrins are used as examples to illustrate the key concepts and players in this process. PMID:28954860

Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

PubMed

Yudowski, Guillermo A; Olsen, Olav; Adesnik, Hillel; Marek, Kurt W; Bredt, David S

2013-01-01

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

Emerging intracellular receptors for hemorrhagic fever viruses.

PubMed

Jae, Lucas T; Brummelkamp, Thijn R

2015-07-01

Ebola virus and Lassa virus belong to different virus families that can cause viral hemorrhagic fever, a life-threatening disease in humans with limited treatment options. To infect a target cell, Ebola and Lassa viruses engage receptors at the cell surface and are subsequently shuttled into the endosomal compartment. Upon arrival in late endosomes/lysosomes, the viruses trigger membrane fusion to release their genome into the cytoplasm. Although contact sites at the cell surface were recognized for Ebola virus and Lassa virus, it was postulated that Ebola virus requires a critical receptor inside the cell. Recent screens for host factors identified such internal receptors for both viruses: Niemann-Pick disease type C1 protein (NPC1) for Ebola virus and lysosome-associated membrane protein 1 (LAMP1) for Lassa virus. A cellular trigger is needed to permit binding of the viral envelope protein to these intracellular receptors. This 'receptor switch' represents a previously unnoticed step in virus entry with implications for host-pathogen interactions and viral tropism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

NASA Astrophysics Data System (ADS)

Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

2013-11-01

Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

In Vitro and In Vivo Measurement of Percutaneous Penetration of Low Molecular Weight Toxins of Military Interest

DTIC Science & Technology

1989-12-15

epidermal surfaces were exposed to ambient air (220C) during the entire length of the experiment. Penetration of [3H]PbTx-3 into skin layers and receptor...bathed by the receptor fluid and the epidermal surface was exposed to ambient conditions in an en- vironmental chamber. Temperature and relative...DMSO or water. The epidermal surfaces were exposed to ambient conditions in the environmental chamber. In order to determine if constituents leaching

Impact of input data uncertainty on environmental exposure assessment models: A case study for electromagnetic field modelling from mobile phone base stations.

PubMed

Beekhuizen, Johan; Heuvelink, Gerard B M; Huss, Anke; Bürgi, Alfred; Kromhout, Hans; Vermeulen, Roel

2014-11-01

With the increased availability of spatial data and computing power, spatial prediction approaches have become a standard tool for exposure assessment in environmental epidemiology. However, such models are largely dependent on accurate input data. Uncertainties in the input data can therefore have a large effect on model predictions, but are rarely quantified. With Monte Carlo simulation we assessed the effect of input uncertainty on the prediction of radio-frequency electromagnetic fields (RF-EMF) from mobile phone base stations at 252 receptor sites in Amsterdam, The Netherlands. The impact on ranking and classification was determined by computing the Spearman correlations and weighted Cohen's Kappas (based on tertiles of the RF-EMF exposure distribution) between modelled values and RF-EMF measurements performed at the receptor sites. The uncertainty in modelled RF-EMF levels was large with a median coefficient of variation of 1.5. Uncertainty in receptor site height, building damping and building height contributed most to model output uncertainty. For exposure ranking and classification, the heights of buildings and receptor sites were the most important sources of uncertainty, followed by building damping, antenna- and site location. Uncertainty in antenna power, tilt, height and direction had a smaller impact on model performance. We quantified the effect of input data uncertainty on the prediction accuracy of an RF-EMF environmental exposure model, thereby identifying the most important sources of uncertainty and estimating the total uncertainty stemming from potential errors in the input data. This approach can be used to optimize the model and better interpret model output. Copyright © 2014 Elsevier Inc. All rights reserved.

An Rgd Sequence in the P2y2 Receptor Interacts with αVβ3 Integrins and Is Required for Go-Mediated Signal Transduction

PubMed Central

Erb, Laurie; Liu, Jun; Ockerhausen, Jonathan; Kong, Qiongman; Garrad, Richard C.; Griffin, Korey; Neal, Chris; Krugh, Brent; Santiago-Pérez, Laura I.; González, Fernando A.; Gresham, Hattie D.; Turner, John T.; Weisman, Gary A.

2001-01-01

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go. PMID:11331301

Beta-adrenergic and atrial natriuretic peptide interactions on human cardiovascular and metabolic regulation

PubMed Central

Birkenfeld, Andreas L.; Boschmann, Michael; Moro, Cedric; Adams, Frauke; Heusser, Karsten; Tank, Jens; Diedrich, André; Schroeder, Christoph; Franke, Gabi; Berlan, Michel; Luft, Friedrich C.; Lafontan, Max; Jordan, Jens

2006-01-01

Context Atrial natriuretic peptide (ANP) has well known cardiovascular effects and modifies lipid and carbohydrate metabolism in humans. Objective To determine the metabolic and cardiovascular interaction of beta-adrenergic receptors and ANP. Design Cross over study, conducted 2004–2005 Setting Academic clinical research center Patients Ten healthy, young, male subjects (BMI 24±1 kg/m2) Intervention We infused intravenously incremental ANP doses (6.25, 12.5, and 25 ng/kg/min) with and without propranolol (0.20 mg/kg in divided doses followed by 0.033 mg/kg/h infusion). Metabolism was monitored through venous blood sampling, intramuscular and subcutaneous microdialysis and indirect calorimetry. Cardiovascular changes where monitored by continuous ECG and beat-by-beat blood pressure recordings. Main outcome measures Venous NEFA, glycerol, glucose, insulin; microdialysate glucose, glycerol, lactate, pyruvate. Results ANP increased heart rate dose dependently. Beta-adrenergic receptor blockade abolished the response. ANP elicited a dose-dependent increase in serum non-esterified fatty acid and glycerol concentrations. The response was not suppressed with propranolol. Venous glucose and insulin concentrations increased with ANP, both, without or with propranolol. ANP induced lipid mobilization in subcutaneous adipose tissue. In skeletal muscle, microdialysate lactate increased while the lactate to pyruvate ratio decreased, both, with and without propranolol. Higher ANP doses increased lipid oxidation while energy expenditure remained unchanged. Propranolol tended to attenuate the increase in lipid oxidation. Conclusions Selected cardiovascular ANP effects are at least partly mediated by beta-adrenergic receptor stimulation. ANP induced changes in lipid mobilization and glycolysis are mediated by another mechanism, presumably stimulation of natriuretic peptide receptors whereas substrate oxidation might be modulated through adrenergic mechanisms. PMID:16984990

Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas.

PubMed

Souazé, Frédérique; Viardot-Foucault, Véronique; Roullet, Nicolas; Toy-Miou-Leong, Mireille; Gompel, Anne; Bruyneel, Erik; Comperat, Eva; Faux, Maree C; Mareel, Marc; Rostène, William; Fléjou, Jean-François; Gespach, Christian; Forgez, Patricia

2006-04-01

Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.

Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression.

PubMed

Coke, Christopher J; Scarlett, Kisha A; Chetram, Mahandranauth A; Jones, Kia J; Sandifer, Brittney J; Davis, Ahriea S; Marcus, Adam I; Hinton, Cimona V

2016-05-06

The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans.

PubMed

Caers, Jelle; Janssen, Tom; Van Rompay, Liesbeth; Broeckx, Valérie; Van Den Abbeele, Jan; Gäde, Gerd; Schoofs, Liliane; Beets, Isabel

2016-03-01

Adipokinetic hormones (AKH) are well known regulators of energy metabolism in insects. These neuropeptides are produced in the corpora cardiaca and perform their hormonal function by interacting with specific G protein-coupled receptors (GPCRs) at the cell membranes of target tissues, mainly the fat body. Here, we investigated the sequences, spatial and temporal distributions, and pharmacology of AKH neuropeptides and receptors in the tsetse fly, Glossina morsitans morsitans. The open reading frames of two splice variants of the Glomo-akh receptor (Glomo-akhr) gene and of the AKH neuropeptide encoding genes, gmmhrth and gmmakh, were cloned. Both tsetse AKHR isoforms show strong sequence conservation when compared to other insect AKHRs. Glomo-AKH prepropeptides also have the typical architecture of AKH precursors. In an in vitro Ca(2+) mobilization assay, Glomo-AKH neuropeptides activated each receptor isoform up to nanomolar concentrations. We identified structural features of tsetse AKH neuropeptides essential for receptor activation in vitro. Gene expression profiles suggest a function for AKH signaling in regulating Glossina energy metabolism, where AKH peptides are released from the corpora cardiaca and activate receptors mainly expressed in the fat body. This analysis of the ligand-receptor coupling, expression, and pharmacology of the two Glomo-AKHR variants facilitates further elucidation of the function of AKH in G. m. morsitans. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interactions of endothelin-1 with dexamethasone in primary cultured human trabecular meshwork cells.

PubMed

Zhang, Xinyu; Clark, Abbot F; Yorio, Thomas

2003-12-01

Concentrations of aqueous humor endothelin (ET)-1 are increased in patients with primary open-angle glaucoma (POAG) as well as in animal models of glaucoma. Glucocorticoids have also been associated with glaucoma, in that topical administration of glucocorticoids can increase intraocular pressure by increasing outflow resistance in the trabecular meshwork (TM) in some individuals. Recent research has shown that dexamethasone (Dex), a synthetic glucocorticoid, can increase the release of ET-1 from human nonpigmented ciliary epithelial (HNPE) cells, a source of aqueous ET-1. In the present study, the downstream interaction of ET-1 with Dex in target TM cells, an action that may alter outflow resistance, was investigated. A normal primary human TM (NTM) cell line and a TM cell line derived from a glaucomatous eye (GTM) were used. The cells were treated with vehicle or Dex. The mRNA levels of prepro-ET-1, endothelin receptor A (ET(A)), and endothelin receptor B (ET(B)) were measured by quantitative RT-PCR (QPCR). The protein expression of ET(A) and ET(B) receptors were investigated by Western blot analysis using polyclonal anti-ET(A) and anti-ET(B) antibodies, respectively, on plasma membrane fractions. Intracellular Ca(2+) ([Ca(2+)](i)) mobilization mediated by ET-1 was measured using the Fura-2 AM fluorescent probe technique as an index of ET receptor function. ET-1-stimulated nitric oxide (NO) release was measured using a Griess colorimetric NO synthase assay kit. Both NTM and GTM cultured cells expressed prepro-ET-1 mRNA less abundantly than did HNPE cells, and Dex treatment had no effect on the mRNA expression of the ET-1 gene. TM cells expressed mRNA of ET(A) receptors as detected by QPCR, whereas the ET(B) message was not clearly delineated. Western blot analysis showed that both ET(A) and ET(B) receptor proteins were present. The ET(A) receptor was linked to calcium mobilization as ET-1 produced an increase in intracellular calcium release, and this increase was blocked with a selective ET(A) receptor antagonist. Dex failed to induce any change in the expression of the ET(A) receptor in both NTM and GTM cells, and this was supported by the absence of a Dex effect on the ET-1-induced calcium response. However, Dex treatment diminished ET(B) receptor protein expression and produced a decrease in ET-1-stimulated release of NO, a response mediated by ET(B) receptors in TM cells. The Dex-induced increase in ET-1 released by HNPE cells coupled to the downstream Dex-induced specific suppression of ET(B) receptor protein expression and declines in ET-1-mediated increase in NO released by TM cells could increase contraction and decrease relaxation of the TM and contribute to the declines in conventional aqueous humor outflow and increases in intraocular pressure that occur with glucocorticoids.

An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport.

PubMed

van der Vorm, E R; van der Zon, G C; Möller, W; Krans, H M; Lindhout, D; Maassen, J A

1992-01-05

In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.

Analysis of Helium Segregation on Surfaces of Plasma-Exposed Tungsten

NASA Astrophysics Data System (ADS)

Maroudas, Dimitrios; Hu, Lin; Hammond, Karl; Wirth, Brian

2015-11-01

We report a systematic theoretical and atomic-scale computational study of implanted helium segregation on surfaces of tungsten, which is considered as a plasma facing component in nuclear fusion reactors. We employ a hierarchy of atomic-scale simulations, including molecular statics to understand the origin of helium surface segregation, targeted molecular-dynamics (MD) simulations of near-surface cluster reactions, and large-scale MD simulations of implanted helium evolution in plasma-exposed tungsten. We find that small, mobile helium clusters (of 1-7 He atoms) in the near-surface region are attracted to the surface due to an elastic interaction force. This thermodynamic driving force induces drift fluxes of these mobile clusters toward the surface, facilitating helium segregation. Moreover, the clusters' drift toward the surface enables cluster reactions, most importantly trap mutation, at rates much higher than in the bulk material. This cluster dynamics has significant effects on the surface morphology, near-surface defect structures, and the amount of helium retained in the material upon plasma exposure.

Functional characterization of c-Mpl ectodomain mutations that underlie congenital amegakaryocytic thrombocytopenia.

PubMed

Varghese, Leila N; Zhang, Jian-Guo; Young, Samuel N; Willson, Tracy A; Alexander, Warren S; Nicola, Nicos A; Babon, Jeffrey J; Murphy, James M

2014-02-01

Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

Surface-water, ground-water, and sediment geochemistry of epizonal and shear-hosted mineral deposits in the Tintina Gold Province--arsenic and antimony distribution and mobility: Chapter G in Recent U.S. Geological Survey studies in the Tintina Gold Province, Alaska, United States, and Yukon, Canada--results of a 5-year project

USGS Publications Warehouse

Mueller, Seth H.; Goldfarb, Richard J.; Verplanck, Philip L.; Trainor, Thomas P.; Sanzolone, Richard F.; Adams, Monique; Gough, Larry P.; Day, Warren C.

2007-01-01

Epigenetic mineral deposits in the Tintina Gold Province are generally characterized by high concentrations of arsenic and antimony in their mineral assemblage. A total of 347 samples (ground water, surface water, and stream sediment) were collected to investigate the distribution and mobility of arsenic and antimony in the environment near known mineral deposits. Samples were collected from east to west at Keno Hill and Brewery Creek, Yukon, Canada; and Cleary Hill, True North, Scrafford Mine, Fairbanks, Ryan Lode, Stampede Creek, Slate Creek, and Donlin Creek, all in Alaska. Surface- and ground-water samples are all slightly acidic to near-neutral in pH (5-8), have a wide range in specific conductance (surface water 17-2,980 microsiemens per centimeter and ground water 170-2,940 microsiemens per centimeter), and show elevated dissolved arsenic and antimony concentrations (arsenic in surface water is less than 1 to 380 micrograms per liter and in ground water is less than 1 micrograms per liter to 1.5 milligrams per liter; antimony in surface water is less than 2 to 660 micrograms per liter and in ground water is less than 2 to 60 micrograms per liter). Stream sediments downstream from these deposits have high concentrations of arsenic and antimony (arsenic median is 1,670 parts per million, maximum is 10,000 parts per million; antimony median is 192 parts per million, maximum is 7,200 parts per million). The mobility of arsenic and antimony is controlled by the local redox environment, with arsenic being less mobile in oxidized surface waters relative to antimony, and arsenic more mobile in reduced ground water. These factors suggest that both antimony and arsenic may be useful pathfinder elements in water and sediment for targeting similar style deposits elsewhere in the Tintina Gold Province.

Mobile-bearing knees reduce rotational asymmetric wear.

PubMed

Ho, Fang-Yuan; Ma, Hon-Ming; Liau, Jiann-Jong; Yeh, Chuan-Ren; Huang, Chun-Hsiung

2007-09-01

Polyethylene wear of bearing components is the most common long-term complication in total knee arthroplasty. One would anticipate differing kinematics would generate different wear patterns (including wear type, degree, and symmetry) on the articulating surface of mobile-bearing and fixed-bearing inserts. Because mobile-bearing designs facilitate movement of the insert relative to the tray when the knee rotates, we hypothesized mobile-bearing designs would reduce the incidence of rotational asymmetric wear. We examined 51 worn tibial inserts, including 15 from mobile-bearing rotating-platform posterior-cruciate-sacrificing dished prostheses and 36 from fixed-bearing posterior-cruciate-retaining flat prostheses, which were retrieved at revision surgery with an average implantation time of 115 months. We divided wear types into low-grade wear (burnishing, abrasion, and cold flow) and high-grade wear (scratching, pitting, metal embedding, and delamination) to assess wear degree of polyethylene. To assess symmetry of wear, the insert surface was divided into medial and lateral sides and each side was further divided into three equal zones along the anteroposterior direction. Low-grade wear was more common in mobile-bearing knees, whereas high-grade wear was more common in fixed-bearing knees. We identified no internal/external rotational asymmetric wear or anteroposterior asymmetric wear in mobile-bearing knees.

Novel Deployment of Mobile Eddy Covariance Tower Observations Across Variations in the Built Environment in a Desert Urban Area

DTIC Science & Technology

2015-11-03

author(s) and should not contrued as an official Department of the Army position, policy or decision, unless so designated by other documentation. 9...Grass; 6 Undeveloped-Open Land (generally gravel or bare soil); Pavement ; Buildings and Concrete; and Residential Buildings (TG-ECT site only...pervious surfaces, while pavement , buildings and concrete, and residential buildings represent impervious surfaces. 2.3. Mobile Eddy Covariance Tower

Segregation Behavior of Sulfur and Other Impurities Onto the Free Surfaces of ED-Ni Deposits

NASA Technical Reports Server (NTRS)

Panda, Binayak; Jerman, Gregory; Gentz, Steven J. (Technical Monitor)

2000-01-01

Most researchers attribute grain boundary embrittlement in electro-deposited Nickel (ED-Ni) to the presence of small quantities of Sulfur as an impurity. It occurs in a highly mobile form that segregates to the grain boundaries. Evaluation of Sulfur segregation requires that a sample be fractured through the grain boundaries. However, this action may not always be possible. ED-Ni is inherently tough at ambient temperature, especially if a low level of Sulfur was intentionally maintained. A new method was developed to study Sulfur and other migrant species to the grain boundaries, which also migrate to free surfaces. A test specimen is heated by a quartz lamp within the sample preparation chamber, allowing the mobile species to migrate to polished free surfaces. There the mobile species are analyzed using X-ray photoelectron spectroscopy (XPS) also known as Electron Spectroscopy for Chemical Analysis (ESCA).

Segregation Behavior of Sulfur and Other Impurities onto the Free Surfaces of ED-NI Deposits

NASA Technical Reports Server (NTRS)

Panda, B.; Jerman, G.

2001-01-01

Most researchers attribute grain boundary embrittlement in electro-deposited nickel (ED-Ni) to the presence of small quantities of sulfur as an impurity. It occurs in a highly mobile form that segregates to the grain boundaries. Evaluation of sulfur segregation requires that a sample be fractured through the grain boundaries. However, this action may not always be possible. ED-Ni is inherently tough at ambient temperature, especially if a low level of sulfur was intentionally maintained. A new method was developed to study sulfur and other migrant species to the grain boundaries, which also migrate to free surfaces. A test specimen is heated by a quartz lamp within the sample preparation chamber, allowing the mobile species to migrate to polished free surfaces. There the mobile species are analyzed using X-ray photoelectron spectroscopy (XPS) also known as Electron Spectroscopy for Chemical Analysis (ESCA).

Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

PubMed

Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

2015-06-01

Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. Copyright © 2015. Published by Elsevier B.V.

Surface Energy Balance System (SEBS) Handbook

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, DR

2011-02-14

A Surface Energy Balance System (SEBS) has been installed collocated with each deployed ECOR system at the Southern Great Plains (SGP), North Slope of Alaska (NSA), Tropical Western Pacific (TWP), ARM Mobile Facility 1 (AMF1), and ARM Mobile Facility 2 (AMF2). The surface energy balance system consists of upwelling and downwelling solar and infrared radiometers within one net radiometer, a wetness sensor, and soil measurements. The SEBS measurements allow the comparison of ECOR sensible and latent heat fluxes with the energy balance determined from the SEBS and provide information on wetting of the sensors for data quality purposes.

Micro faraday-element array detector for ion mobility spectroscopy

DOEpatents

Gresham, Christopher A [Albuquerque, NM; Rodacy, Phillip J [Albuquerque, NM; Denton, M Bonner [Tucson, AZ; Sperline, Roger [Tucson, AZ

2004-10-26

An ion mobility spectrometer includes a drift tube having a collecting surface covering a collecting area at one end of the tube. The surface comprises a plurality of closely spaced conductive elements on a non-conductive substrate, each conductive element being electrically insulated from each other element. A plurality of capacitive transimpedance amplifiers (CTIA) adjacent the collecting surface are electrically connected to the plurality of elements, so charge from an ion striking an element is transferred to the capacitor of the connected CTIA. A controller counts the charge on the capacitors over a period of time.

Quantitative Characterization of Shear-Induced Platelet Receptor Shedding: Glycoprotein Ibα, Glycoprotein VI, and Glycoprotein IIb/IIIa.

PubMed

Chen, Zengsheng; Koenig, Steven C; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

2017-11-07

The structural integrity of platelet receptors is essential for platelets to play the normal hemostatic function. The high non-physiologic shear stress (NPSS) commonly exists in blood-contacting medical devices and has been shown to cause platelet receptor shedding. The loss of platelet receptors may impair the normal hemostatic function of platelets. The aim of this study was to quantify NPSS-induced shedding of three key receptors on the platelet surface. Human blood was subjected to the matrix of well-defined shear stresses and exposure times, generated by using a custom-designed blood-shearing device. The expression of three key platelet receptors, glycoprotein (GP) Ibα, GPVI, and GPIIb/IIIa, in sheared blood was quantified using flow cytometry. The quantitative relationship between the loss of each of the three receptors on the platelet surface and shear condition (shear stress level and exposure time) was explored. It was found that these relationships followed well the power law functional form. The coefficients of the power law models for the shear-induced shedding of these platelet receptors were derived with coefficients of determination (R) of 0.77, 0.73, and 0.78, respectively. The power law models with these coefficients may be potentially used to predict the shear-induced platelet receptor shedding of human blood.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

PubMed

Moringo, Nicholas A; Shen, Hao; Tauzin, Lawrence J; Wang, Wenxiao; Bishop, Logan D C; Landes, Christy F

2017-10-17

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content. Single-molecule tracking indicates lysozyme loading capacities, and surface mobility at the polymer interface is hindered as a result of all functionalization techniques. Adsorption dynamics of lysozyme depend on the extent and the specificity of the oxygen functionalities introduced to the polystyrene surface. Hindered adsorption and mobility are dominated by hydrophobic effects attributed to water hydration layer formation at the functionalized polystyrene surfaces.

Juno is the egg Izumo receptor and is essential for mammalian fertilisation

PubMed Central

Bianchi, Enrica; Doe, Brendan; Goulding, David; Wright, Gavin J.

2014-01-01

Fertilisation occurs when sperm and egg recognise each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell surface protein, but its egg receptor has remained a mystery. Here, we identify Juno as the receptor for Izumo1 on mouse eggs, and show this interaction is conserved within mammals. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilisation suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives. PMID:24739963

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yue; Sheng, Ju; Baggen, Jim

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells.

PubMed

Babakinejad, Babak; Jönsson, Peter; López Córdoba, Ainara; Actis, Paolo; Novak, Pavel; Takahashi, Yasufumi; Shevchuk, Andrew; Anand, Uma; Anand, Praveen; Drews, Anna; Ferrer-Montiel, Antonio; Klenerman, David; Korchev, Yuri E

2013-10-01

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.

Three cysteine residues of SLC52A1, a receptor for the porcine endogenous retrovirus-A (PERV-A), play a critical role in cell surface expression and infectivity.

PubMed

Colon-Moran, Winston; Argaw, Takele; Wilson, Carolyn A

2017-07-01

Porcine endogenous retrovirus-A (PERV-A), a gammaretrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation. Published by Elsevier Inc.

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaramillo, Maria L.; Leon, Zully; Grothe, Suzanne

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of {sup 125}I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized {sup 125}I-225 mAb is recycled to the surface much more efficiently than internalized {sup 125}I-EGF. Also, we found that internalization of {sup 125}I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidencedmore » by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.« less

Autoantibodies to Synaptic Receptors and Neuronal Cell Surface Proteins in Autoimmune Diseases of the Central Nervous System

PubMed Central

Geis, Christian; Graus, Francesc

2017-01-01

Investigations in the last 10 years have revealed a new category of neurological diseases mediated by antibodies against cell surface and synaptic proteins. There are currently 16 such diseases all characterized by autoantibodies against neuronal proteins involved in synaptic signaling and plasticity. In clinical practice these findings have changed the diagnostic and treatment approach to potentially lethal, but now treatable, neurological and psychiatric syndromes previously considered idiopathic or not even suspected to be immune-mediated. Studies show that patients' antibodies can impair the surface dynamics of the target receptors eliminating them from synapses (e.g., NMDA receptor), block the function of the antigens without changing their synaptic density (e.g., GABAb receptor), interfere with synaptic protein-protein interactions (LGI1, Caspr2), alter synapse formation (e.g., neurexin-3α), or by unclear mechanisms associate to a new form of tauopathy (IgLON5). Here we first trace the process of discovery of these diseases, describing the triggers and symptoms related to each autoantigen, and then review in detail the structural and functional alterations caused by the autoantibodies with special emphasis in those (NMDA receptor, amphiphysin) that have been modeled in animals. PMID:28298428

Crystal Structure of an LSD-Bound Human Serotonin Receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wacker, Daniel; Wang, Sheng; McCorvy, John D.

The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR—a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatlymore » accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.« less

Crystal Structure of an LSD-Bound Human Serotonin Receptor.

PubMed

Wacker, Daniel; Wang, Sheng; McCorvy, John D; Betz, Robin M; Venkatakrishnan, A J; Levit, Anat; Lansu, Katherine; Schools, Zachary L; Che, Tao; Nichols, David E; Shoichet, Brian K; Dror, Ron O; Roth, Bryan L

2017-01-26

The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT 2B . The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD's key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT 2B R and 5-HT 2A R-a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD's slow binding kinetics may be due to a "lid" formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD's binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD's actions at human serotonin receptors. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

PubMed Central

Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J

2017-01-01

Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with AMPARs in artificial expression systems, but it is unknown whether Shisa7 has a functional role in glutamatergic synapses. We show that Shisa7 physically interacts with synaptic AMPARs in mouse hippocampus. Shisa7 gene deletion resulted in faster AMPAR currents in CA1 synapses, without affecting its synaptic expression. Shisa7 KO mice showed reduced initiation and maintenance of long-term potentiation of glutamatergic synapses. In line with this, Shisa7 KO mice showed a specific deficit in contextual fear memory, both short-term and long-term after conditioning, whereas auditory fear memory and anxiety-related behavior were normal. Thus, Shisa7 is a bona-fide AMPAR modulatory protein affecting channel kinetics of AMPARs, necessary for synaptic hippocampal plasticity, and memory recall. PMID:29199957

Nutritional Signaling via Free Fatty Acid Receptors

PubMed Central

Miyamoto, Junki; Hasegawa, Sae; Kasubuchi, Mayu; Ichimura, Atsuhiko; Nakajima, Akira; Kimura, Ikuo

2016-01-01

Excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises. Dysfunction of energy balance by excess food intake leads to metabolic diseases, such as obesity and diabetes. Free fatty acids (FFAs) provided by dietary fat are not only important nutrients, but also contribute key physiological functions via FFA receptor (FFAR)-mediated signaling molecules, which depend on FFAs’ carbon chain length and the ligand specificity of the receptors. Functional analyses have revealed that FFARs are critical for metabolic functions, such as peptide hormone secretion and inflammation, and contribute to energy homeostasis. In particular, recent studies have shown that the administration of selective agonists of G protein-coupled receptor (GPR) 40 and GPR120 improved glucose metabolism and systemic metabolic disorders. Furthermore, the anti-inflammation and energy metabolism effects of short chain FAs have been linked to the activation of GPR41 and GPR43. In this review, we summarize recent progress in research on FFAs and their physiological roles in the regulation of energy metabolism. PMID:27023530

The rigidity and mobility of screw dislocations in a thin film

NASA Astrophysics Data System (ADS)

Wang, Fei

2018-07-01

An equation of screw dislocations in a thin film is derived for arbitrary boundary conditions. The boundary conditions can be the free surface, the fixed surface or the gradient loading imposed on the surface. The new equation makes it possible to study changes in the dislocation structure under various gradient stress applied to the surface. The rigidity and mobility of screw dislocations in a thin film are explored by using the equation. It is found that the screw dislocation core in a thin film is like a Hookean body with a specific shear stress applied to the surface. Free-surface effects on the Peierls stress are investigated and compared with previous studies. An abnormal behavior of the Peierls stress of screw dislocations in a soft-inclusion film between two rigid films is predicted theoretically.

Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4.

PubMed

Ma, Fei; Kouzoukas, Dimitrios E; Meyer-Siegler, Katherine L; Westlund, Karin N; Hunt, David E; Vera, Pedro L

2017-05-25

Bladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes. Disulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 μg/150 μl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 μg/150 μl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 μg/150 μl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 μg/150 μl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect. The disulfide form of HMGB1 mediates bladder pain directly (not secondary to inflammation or injury) through activation of TLR4 receptors in the bladder. Thus, TLR4 receptors are a specific local target for bladder pain.

Effects of the surface mobility on the oxidation of adsorbed CO on platinum electrodes in alkaline media. The role of the adlayer and surface defects.

PubMed

Herrero, Enrique; Chen, Qing-Song; Hernández, Javier; Sun, Shi-Gang; Feliu, Juan M

2011-10-06

The oxidation of adsorbed CO on Pt single crystal electrodes has been studied in alkaline media. The surfaces used in this study were the Pt(111) electrode and vicinal stepped and kinked surfaces with (111) terraces. The kinked surfaces have either (110) steps broken by (100) kinks or (100) steps broken by (110) kinks and different kink densities. The voltammetric profiles for the CO stripping on those electrodes show peaks corresponding to the oxidation of CO on the (111) terraces, on the (100) steps/kinks and on the (110) steps/kinks at very distinctive potentials. Additionally, the stripping voltammograms always present a prewave. The analysis of the results with the different stepped and kinked surfaces indicates that the presence of the prewave is not associated with defects or kinks in the electrode surface. Also, the clear separation of the CO stripping process in different peak contributions indicates that the mobility of CO on the surface is very low. Using partial CO stripping experiments and studies at different pH, it has been proposed that the low mobility is a consequence of the negative absolute potential at which the adlayers are formed in alkaline media. Also, the surface diffusion coefficient for CO in these media has been estimated from the dependence of the stripping charge of the peaks with the scan rate of the voltammetry.

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells.

PubMed

Boensch, C; Huang, S S; Connolly, D T; Huang, J S

1999-04-09

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.

Clear microstructure-performance relationships in Mn-containing perovskite and hexaaluminate compounds prepared by activated reactive synthesis.

PubMed

Laassiri, Said; Bion, Nicolas; Duprez, Daniel; Royer, Sébastien; Alamdari, Houshang

2014-03-07

Microstructural properties of mixed oxides play essential roles in their oxygen mobility and consequently in their catalytic performances. Two families of mixed oxides (perovskite and hexaaluminate) with different microstructural features, such as crystal size and specific surface area, were prepared using the activated reactive synthesis (ARS) method. It was shown that ARS is a flexible route to synthesize both mixed oxides with nano-scale crystal size and high specific surface area. Redox properties and oxygen mobility were found to be strongly affected by the material microstructure. Catalytic activities of hexaaluminate and perovskite materials for methane oxidation were discussed in the light of structural, redox and oxygen mobility properties.

Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors.

PubMed

Sánchez-Más, Jesús; Guillo, Lidia A; Zanna, Paola; Jiménez-Cervantes, Celia; García-Borrón, José C

2005-04-01

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2

PubMed Central

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V1) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys8]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg8]-vasopressin (AVP) at V1 and vasopressin-2 (V2) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V1 and V2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [3H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V1) and cyclic adenosine monophosphate (V2). Binding potency at V1 and V2 was AVP>LVP>>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V1 than for V2. Cellular activity potency was also AVP>LVP>>terlipressin. Terlipressin was a partial agonist at V1 and a full agonist at V2; LVP was a full agonist at both V1 and V2. The in vivo response to terlipressin is likely due to the partial V1 agonist activity of terlipressin and full V1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors. PMID:29302194